Application of Chinese herbaceous peony extract in inhibition of AGEs-RAGE combination and preparation method of preparation containing Chinese herbaceous peony extract

Abstract

The invention provides an application of a paeonia lactiflora extract in preparation of a preparation for inhibiting combination of advanced glycation end products (AGEs) and receptors (RAGE) thereof, the application is also used for inhibiting expression of RAGE genes, and the paeonia lactiflora extract is prepared by the following steps: extracting from roots of paeonia lactiflora by using a 1, 3-butanediol solution as an extraction solvent. According to the present invention, the combination of AGEs and RAGE can be inhibited, and the novel preparation is mainly suitable for human skin and the like.

Description

Technical Field

[0001] The present invention relates to the application of paeony extract in inhibiting the binding of AGEs-RAGE and a preparation method of a preparation containing the same. Background Art

[0002] Advanced glycation end products (AGEs, hereinafter also referred to as "AGEs") are a general term for substances formed by the binding of proteins or lipids to reducing sugars such as glucose and fructose through various non-enzymatic reactions such as the Maillard reaction (Non-Patent Document 1). This process is called glycation. Glycated proteins in living organisms are difficult to decompose due to their structural changes and tend to accumulate by deviating from the normal decomposition pathway. The association between the accumulation of AGEs and diseases such as diabetes and atherosclerosis has been reported (Non-Patent Document 2).

[0003] In the skin, collagen, elastin, etc. with a relatively long half-life in the dermis layer are glycated, and keratin, etc. in the epidermis layer are glycated. This glycation leads to a decrease in the quality of these substances, and further causes problems such as wrinkles and thickening of the cutin. In addition, some AGEs are yellowish, and are considered to be one of the reasons for dull and yellowish skin. Therefore, cosmetic raw materials that inhibit the production of AGEs for the purpose of improving beauty are being studied (Patent Document 1).

[0004] AGEs not only reduce the quality of proteins, but also bind to the receptor for AGEs (RAGE, hereinafter also referred to as "RAGE"), triggering biological reactions such as diabetic complications, chronic inflammation (Non-Patent Document 3), melanogenesis (Non-Patent Document 4), cell proliferation inhibition, and adhesion inhibition (Non-Patent Document 5, Non-Patent Document 6). Therefore, research on cosmetic raw materials with the effect of inhibiting the production of AGEs is constantly advancing.

[0005] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2020-193182

[0006] Non-Patent Document 1: Ott C, Jacobs K, Haucke E, Navarrete Santos A, Grune T, Simm A. Role of advanced glycation end products in cellular signaling. Redox Biol. 2014;2(1):411-429. doi:10.1016 / j.redox.2013.12.016 Non - Patent Document 2: Reddy VP, Aryal P, Darkwah EK. Advanced Glycation EndProducts in Health and Disease. Microorganisms. 2022;10(9). doi:10.3390 / microorganisms10091848 Non - Patent Document 3: Park HJ, Boyington JC. The 1.5A crystal structure of humanreceptor for advanced glycation endproducts (RAGE) ectodomains reveals uniquefeatures determining ligand binding. J Biol Chem. 2010;285(52):40762 - 40770. doi:10.1074 / jbc.M110.169276 Non - Patent Document 4: Eun Jung Lee, Ji Young Kim & Sang Ho Oh. Advanced glycationend products (AGEs) promote melanogenesis through receptor forAGEs. Sci.Rep. 2016;6, doi:10.1038 / srep27848 Non - Patent Document 5: Huijuan Liao, M.D. / Ph.D., Julia Zakhaleva, M.D., and WeiliamChen, Ph.D., R.Ph.. Cells and Tissue Interactions with Glycated Collagen andthei r Relevance to Delayed Diabetic Wound Healing. Biomaterials. 2009March;30(9):1689 - 1696. doi:10.1016 / j.biomaterials.2008.11.038. Non-patent Document 6: Lennert Van Putte, Sofie De Schrijverl and Peter Moortgat, The effects of advanced glycation end products (AGEs) on dermal wound healing and scar formation: a systematic review., Scars, Burns&Healing, 2016; 2 doi:10.1177 / 2059513116676828

[0007] However, there is less material focusing on the signal transduction triggered by the binding of AGEs and RAGE.

[0008] Therefore, the object of the present invention is to provide an application of paeony extract in inhibiting the binding of AGEs-RAGE and a preparation method of a preparation containing the same, which can inhibit the binding of AGEs and RAGE and can prepare a new preparation mainly applicable to human skin, etc. Summary of the Invention

[0009] In order to achieve the above object, the first aspect of the present invention is to provide an application of paeony extract in preparing a preparation for inhibiting the binding of advanced glycation end products (AGEs) to its receptor (RAGE).

[0010] The second aspect of the present invention is based on the first aspect, and the above application is also used to inhibit the expression of RAGE gene. The third aspect of the present invention is based on the first aspect, and the paeony extract is prepared by the following steps: using a 1,3-butanediol solution as an extraction solvent and extracting from the roots of paeony.

[0011] The fourth aspect of the present invention is to provide a method for preparing a preparation for inhibiting AGEs-RAGE binding, the preparation contains paeony extract, and the method includes the following steps: mixing the paeony extract with a pharmaceutically acceptable carrier. The fifth aspect of the present invention is based on the fourth aspect, and the above preparation is also used to inhibit the expression of RAGE gene. The sixth aspect of the present invention is based on the fourth aspect, and the paeony extract is prepared by the following steps: using a 1,3-butanediol solution as an extraction solvent and extracting from the roots of paeony.

[0012] According to the present invention, for example, it can inhibit the binding of AGEs and RAGE, and mainly can provide a new preparation applicable to human skin, etc. Brief Description of the Drawings

[0013] Figure 1 It is a graph showing the inhibitory effect of AGEs and RAGE binding when adding various plant extracts in Example 1. Figure 2 It is a graph showing the amount of melanin production per cell when adding various plant extracts in Example 2. Figure 3 It is a graph showing the amount of melanin production per cell when adding various Ruscus aculeatus extracts in Example 3. Figure 4 It is a graph showing the expression levels of various genes when adding Paeonia lactiflora extract to NBNHDF cells in Example 4. Figure 5 It is a photograph showing the results of observing the effect of glycated epidermal basement membrane components on the proliferation of NBNHEK cells after adding Paeonia lactiflora extract in Example 5. Figure 6 It is a photograph of the image of the subject's cheek skin and the image after color integration in Example 6. Figure 7 It is a graph showing the correlation between the skin age of the subject and the value of the color non-uniformity index in Example 6. Figure 8 It is a graph showing the results of the human skin monitoring test in Example 6. Figure 9 It is a graph showing the expression levels of various genes when adding Paeonia lactiflora extract to NBNHEK cells in Example 7. Figure 10 It is a graph showing the amount of melanin production per cell when adding Paeonia lactiflora extract in Example 8. Sequence Listing: An electronic file containing the biological sequences related to the present invention (file name: SequenceListing63-19Y1_P24145-02.xml) Detailed Description of the Invention

[0014] <Definition> In this specification, "Paeonia lactiflora" refers to, for example, a plant of the family Paeoniaceae, genus Paeonia, or its related plants.

[0015] In this specification, "Ruscus aculeatus", also known as Butcher's Broom, refers to a plant of the family Asparagaceae, subfamily Nolinoideae, genus Ruscus, or its related plants.

[0016] In this specification, "Malva sylvestris", also known as blue mallow, refers to a plant of the Malvaceae family, Malva genus, or its related plants. Examples of such related plants include Malva mauritiana, etc.

[0017] In this specification, "Rosa multiflora" refers to a plant of the Rosaceae family, Rosa genus, or its related plants. The fruit of Rosa multiflora is also known as Rosa laevigata Michx.

[0018] In this specification, "Equisetum arvense" refers to a plant of the Equisetaceae family, Equisetum genus, or its related plants.

[0019] In this specification, "epidermal keratinocytes" refer to cells capable of differentiating into basal cells, spinous cells, granular cells, and keratinocytes that make up the epidermis. These epidermal keratinocytes ultimately form keratinocytes of the stratum corneum through differentiation, and this process is called keratinization.

[0020] In this specification, "dermal fibroblast" is a cell differentiated from cells called mesenchymal stem cells. In the dermis of the skin, such cells have the property of producing components such as collagen, hyaluronic acid, and elastin.

[0021] In this specification, "melanocyte" is also known as a pigment cell. It is a cell derived from the neural crest and is present in the epidermal basement membrane and dermal papilla in the skin and is the cell that produces melanin.

[0022] In this specification, "TNF (tumor necrosis factor)-α" is a multifunctional inflammatory cytokine belonging to the TNF superfamily. The TNF-α is produced by macrophages, lymphocytes, fibroblasts, epidermal keratinocytes, etc. during inflammation.

[0023] In this specification, "IL-8" (Interleukin-8) is a cytokine (chemokine) that chemotactically attracts neutrophils. IL-8 is known to be one of the triggers of the inflammatory response. IL-8 is also known as CXCL8.

[0024] In this specification, "TGM1" (Transglutaminase 1) is an epidermal enzyme involved in protein cross-linking. TGM1 is expressed during the final differentiation of keratinized squamous epithelium and forms a cornified cell envelope in differentiated keratinocytes.

[0025] In this specification, "K10 (KRT10)" (Keratin 10) is a keratin of intermediate filaments (about 10 nm in diameter). K10 is expressed in the differentiated stratum spinosum and stratum granulosum.

[0026] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples and the like, and can be implemented with any modifications. In addition, in the present invention and each embodiment, unless otherwise specified, the descriptions of each content can be cited from each other. In this specification, when the expression "~" is used, it means including the numerical values or physical values before and after it. In addition, in this specification, the expression "A and / or B" includes the cases of "A only", "B only", and "both A and B".

[0027] <Preparation or composition for inhibiting the binding of AGEs and RAGE> In certain embodiments, the present invention provides a preparation or composition for inhibiting the binding of AGEs (advanced glycation end products) and RAGE (receptor for AGEs). The preparation for inhibiting the binding of AGEs and RAGE according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. In addition, the composition for inhibiting the binding of AGEs and RAGE according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. In the following description, unless otherwise specified, the description of each preparation in the present invention can be applied to the corresponding compositions.

[0028] The inventors of the present invention have found through in-depth research that in epidermal keratinocytes and fibroblasts, peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract can inhibit the binding of AGEs and RAGE, and thus the present invention has been established. Therefore, according to the preparation or composition for inhibiting the binding of AGEs and RAGE of the present invention, it is expected to obtain effects such as inhibiting TNF-α gene expression, inhibiting IL-8 gene expression, promoting TGM1 gene expression, promoting K10 gene expression, inhibiting melanogenesis, anti-inflammation, promoting cell proliferation, promoting cell adhesion, normalizing metabolism and / or moisturizing, etc., and these effects all originate from inhibiting the signal transduction triggered by the binding of AGEs and RAGE.

[0029] The above-mentioned peony extract, ruscus extract, mallow extract, wild rose extract and / or equisetum extract (hereinafter collectively referred to as "plant extracts") can be prepared by solvent extraction of the corresponding plants. One kind of the plants can be used, or two or more kinds can be used. The plant materials for extraction can be plant individuals or parts of plants. The plant parts include, for example, roots, rhizomes, leaves, stems, flowers, fruits, pericarp, seeds or their mixtures. The materials can be the collected plants themselves or products processed by freezing, drying and / or pulverizing, etc. When the plant is peony, the material is preferably the root. When the plant is ruscus, the material is preferably the root and / or rhizome. When the plant is wild rose, the material is preferably the fruit (rosa laevigata michx). When the plant is equisetum, the material is preferably the whole plant (herb).

[0030] The solvents include, for example, water, aqueous solvents such as buffer solutions; lower alcohols or water-containing lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol; polyols or water-containing polyols such as propylene glycol, 1,3-butanediol, 1,2-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,3,5-pentanetriol, glycerol, polyethylene glycol (for example, molecular weight 100 - 100,000); organic solvents such as acetone, ethyl acetate, diethyl ether, dimethyl ether, ethyl methyl ether, dioxane, hexane, acetonitrile, xylene, benzene, chloroform, carbon tetrachloride, phenol, toluene; acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) or bases (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) prepared at a specified concentration as required. The solvent is preferably ethanol or 1,3-butanediol, or a mixed solvent thereof. The solvents can be used alone or in combination of two or more. The extraction can be carried out, for example, by soaking the plants in the solvent at room temperature (about 25°C) for 1 - 20 days, preferably 7 - 14 days.

[0031] The above-mentioned plant extracts can be, for example, crude products, dried products, freeze-dried products of plant extracts, or products after treatments such as spray drying.

[0032] The treatment methods in the above-mentioned treatment products include, but are not limited to: decomposition by adding acids (such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acids, etc.) or bases (such as sodium hydroxide, calcium hydroxide, ammonia, etc.); microbial fermentation or metabolic transformation; using adsorbents such as ion exchange resins, activated carbon, diatomaceous earth, etc.; fractionation by chromatography with various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.); filtration using filter paper, membrane filters or ultrafiltration membranes, etc.; pressurization or depressurization; heating or cooling; drying or freeze-drying; adjusting the pH value; deodorization; decolorization; long-term static storage, etc. These treatment methods can be carried out alone or two or more of them can be carried out simultaneously.

[0033] "Inhibition of the binding of AGEs and RAGE" means that the binding of AGEs and RAGE is inhibited, reduced or hindered, which can represent either a state where the binding of AGEs and RAGE completely disappears or a state where the binding rate of AGEs and RAGE decreases. The decrease in the binding rate of AGEs and RAGE can be evaluated by comparing the binding rate of AGEs and RAGE when using the preparation of the present invention for inhibiting the binding of AGEs and RAGE with the case where the preparation is not used. In this evaluation, when the binding rate of AGEs and RAGE in the control group without using the preparation of the present invention is 5% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less or 99% or less, it can be considered that the preparation for inhibiting the binding of AGEs and RAGE has the effect of inhibiting the binding of AGEs and RAGE. The inhibitory effect of the binding of AGEs and RAGE can be evaluated, for example, by measuring the binding between AGEs and RAGE using methods such as ELISA in a cell-free assay using recombinant proteins according to Example 1 described below.

[0034] The preparation of the present invention for inhibiting the binding of AGEs and RAGE can inhibit the downstream signal transduction of RAGE. Therefore, the evaluation of the inhibition of the binding of AGEs and RAGE in the present invention can also be carried out by evaluating the downstream signals of RAGE. Specifically, it can be evaluated by measuring the degree of inhibition of TNF-α gene expression, the degree of inhibition of IL-8 gene expression, the degree of promotion of TGM1 gene expression, the degree of promotion of K10 gene expression, the degree of inhibition of melanin production, the degree of anti-inflammation, the degree of promotion of cell proliferation, the degree of promotion of cell adhesion, etc.

[0035] The above-mentioned RAGE gene, for example, in NCBI Gene, the gene with Gene ID: 177 is the RAGE gene. As an example of the RAGE gene, the mRNA encoded by the human RAGE gene, in Genbank, the polynucleotides composed of the base sequences have accession numbers NM_001136.5, NM_001206929.2, NM_001206932.2, NM_001206934.2, NM_001206936.2, NM_001206940.2, NM_172197.3, NM_001206954.2, etc. The expression level of the RAGE gene can measure the expression level of any one or two or more RAGE gene isomers, or can measure the expression levels of all isomers.

[0036] As an example of the TNF-α gene, the mRNA encoded by the human TNF-α gene, in Genbank, the polynucleotide composed of the base sequence with accession number NM_000594.4 is one of them. The expression level of the TNF-α gene can measure the expression level of any one or two or more TNF-α gene isomers, or can measure the expression levels of all isomers.

[0037] As an example of the IL-8 gene, the mRNA encoded by the human IL-8 gene, in Genbank, the polynucleotide composed of the base sequence with accession number NM_001354840.3 is one of them. The expression level of the IL-8 gene can measure the expression level of any one or two or more IL-8 gene isomers, or can measure the expression levels of all isomers.

[0038] As an example of the TGM1 gene, the mRNA encoded by the human TGM1 gene, in Genbank, the polynucleotide composed of the base sequence with accession number NM_0003593 is one of them. The expression level of the TGM1 gene can measure the expression level of any one or two or more TGM1 gene isomers, or can measure the expression levels of all isomers.

[0039] As an example of the K10 gene, the mRNA encoded by the human K10 gene (KRT10 gene), in Genbank, the polynucleotide composed of the base sequence with accession number NM_000421.5 is one of them. The expression level of the K10 gene can measure the expression level of any one or two or more K10 gene isomers, or can measure the expression levels of all isomers.

[0040] The above-mentioned melanin production can be evaluated by extracting the melanin contained in the cells and measuring the absorbance according to Example 3 described below.

[0041] The above anti-inflammatory effect can be evaluated by measuring the expression levels of the above TNF-α, IL-8, etc.

[0042] The preparation of the present invention for inhibiting the binding of AGEs and RAGE can inhibit the binding of AGEs and RAGE by being used on a subject to be administered. The usage conditions (administration conditions) of this preparation are not particularly limited, and the administration method, administration time, administration dose, etc. can be appropriately set according to the type of the subject to be administered, etc.

[0043] The preparation of the present invention for inhibiting the binding of AGEs and RAGE can be used in vivo or in vitro.

[0044] The subject to be administered with the preparation of the present invention for inhibiting the binding of AGEs and RAGE is not particularly limited. When using this preparation in vivo, the subject to be administered can be a human, or a non-human animal other than a human, such as a mammal such as a mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, guinea pig, etc., and a bird, etc. When using the preparation of the present invention for inhibiting the AGEs-RAGE binding in vitro, the subject to be administered can be a cell, tissue, organ, etc., where the cell can be a cell collected from a living body, a cultured cell, etc., and the tissue or organ can be a tissue (biological tissue) or organ collected from a living body, etc.

[0045] The administration methods of the preparation of the present invention for inhibiting the binding of AGEs and RAGE include oral administration and non-oral administration. Non-oral administration includes transdermal administration, skin application (contact), etc. Skin application can include oral mucosal application, that is, applying or contacting the epithelial cells in the oral cavity. In addition, skin application, in addition to being applied on the skin surface, can also mean administering or injecting through the skin surface into the skin or subcutaneous tissue. The administration or injection through the skin surface into the skin can be implemented by means such as microneedles.

[0046] The dosage form of the preparation of the present invention for inhibiting the binding of AGEs and RAGE is not particularly limited and can be appropriately determined according to the administration method. The dosage form is, for example, liquid, solid, etc. When the administration method is oral administration, the dosage form is, for example, tablets, pills, capsules, granules, powders, liquid preparations, etc.

[0047] The preparation of the present invention for inhibiting the binding of AGEs and RAGE may contain additives as required. When used as a composition, the additives preferably include pharmaceutically acceptable additives or pharmaceutically acceptable carriers. The additives are not particularly limited. For example, there are matrix materials, excipients, colorants, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring and odor-masking agents, etc. In the present invention, as long as the compounding amount of the additives does not interfere with the functions of the extracts of one or more of Paeonia lactiflora, Ruscus aculeatus, Malva sylvestris, Rosa multiflora, and Equisetum arvense, there is no particular limitation.

[0048] Examples of the above excipients include sugar derivatives such as lactose, lactose hydrate, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; sulfates such as calcium sulfate, etc. inorganic excipients. Examples of the above colorants include yellow ferric oxide, etc. Examples of the above lubricants include metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; polyethylene glycol; silicon dioxide; hydrogenated vegetable oil, etc. Examples of the above flavoring and odor-masking agents include spices such as cocoa powder, menthol, aromatic powder, peppermint oil, borneol, and cinnamon powder, as well as sweeteners, acidulants, etc. Examples of the above binders include hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyethylene glycol, etc. Examples of the above disintegrants include cellulose derivatives such as carboxymethyl cellulose and carboxymethyl cellulose calcium; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, and sodium starch glycolate, etc. Examples of the above stabilizers include parabens such as methyl p-hydroxybenzoate and propyl p-hydroxybenzoate; alcohols such as chlorobutanol, benzyl alcohol, and phenethyl alcohol; benzyl ammonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; sorbic acid, etc. Examples of the above coating agents include hydroxypropyl methyl cellulose, polyethylene glycols such as polyethylene glycol 6000, talc, titanium dioxide, etc.

[0049] When the composition of the present invention for inhibiting the binding of AGEs and RAGE is an oral administration composition, specific examples thereof include beverages, foods, pharmaceuticals (categories), quasi-drugs (categories), etc.

[0050] When the preparation or composition of the present invention for inhibiting the binding of AGEs and RAGE is used for transdermal administration or skin application (hereinafter also referred to as "skin topical agent"), depending on the form of use, the form of the skin topical agent includes ampoules, capsules, powders, granules, liquids, gels, foams, emulsions, sheets, sprays, aerosol sprays, etc. Forms of use include, for example, pharmaceuticals (types); quasi-drugs (types); skin topical agents for local or systemic use; pharmaceutical and / or cosmetic preparations applicable to the scalp and hair; bath agents used by adding to bath water; other preparations, etc. Skin topical agents for local or systemic use include, for example, basic cosmetics such as lotions, emulsions, creams, ointments, lotions, oils, face masks, etc., cleansing products or skin cleansers such as solid soaps, liquid soaps, hand sanitizers, etc., makeup cosmetics such as massage agents, makeup removers, hair removal agents, shaving treatment agents, aftershaves, pre-shave lotions, shaving creams, foundations, lipsticks, blushes, eyeshadows, eyeliners, mascaras, etc., perfumes, nail agents, nail enamels, nail enamel removers, cataplasms, plasters, tape agents, sheet agents, patches, aerosols, dentifrices, mouthwashes, etc. Pharmaceutical and / or cosmetic preparations applicable to the scalp and hair include, for example, shampoos, conditioners, hair care agents, hair pretreatment agents, perming liquids, hair dyes, hair styling agents, hair growth agents, hair nourishing / conditioning agents, cataplasms, plasters, tape agents, sheet agents, aerosols, etc. Other preparations include, for example, axillary odor preventives or deodorants, antiperspirants, sanitary products, sanitary cotton products, wet wipes, etc.

[0051] The above-mentioned skin topical agent can be arbitrarily selected and / or used in combination with the components and / or additives exemplified below as needed within the range that does not affect the effects of extracts of one or more of Paeonia lactiflora, Ruscus aculeatus, Malva sylvestris, Rosa multiflora, Equisetum arvense.

[0052] (1) Various oils and fats: Avocado oil, almond oil, Evodia rutaecarpa oil, perilla oil, olive oil, orange oil, neroli oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, tallow fatty acid, macadamia oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peach kernel oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, beeswax, coconut oil, tallow, lard, squalene, squalane, pristane, or hydrogenated products (hardened oils, etc.) of these oils and fats, etc.

[0053] (2) Waxes: Beeswax, carnauba wax, cetyl wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, lignite wax, shellac wax, rice bran wax, etc.

[0054] (3) Mineral oils: Liquid paraffin, petrolatum, paraffin wax, ozokerite, microcrystalline wax, microcrystalline paraffin wax, etc.

[0055] (4) Fatty acids: Lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid and other natural fatty acids; isononanoic acid, caproic acid, 2-ethylbutyric acid, isovaleric acid, 2-methylvaleric acid, 2-ethylhexanoic acid, isovaleric acid and other synthetic fatty acids.

[0056] (5) Alcohols: Ethanol, isopropanol, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol and other natural alcohols; 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol and other synthetic alcohols.

[0057] (6) Polyhydric alcohols: Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butanediol, pentanediol, glycerol, pentaerythritol, xylitol, arabinitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[0058] (7) Esters: Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyl decyl dimethyl octanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

[0059] (8) Metal soaps: Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0060] (9) Gums, sugars or water-soluble high molecular compounds: Gum arabic, benzoin gum, dammar resin, guaiac resin, Irish moss, karaya gum, tragacanth gum, carob gum, kelp, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitosan or chitosan, hydroxyalkyl (C2-C4) chitosan or chitosan added with alkylene (C2-C4) oxides (such as ethylene oxide), low molecular weight chitosan or chitosan salts, sulfated chitosan or chitosan, phosphorylated chitosan or chitosan, alginic acid or its salts, hyaluronic acid or its salts, chondroitin sulfate or its salts, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylate, polyalkylene oxides such as polyethylene oxide or polypropylene oxide or their cross-linked polymers, carboxyvinyl polymer, polyethyleneimine, etc.

[0061] (10) Surfactants: Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkyl amine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, titanium- and silicon-containing surfactants, fluorocarbon surfactants), etc.

[0062] (11) Various vitamins: Vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, niacins, pantothenics, biotins, choline, inositols; vitamin C group: ascorbic acid or its derivatives; vitamin D group: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol; vitamin E group: vitamin E or its derivatives, ubiquinones; vitamin K group: phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4); others, such as essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin P group (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0063] (12) Various amino acids: Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., and their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidonecarboxylic acid, etc.

[0064] (13) Additives: The above-mentioned skin external preparations may also be added with various additives from animals or plants. These additives may be processed according to conventional methods according to the type and form of the product to be added, and may be arbitrarily selected and added from various materials. The processing may be, for example, pulverizing, milling, cleaning, hydrolysis, fermentation, refining, squeezing, extraction, fractionation, filtering, drying, powdering, granulation, dissolving, sterilization, pH adjustment, deodorization, decolorization, etc., and these processing methods may be arbitrarily selected and / or combined.

[0065] The solvent used for extraction can be selected according to the purpose of use, type or subsequent processing of the product. The extraction solvent, for example, is expected to be selected from various organic solvents such as water, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, etc., lower alcohols or aqueous lower alcohols, propylene glycol, 1,3-butanediol, glycerol, etc., polyols or aqueous polyols, acetone, ethyl acetate, etc., or a mixture of two or more. However, for example, in some uses, if it is not desired to contain an organic solvent, only water can be used, or ethanol that is easy to remove after extraction can be used, used alone or mixed with water in any proportion, and the product of squeeze extraction can also be used.

[0066] When additives derived from plant or animal raw materials are used in external preparations or cosmetics for systemic or local use, the above-mentioned skin external preparations can be expected to have beauty effects such as protecting skin and hair, moisturizing, improving touch and texture, imparting softness, relieving irritation, relieving stress through fragrance, activating cells (preventing cell aging), inhibiting inflammation, improving skin and hair quality, preventing and improving skin roughness, promoting hair growth, preventing hair loss, imparting gloss, cleansing effect, relieving fatigue, promoting blood circulation, and warm bath effect, in addition to having fragrance enhancement, deodorization, thickening, antiseptic, buffering and other effects.

[0067] The above-mentioned external skin preparation can also be expected to obtain various cosmetic and medicinal effects from various known raw materials. By combining these effects, the expected effects of the present invention can be enhanced to produce a product with multifunctional effects.

[0068] The preparation for inhibiting the binding of AGEs and RAGE according to the present invention, for example, can inhibit the binding of AGEs and RAGE. Therefore, the preparation for inhibiting the binding of AGEs and RAGE according to the present invention can also be said to be a preparation for inhibiting the downstream signals of RAGE.

[0069] <Preparation or composition for inhibiting TNF-α gene expression> In another embodiment, the present invention provides a preparation or composition for inhibiting the expression of TNF-α gene. The preparation for inhibiting the expression of TNF-α gene according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The composition for inhibiting the expression of TNF-α gene according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. According to the preparation or composition for inhibiting the expression of TNF-α gene of the present invention, the effect of inhibiting fibroblast inflammation can be obtained.

[0070] The above-mentioned "inhibition of TNF-α gene expression" means that the expression level of TNF-α gene is inhibited or reduced, and it can also represent the change from the state of TNF-α gene expression to the state of no expression. The expression of TNF-α gene can be evaluated, for example, by measuring the mRNA expression level of TNF-α gene according to Example 4 described below.

[0071] The preparation for inhibiting the expression of TNF-α gene according to the present invention can inhibit the expression of TNF-α gene by using it on the administration object. Thus, the preparation for inhibiting the expression of TNF-α gene according to the present invention can obtain the effect of inhibiting dermal fibroblast inflammation. The usage conditions (administration conditions) of the preparation for inhibiting the expression of TNF-α gene according to the present invention can refer to the description of the usage conditions of the preparation for inhibiting the binding of AGEs and RAGE according to the present invention.

[0072] <Preparation or composition for inhibiting IL-8 gene expression> In another embodiment, the present invention provides a preparation or composition for inhibiting the expression of IL-8 gene. The preparation for inhibiting the expression of IL-8 gene according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The composition for inhibiting the expression of IL-8 gene according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. According to the preparation or composition for inhibiting the expression of IL-8 gene of the present invention, the effect of inhibiting fibroblast inflammation can be obtained.

[0073] The above-mentioned "inhibition of IL-8 gene expression" means that the expression level of the IL-8 gene is inhibited or reduced, and it can also represent the change from the state where the IL-8 gene is expressed to the state where it is not expressed. The expression of the IL-8 gene can be evaluated, for example, by measuring the mRNA expression level of the IL-8 gene according to Example 4 described below.

[0074] The preparation of the present invention for inhibiting the expression of the IL-8 gene can inhibit the expression of the IL-8 gene by being used on the administration subject. Thus, the preparation of the present invention for inhibiting the expression of the IL-8 gene can obtain the effect of inhibiting the inflammation of dermal fibroblasts. The usage conditions (administration conditions) of the preparation of the present invention for inhibiting the expression of the IL-8 gene can refer to the description of the usage conditions of the preparation of the present invention for inhibiting the binding of AGEs and RAGE.

[0075] <Preparation or composition for promoting TGM1 gene expression> In another embodiment, the present invention provides a preparation or composition for promoting the expression of the TGM1 gene. The preparation of the present invention for promoting the expression of the TGM1 gene contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum arvense extract. The composition of the present invention for promoting the expression of the TGM1 gene contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum arvense extract. According to the preparation or composition of the present invention for promoting the expression of the TGM1 gene, the effect of normalizing metabolism and / or moisturizing can be obtained.

[0076] The above-mentioned "promotion of TGM1 gene expression" means that the expression level of the TGM1 gene is promoted or increased, and it can also represent the change from the state where the TGM1 gene is not expressed to the state where it is expressed. The expression of the TGM1 gene can be evaluated, for example, by measuring the mRNA expression level of the TGM1 gene according to Example 7 described below.

[0077] The preparation of the present invention for promoting the expression of the TGM1 gene can promote the expression of the TGM1 gene by being used on the administration subject. Thus, the preparation of the present invention for promoting the expression of the TGM1 gene can obtain the effect of normalizing metabolism and / or moisturizing. The usage conditions (administration conditions) of the preparation of the present invention for promoting the expression of the TGM1 gene can refer to the description of the usage conditions of the preparation of the present invention for inhibiting the binding of AGEs and RAGE.

[0078] <Preparation or composition for promoting K10 gene expression> In another embodiment, the present invention provides a preparation or composition for promoting K10 gene expression. The preparation for promoting K10 gene expression according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and / or equisetum extract. The composition for promoting K10 gene expression according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and / or equisetum extract. According to the preparation or composition for promoting K10 gene expression of the present invention, the effects of normalizing metabolism and / or moisturizing can be obtained.

[0079] The above-mentioned "promotion of K10 gene expression" means that the expression level of the K10 gene is promoted or increased, and it can also indicate a change from a state of no expression of the K10 gene to a state of having expression. The expression of the K10 gene can be evaluated, for example, by measuring the mRNA expression level of the K10 gene according to Example 7 described below.

[0080] The preparation for promoting K10 gene expression according to the present invention can promote the expression of the K10 gene by being used on a subject to be administered. Thus, the preparation for promoting K10 gene expression according to the present invention can obtain the effects of normalizing metabolism and / or moisturizing. The usage conditions (administration conditions) of the preparation for promoting K10 gene expression according to the present invention can refer to the description of the usage conditions of the preparation for inhibiting the binding of AGEs and RAGE.

[0081] <Melanin production inhibitor or composition> In another embodiment, the present invention provides a preparation or composition capable of inhibiting melanin production in melanocytes. The melanin production inhibitor according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and / or equisetum extract. The melanin production inhibitory composition according to the present invention contains peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and / or equisetum extract. According to the melanin production inhibitor or composition of the present invention, the melanin production in melanocytes can be inhibited, and thus a whitening effect can be obtained.

[0082] The melanin production inhibitor according to the present invention can inhibit the melanin production in melanocytes by being used on a subject to be administered. The usage conditions (administration conditions) of the melanin production inhibitor according to the present invention can refer to the description of the usage conditions of the preparation for inhibiting the binding of AGEs and RAGE.

[0083] <Inflammation inhibitor or composition> In another embodiment, the present invention provides a preparation or composition capable of inhibiting inflammation in epidermal keratinocytes. The anti-inflammatory agent of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The anti-inflammatory composition of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The anti-inflammatory agent or composition according to the present invention can inhibit the inflammation of epidermal keratinocytes.

[0084] The anti-inflammatory agent of the present invention can inhibit the inflammation of epidermal keratinocytes by being used on a subject to be administered. The usage conditions (administration conditions) of the anti-inflammatory agent of the present invention can refer to the description of the usage conditions of the preparation of the present invention for inhibiting the binding of AGEs and RAGE.

[0085] <Cell proliferation promoter or composition> In another embodiment, the present invention provides a preparation or composition capable of promoting cell proliferation. The cell proliferation promoter of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The cell proliferation promoting composition of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The cell proliferation promoter or composition according to the present invention can, for example, promote the proliferation of cells such as epidermal keratinocytes and dermal fibroblasts.

[0086] The cell proliferation promoter of the present invention can, for example, promote the proliferation of cells such as epidermal keratinocytes and dermal fibroblasts by being used on a subject to be administered. The usage conditions (administration conditions) of the cell proliferation promoter of the present invention can refer to the description of the usage conditions of the preparation of the present invention for inhibiting the binding of AGEs and RAGE.

[0087] <Cell adhesion promoter or composition> In another embodiment, the present invention provides a preparation or composition capable of promoting cell adhesion. The cell adhesion promoter of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The cell adhesion promoting composition of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum extract. The cell adhesion promoter or composition according to the present invention can, for example, promote the adhesion of cells such as epidermal keratinocytes and dermal fibroblasts.

[0088] The cell adhesion promoter of the present invention can, for example, promote the adhesion of cells such as epidermal keratinocytes and dermal fibroblasts by being used on a subject to be administered. The usage conditions (administration conditions) of the cell proliferation adhesion promoter of the present invention can refer to the description of the usage conditions of the preparation of the present invention for inhibiting the binding of AGEs and RAGE.

[0089] <Method for inhibiting the binding of AGEs and RAGE> In another embodiment, the present invention discloses a method capable of inhibiting the binding of AGEs and RAGE. The method for inhibiting the binding of AGEs and RAGE of the present invention uses the inhibitor and / or composition for inhibiting the binding of AGEs and RAGE of the present invention. According to the method for inhibiting the binding of AGEs and RAGE of the present invention, for example, it can inhibit the binding of AGEs and RAGE, and is expected to obtain the effect of inhibiting the downstream signals of RAGE.

[0090] The method for inhibiting the binding of AGEs and RAGE of the present invention includes the step of using the preparation and / or composition for inhibiting the binding of AGEs and RAGE of the present invention for an object. The above use can be, for example, contacting with the skin or the like, or administering.

[0091] In the method for inhibiting the binding of AGEs and RAGE of the present invention, the above use step can be carried out in vitro or in vivo. For the object (administration object) and administration conditions of the method for inhibiting the binding of AGEs and RAGE of the present invention, reference can be made to the description of the administration object and administration conditions in the preparation and / or composition for inhibiting the binding of AGEs and RAGE of the present invention.

[0092] <Method for inhibiting TNF-α gene expression> In another embodiment, the present invention discloses a method capable of inhibiting TNF-α gene expression. The method for inhibiting TNF-α gene expression of the present invention uses the preparation and / or composition for inhibiting TNF-α gene expression of the present invention. According to the method for inhibiting TNF-α gene expression of the present invention, for example, it can obtain the effect of inhibiting fibroblast inflammation.

[0093] The method for inhibiting TNF-α gene expression of the present invention includes the step of using the preparation and / or composition for inhibiting TNF-α gene expression of the present invention for an object. The above use can be, for example, contacting with the skin or the like, or administering.

[0094] In the method for inhibiting TNF-α gene expression of the present invention, the above use step can be carried out in vitro or in vivo. For the object (administration object) and administration conditions of the method for inhibiting TNF-α gene expression of the present invention, reference can be made to the description of the administration object and administration conditions in the preparation and / or composition for inhibiting TNF-α gene expression of the present invention.

[0095] <Method for inhibiting IL-8 gene expression> In another embodiment, the present invention discloses a method capable of inhibiting the expression of the IL-8 gene. The method for inhibiting the expression of the IL-8 gene of the present invention uses the preparation and / or composition of the present invention for inhibiting the expression of the IL-8 gene. According to the method for inhibiting the expression of the IL-8 gene of the present invention, for example, the effect of inhibiting fibroblast inflammation can be obtained.

[0096] The method for inhibiting the expression of the IL-8 gene of the present invention includes the step of using the preparation and / or composition of the present invention for inhibiting the expression of the IL-8 gene on an object. The above use can be, for example, contact with the skin or the like, or administration.

[0097] In the method for inhibiting the expression of the IL-8 gene of the present invention, the above use step can be carried out in vitro or in vivo. The object (administration object) and administration conditions of the method for inhibiting the expression of the IL-8 gene of the present invention can refer to the description of the administration object and administration conditions in the preparation and / or composition of the present invention for inhibiting the expression of the IL-8 gene.

[0098] <Method for promoting TGM1 gene expression> In another embodiment, the present invention discloses a method capable of promoting the expression of the TGM1 gene. The method for promoting the expression of the TGM1 gene of the present invention uses the preparation and / or composition of the present invention for promoting the expression of the TGM1 gene. According to the method for promoting the expression of the TGM1 gene of the present invention, for example, the effects of normalizing metabolism and / or moisturizing can be obtained.

[0099] The method for promoting the expression of the TGM1 gene of the present invention includes the step of using the preparation and / or composition of the present invention for promoting the expression of the TGM1 gene on an object. The above use can be, for example, contact with the skin or the like, or administration.

[0100] In the method for promoting the expression of the TGM1 gene of the present invention, the above use step can be carried out in vitro or in vivo. The object (administration object) and administration conditions of the method for promoting the expression of the TGM1 gene of the present invention can refer to the description of the administration object and administration conditions in the preparation and / or composition of the present invention for promoting the expression of the TGM1 gene.

[0101] <Method for promoting K10 gene expression> In another embodiment, the present invention discloses a method capable of promoting the expression of the K10 gene. The method for promoting the expression of the K10 gene of the present invention uses the preparation and / or composition of the present invention for promoting the expression of the K10 gene. According to the method for promoting the expression of the K10 gene of the present invention, for example, the effects of normalizing metabolism and / or moisturizing can be obtained.

[0102] The method for promoting K10 gene expression of the present invention includes the step of using the preparation and / or composition for promoting K10 gene expression of the present invention on an object. The above-mentioned use can be, for example, contact with the skin or the like, or administration.

[0103] In the method for promoting K10 gene expression of the present invention, the above-mentioned use step can be carried out in vitro or in vivo. For the object (administration object) and administration conditions of the method for promoting K10 gene expression of the present invention, reference can be made to the description of the administration object and administration conditions in the preparation and / or composition for promoting K10 gene expression of the present invention.

[0104] <Method for inhibiting melanogenesis> In another embodiment, the present invention discloses a method capable of inhibiting melanogenesis. The method for inhibiting melanogenesis of the present invention uses the melanogenesis inhibitor and / or melanogenesis inhibitory composition of the present invention. According to the method for inhibiting melanogenesis of the present invention, the melanogenesis of melanocytes can be inhibited, and thus a whitening effect can be obtained.

[0105] The method for inhibiting melanogenesis of the present invention includes the step of using the melanogenesis inhibitor and / or composition of the present invention on an object. The above-mentioned use can be, for example, contact with the skin or the like, or administration.

[0106] <Method for inhibiting inflammation> In another embodiment, the present invention discloses a method capable of inhibiting inflammation. The method for inhibiting inflammation of the present invention uses the inflammation inhibitor and / or inflammation inhibitory composition of the present invention. According to the method for inhibiting inflammation of the present invention, for example, the inflammation of fibroblasts can be inhibited.

[0107] The method for inhibiting inflammation of the present invention includes the step of using the inflammation inhibitor and / or composition of the present invention on an object. The above-mentioned use can be, for example, contact with the skin or the like, or administration.

[0108] In the method for inhibiting inflammation of the present invention, the above-mentioned use step can be carried out in vitro or in vivo. For the object (administration object) and administration conditions of the method for inhibiting inflammation of the present invention, reference can be made to the description of the administration object and administration conditions in the preparation and / or composition for inhibiting the binding of AGEs and RAGE of the present invention.

[0109] <Method for promoting cell proliferation> In another embodiment, the present invention discloses a method capable of promoting cell proliferation. The cell proliferation promoting method of the present invention uses the cell proliferation promoter and / or cell proliferation promoting composition of the present invention. According to the cell proliferation promoting method of the present invention, for example, it can promote the proliferation of cells such as epidermal keratinocytes and dermal fibroblasts.

[0110] The cell proliferation promoting method of the present invention includes a step of using the cell proliferation promoter and / or composition of the present invention on a subject. The above use can be, for example, contact with the skin or the like, or administration.

[0111] In the cell proliferation promoting method of the present invention, the above use step can be carried out in vitro or in vivo. The subject (administration subject) and administration conditions of the cell proliferation promoting method of the present invention can refer to the description of the administration subject and administration conditions in the preparation and / or composition for inhibiting the binding of AGEs and RAGE of the present invention.

[0112] <Method for promoting cell adhesion> In another embodiment, the present invention discloses a method capable of promoting cell adhesion. The cell adhesion promoting method of the present invention uses the cell adhesion promoter and / or cell adhesion promoting composition of the present invention. According to the cell adhesion promoting method of the present invention, for example, it can promote the adhesion of cells such as epidermal keratinocytes and dermal fibroblasts.

[0113] The cell adhesion promoting method of the present invention includes a step of using the cell adhesion promoter and / or composition of the present invention on a subject. The above use can be, for example, contact with the skin or the like, or administration.

[0114] In the cell adhesion promoting method of the present invention, the above use step can be carried out in vitro or in vivo. The subject (administration subject) and administration conditions of the cell adhesion promoting method of the present invention can refer to the description of the administration subject and administration conditions in the preparation and / or composition for inhibiting the binding of AGEs and RAGE of the present invention.

[0115] <Cosmetics> In another embodiment, the present invention provides a cosmetic. The cosmetic of the present invention comprises a preparation and / or composition for inhibiting the binding of AGEs and RAGE, a preparation and / or composition for inhibiting the expression of TNF-α gene, a preparation and / or composition for inhibiting the expression of IL-8 gene, a preparation and / or composition for promoting the expression of TGM1 gene, a preparation and / or composition for promoting the expression of K10 gene, a melanogenesis inhibitor and / or composition, an inflammation inhibitor and / or composition, a cell proliferation promoter and / or composition, and a cell adhesion promoter and / or composition. That is, the cosmetic of the present invention comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum arvense extract. The cosmetic of the present invention can inhibit melanogenesis caused by the binding of AGEs and RAGE, and thus can also be called a whitening cosmetic. Therefore, according to the cosmetic of the present invention, a whitening effect can be obtained.

[0116] <Use> In another embodiment, the present invention relates to the use of a preparation and / or composition for inhibiting the binding of AGEs and RAGE; the use of a preparation and / or composition for inhibiting the expression of TNF-α gene; the use of a preparation and / or composition for inhibiting the expression of IL-8 gene; the use of a preparation and / or composition for promoting the expression of TGM1 gene; the use of a preparation and / or composition for promoting the expression of K10 gene; the use of a preparation and / or composition for inhibiting melanogenesis; the use of a preparation and / or composition for inhibiting inflammation; the use of a preparation and / or composition for promoting cell proliferation; and the use of a preparation and / or composition for promoting cell adhesion.

[0117] The present invention also relates to the use for manufacturing a preparation and / or composition for inhibiting the binding of AGEs and RAGE; the use for manufacturing a preparation and / or composition for inhibiting the expression of TNF-α gene; the use for manufacturing a preparation and / or composition for inhibiting the expression of IL-8 gene; the use for manufacturing a preparation and / or composition for promoting the expression of TGM1 gene; the use for manufacturing a preparation and / or composition for promoting the expression of K10 gene; the use for manufacturing a preparation and / or composition for inhibiting melanogenesis; the use for manufacturing a preparation and / or composition for inhibiting inflammation; the use for manufacturing a preparation and / or composition for promoting cell proliferation; and the use for manufacturing a preparation and / or composition for promoting cell adhesion.

Examples

[0118] Next, the examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents are used according to their operating procedures unless otherwise specified. It should be noted that the unit "%" of the addition amount of various extracts and the like described in this example represents %(w / v).

[0119] <Preparation Example 1 of Paeonia lactiflora Extract> To 100 g of dried Paeonia lactiflora roots, 1.5 kg of a 50% 1,3 - butanediol solution was added. After the addition, it was soaked at 10°C - 30°C for 7 - 14 days. After soaking, filtration was carried out using a filter material. After filtration, it was allowed to stand at - 30°C - 10°C for 7 - 14 days. After standing, filtration was carried out again using a filter material.

[0120] <Preparation Example 1 of Ruscus aculeatus Extract> To 100 g of dried Ruscus aculeatus underground parts (roots and / or rhizomes), 1 kg of a 50% 1,3 - butanediol solution was added. After the addition, it was soaked at 10°C - 30°C for 7 - 14 days. After soaking, filtration was carried out using a filter material. After filtration, it was allowed to stand at - 30°C - 10°C for 7 - 14 days. After standing, filtration was carried out using a filter material.

[0121] <Preparation Example 1 of Malva sylvestris Extract> To 100 g of dried Malva sylvestris flowers, 2 kg of a 50% 1,3 - butanediol solution was added. After the addition, it was soaked at 10°C - 30°C for 7 - 14 days. After soaking, filtration was carried out using a filter material. After filtration, it was allowed to stand at - 30°C - 10°C for 7 - 14 days. After standing, filtration was carried out using a filter material.

[0122] <Preparation Example 1 of Rosa multiflora Fruit Extract> To 100 g of dried Rosa multiflora fruits (Rosa laevigata Michx.), 1 kg of a 50% 1,3 - butanediol solution was added. After the addition, it was soaked at 10°C - 30°C for 7 - 14 days. After soaking, filtration was carried out using a filter material. After filtration, it was allowed to stand at - 30°C - 10°C for 7 - 14 days. After standing, filtration was carried out using a filter material.

[0123] <Preparation Example 1 of Equisetum arvense Extract> To 100 g of dried Equisetum arvense whole plants, 1 kg of a 50% 1,3 - butanediol solution was added. After the addition, it was soaked at 10°C - 30°C for 7 - 14 days. After soaking, filtration was carried out using a filter material. After filtration, it was allowed to stand at - 30°C - 10°C for 7 - 14 days. After standing, filtration was carried out using a filter material.

[0124] The dry solid content of each extract obtained in the above Preparation Example 1 was 0.1 - 5%.

[0125] <Preparation Example 2 of Paeonia lactiflora Extract> To 100 g of dried peony roots, 1.5 kg of a 50% ethanol solution was added. After the addition, it was macerated for 7 - 14 days under the condition of 10°C - 30°C. After maceration, filtration was carried out using a filtering material. After filtration, it was allowed to stand for 7 - 14 days under the condition of -30°C - 10°C. After standing, filtration was carried out using a filtering material.

[0126] <Preparation Example 2 of Ruscus aculeatus extract> To 100 g of dried material of the underground part (roots and / or rhizomes) of Ruscus aculeatus, 1 kg of a 50% ethanol solution was added. After the addition, it was macerated for 7 - 14 days under the condition of 10°C - 30°C. After maceration, filtration was carried out using a filtering material. After filtration, it was allowed to stand for 7 - 14 days under the condition of -30°C - 10°C. After standing, filtration was carried out using a filtering material.

[0127] <Preparation Example 2 of Malva sylvestris extract> To 100 g of dried Malva sylvestris flowers, 2 kg of a 50% ethanol solution was added. After the addition, it was macerated for 7 - 14 days under the condition of 10°C - 30°C. After maceration, filtration was carried out using a filtering material. After filtration, it was allowed to stand for 7 - 14 days under the condition of -30°C - 10°C. After standing, filtration was carried out using a filtering material.

[0128] <Preparation Example 2 of Rosa multiflora extract> To 100 g of dried Rosa laevigata Michx. fruits, 1 kg of a 50% ethanol solution was added. After the addition, it was macerated for 7 - 14 days under the condition of 10°C - 30°C. After maceration, filtration was carried out using a filtering material. After filtration, it was allowed to stand for 7 - 14 days under the condition of -30°C - 10°C. After standing, filtration was carried out using a filtering material.

[0129] <Preparation Example 2 of Equisetum arvense extract> To 100 g of dried Equisetum arvense L. whole herb, 1 kg of a 50% ethanol solution was added. After the addition, it was macerated for 7 - 14 days under the condition of 10°C - 30°C. After maceration, filtration was carried out using a filtering material. After filtration, it was allowed to stand for 7 - 14 days under the condition of -30°C - 10°C. After standing, filtration was carried out using a filtering material.

[0130] The dry solid content of each extract obtained in the above Preparation Example 2 was 0.1 - 5%.

[0131] <Preparation Example 3 of Ruscus aculeatus extract> To 100 g of the dried rhizome and / or root of Ruscus aculeatus, 1 kg of a 50% ethanol solution was added. After the addition, it was macerated at 10°C - 30°C for 7 - 14 days. After maceration, the Ruscus aculeatus was recovered, and 1 kg of a 50% ethanol solution was added again. After the addition, it was macerated again at 10°C - 30°C for 7 - 14 days. The extracts after each maceration were mixed and adsorbed onto a column packed with Diaion HP20. After adsorption, it was washed with 50% ethanol and then eluted with 100% ethanol. After elution, the obtained extract was treated with activated carbon in an amount equal to the solid content and filtered through a filter material.

[0132] The dry solid content of each extract obtained in Preparation Example 3 above was 0.1 - 5%.

[0133] [Example 1] A study was conducted on whether each plant extract has an inhibitory effect on the binding of AGEs and RAGE.

[0134] (1) Sample preparation Recombinant human RAGE Fc chimeric protein (manufactured by R&D Systems, 1145 - RG) (hereinafter referred to as RAGE - Fc) was prepared as a 2 μg / mL solution with PBS. Paeonia extract (Falcorex Paeonia B, Ichimaru Pharcos Co., Ltd., an extract obtained by extracting from the roots of Paeonia lactiflora Pall. (Paeonia albiflora Pall. var. trichocarpa Bunge) (Paeoniaceae) or other related plants (Paeoniaceae) with a 1,3 - butanediol solution) was prepared as solutions with final concentrations of 0.25%, 0.5%, and 1% with PBS containing 0.1% BSA. Ruscus aculeatus extract (the above Preparation Example 1), Malva sylvestris extract (Falcorex Malva B, Ichimaru Pharcos Co., Ltd.), Rosa laevigata extract (Falcorex Rosa laevigata B, Ichimaru Pharcos Co., Ltd.), Equisetum arvense extract (the above Preparation Example 1) were each prepared as solutions with final concentrations of 0.125%, 0.25%, and 0.5% with PBS containing 0.1% BSA. Biotin - labeled AGE - BSA (manufactured by R&D Systems, BT4127) (hereinafter referred to as AGEs - Biotin) was prepared as a solution with a final concentration of 20 μg / mL with PBS containing 0.1% BSA.

[0135] (2) AGEs and RAGE binding inhibition assay In a 96-well plate, add 50 μL of the RAGE-Fc solution prepared in the above Example 1(1) to each well, and let it stand at 4 °C to allow RAGE-Fc to coat the well plate. Then, let it stand overnight, and restore the well plate to room temperature. Add 200 μL of PBS containing 1% BSA to each well for blocking. After blocking for 2 - 4 hours, wash the well plate with PBS containing 0.1% Tween (PBS-T). After washing, add 50 μL of each plant extract prepared in the above Example 1(1) to each well. For the control group without added extract, add PBS containing 0.1% BSA with 50% BG at the position of each plant extract. After addition, react for 1 - 2 hours, and wash the well plate with PBS-T. After washing, add 50 μL of AGEs-Biotin prepared in the above Example 1(1) to each well. After addition, react for 30 minutes - 1 hour, and wash the well plate with PBS-T. After washing, dilute streptavidin-HRP with PBS containing 0.1% BSA at 1 / 1000, and add 50 μL to each well. After addition, react for 30 minutes - 1 hour, and wash the well plate with PBS-T. After washing, detect with TMB, terminate the reaction with 1 / 10 diluted sulfuric acid, and measure the absorbance at 450 nm. Each experimental group was tested with 1 sample (n = 1). The results are as Figure 1 shown.

[0136] Figure 1 is a graph of the inhibitory effect of AGEs and RAGE binding after the addition of each plant extract. In Figure 1 , (A) represents the result of adding peony extract, (B) represents the result of adding butcher's broom extract, (C) represents the result of adding mallow extract, (D) represents the result of adding multiflora rose extract, and (E) represents the result of adding horsetail extract. Figure 1 In, the vertical axis represents the inhibition rate (%) of AGEs and RAGE binding, and the horizontal axis represents the concentration of each plant extract. As Figure 1 shown, after adding each plant extract, the inhibition rate of AGEs and RAGE binding increased in a concentration-dependent manner. It can be seen that peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and horsetail extract all have the effect of inhibiting AGEs and RAGE binding.

[0137] [Example 2] It is known that AGEs can promote melanogenesis through RAGE. Therefore, a study was conducted on whether each plant extract has the effect of inhibiting AGEs-induced melanogenesis.

[0138] (1) Specimen preparation Paeonia extract (Falcorex Paeonia B, Ichimaru Pharcos Co., Ltd.), Ruscus extract (Preparation Example 1 above), Malva extract (Falcorex Malva B, Ichimaru Pharcos Co., Ltd.), Rosa multiflora extract (Falcorex Rosa laevigata B, Ichimaru Pharcos Co., Ltd.), Equisetum extract (Preparation Example 1 above) were each prepared at the final concentrations shown in Table 1 below when added to the medium using MGM (trademark)-4 Melanocyte Growth Medium-4 BulletKit (trademark) (MGM, manufactured by Lonza). AGEs (AGE-BSA, manufactured by CalbioChem) was prepared as a 10 mg / mL solution in PBS.

[0139]

Table 1

[0140] (2) Cultivation of melanocytes In a 12-well plate, melanocytes (epidermal melanocytes derived from normal human neonates, manufactured by LIFELINE, Lot: 07965, passage number 5) were inoculated at a density of 1.4×10 5 cells per well and cultured for 2 days under the conditions of 5% CO 2 , 37°C. MGM medium was used during the cultivation. After the cultivation, the medium in the well plate was aspirated and replaced with MGM medium without fetal bovine serum (FBS) (MGM FBS-). After replacing the medium, the cells were cultured overnight and then replaced with MGM FBS- medium containing each of the plant extracts prepared in Example 2(1) above, and the melanocytes were cultured for 1 hour. The untreated group (n.t. group) and the negative control group (NC group) were replaced with MGM FBS- medium containing the same amount of solvent as the extract-added group. After the cultivation, AGEs prepared in Example 2(1) above were added to the medium to a final concentration of 400 μg / mL. The n.t. group was added with an equal amount of PBS instead of AGEs. After the addition, the cells were continuously cultured for 72 hours under the conditions of 5% CO 2 , 37°C. After the cultivation, each plant extract treatment and AGEs addition operation were performed again, and the cells were continuously cultured for 72 hours under the same conditions.

[0141] (3) Evaluation of cell number After the culture in the above Example 2(2) was completed, the culture medium was removed. Then, Cell Counting Kit-8 (DOJINDO) diluted 1 / 20 with MGM FBS was added, and the number of viable cells was evaluated by measuring the absorbance at 450 nm. Three sample tests were performed for each experimental group (n = 3). Statistical analysis was performed using Dunnett’s test (*: p < 0.05, ***: p < 0.001 vs n.t.).

[0142] (4) Evaluation of melanin content The cells cultured in the above Example 2(2) were digested with trypsin and collected. After collection, a mixture of ethanol:diethyl ether = 3:1 (volume ratio) was added, and centrifugation was performed to remove the supernatant, obtaining a melanin precipitate. The precipitate was dried overnight, and the obtained dried melanin was dissolved in 25 μL of a 1N NaOH / 10% DMSO mixture. After dissolution, the absorbance at 420 nm was measured, and the amount of melanin production was calculated. Three sample tests were performed for each experimental group (n = 3). Statistical analysis was performed using Dunnett’s test (*: p < 0.05, ***: p < 0.001 vs NC). The results are as Figure 2 shown.

[0143] Figure 2 is a graph of the average melanin production of cells after the addition of each plant extract. In Figure 2 , the vertical axis represents the relative value with respect to the n.t. group, and the horizontal axis represents the experimental groups. Figure 2 In (A), the results of adding peony extract are shown (in the figure, the value of the n.t. group is set to 1, AGEs group: 1.22, 0.3% addition group: 1.19, 1% addition group: 1.08), (B) shows the results of adding butcher's broom extract (in the figure, the value of the n.t. group is set to 1, AGEs group: 1.22, 0.3% addition group: 1.08), (C) shows the results of adding mallow extract (in the figure, the value of the n.t. group is set to 1, AGEs group: 1.27, 0.5% addition group: 0.96), (D) shows the results of adding multiflora rose extract (in the figure, the value of the n.t. group is set to 1, AGEs group: 1.21, 1% addition group: 1.02), (E) shows the results of adding equisetum extract (in the figure, the value of the n.t. group is set to 1, AGEs group: 1.18, 0.3% addition group: 1.06). As Figure 2 shown, after the addition of each plant extract, the relative values were all decreased compared with the non-added group (negative control group) of each plant extract. These results indicate that peony extract, butcher's broom extract, mallow extract, multiflora rose extract, and equisetum extract may all inhibit melanin production by inhibiting the binding of AGEs and RAGE and inhibiting the downstream signals of RAGE.

[0144] [Example 3] It was investigated whether the extract obtained from Ruscus aculeatus L. during the extraction process could inhibit AGEs-induced melanogenesis.

[0145] (1) Specimen preparation In Preparation Example 3 of the above-mentioned Ruscus aculeatus L. extract, the solution obtained after washing with 50% ethanol was designated as Ruscus aculeatus L. HP20W, the solution obtained by eluting with 100% ethanol was designated as Ruscus aculeatus L. HP20E, and the solution obtained by treating HP20E with activated carbon in an amount equal to the solid content and filtering was designated as Ruscus aculeatus L. HP20E charcoal. The above-mentioned Ruscus aculeatus L. extracts were respectively formulated into the final concentrations shown in Table 2 below when added to the medium using MGM (trademark)-4 Melanocyte Growth Medium-4 BulletKit (trademark) (hereinafter referred to as MGM, manufactured by Lonza). Hereinafter, Ruscus aculeatus L. HP20W, Ruscus aculeatus L. HP20E, and HP20E charcoal are also collectively referred to as Ruscus aculeatus L. extracts. AGEs (AGE-BSA, manufactured by CalbioChem) was formulated into a 10 mg / mL solution with PBS.

[0146]

Table 2

[0147] (2) Cultivation of melanocytes In a 12-well plate, melanocytes (epidermal melanocytes derived from normal human neonates, manufactured by LIFELINE, Lot: 07965, passage number 5) were inoculated at a density of 1.4×10 5 cells per well and cultured for 2 days under the conditions of 5% CO 2 and 37 °C. MGM medium was used during the cultivation process. After the cultivation, the medium in the well plate was aspirated and replaced with MGM FBS-medium. After replacing the medium, the cells were cultured overnight and then replaced with MGM FBS-medium containing the Ruscus aculeatus L. HP20W, Ruscus aculeatus L. HP20E, or Ruscus aculeatus L. HP20E charcoal prepared in (1) of Example 3 above, and the melanocytes were cultured for 1 hour. The non-added group (n.t. group) and the negative control group (NC group) were replaced with MGM FBS-medium containing the same amount of solvent as the Ruscus aculeatus L. extract-added group. After cultivation, AGEs prepared in (1) of Example 3 above was added to the medium to make the final concentration reach 400 μg / mL. The n.t. group added an equal amount of PBS instead of AGEs. After the addition, the cells were continuously cultured for 72 hours under the conditions of 5% CO 2 and 37 °C. After the cultivation, the pretreatment with the Ruscus aculeatus L. extract and the addition of AGEs were performed again, and the cells were continuously cultured for 72 hours under the same conditions.

[0148] (3) Evaluation of cell number After the culture in Example 3(2) above was completed, the culture medium was removed. Then, Cell Counting Kit-8 (DOJINDO) diluted 1 / 20 with MGM FBS was added, and the number of viable cells was evaluated by measuring the absorbance at 450 nm. Three sample tests were performed for each experimental group (n = 3). Statistical analysis was performed using Dunnett’s test (*: p < 0.05, ***: p < 0.001 vs n.t.).

[0149] (4) Evaluation of melanin content The cells cultured in Example 3(2) above were digested with trypsin and collected. After collection, a mixture of ethanol:diethyl ether = 3:1 (volume ratio) was added, and centrifugation was performed to remove the supernatant, obtaining a melanin precipitate. The precipitate was dried overnight, and the obtained dried melanin was dissolved in 25 μL of a 1N NaOH / 10% DMSO mixture. After dissolution, the absorbance at 420 nm was measured, and the amount of melanin production was calculated. Three sample tests were performed for each experimental group (n = 3). Statistical analysis was performed using Dunnett’s test (*: p < 0.05, ***: p < 0.001 vs NC). The results are as Figure 3 shown.

[0150] Figure 3 is a graph of the average melanin production per cell after the addition of each ruscus extract. In Figure 3 , the vertical axis represents the relative value with respect to the n.t. group, and the horizontal axis represents the experimental group and the addition concentration of the ruscus extract. Figure 3 In, (A) shows the results of adding ruscus HP20W (the value of the n.t. group in the figure is set to 1, AGEs group: 1.30, 0.1% addition group: 1.28, 0.3% addition group: 1.23, 1% addition group: 1.32), (B) shows the results of adding ruscus HP20E (the value of the n.t. group in the figure is set to 1, AGEs group: 1.30, 0.001% addition group: 1.43, 0.003% addition group: 1.07, 0.01% addition group: 1.07), (C) shows the results of adding HP20E charcoal (the value of the n.t. group in the figure is set to 1, AGEs group: 1.30, 0.001% addition group: 1.18, 0.003% addition group: 1.36, 0.01% addition group: 1.19). As Figure 3 shown, after adding ruscus HP20E or HP20E charcoal, the relative value decreased compared with the non-addition group (negative control group) of each plant extract. These results suggest that both ruscus HP20E and ruscus HP20E charcoal may inhibit melanin production by inhibiting the binding of AGEs and RAGE and inhibiting the downstream signals of RAGE.

[0151] [Example 4] It was investigated whether the peony extract could inhibit AGEs-induced inflammation.

[0152] (1) Specimen preparation The peony extract (Falcorex Peony B, -Maru, manufactured by Falcos Co., Ltd.) was prepared as a solution with a final concentration of 0.4% when added to the medium using D-MEM medium (manufactured by Fujifilm Wako Pure Chemical Corporation) containing 0.25% FBS. AGEs were prepared as a 10 mg / mL solution in PBS.

[0153] (2) Cultivation of normal human-derived dermal fibroblasts (NBNHDF) In a 35 mm culture dish, NBNHDF (normal human neonatal-derived dermal fibroblasts, manufactured by KURABO Industries Ltd., Lot: 5884, passage number 5) were inoculated at a density of 1×10 5 cells per dish and cultured for 2 days under the conditions of 5% CO 2 ₂ and 37 °C. During the cultivation, D-MEM medium containing 10% FBS was used. After the cultivation was completed, the medium in the culture dish was aspirated and replaced with D-MEM medium containing 0.25% FBS. After replacing the medium, the cells were cultured overnight and then replaced with D-MEM medium containing 0.25% FBS with the peony extract prepared in Example 4(1) above, and the NBNHDF were cultured for 1 hour. The non-added group (n.t. group) and the negative control group (NC group) were replaced with D-MEM medium containing the same amount of solvent as the peony extract-added group and 0.25% FBS. After cultivation, the AGEs prepared in Example 4(1) above were added to the medium to a final concentration of 400 μg / mL. The n.t. group was added with an equal amount of PBS instead of AGEs. After the addition, the cells were continuously cultured for 16 hours under the conditions of 5% CO 2 ₂ and 37 °C. After the cultivation was completed, total RNA was extracted using the RNeasy-Kit (manufactured by QIAGEN).

[0154] (3) Expression analysis Measure the concentration of total RNA extracted in Example 4(2) above, and prepare cDNA using ReverTra Ace (registered trademark) qPCR RT Master Mix (manufactured by TOYOBO Co., Ltd.). After preparation, use THUNDER BIRD (registered trademark) Next SYBR (registered trademark) qPCR (manufactured by TOYOBO Co., Ltd.) to quantitatively analyze gene expression levels by real-time PCR. In the quantitative analysis, interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and receptor for advanced glycation end products (RAGE) are used as target genes, and ribosomal protein S18 (RPS18) is used as an internal reference gene. The following Table 3 lists the primers used in the quantitative analysis. Three sample detections are performed for each experimental group (n = 3). Statistical analysis is carried out using Dunnett’s test (*: p < 0.05, ***: p < 0.001 vs NC). The results are as Figure 4 shown.

[0155]

Table 3

[0156] Figure 4 is a graph showing the expression levels of each gene in NBNHDF cells after adding the peony extract. In Figure 4 , the vertical axis represents the relative value of the expression level of each gene relative to the n.t. group, and the horizontal axis represents the experimental groups. Figure 4 , (A) shows the results of IL-8 gene expression (the value of the n.t. group in the figure is set to 1, AGEs group: 1.27, 0.4% addition group: 0.56), (B) shows the results of TNF-α gene expression (the value of the n.t. group in the figure is set to 1, AGEs group: 1.42, 0.4% addition group: 0.95), and (C) shows the results of RAGE gene expression (the value of the n.t. group in the figure is set to 1, AGEs group: 1.17, 0.4% addition group: 0.84). As Figure 4 shown, after adding the peony extract, the relative value decreased compared with the non-added group (negative control group) of each plant extract. These results indicate that the peony extract can inhibit the gene expression of IL-8, TNF-α, and RAGE. In addition, the peony extract may inhibit the expression of IL-8 and TNF-α by inhibiting the binding of AGEs and RAGE and suppressing the downstream signals of RAGE.

[0157] [Example 5] Study whether the peony extract can inhibit the delay in cell proliferation caused by the glycation of epidermal basement membrane components.

[0158] (1) Specimen preparation The peony extract (Falcorex Peony B, manufactured by Ichimaru Pharcos Co., Ltd.) was formulated into a solution with a final concentration of 0.4% when added to the culture medium. During the preparation process, the additives attached to KG (trademark) Gold Keratinocyte Growth Medium BulletKit (trademark), Calcium Free (hereinafter referred to as KG, manufactured by Lonza) were used. Among them, 1 / 5 of the specified amounts of BPE and EGF were added, and other additives were added according to the specified amounts, and the calcium ion concentration was adjusted to 0.06 mmol / l in the culture medium (hereinafter referred to as the test KG).

[0159] (2) Coating of the culture plate and glycation reaction The epidermal basement membrane components Cultrex Ultimatrix BME (hereinafter referred to as BME, manufactured by R&D, 3433 - 005 - 01), iMatrix332 (manufactured by nippi, 892031), iMatrix511 (manufactured by nippi, 892011), and Fibronectin (manufactured by Corning, 354008) were diluted according to Table 4 below. After dilution, coating was performed on a 96 - well culture plate according to the operating procedures of each product. After coating, D(+)-glucose (manufactured by Kishida, 000 - 34105) dissolved in PBS was added thereto to make the final concentration reach 100 mg / ml, and it was incubated at 37°C for 7 days to glycate the basement membrane components. The n.t. group was added with an equal amount of PBS.

[0160]

Table 4

[0161] (3) Culture of normal human neonatal - derived epidermal keratinocytes (NBNHEK) The 96 - well culture plate prepared in Example 5(2) above was washed twice with PBS. After washing, NBNHEK (manufactured by LIFELlNE, Lot: 10232, passage number 4) was inoculated at a density of 1.5×10 3 cells / 100 μl per well. After inoculation, culture was carried out under the conditions of 5% CO 2 , 37°C. During the culture process, KG medium containing all the specified amounts of additives and 0.06 mmol / l Ca 2+ was used. After culturing for 24 hours, it was changed to the culture medium prepared in Example 5(1) above and cultured for another 72 hours. The n.t. group and the NC group were changed to the test KG medium containing an equal amount of solvent. After the culture was completed, each well of cells was photographed using an inverted microscope (manufactured by OLYMPUS, main unit: IX70, camera: DP70). The results are as followsFigure 5 as shown

[0162] Figure 5 It is a photograph of the results of observing the effect of glycated epidermal basement membrane components on the proliferation of NBNHEK cells after adding peony extract. Figure 5 Among them, (A) is the result of the control group, (B) is the result of the glycation model group, and (C) is the result after adding peony extract. As Figure 5 shown, cell proliferation was inhibited in the glycation model group, while no cell proliferation inhibition was observed after adding peony extract. These results suggest that peony extract may inhibit the binding of AGEs and RAGE and downstream signals of RAGE, thereby improving cell proliferation inhibition caused by glycation.

[0163] [Example 6] Study whether peony extract has the effect of inhibiting skin pigmentation.

[0164] (1) Correlation between the color non-uniformity index value and human skin age The possibility of peony extract inhibiting human skin pigmentation was studied, and for skin pigmentation, the information processing method developed by the present inventor was used. First, it was confirmed whether this information processing method can evaluate the correlation with skin pigmentation caused by aging. Specifically, the skin non-uniformity index value calculated from the image containing the skin was used as an index of skin pigmentation, and the correlation between this color non-uniformity index value and human skin age was confirmed. First, for the same subject, at specific ages (38, 40, 41, 42, 43, 44, and 47 years old), images of approximately the same part of the cheek skin were obtained ( Figure 6 (A)). For the cheek skin images of each age, SLIC registered in OpenCV was used for segmentation to make the number of color types (number of segments) in the image less than 100 colors, thereby integrating the image colors and dividing the regions ( Figure 6 (B)).

[0165] For the obtained images ( Figure 6 (B)), the dispersion degree (standard deviation) of the color (Lab) of each region, the area of each region (segment), the color change rate (color gradient) between each region and adjacent regions, and the average value of the lightness (L value) of each region were calculated. Then, using a large language model, variables and formulas related to the target skin age in the evaluation items were set according to the obtained index values.

[0166] The results show that the variables of the L value (wL), a value (wa), b value (ωb), the area of each region, the color gradient (ωgrad), and the average L value (correction coefficient) are set as L value: a value: b value: the area of each region: color gradient = 3:2:1.5:1:2:0.03. The color non-uniformity index value (corrected mixed score) calculated by the following formulas (3)-(12) is related to the target skin age. It should be noted that the values in the following formulas (6)-(12) are calculated using the corrected L value (Li’), a value (ai’), and b value (bi’). In addition, the average L value in formula (11) is the average of the corrected L values (Li’) of each segment. The results are as Figure 7 shown.

[0167]

Equation 1

[0168] Figure 7 is a correlation chart of the target skin age and the color non-uniformity index value. Figure 7 In, the horizontal axis represents the target skin age, and the vertical axis represents the color non-uniformity index value. As Figure 7 shown, the color non-uniformity index value and the target skin age show a high degree of correlation (r = 0.7196) (age / score: 38 / 4.68, 40 / 4.78, 41 / 5.12, 42 / 5.66, 43 / 6.85, 44 / 7.42, 47 / 9.06). These results confirm that the correlation with skin pigmentation caused by age growth can be evaluated through the above information processing method.

[0169] (2) Human skin monitoring experiment Using the above information processing method, study whether paeony extract can inhibit human skin pigmentation. Before the study, emulsions and placebo emulsions for human skin monitoring experiments were prepared. The emulsion for human skin monitoring experiment (Example 6) was formulated according to the components and content ratios in Table 5 below. The placebo emulsion (Comparative Example 6) was formulated according to the components and content ratios in Table 6 below.

[0170]

Table 5

[0171]

Table 6

[0172] On the designated parts of the faces of normal human subjects (4 women with an average age of 46 years), the lotion in Table 5 above and the placebo lotion in Table 6 were applied, and a monitoring test was conducted. This monitoring test was carried out from November 18, 2024 to December 24, 2024. The lotion in Table 5 was applied to the designated part on one side of the subject's face (Example 6), and the lotion in Table 6 was applied to the designated part on the other side of the subject's face (Comparative Example 6). The application operation was carried out 2 times a day (once in the morning and once in the evening) for 4 weeks. In the monitoring test, on the day of the start of application, 2 weeks after application, and 4 weeks after application, the designated parts were photographed using a camera. For the obtained images, the color non-uniformity index value was calculated in the same manner as the information processing method in Example 6(1) above. Based on the color non-uniformity index value on the day of the start of application (0w) as the benchmark (100), the relative value of color non-uniformity was calculated. The results are as Figure 8 shown.

[0173] Figure 8 is a chart of the results of the human skin monitoring test. Figure 8 In it, the vertical axis represents the relative value when the color non-uniformity index value on the day of the start of application (0w) is 100, and the horizontal axis represents the number of weeks (w) after the start of application. Figure 8 In it, the value of Comparative Example 6 after 2 weeks is 107.2, and the value after 4 weeks is 107.5; the value of Example 6 after 2 weeks is 88.7, and the value after 4 weeks is 77.5. As Figure 8 shown, compared with Comparative Example 6 after 4 weeks of application, the color non-uniformity index value of Example 6 after 4 weeks of application is lower. These results indicate that the peony extract can inhibit skin pigmentation.

[0174] [Example 7] Study whether the peony extract can inhibit the differentiation delay caused by the glycosylation of epidermal basement membrane components.

[0175] (1) Specimen preparation The peony extract (Falcorex Peony B, Ichimai Falcos Co., Ltd.) was respectively formulated into solutions with a final concentration of 0.2% or 0.4% when added to the culture medium. During the preparation process, using the additives attached to KG (trademark) Gold Keratinocyte Growth Medium BulletKit (trademark), Calcium Free (hereinafter referred to as KG, manufactured by Lonza), BPE, EGF, and Insulin were added in the specified amounts, and the calcium ion concentration was adjusted to 0.15 mmol / | of the culture medium (hereinafter referred to as the KG for differentiation test).

[0176] (2) Coating of the culture plate and glycosylation reaction Dilute the epidermal basement membrane component iMatrix332 (manufactured by Nippi, Inc., 892031) to 4.2 μg / cm 2 . After dilution, coat a 12-well culture plate according to the operating procedures. After coating, add D(+)-glucose (manufactured by Kishida Co., Ltd., 000-34105) dissolved in PBS to a final concentration of 100 mg / ml, and incubate at 37 °C for 14 days to glycosylate the basement membrane component. The negative control group adds an equal amount of PBS.

[0177] (3) Culture of normal human neonatal-derived epidermal keratinocytes (NBNHEK) Wash the 12-well culture plate prepared in Example 7(2) above twice with PBS. After washing, inoculate NBNHEK (manufactured by LIFELINE, Lot: 10232, passage number 4) at a density of 1.8×10 5 cells / ml per well. After inoculation, culture under the conditions of 5% CO 2 and 37 °C. During the culture process, use KG medium containing all the specified additives and 0.06 mmol / l Ca 2+ . After culturing for 24 hours, change to the medium prepared in Example 7(1) above (KG for differentiation test), and continue culturing for 24 hours. Then, change to the KG medium for differentiation test again and culture under the conditions of 5% CO 2 and 37 °C for 6 hours. After the culture is completed, extract total RNA using the RNeasy-Kit (QIAGEN).

[0178] (4) Expression analysis Measure the concentration of the total RNA extracted in Example 7(3) above, and prepare cDNA using ReverTra Ace (registered trademark) qPCR RT Master Mix (TOYOBO). After preparation, use THUNDER BIRD (registered trademark) Next SYBR (registered trademark) qPCR (TOYOBO) to quantitatively analyze the gene expression level by real-time PCR. In the quantitative analysis, use transglutaminase 1 (TGM1) and keratin 10 (K10) as target genes, and use RPS18 as an internal reference gene. The following Table 7 lists the primers used in the quantitative analysis (all manufactured by Takara Bio Inc.). Three sample detections are performed for each experimental group (n = 3). Statistical analysis uses Dunnett’s test (*: p < 0.05, **: p < 0.001 vs NC). The results are as Figure 9 shown.

[0179]

Table 7

[0180] Figure 9 It is a graph of the expression levels of each gene after adding peony extract to NBNHEK cells. In Figure 9 , the vertical axis represents the relative value of the expression level of each gene relative to the n.t. group, and the horizontal axis represents the test groups. Figure 9 In , (A) shows the results of TGM1 gene expression (in the figure, the value of the n.t. group is set to 1, PC group: 2.7, NC group: 2.0, 0.2% addition group: 2.1, 0.4% addition group: 0.4), and (B) shows the results of K10 gene expression (in the figure, the value of the n.t. group is set to 1, PC group: 3.9, NC group: 1.7, 0.2% addition group: 2.3, 0.4% addition group: 2.6). As Figure 9 shown, after adding peony extract, the relative value increased compared with the non-added group (negative control group) of peony extract. These results indicate that peony extract can increase the gene expression of TGM1 and K10. It is speculated from this that peony extract has the effect of normalizing metabolism, barrier function and / or moisturizing during epidermal differentiation.

[0181] [Example 8] Study whether peony extract can inhibit melanogenesis.

[0182] (1) Component fractionation Analyze the components contained in peony extract. The specific operation is as follows: Dilute 200 g of the peony extract prepared in Preparation Example 1 above 5 times with water to prepare a sample solution. After adjustment, add 200 g of HP-20 gel to the sample solution, stir and adsorb overnight. At the same time, swell 200 g of HP-20 gel with a 10% ethanol solution and load it into a column. Add the adsorbed gel and the non-adsorbed solution to the column. After addition, add the batch solution to the column, and then elute with 1 L of 10% ethanol solution to remove BG. Gradually increase the ethanol concentration in a 10% gradient, and collect 250 ml of eluate each time (a total of 1 L is collected). After collection, according to the HPLC analysis results, divide the eluate into 5 fractions (Fr1-Fr5), and adjust it to a solution suitable for cell experiments with 200 g of 50% ethanol solution.

[0183] (2) Study on inhibiting melanogenesis Except for adding peony extract (final concentration 0.4%, Falcorex Peony B, Ichimaru Pharcos Co., Ltd.) or Fr1-Fr5 fractionated in Example 8(1) above (final concentration 0.48%) to the medium, cultivate melanocytes and evaluate the melanin content according to the same method as in Example 2. The results are as Figure 10 shown.

[0184] Figure 10 It is a graph of the average melanin production of cells after adding peony extract. In Figure 10 , the vertical axis represents the relative value with respect to the AGEs group, and the horizontal axis represents the test groups. Figure 10 The respective values in Figure 10 are as follows (the value of the AGEs group in the figure is set to 1): n.t. group: 0.77, 0.4% peony extract group: 0.87, Fr1 addition group: 1.01, Fr2 addition group: 1.02, Fr3 addition group: 0.97, Fr4 addition group: 0.99, Fr5 addition group: 0.99. As

[0185] shown, after adding peony extract, the relative value decreased compared to Fr1 - Fr5. These results indicate that, compared to using each fraction, using peony extract as a whole is more effective in inhibiting melanin production.

[0186] Based on the above results, it can be seen that each extract of peony, butcher's broom, mallow, wild rose, and equisetum can inhibit the binding of AGEs and RAGE. Moreover, these extracts have the potential to inhibit AGEs - induced melanin production by inhibiting the binding of AGEs and RAGE. In addition, peony extract has effects such as inhibiting inflammation, improving cell proliferation inhibition caused by basement membrane glycation, improving cell adhesion inhibition, improving skin pigmentation, normalizing metabolism during epidermal differentiation, normalizing barrier function, and / or moisturizing by inhibiting the binding of AGEs and RAGE. Extracts with AGEs and RAGE binding inhibitory activity (each extract of butcher's broom, mallow, wild rose, and equisetum), by inhibiting RAGE signaling, are also expected to exert effects such as inhibiting inflammation, improving cell proliferation inhibition caused by basement membrane glycation, improving cell adhesion inhibition, improving skin pigmentation, normalizing metabolism during epidermal differentiation, normalizing barrier function, and / or moisturizing, similar to peony extract.

[0186] As described above, the present invention has been described through embodiments and examples, but the present invention is not limited to the above - mentioned embodiments and examples. Within the scope of the present invention, those skilled in the art can make various modifications to its structure and details.

[0187] This application claims priority based on Japanese Patent Application No. 2024 - 131958 filed on August 8, 2024, the entire disclosure of which is incorporated herein by reference.

[0188] <Supplementary Note> Some or all of the above - mentioned embodiments and examples can be as described in the following supplementary note, but are not limited thereto. <Preparation for Inhibiting the Binding of AGEs and RAGE> (Supplementary Note 1) A preparation for inhibiting the binding of AGEs (advanced glycation end products) and RAGE (receptor for AGEs), which comprises peony extract, butcher's broom extract, mallow extract, multiflora rose extract and / or equisetum arvense extract. (Supplementary Note 2) The preparation according to Supplementary Note 1, which is used for inhibiting RAGE gene expression, inhibiting TNF-α gene expression, inhibiting IL-8 gene expression, promoting TGM1 gene expression and / or promoting K10 gene expression. (Supplementary Note 3) The preparation according to Supplementary Note 1, which is used for inhibiting melanogenesis, anti-inflammation, promoting cell proliferation and / or promoting cell adhesion. <Skin topical preparation> (Supplementary Note 4) A preparation for skin topical application, which comprises the preparation described in any one of Supplementary Notes 1 to 3. <Cosmetic> (Supplementary Note 5) A cosmetic, which comprises the preparation described in any one of Supplementary Notes 1 to 4. (Supplementary Note 6) The cosmetic according to Supplementary Note 5, which is a whitening cosmetic.

[0189] In summary, according to the content of the present invention, a preparation that can inhibit the binding of AGEs and RAGE and is mainly applicable to human skin and the like can be provided. Therefore, the present invention has extremely high application value in the fields of cosmetics and the like.

Sequence Listing

Claims

1. Use of a peony extract in the preparation of a preparation for inhibiting the binding of advanced glycation end products (AGEs) to their receptors (RAGE).

2. The use according to claim 1, characterized in that The use is also used to inhibit the expression of RAGE gene.

3. The use according to claim 1, characterized in that The peony extract is prepared by the following steps: 1,3-butanediol solution is used as an extraction solvent and the extract is obtained by extraction from peony roots.

4. A method for preparing a preparation for inhibiting the binding of AGEs to RAGE, the preparation comprising a peony extract, the method comprising the following steps: The peony extract is mixed with a pharmaceutically acceptable carrier.

5. The preparation method according to claim 4, characterized in that: The formulation is also useful for inhibiting the expression of the RAGE gene.

6. The preparation method according to claim 4, characterized in that: The peony extract is prepared by the following steps: 1,3-butanediol solution is used as an extraction solvent and the extract is obtained by extraction from peony roots.

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