Method for screening skin condition-improving component

Abstract

To provide a novel method for screening skin condition-improving components.SOLUTION: Provided is a method for screening skin condition-improving components, using upregulation of pleiotrophin expression as an index.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for screening ingredients for improving skin conditions. [Background technology]

[0002] Good skin condition not only increases self-satisfaction and self-confidence and has a positive impact on daily life, but also affects one's impression and trustworthiness, so there is a demand for products that help keep skin in good condition.

[0003] Skin viscoelasticity / elasticity is used as one of the indicators of skin condition (Non-Patent Document 1). Therefore, topical preparations that improve skin viscoelasticity / elasticity have been developed (Patent Document 1).

[0004] It is also known that the dermis influences the viscoelasticity / elasticity of the skin (Non-Patent Document 2). The dermis deteriorates with age, and various mechanisms for regenerating the dermis have been proposed (Patent Document 2). However, no satisfactory solution has yet been provided.

[0005] Here, one example of a natural phenomenon in which new dermis is created in the living body is when a hair follicle regresses into the catagen phase and then regenerates in the dermal area where the hair follicle once existed (Figure 1). The catagen phase is one of three stages in the "hair cycle," which is a cycle of repeated hair growth and shedding, and is divided into the anagen phase, catagen phase, and telogen phase (Non-Patent Document 3).

[0006] Recently, it has been reported that changes in gene expression in various cells around hair follicles are linked to the regeneration of the dermis around the hair follicle during hair follicle regression (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2021-116276 [Non-patent literature]

[0008] [Non-Patent Document 1] Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity[online], February 2007, [Search Date: April 24, 2023], <URL:https: / / doi.org / 10.1111 / j.1600-0846.2007.00224.x> [Non-Patent Document 2] Molecular Mechanisms of Dermal Aging and Antiaging Approaches[online], May 2019, [Search Date: April 24, 2023], <URL:https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6540032 / > [Non-Patent Document 3] New Dermatology, 3rd Edition (Nakayama Shoten)[online], February 2018, [Search Date: April 24, 2023], <URL:https: / / www.derm-hokudai.jp / wp / wp-content / uploads / 2021 / 12 / 1-08.pdf> [Non-Patent Document 4] Constructing a Single-Cell Transcriptome Atlas of the Hair Cycle to Decipher Tissue Regeneration Dynamics in Human Skin[online], January 2024, [Search Date: April 24, 2023], <URL:http: / / dx.doi.org / 10.2139 / ssrn.<4690956> [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] The present inventors independently studied the effects of various ligands described in Non-Patent Document 4 on dermal regeneration and found that pleiotrophin (PTN) is particularly effective in dermal regeneration (Figure 2). In other words, it was found that dermal regeneration can be induced by enhancing the expression of pleiotrophin or pleiotrophin receptor.

[0010] An objective of the present invention is to provide a novel composition that enhances the expression of pleiotrophin.

[0011] An object of the present invention is to provide a novel method for screening ingredients that improve skin conditions. [Means for solving the problem]

[0012] The present invention, which solves the above-mentioned problems, provides a method for screening for ingredients that improve skin conditions, using the effect of enhancing pleiotrophin expression as an indicator.

[0013] In a preferred embodiment of the present invention, the test substance is judged to be a more excellent skin condition-improving ingredient as the test substance exhibits a higher effect of enhancing the expression of pleiotrophin cultured in the presence of the test substance.

[0014] In a preferred embodiment of the present invention, the method comprises adding a test substance to a cell culture system and measuring the effect of enhancing pleiotrophin expression in the cells.

[0015] In addition, in a preferred embodiment of the present invention, when the effect of enhancing pleiotrophin expression in cells cultured with the addition of a test substance is statistically significantly greater than the effect of enhancing pleiotrophin expression in cells cultured without the addition of the test substance, the test substance is determined to be a candidate for a skin condition-improving component.

[0016] In addition, a preferred embodiment of the present invention comprises the steps of culturing cells in the presence of a test substance, measuring the effect of enhancing pleiotrophin expression in the cells, and determining that the test substance is a skin condition-improving ingredient when the effect of enhancing pleiotrophin expression is greater than that of cells cultured in the absence of the test substance.

[0017] The present invention also relates to a pleiotrophin expression enhancer containing a brown algae extract as an active ingredient.

[0018] The present invention preferably relates to a pleiotrophin expression enhancer for improving skin conditions.

[0019] The present invention is preferably a pleiotrophin expression enhancer for improving one or more selected from wrinkles, sagging skin, complexion, dullness, and skin viscoelasticity and / or elasticity.

[0020] The present invention also relates to an agent for inducing degradation and / or neoplasia of extracellular matrix, which comprises a brown algae extract as an active ingredient.

[0021] The present invention also relates to a skin fiber structure improving agent containing a brown algae extract as an active ingredient.

[0022] The present invention also relates to an angiogenesis inducer containing a brown algae extract as an active ingredient.

[0023] The present invention also relates to an agent for enhancing the phagocytic activity of leukocytes, which contains a brown algae extract as an active ingredient.

[0024] The present invention also relates to an external skin composition for enhancing pleiotrophin expression, which contains a brown algae extract as an active ingredient.

[0025] The present invention also relates to an external skin composition for improving skin conditions, which comprises a pleiotrophin expression enhancer and a pleiotrophin receptor expression enhancer, and which contains a brown algae extract as an active ingredient. [Effects of the Invention]

[0026] According to the present invention, it is possible to screen for ingredients that improve skin conditions. [Brief explanation of the drawings]

[0027] [Figure 1] Diagram of the hair cycle and dermal regeneration after hair follicle regression [Figure 2] Conceptual diagram of pleiotrophin and dermal regeneration [Figure 3] Graph showing the results of functional analysis of each pleiotrophin gene group [Figure 4] Graph showing verification results of collagen gene expression levels [Figure 5] Image showing the results of observing collagen structure [Figure 6] Graph showing the results of measuring the state of angiogenesis [Figure 7] Graph showing the measurement results of macrophage phagocytic activity [Figure 8] Graph showing the results of verifying the gene expression of syndecan-4 by plant extracts [Figure 9] Graph showing the results of verifying the expression of syndecan-4 genes by the complex extract [Figure 10] Graph showing the results of verifying the effect of brown algae extract 2 on pleiotrophin gene expression [Figure 11] Graph showing the results of verifying pleiotrophin gene expression by the composite extract [Figure 12] Graph showing the results of verifying pleiotrophin gene expression by plant extracts DETAILED DESCRIPTION OF THE INVENTION

[0028] Preferred embodiments of the present invention will be described below, but it goes without saying that the technical scope of the present invention is not limited to the following embodiments.

[0029] <1> Screening method for ingredients that improve skin conditions The present invention relates to a method for screening for a skin condition-improving ingredient using the effect of enhancing pleiotrophin expression as an index. Here, screening includes searching for a skin condition-improving ingredient or a candidate thereof.

[0030] As described above, skin conditions can be improved by increasing the expression of pleiotrophin. Therefore, ingredients that improve skin conditions can be screened using the effect of increasing pleiotrophin expression as an indicator.

[0031] In the screening method of the present invention, the skin condition-improving components to be screened are not particularly limited, and examples of test substances include commercially available compounds (including peptides), known compounds (including peptides), groups of compounds obtained by combinatorial chemistry techniques, extracts derived from living organisms, or mixtures thereof.

[0032] The term "biological extract" refers to not only the extract itself derived from animals or plants, but also a collective term for fractions of extracts, purified fractions, and solvent-removed extracts, fractions, and purified products. Examples of plant extracts include extracts made from wild or cultivated plants, extracts sold as herbal medicine ingredients, and commercially available extracts.

[0033] In the present invention, the test substance is preferably judged to be a superior skin condition-improving ingredient as the test substance exhibits a higher effect of enhancing the expression of pleiotrophin cultured in the presence of the test substance.

[0034] The present invention preferably comprises adding a test substance to a cell culture system and measuring the effect of enhancing pleiotrophin expression in the cells, preferably epidermal keratinocytes.

[0035] Specifically, the screening method of the present invention enables a test substance to be determined to be a superior skin condition-improving ingredient when the test substance exhibits a higher pleiotrophin expression enhancing effect in cells cultured in the presence of the test substance, preferably epidermal keratinocytes.

[0036] Furthermore, preferably, when the effect of enhancing pleiotrophin expression in cells cultured with the addition of the test substance is statistically significantly greater than the effect of enhancing pleiotrophin expression in cells cultured without the addition of the test substance, the test substance can be determined to be a candidate for a skin condition-improving ingredient. More specifically, when the effect of enhancing pleiotrophin expression in cells cultured with the addition of a test substance is more than 1-fold greater than the effect of enhancing pleiotrophin expression in cells cultured without the addition of the test substance, the test substance can be selected as a candidate ingredient for improving skin conditions. The cells are preferably epidermal keratinocytes.

[0037] The present invention may also be implemented by comprising the steps of culturing cells in the presence of a test substance, measuring the effect of the test substance on enhancing pleiotrophin expression in the cells, and determining that the test substance is a skin condition-improving ingredient when the effect of the test substance on enhancing pleiotrophin expression is greater than that of cells cultured in the absence of the test substance. The cells are preferably epidermal keratinocytes.

[0038] Each step in the embodiment of the present invention will be described in detail below.

[0039] (1)Culture process The culture step is a step of culturing epidermal keratinocytes in the presence of a test substance. The cell culture method is not particularly limited, and adherent culture or suspension culture can be appropriately selected depending on the properties of the cells used.

[0040] The duration of the culture step is not particularly limited, but is preferably 3 hours or more, more preferably 6 hours or more. The upper limit of the culture period is also not particularly limited, but as a guideline, it is preferably 1 month or less, more preferably 1 week or less, even more preferably 1 day or less, and particularly preferably 12 hours or less.

[0041] In the culturing step, culturing is carried out in the presence of a test substance. Specific examples include an embodiment in which the test substance is added to a medium and then cultured. The type of test substance is not limited, and any low molecular weight compound, high molecular weight compound, protein, peptide, nucleic acid, or the like can be applied. The test substance may be a pure compound or a mixture containing two or more components, such as a plant or animal extract.

[0042] In the culturing step, one or more test substances may be used, and when evaluating a synergistic effect due to a combination of test substances, it is preferable to add two or more test substances.

[0043] In the culturing step, cells may be cultured in the absence of the test substance as a control in parallel with the culture in the presence of the test substance. In this case, it is preferable to keep the culture conditions the same except for the presence or absence of the test substance.

[0044] (2) Measurement process The measuring step is a step of measuring the pleiotrophin expression enhancing effect in epidermal keratinocytes that have been subjected to cell culture. The method for measuring the enhancing effect of pleiotrophin expression in epidermal keratinocytes is not particularly limited.

[0045] (3) Judgment process The determination step is a step of determining whether or not the test substance is a skin condition-improving ingredient based on the pleiotrophin expression-enhancing effect measured in the measurement step. Specifically, the test substance is determined to be a skin condition-improving ingredient when the pleiotrophin expression-enhancing effect in epidermal keratinocytes cultured in the presence of the test substance is greater than the pleiotrophin expression-enhancing effect in epidermal keratinocytes cultured in the absence of the test substance.

[0046] Here, the test substance determined to be a skin condition-improving component in the determination step can be used as a pleiotrophin expression enhancer, an extracellular matrix degradation and / or neoplasia inducer, a skin fiber structure improver, an angiogenesis inducer, or an agent for enhancing the phagocytic activity of leukocytes.

[0047] Here, the test substance determined in the determination step may be determined as an active ingredient as is, or may be determined as a candidate active ingredient to be subjected to secondary screening.

[0048] The skin condition-improving ingredient obtained by the screening method of the present invention can be used to improve one or more selected from wrinkles, sagging skin, and skin viscoelasticity and / or elasticity.

[0049] <2> Agent The present invention relates to a pleiotrophin expression enhancer containing a brown algae extract as an active ingredient. According to the above invention, the expression of pleiotrophin can be enhanced, and by enhancing the expression of pleiotrophin, regeneration of the dermis can be induced.

[0050] <2-1> Active ingredient The brown algae extract may be an extract of one or more brown algae selected from Fucus serratus, Sargassum fuluvellum, Cystoseira tamariscifolia, Ascophyllum nodosum, Hizikia fusiforme, and Sargassum horneri.

[0051] The extract used in the present invention can be extracted using the whole plant or other parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, spikes, flower buds, and seeds, although it is preferable to crush or shred these beforehand to improve extraction efficiency. Suitable examples of the extraction solvent include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butylene glycol and propylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. The extraction solvent is preferably water, an alcohol, or a mixture of water and an alcohol.

[0052] Specific extraction methods include, for example, adding 1 to 30 parts by mass of solvent to 1 mass of the part of the plant body or its dried material used for extraction, immersing for several days at room temperature or for several hours at a temperature near the boiling point, cooling to room temperature, removing insoluble matter and / or solvent as desired, and fractionating and purifying by column chromatography or the like, but the extraction method is not limited to this.

[0053] As a specific extraction method, lixiviation can be used, in which freeze-dried brown algae are exposed to refluxing water, allowing minerals and active ingredients to diffuse into the extraction solvent.

[0054] One specific extraction method that can be used is Vacuum Microwave Hydro Distillation (VMHD), which uses heat and reduced pressure generated by microwaves to destroy brown algae cells, and then collects and concentrates the volatile components within the cells that rise in the vapor stream.

[0055] As a specific extraction method, supercritical carbon dioxide extraction can also be used. In supercritical carbon dioxide extraction, carbon dioxide is subjected to pressure and temperature to turn it into a supercritical gas, and components are extracted from brown algae using the carbon dioxide in the supercritical gas state.

[0056] The extract of Fucus serratus is preferably an extract of the seaweed part of Fucus serratus.

[0057] The extract of Sargassum fuluvellum is preferably an extract of the seaweed part of Sargassum fuluvellum.

[0058] The extract of Cystoseira tamariscifolia is preferably an extract of the seaweed part of Cystoseira tamariscifolia.

[0059] The extract of Ascophyllum nodosum is preferably an extract of the seaweed part of Ascophyllum nodosum.

[0060] The extract of Hizikia fusiforme is preferably an extract of the seaweed part of Hizikia.

[0061] The extract of Akamoku (Sargassum horneri) is preferably an extract of the seaweed part of Akamoku.

[0062] <2-2>Applications The present invention relates to a pleiotrophin expression enhancer containing a brown algae extract as an active ingredient.

[0063] Enhanced expression of pleiotrophin leads to the synthesis and / or degradation of extracellular matrix in fibroblasts, angiogenesis by vascular endothelial cells, and phagocytosis by leukocytes. These actions can improve skin condition.

[0064] Specifically, the active ingredient can improve one or more of the following: wrinkles, sagging skin, complexion, dullness, and skin viscoelasticity and / or elasticity.

[0065] The present invention also relates to an agent for inducing degradation and / or neoplasia of extracellular matrix, which comprises a brown algae extract as an active ingredient.

[0066] In a preferred embodiment of the present invention, the present invention also provides an agent for inducing collagen degradation and / or new collagen synthesis, which contains brown algae extract as an active ingredient.

[0067] The present invention also relates to a skin fiber structure improving agent containing a brown algae extract as an active ingredient.

[0068] In a preferred embodiment of the present invention, the present invention is also a collagen improving agent containing brown algae extract as an active ingredient.

[0069] Here, the fibrous structure of the skin is composed of fibers such as collagen, as shown in Figure 5. In this specification, improving the fibrous structure of the skin means making this fibrous structure oriented to some extent.

[0070] The skin fiber structure improving agent can enhance the expression of pleiotrophin, thereby improving the skin fiber structure by making fibroblasts more susceptible to pleiotrophin.

[0071] The present invention also relates to an angiogenesis inducer containing a brown algae extract as an active ingredient.

[0072] The present invention also relates to an agent for enhancing the phagocytic activity of leukocytes, which contains a brown algae extract as an active ingredient.

[0073] Examples of leukocytes include macrophages, neutrophils, eosinophils, and dendritic cells, and preferred are agents that enhance the phagocytic activity of macrophages. That is, the present invention is preferably a macrophage phagocytosis enhancer containing a brown algae extract as an active ingredient.

[0074] The present invention also relates to an external skin composition for enhancing pleiotrophin expression, which contains a brown algae extract as an active ingredient.

[0075] <2-3>Usage / Capacity In the present invention, the undiluted extract obtained by extraction with any solvent may be used as is as the agent of the present invention, or the undiluted extract may be diluted to any concentration and then used as the agent of the present invention. The agent according to the present invention is preferably applied to the dermis.

[0076] The total content of one or more extracts selected from the extracts of the present invention in the agent of the present invention is typically 0.00001% by mass or more, based on the dry mass, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more.

[0077] Furthermore, the total content of one or more extracts selected from the extracts of the present invention in the agent of the present invention is typically 80% by mass or less, based on the dry mass, preferably 50% by mass or less, more preferably 30% by mass or less, even more preferably 10% by mass or less, and even more preferably 1% by mass or less.

[0078] Furthermore, the total content of one or more active ingredients selected from the extract of the present invention depends on the dosage form, and the amount applied to the skin per application (application amount) is usually 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less, as the dry mass of the extract.

[0079] <3> Composition for topical application to the skin for enhancing pleiotrophin expression A preferred embodiment of the present invention is a composition for external application to the skin for enhancing pleiotrophin expression, which contains a brown algae extract as an active ingredient.

[0080] The composition for external application to skin for enhancing pleiotrophin expression of the present invention is preferably formulated into an external or oral preparation in appropriate combination with any ingredients used in formulation. Examples of external preparations include cosmetics, quasi-drugs, external skin medicines, etc. There are no particular limitations on the dosage form.

[0081] Furthermore, when the present invention is in the form of a composition for external use on the skin, it is also preferable that the present invention be in a form labeled with an indication of use, such as "for enhancing pleiotrophin expression," "for improving skin wrinkles," "for improving skin sagging," "for improving skin viscoelasticity," "for improving skin elasticity," "for inducing degradation and / or regeneration of extracellular matrix," "for improving skin fiber structure," "for inducing angiogenesis," or "for enhancing phagocytic activity of leukocytes."

[0082] The "indication" includes all indications that have the function of informing consumers of the intended use. In other words, any indication that can recall or infer the intended use falls under the category of "indication," regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. Furthermore, the phrase "labeled" means that there is a labeling action that aims to associate the label with the topical skin composition (product) etc.

[0083] The act of labeling is preferably one that allows consumers to directly recognize the intended use. Specific examples include the act of describing the intended use on the product or its packaging according to the present invention, and the act of describing the intended use in product advertisements, price lists, or transaction documents (including those provided by electromagnetic means).

[0084] When the external skin composition for inhibiting gene expression of the present invention is used as a cosmetic, in addition to the above-mentioned components, it may contain whitening components, components for improving wrinkles and / or sagging, anti-inflammatory components, etc.

[0085] There are no particular limitations on the whitening ingredient, as long as it is one that is commonly used in cosmetics. Examples of water-soluble whitening ingredients include 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethyl ascorbic acid, tranexamic acid, ellagic acid, arbutin, nicotinamide, and pantothenyl alcohol. Examples of oil-soluble whitening ingredients include 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2-(triphenylmethyloxy)ethanol, 2-(triphenylmethylamino)ethanol, 2-(triphenylmethyloxy)ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane, aminodiphenylmethane, N-(o-toluoyl)cysteic acid, N-(m-toluoyl)cysteic acid, N-(p-toluoyl)cysteic acid, and N-(p-methoxybenzoyl)cysteic acid. Further preferred examples of other whitening ingredients include N-benzoylserine, N-(p-methylbenzoyl)serine, N-(p-ethylbenzoyl)serine, N-(p-methoxybenzoyl)serine, N-(p-fluorobenzoyl)serine, N-(p-trifluoromethylbenzoyl)serine, N-(2-naphthoyl)serine, N-(4-phenylbenzoyl)serine, N-(p-methylbenzoyl)serine methyl ester, N-(p-methylbenzoyl)serine ethyl ester, N-(2-naphthoyl)serine methyl ester, N-benzoyl-O-methylserine, N-(p-methylbenzoyl)-O-methylserine, N-(p-methylbenzoyl)-O-acetylserine, and N-(2-naphthoyl)-O-methylserine.

[0086] The other anti-wrinkle ingredients are not particularly limited as long as they are commonly used in cosmetics. Examples include vitamin A or its derivatives such as retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, and retinol acetate. Other examples include ursolic acid benzyl ester, ursolic acid phosphate ester, betulinic acid benzyl ester, benzilic acid phosphate ester, and nicotinamide. The content of other anti-wrinkle ingredients in cosmetics besides the neutrophil chemotactic factor gene expression inhibitor is typically 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (dry mass in the case of an extract).

[0087] Examples of anti-inflammatory components include kurarinone, glabridin, glycyrrhizinic acid, dipotassium glycyrrhizinate, glycyrrhetinic acid, alkyl glycyrrhetinate, pantothenyl alcohol, etc. Among these, preferred examples of oil-soluble components include glabridin, glycyrrhetinic acid and salts thereof, and alkyl glycyrrhetinate and salts thereof. The content of the anti-inflammatory component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry mass in the case of an extract).

[0088] It is also preferable to use extracts derived from animals and plants that are generally used in medicines, cosmetics, foods, etc.For example, Akebia extract, Asunaro extract, Asparagus extract, Avocado extract, Hydrangea extract, Almond extract, Arnica extract, Aronia extract, Apricot extract, Ginkgo extract, Fennel extract, Aralia extract, Aralia udo extract, Eleuthero extract, Enmeiso extract, Phellodendron bark extract, Panax ginseng extract, Nettle extract, Pueraria lobata extract, Pueraria lobata extract, Chamomilla recutita extract, Carrot extract, Artemisia capillaris extract, Licorice extract, Kiwi extract, Cucumber extract, Guava extract, Gardenia extract, Sasa veitchii extract, Kuru extract Rice extract, black rice extract, chlorella extract, mulberry extract, cinnamon root extract, Alpinia zerumbet extract, gentian extract, geranium extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, salvia extract, soapwort extract, bamboo extract, sanshou extract, Japanese pepper extract, shiitake mushroom extract, rehmannia root extract, lithospermum root extract, perilla extract, linden extract, meadowsweet extract, ginger extract, calamus root extract, horsetail extract, stevia extract, fermented stevia, elderberry extract, yaw wood Lysom extract, peppermint extract, sage extract, mallow extract, cnidium extract, swertia bristle extract, mulberry extract, rhubarb extract, soybean extract, tallow extract, thyme extract, dandelion extract, clove extract, chili pepper extract, angelica extract, calendula extract, peach kernel extract, tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, burdock extract, lotus extract, parsley extract, birch extract, witch hazel extract, jasmine extract, cypress extract, loquat extract, Preferred examples of extracts include dandelion extract, butterbur extract, poria extract, loofah extract, peppermint extract, linden extract, pine extract, skunk cabbage extract, melissa extract, mozuku extract, cornflower extract, eucalyptus extract, lily extract, coix seed extract, mugwort extract, lavender extract, apple extract, rooibos tea extract, lychee extract, lettuce extract, forsythia extract, astragalus extract, rosemary extract, Roman chamomile extract, royal jelly extract, and burnet extract.

[0089] The content (dry mass) of the optional animal and plant-derived extract in the cosmetic is usually 0.01 to 30 mass %, preferably 0.1 to 10 mass %, more preferably 0.3 to 3 mass %.

[0090] In addition to the above-mentioned components, optional components commonly used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, polyols such as 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol, and cyclohexylglycerin, fatty acid soaps ( Anionic surfactants such as sodium laurate, sodium palmitate, potassium lauryl sulfate, and alkyl sulfate triethanolamine ether; cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide; imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.); betaine-based surfactants (alkyl betaine, amido betaine, sulfobetaine, etc.); amphoteric surfactants such as acyl methyl taurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.); glycerin fatty acids (glycerin monostearate, etc.); propylene glycol fatty acid esters (propylene glycol monostearate, etc.); hydrogenated castor oil derivatives; glycerin alkyl ether; POE sorbitan Tan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitan fatty acid esters (POE-sorbitan monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE·POP alkyl ethers (POE·POP 2-decyltetradecyl ether, etc.), Tetronics, POE castor oil·hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, sodium pyrrolidone carboxylate, lactic acid,Moisturizing ingredients such as sodium lactate; powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate, which may be surface-treated; inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, and zinc oxide, which may be surface-treated; composite pigments such as sintered iron oxide and titanium dioxide, which may be surface-treated; pearlizing agents such as titanium mica, fish phosphate foil, and bismuth oxychloride, which may be surface-treated; Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, and Blue No. 505, which may be laked. Examples of suitable organic pigments include organic pigments such as No. 1, Green No. 201, Purple No. 201, and Red No. 204; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomer; lower alcohols such as ethanol and isopropanol; vitamin A or a derivative thereof, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof; vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0091] <4> External skin composition for improving skin conditions The present invention also relates to an external skin composition for improving skin conditions, which comprises a pleiotrophin expression enhancer and a pleiotrophin receptor expression enhancer, and which contains a brown algae extract as an active ingredient. By including both a pleiotrophin expression enhancer and a pleiotrophin receptor expression enhancer, the effect of pleiotrophin can be further improved. Specifically, it is possible to obtain higher effects of improving skin condition, inducing degradation and / or regeneration of extracellular matrix, and improving skin fiber structure.

[0092] The pleiotrophin expression enhancer is as described above.

[0093] The pleiotrophin receptor is preferably a proteoglycan, more preferably a syndecan family protein, and even more preferably syndecan 4 (SDC4).

[0094] In the topical skin composition for improving skin condition of the present invention, it is preferable to use, as an active ingredient, one or more plant extracts selected from Ruscus aculeatus, Olea europaea, and Plantago major as a pleiotrophin receptor expression enhancer.

[0095] The extract used in the present invention can be extracted using the whole plant or parts such as the plant, above-ground parts, rhizomes, trunks, leaves, stems, spikes, flower buds, and seeds, although it is preferable to crush or shred these beforehand to improve extraction efficiency. Suitable examples of the extraction solvent include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butylene glycol and propylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. The extraction solvent is preferably water, an alcohol, or a mixture of water and an alcohol.

[0096] Specific extraction methods include, for example, a method in which 1 to 30 parts by mass of solvent is added to 1 mass of the part to be extracted or its dried product, followed by immersion for several days at room temperature or for several hours at a temperature near the boiling point, and then cooling to room temperature, followed by removal of insoluble matter and / or solvent as desired, and fractionation and purification by column chromatography or the like, but the extraction method is not limited to this.

[0097] The plant extract of Ruscus aculeatus is preferably Ruscus aculeatus root extract. As the extraction solvent, water or an ethanol solution can be preferably used.

[0098] The plant extract of olive (Olea europaea) is preferably olive leaf extract. As the extraction solvent, water or an ethanol solution can be preferably used.

[0099] The plant extract of Plantago major is preferably Plantago major seed extract. As the extraction solvent, water or an ethanol solution can be preferably used. [Example]

[0100] Example 1 The functions of each gene group whose expression was enhanced by pleiotrophin were analyzed by the following procedure. 1) Human skin fibroblasts were seeded and cultured for 24 hours. 2) Pleiotrophin (final concentration: 300 ng / mL) was added, and the cells were cultured for 6 hours. 3) Total RNA was extracted. 4) Gene expression information was obtained using RNA sequencing. 5) Genes with differential expression were extracted (|rate of change|≧2, p<0.05). 6) The functions of each gene group were analyzed using enrichment analysis.

[0101] The results are shown in Figure 3. As shown in Figure 3, pleiotrophin induces the synthesis / degradation of extracellular matrix in fibroblasts.

[0102] Since pleiotrophin induces the synthesis / degradation of extracellular matrix in fibroblasts, it has been shown that pleiotrophin expression can be used as an indicator to screen for substances that can induce the degradation and / or synthesis of extracellular matrix.

[0103] <Example 2> Changes in collagen gene expression due to pleiotrophin were examined using the following procedure. 1) Human skin fibroblasts were seeded and cultured for 24 hours. 2) Pleiotrophin (final concentration: 300 ng / mL) was added, and the cells were cultured for 48 hours. 3) Total RNA was extracted. 4) The gene expression level of collagen was verified using qPCR.

[0104] The results are shown in Figure 4. As shown in FIG. 4, the addition of pleiotrophin increased the collagen gene expression level.

[0105] Since the action of pleiotrophin increases the expression level of collagen genes, it was shown that substances that increase the expression level of collagen genes can be screened using pleiotrophin expression as an indicator.

[0106] Example 3 The dermal collagen structure was observed using the following procedure. 1) Human facial skin was removed. 2) The dermis with epidermis was immediately cultured at the air-liquid interface in a medium containing pleiotrophin (final concentration 300 ng / mL). 3) After one week of culture, the dermal collagen structure was observed using a scanning electron microscope.

[0107] The results are shown in Figure 5. As shown in Figure 5, the collagen structure is more organized when pleiotrophin is added.

[0108] The improvement of skin fiber structure due to the action of pleiotrophin indicates that substances that improve skin fiber structure can be screened using pleiotrophin expression as an indicator.

[0109] Example 4 The state of angiogenesis was observed according to the following procedure. 1) A Geltrex coated plate was prepared. 2) Human skin-derived vascular endothelial cells were seeded. 3) Pleiotrophin was added and the cells were cultured for 20 hours. 4) Calcein staining was performed and fluorescent images were obtained. 5) Lumen formation was evaluated by calculating the following indices. Total lumen length Number of branching points

[0110] The results are shown in Figure 6. As shown in FIG. 6, when vascular endothelial cells were cultured in the presence of pleiotrophin, the total lumen length and the number of branch points increased significantly and in a concentration-dependent manner.

[0111] The fact that angiogenesis was promoted by the action of pleiotrophin indicated that substances that improve the structure of angiogenesis can be screened using pleiotrophin expression as an indicator.

[0112] <Example 5> The phagocytic activity of macrophages was evaluated by the following procedure. 1) Human monocytic leukemia cell line (THP-1) was inoculated. 2) The cells were cultured in 150 nM PMA-containing medium for 24 hours, and then cultured in normal medium for 24 hours to induce differentiation into macrophages. 3) Pleiotrophin was added and the cells were cultured for 24 hours. 4) Beads conjugated with a fluorescent dye that emits fluorescence when phagocytosed were added, and fluorescence images were taken after 3 hours of incubation. 5) Phagocytic activity was evaluated by calculating the fluorescent signal intensity.

[0113] The results are shown in Figure 7. As shown in Figure 7, culturing macrophages in the presence of pleiotrophin significantly increased phagocytosis.

[0114] Since the action of pleiotrophin enhanced the phagocytic ability of macrophages, it was shown that substances that enhance the phagocytic activity of macrophages (white blood cells) can be screened using pleiotrophin expression as an indicator.

[0115] Example 6 The gene expression of syndecan-4 by the plant extract was verified by the following procedure. 1) Human skin fibroblasts were seeded and cultured for 24 hours. 2) Plant extracts (final concentration of each: 0.1%) were added and the mixture was cultured for 24 hours. 3) Total RNA was extracted. 4) Syndecan 4 (SDC4) gene expression was verified using qPCR.

[0116] The plant extracts used were Butcher's Broom Root Extract (Butcher's Broom Extract / Maruzen Pharmaceutical Co., Ltd.), Olive Leaf Extract (Olive Leaf Extract BG / Maruzen Pharmaceutical Co., Ltd.), and Plantago Major Seed Extract (Absolage / Ichimaru Pharcos Co., Ltd.).

[0117] The results are shown in Figure 8. As shown in FIG. 8, enhanced gene expression of syndecan-4 was observed in all of the extracts of the root of the kaede, the olive leaf extract, and the seed extract of the plantago major.

[0118] Example 7 The gene expression of syndecan-4 by the composite extract was verified by the following procedure. 1) Human skin fibroblasts were seeded and cultured for 24 hours. 2) The composite extract was added and cultured for 24 hours. 3) Total RNA was extracted. 4) Syndecan-4 gene expression was verified using qPCR.

[0119] The composite extract used was a mixture of Butcher's Broom Root Extract (Maruzen Pharmaceutical Co., Ltd.) and Brown Algae Extract 1 (final concentration of each 0.1%).

[0120] The results are shown in Figure 9. As shown in FIG. 9, enhanced gene expression of syndecan-4 was observed in the combined extract of the root extract of Hagfish and brown algae extract.

[0121] Example 8 The effects of brown algae extract 2 and the composite extract on pleiotrophin gene expression were examined using the following procedure. 1) Normal human epidermal keratinocytes were seeded and cultured for 24 hours. 2) Brown algae extract 2 (final concentration 0.1%) or the composite extract was added and cultured for 6 hours. 3) Total RNA was extracted. 4) Pleiotrophin gene expression was verified using qPCR.

[0122] As a composite extract, a mixture of brown algae extract 2 and kadzu root extract (Butcher's Broom extract / Maruzen Pharmaceutical Co., Ltd.) (final concentration of each 0.1%) was used.

[0123] The results are shown in Figure 10. As shown in FIG. 10, increased pleiotrophin gene expression was observed in brown algae extract 2 and the composite extract.

[0124] Example 9 The gene expression of pleiotrophin by the composite extract was verified by the following procedure. 1) Normal human epidermal keratinocytes were seeded and cultured for 24 hours. 2) The composite extract (final concentration of each 0.1%) was added and cultured for 6 hours. 3) Total RNA was extracted. 4) Pleiotrophin (PTN) gene expression was verified using qPCR.

[0125] The composite extracts used were a mixed extract of brown algae extract 1 and kaede root extract (butcher's broom extract / Maruzen Pharmaceutical Co., Ltd.) (final concentration of both: 0.1%), and a mixed extract of brown algae extract 2 and kaede root extract (butcher's broom extract / Maruzen Pharmaceutical Co., Ltd.) (final concentration of both: 0.1%).

[0126] The results are shown in Figure 11. As shown in FIG. 11, enhanced pleiotrophin gene expression was observed in the mixed extract of brown algae extract 1 and thrush root extract, and in the mixed extract of brown algae extract 2 and thrush root extract.

[0127] Example 10 The gene expression of pleiotrophin by plant extract was verified by the following procedure. 1) Normal human epidermal keratinocytes were seeded and cultured for 24 hours. 2) Plant extract (final concentration 0.1%) was added and cultured for 6 hours. 3) Total RNA was extracted. 4) Pleiotrophin gene expression was verified using qPCR.

[0128] The plant extract used was Butcher's Broom Root Extract (Maruzen Pharmaceutical Co., Ltd.).

[0129] The results are shown in Figure 12. As shown in FIG. 12, no significant effect on pleiotrophin gene expression was observed in the Nagiikada root extract. From this, it is considered that the enhancement of pleiotrophin gene expression by the composite extract in Example 9 is due to the brown algae extract. [Industrial Applicability]

[0130] According to the present invention, a method for screening ingredients that improve skin conditions can be provided.

Claims

1. A method for screening ingredients that improve skin conditions using the effect of enhancing pleiotrophin expression as an indicator.

2. The test substance is judged to be a superior skin condition-improving ingredient when the test substance exhibits a higher pleiotrophin expression enhancing effect when cultured in the presence of the test substance. The screening method according to claim 1.

3. adding a test substance to a cell culture system and measuring the effect of enhancing pleiotrophin expression in the cells; The screening method according to claim 1 or 2.

4. determining that the test substance is a candidate for a skin condition-improving component when the effect of enhancing pleiotrophin expression in cells cultured with the addition of the test substance is statistically significantly greater than the effect of enhancing pleiotrophin expression in cells cultured without the addition of the test substance; The screening method according to claim 1 or 2.

5. Culturing the cells in the presence of a test substance; measuring the effect of enhancing pleiotrophin expression in cells; determining that the test substance is a skin condition-improving ingredient when the pleiotrophin expression-enhancing effect is higher than the pleiotrophin expression-enhancing effect of cells cultured in the absence of the test substance. The screening method according to claim 1 or 2.

6. A pleiotrophin expression enhancer containing brown algae extract as an active ingredient.

7. To improve skin condition, The pleiotrophin expression enhancer according to claim 6.

8. For improving one or more of the following: wrinkles, sagging skin, complexion, dullness, and skin viscoelasticity and / or elasticity; The pleiotrophin expression enhancer according to claim 6 or 7.

9. Contains brown algae extract as an active ingredient. An agent that induces degradation and / or neoplasia of the extracellular matrix.

10. Contains brown algae extract as an active ingredient. Skin fiber structure improver.

11. Contains brown algae extract as an active ingredient. Angiogenesis inducer.

12. Contains brown algae extract as an active ingredient. An agent that enhances phagocytosis by white blood cells.

13. A topical skin composition for enhancing pleiotrophin expression, comprising a brown algae extract as an active ingredient.

14. A composition for external application to the skin for improving skin conditions, comprising the pleiotrophin expression enhancer according to claim 6 and a pleiotrophin receptor expression enhancer.

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