Promoter for HOXA9 expression

Abstract

To identify a gene specifically expressed in dermal fibroblasts of body regions such as the trunk and limbs, and to identify a substance capable of regulating the expression of the gene and provide it as a drug that can efficiently enhance body firmness and elasticity.SOLUTION: A promoter for HOXA9 expression comprises, as an active ingredient, an extract of Kochia scoparia.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a HOXA9 expression promoter. [Background technology]

[0002] Skin is the largest organ covering the entire body and is composed of three layers: the epidermis, the dermis, and the subcutaneous tissue. Differences in the three-layer structure of the skin have been observed between different regions, and its properties are not uniform. For example, it is known that the stratum corneum of the epidermis is particularly developed on the palms of the hands and the soles of the feet, providing a strong barrier function (Non-Patent Documents 1 and 2), and that the skin elasticity is higher on the trunk and extremities than on the face (Non-Patent Documents 3 and 4). Thus, by providing appropriate structures and properties depending on the region, the skin is thought to adequately protect the body from external physical and chemical stimuli and maintain the homeostasis necessary for vital activities.

[0003] Although the mechanisms that generate region-specific structures and properties are not fully understood, the involvement of homeobox (HOX) genes has been suggested for the dermis (Non-Patent Document 5). The HOX gene group plays an important role in the formation of the anterior-posterior axis and somitogenesis during the morphogenesis of organisms, and 39 genes have been identified in humans. For example, the HOXA13 gene, one of the HOX gene group, is specifically expressed in dermal fibroblasts in the distal limbs, induces WNT5A gene expression, and is known to be involved in the induction and maintenance of epidermal differentiation via WNT5A (Non-Patent Document 6). This suggests that the strong barrier function specific to the palms and soles is controlled by HOX genes. However, the genes associated with regional differences in skin elasticity have not been identified, and the control mechanism has not yet been elucidated. Like the face, the trunk and limbs of the body lose firmness with age, resulting in sagging and wrinkles. Therefore, if we can find genes that are specifically expressed in the dermis of the trunk and limbs, it is thought that this will lead to the development of agents that efficiently improve the firmness and elasticity of the trunk and limbs.

[0004] Bassia scoparia (scientific name: Bassia scoparia) is an annual plant belonging to the Bassia genus of the Amaranthaceae family, and its dried fruit is used as a medicinal herb known as jifushi. It has been reported that kochia extracts are used in whitening cosmetic compositions (Patent Document 1), preadipocyte differentiation inhibitors (Patent Document 2), polyphenol absorption enhancers (Patent Document 3), and the like, but no research has been done on their application to site-specific properties of the skin. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 7-25746 [Patent Document 2] Japanese Patent Application Laid-Open No. 2002-138044 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-109991 [Non-patent literature]

[0006] [Non-Patent Document 1] Holbrook et al. Regional differences in the thickness(cell layers) of the human stratum corneum: an ultrastructural analysis. J Invest Dermatol. 1974;62(4):415-422. [Non-patent document 2] Ya-Xian et al. Number of cell layers of the stratum corneum in normal skin-relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291(10):555-559. [Non-patent document 3] Krueger et al. Age-related changes in skin mechanical properties: a quantitative evaluation of 120 female subjects. Skin Res Technol. 2011;17(2):141-148. [Non-patent document 4] Nedelec et al. Skin characteristics: normative data for elasticity, erythema, melanin, and thickness at 16 different anatomical locations. Skin Res Technol. 2016;22(3):263-275. [Non-patent document 5] Rinn et al. Anatomic demarcation by positional variation in fibroblast gene expression programs. PLoS Genet. 2006;2(7):e119. [Non-patent document 6] Rinn et al. A dermal HOX transcriptional program regulates site-specific epidermal fate. Genes Dev. 2008;22(3):303-307. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above-mentioned circumstances, the present invention aims to find genes that are specifically expressed in dermal fibroblasts of the body, such as the trunk and limbs, and to find substances that can control the expression of the genes, and to provide them as drugs that can efficiently improve the firmness and elasticity of the body. [Means for solving the problem]

[0008] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that HOXA9, a member of the HOX gene group, is a gene that is specifically expressed in dermal fibroblasts of the body, that HOXA9 positively regulates the production of extracellular matrix-related factors that make up the dermis, and that extracts of Kochia annua can significantly increase the expression level of the HOXA9 gene in dermal fibroblasts, thereby completing the present invention.

[0009] That is, the present invention includes the following inventions. (1) A HOXA9 expression promoter containing Kochia japonica extract as an active ingredient. (2) The agent described in (1), wherein the promotion of HOXA9 expression is promotion of HOXA9 expression in dermal fibroblasts. (3) The agent according to (2), wherein the dermal fibroblasts are present in the skin of the body. (4) A composition for promoting HOXA9 expression, comprising the agent according to any one of (1) to (3). (5) The composition for promoting HOXA9 expression according to (4), wherein the composition is a cosmetic, a quasi-drug, a pharmaceutical, or a food or beverage. [Effects of the Invention]

[0010] The HOXA9 expression promoter of the present invention is specifically expressed in dermal fibroblasts of the body and can increase the expression level of HOXA9, which positively regulates extracellular matrix-related factors that make up the dermis. Therefore, the HOXA9 expression promoter of the present invention is effective in improving and preventing loss of body firmness and elasticity. DETAILED DESCRIPTION OF THE INVENTION

[0011] The invention will be described in detail below. 1. HOXA9 expression promoter The HOXA9 expression promoter of the present invention contains an extract of Kochia spp. as an active ingredient. "Promotion of HOXA9 expression" in this invention refers to promoting HOXA9 expression at the living body level or culture level. Furthermore, "promotion of HOXA9 expression" in this invention refers to promoting HOXA9 mRNA expression and protein expression.

[0012] The HOXA9 expression promoter of the present invention has the effect of promoting HOXA9 expression, particularly in dermal fibroblasts of the body. Here, "body" refers to the chest, abdomen, back, buttocks, arms, legs, and neck. The origin of the dermal fibroblasts is not particularly limited as long as they are derived from mammals, including humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs.

[0013] Furthermore, the HOXA9 expression promoter according to the present invention also functions to positively regulate the production of extracellular matrix-related factors that constitute the dermis and play an important role in maintaining the shape and elasticity of the skin. Examples of extracellular matrix-related factors that constitute the dermis include collagen (particularly type I collagen, type III collagen, and type V collagen), elastin, fibronectin, laminin, etc.

[0014] HOXA9 (Homeobox A9) is a member of the homeobox gene family. Homeobox genes are involved in axis formation in vertebrates and are present in four chromosomal clusters: HOXA, B, C, and D. The HOXA9 gene is part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor that is thought to regulate gene expression, morphogenesis, and differentiation. The sequences of HOXA9 mRNA and protein are registered in GenBank under Nucleotide NM_152739 and Protein NP_689952, respectively, for humans.

[0015] The Kochia scoparia used in this invention (scientific name: Bassia scoparia) is an annual plant belonging to the Bassia genus of the Amaranthaceae family. It is characterized by its thin, broom-like stems, which turn red when the leaves turn red in autumn. The fruits are used for medicinal and edible purposes, and the dried fruits are used as a herbal medicine called jifushi. Kochia scoparia is a synonym of Bassia scoparia.

[0016] In the present invention, the extract of Kochia lanatum refers to an extract of parts of the plant, such as flowers, fruits, seeds, leaves, stems, and roots, or the whole plant (whole plant), or an extract of a mixture thereof. In the present invention, the part used as the extraction raw material is preferably the fruit in terms of effectiveness. Furthermore, for extraction, these plants may be used as they are, or may be processed by drying, crushing, shredding, etc.

[0017] Examples of solvents used for extracting Kochia include water or hot water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.), etc. These solvents may be used alone or in combination. Among these solvents, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are preferred, with water, ethanol, 1,3-butylene glycol, or propylene glycol being more preferred, and water, a mixed polar solvent of water-ethanol, or a mixed polar solvent of water-1,3-butylene glycol being even more preferred. In the case of a mixed polar solvent of water-ethanol or a mixed polar solvent of water-1,3-butylene glycol, the mixed polar solvent preferably contains 20 to 99 wt % of ethanol or 1,3-butylene glycol, more preferably 50 to 99 wt %. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent can also be used.

[0018] The amount of solvent used is not particularly limited. For example, it may be at least 5 times, preferably at least 10 times, the dry weight of the kochia fruit. However, for convenience of post-extraction concentration and isolation, it is preferable to use a solvent not exceeding 100 times. The extraction temperature is not particularly limited and is a temperature below the boiling point of the solvent used for extraction. It may be heated or extracted at room temperature. The extraction time is 1 to 2 weeks for room temperature extraction and 30 minutes to 24 hours, preferably 1 to 10 hours for heated extraction, but can be adjusted appropriately depending on conditions such as the type of extraction solvent and extraction temperature. More specifically, a kochia extract can be obtained by adding a lower alcohol (e.g., ethanol) or a liquid polyhydric alcohol (e.g., 1,3-butylene glycol) to the kochia fruit and extracting at room temperature (e.g., 15 to 35°C). The number of extraction operations is not particularly limited. It may be performed once, or fresh extraction solvent may be added after the first extraction to perform a second or subsequent extraction. The extraction procedure may be carried out multiple times using the same extraction solvent.

[0019] The extract may be used as is in the form of an extracted solution, or, if necessary, may be subjected to treatment such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc., to an extent that does not affect the effect of the extract. Furthermore, the extracted solution may be subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0020] The content of Kochia japonica extract in the agent of the present invention varies depending on the nature of the extract (liquid extract, concentrate, or dried product) and is not particularly limited, but for example, it is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 1% by weight, calculated as dry matter, of the total amount of the agent.

[0021] The agent of the present invention can be used as a culture medium additive for activating dermal fibroblasts through promoting HOXA9 expression, for example, when culturing the cells for use in regenerative medicine. The dermal fibroblasts may be primary cultured cells, subcultured cells, or frozen cells.

[0022] 2. Composition for promoting HOXA9 expression When the HOXA9 expression promoter of the present invention (hereinafter referred to as "the agent of the present invention") is used in vivo, it can be used as is, but it can also be incorporated into various compositions such as cosmetics, pharmaceuticals, quasi-drugs, food and beverages, etc. together with appropriate additives within a range that does not impair the effects of the present invention, and provided as a composition for promoting HOXA9 expression.

[0023] When the agent of the present invention is used for the purpose of, for example, improving body firmness and elasticity, or reducing wrinkles, blemishes, roughness, and dark spots, it is preferably in the form of a cosmetic or quasi-drug, or in the form of a food or beverage such as a beauty drink. Furthermore, when the agent of the present invention is used for the purpose of, for example, treating and / or preventing diseases or pathologies associated with a decrease or decreased function of extracellular matrix-related factors that constitute the dermis, it is preferably used in the form of a pharmaceutical. Examples of diseases or pathologies associated with a decrease or decreased function of extracellular matrix-related factors that constitute the dermis include stria extensor (linear atrophy of the skin), senile atrophy of the skin, solar elastosis, scleroderma, wounds, burns, pressure ulcers, scars, acquired dermal melanocytosis (ADM), and nevus of Ota.

[0024] When the agent of the present invention is incorporated into cosmetics or quasi-drugs, topical skin compositions are preferred, and may be in any of the following dosage forms: aqueous solution, solubilized, emulsion, powder, powder dispersion, oil, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. Furthermore, such cosmetics or quasi-drugs can be produced by appropriately blending various ingredients, additives, bases, etc. commonly used in topical skin compositions together with the Kochia angustifolium extract, according to their type, according to methods known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients included in the topical skin composition include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hydrogenated castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate, octanoic acid, Cetyl, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins (β-carotene (vitamin A), vitamin C, vitamin E, etc.), plant and animal extracts, various surfactants, moisturizers, UV absorbers, pH adjusters, stabilizers, preservatives, disinfectants, fragrances, etc.

[0025] Examples of types of cosmetics and quasi-drugs include, but are not limited to, lotions, emulsions, gels, beauty serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, powders, bath additives, body lotions, and body shampoos.

[0026] When the agent of the present invention is incorporated into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various formulations suitable for application to the affected area. Pharmacologically and pharmaceutically acceptable additives include formulation bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonicity agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, and flavoring agents, and can be added as appropriate depending on the dosage form and intended use. Various formulations suitable for oral or parenteral systemic or local administration can be prepared using various known methods. When providing the pharmaceutical product of the present invention in the above-mentioned forms, it can be manufactured using methods commonly used by those skilled in the art, such as the methods set forth in the General Provisions for Preparations [2] of the Japanese Pharmacopoeia.

[0027] For oral administration formulations, for example, excipients such as starch, glucose, sucrose, fructose, lactose, sorbitol, mannitol, crystalline cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, or dextrin; disintegrants or disintegration aids such as carboxymethylcellulose, carboxymethylcellulose calcium, starch, or hydroxypropylcellulose; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, or titanium oxide; and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used, but are not limited to these.

[0028] For parenteral administration, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum water, and vegetable oils can be used; isotonicity agents such as glucose, sodium chloride, and D-mannitol; and pH adjusters such as inorganic acids, organic acids, inorganic bases, and organic bases, but these are not limited to these.

[0029] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs, and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, suppositories, transdermal absorption preparations, transmucosal absorption preparations, and patches. The pharmaceutical may also be provided as a dried product that is reconstituted when used, and in the case of an injectable preparation, it is provided in the form of a unit-dose ampule or a multi-dose container.

[0030] Furthermore, when used as a pharmaceutical for treating, ameliorating, or preventing diseases or pathological conditions associated with a decrease or functional impairment of the extracellular matrix-related factors that constitute the dermis, the appropriate form is a topical preparation, such as an ointment, cream, gel, liquid, patch (poultice, plaster), foam, spray, or atomized preparation. Ointments refer to homogeneous, semi-solid topical preparations, including oleaginous ointments, emulsion ointments, and water-soluble ointments. Gels refer to topical preparations in which a water-insoluble component, a hydrated compound, is suspended in an aqueous liquid. Solutions refer to liquid topical preparations, including lotions, suspensions, emulsions, liniments, and the like.

[0031] The agent of the present invention can also be incorporated into food and beverage products. In the present invention, the term "food and beverage product" refers not only to general food and beverage products but also to foods other than pharmaceuticals that can be consumed for the purpose of maintaining or improving health, such as health foods, functional foods, health-promoting foods, and foods for special dietary uses. Health foods include foods offered under names such as dietary supplements, health supplements, and supplements. Health-promoting foods are defined by the Food Sanitation Act and the Food Promotion Act and include foods for specified health uses and foods with nutrient functions, which can display specific health benefits, nutritional component functions, and disease risk reduction, as well as foods with functional claims, which can display scientifically based functionality reported to the Commissioner of the Consumer Affairs Agency. Special dietary foods also include foods for patients, elderly people, infants, and pregnant women, which are labeled as suitable for specific subjects or patients with specific diseases. The agent of the present invention can be suitably used in the above-mentioned health foods, etc., because it can be taken continuously on a daily basis, especially when long-term administration is required to improve and prevent various symptoms such as loss of body firmness and elasticity, wrinkles, and sagging skin. Here, the indication of specific health benefits or nutritional functions attached to food and beverages can be made on product containers, packaging, instructions, attached documents, etc., product flyers and pamphlets, product advertisements in newspapers and magazines, etc.

[0032] The form of the food or drink may be any form suitable for consumption, such as solid, liquid, granular, particulate, powder, capsule, cream, paste, etc. Types of food or drink include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock foods, oils and fats and processed oils and fats, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated concentrates and powders for adjusting such beverages.

[0033] The food and drink products of the present invention may be appropriately blended with commonly used additives depending on the type of product. Any additives that are acceptable from the standpoint of food hygiene can be used, including, for example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0034] When the Kochia sieboldii extract is incorporated into cosmetics, quasi-drugs, or pharmaceuticals, its content is not particularly limited as long as it exerts the HOXA9 expression-promoting effect. However, for external use, the content of the extract, calculated as solid matter, is preferably 0.0001 wt% or more, more preferably 0.001 to 10 wt%, based on the total weight of the formulation (composition). A content below 0.0001 wt% is unlikely to be sufficient. A content above 10 wt% is uneconomical, as it is unlikely to enhance the effect. For internal use, the intake amount varies depending on age, weight, symptoms, therapeutic effect, administration method, treatment time, etc. Typically, the daily intake per adult is preferably 5 mg or more, more preferably 10 mg to 5 g, and most preferably 20 mg to 2 g. The above amounts are merely examples and can be appropriately determined or adjusted taking into account the type and form of the composition, typical dosage, efficacy, and other factors. Furthermore, the active ingredient may be added in advance or during the manufacturing process, as appropriate, taking into account workability. [Example]

[0035] In the following, in order to explain the present invention in detail, production examples and experimental examples of the extract used in the present invention are given as examples, but the present invention is not limited to these. % in the production examples means % by weight.

[0036] [Example 1] Example of production of Kochia spp. extract An extract of Kochia scoparia was produced as follows: In Production Examples 1 to 4, Kochia scoparia fruit was used as the extraction material.

[0037] (Production Example 1) Preparation of hot water extract of Kochia 200 mL of water was added to 10 g of dried Kochia angustifolium, and the mixture was extracted for 2 hours at 95-100° C. The resulting extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 1.5 g of a hot water extract of Kochia angustifolium.

[0038] (Production Example 2) Preparation of 50% ethanol extract of Kochia 10 g of dried Kochia spp. was soaked in 200 mL of 50% ethanol solution at room temperature for 7 days for extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 1.3 g of 50% ethanol extract of Kochia spp.

[0039] (Production Example 3) Preparation of ethanol extract of Kochia 10 g of dried Kochia spp. was soaked in 200 mL of ethanol at room temperature for 7 days for extraction. The resulting extract was filtered and then concentrated to dryness using an evaporator to obtain 0.076 g of ethanol extract of Kochia spp.

[0040] (Production Example 4) Preparation of 1,3-butylene glycol extract of Kochia 10 g of dried Kochia spp. was soaked in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction, and the resulting extract was filtered to obtain 193 g of 1,3-butylene glycol extract of Kochia spp.

[0041] [Example 2] (Experimental Example 1) Search for body-specific genes We compared the HOXA9 mRNA expression levels in dermal fibroblasts derived from skin collected from the face, abdomen, buttocks, and thighs. Dermal fibroblasts derived from skin from each site were collected from four adults. 5 × 10 dermal fibroblasts derived from skin from each site were collected. 4Cells were seeded in 12-well plates (Falcon) at 10% FBS-containing DMEM medium at 37°C and 5% CO2 for 3 days. After incubation, total RNA was extracted. Total RNA was extracted from the cells using RNAiso Plus (Takara Bio), and the total RNA content was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). mRNA expression levels were measured by real-time RT-PCR. Specifically, 500 ng of total RNA extracted from the cells was reverse transcribed to cDNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems), followed by real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) using the following primer set with SYBR Select Master Mix (Thermo Fisher Scientific). Other procedures were performed according to established procedures.

[0042] Primer set for HOXA9 5'-AGAATGAGAGCGGCGGAGA-3' (SEQ ID NO: 1) 5'-TTCCAGGGTCTGGTGTTTTG-3' (SEQ ID NO: 2) Primer set for GAPDH (internal standard) 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 3) 5'-TCTTCTGGGTGGCAGTGATG-3' (SEQ ID NO: 4)

[0043] The expression level of HOXA9 mRNA was evaluated by calculating the relative expression level of HOXA9 mRNA (HOXA9 mRNA expression level / GAPDH mRNA expression level), which was calculated as a ratio of the expression level of HOXA9 mRNA in abdominal-derived dermal fibroblasts to the expression level of GAPDH mRNA, an internal standard, and setting the relative expression level of HOXA9 mRNA in dermal fibroblasts from other sites as 1. The test results are shown in Table 1 below.

[0044] [Table 1]

[0045] As shown in Table 1, the expression levels of HOXA9 mRNA in dermal fibroblasts derived from the abdomen, buttocks, and thigh were similar, but were significantly lower in dermal fibroblasts derived from the face. These results demonstrate that HOXA9 is specifically expressed in the body.

[0046] (Experimental Example 2) Effect of HOXA9 on the extracellular matrix 5 × 10 abdominal dermal fibroblasts 4 Cells were seeded into 12-well plates at 1000 x g for 4 h and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 until 80% confluent. After washing twice with PBS, cells were added to OPTI-MEM (Thermo Fisher Scientific) containing Lipofectamine RNAiMAX (Thermo Fisher Scientific) and a 100 nM final concentration of DsiRNA targeting HOXA9 (Integrated DNA Technologies) or a negative control DsiRNA (Integrated DNA Technologies) to knockdown HOXA9. After 4 hours, the cells were washed with PBS and cultured in DMEM medium for an additional 3 days. After incubation, mRNA expression levels of HOXA9 and the extracellular matrix-associated factor genes COL1A1, COL3A1, COL5A1, ELN, and FBLN5 were measured as described in Experimental Example 1. The following primer set was used for PCR reactions.

[0047] Primer set for HOXA9 5'-AGAATGAGAGCGGCGGAGA-3' (SEQ ID NO: 1) 5'-TTCCAGGGTCTGGTGTTTTG-3' (SEQ ID NO: 2) Primer set for COL1A1 5'-AGGACAAGAGGCATGTCTGGTT-3' (SEQ ID NO: 5) 5'-TTGCAGTGGTAGGTGATGTTCTG-3' (SEQ ID NO: 6) Primer set for COL3A1 5'-GGTTTTGCCCCGTATTATGGA-3' (SEQ ID NO: 7) 5'-GTGAAGTCATAATCTCATCGGTGTTG-3' (SEQ ID NO: 8) Primer set for COL5A1 5'-GCCCATCGTGGACATCAT-3' (SEQ ID NO: 9) 5'-GCCCCACTTCAAATCCAA-3' (SEQ ID NO: 10) Primer set for ELN 5'-ACCTGGGACAACTGGAATCC-3' (SEQ ID NO: 11) 5'-AAAGCAGCAGCAAAGTTCGG-3' (SEQ ID NO: 12) Primer set for FBLN5 5'-GCTGCCCCACCACTCTCA-3' (SEQ ID NO: 13) 5'-TCTGGTATCCAAAGCGGCAT-3' (SEQ ID NO: 14) Primer set for GAPDH (internal standard) 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 3) 5'-TCTTCTGGGTGGCAGTGATG-3' (SEQ ID NO: 4)

[0048] The mRNA expression levels of the HOXA9, COL1A1, COL3A1, COL5A1, ELN, and FBLN5 genes were evaluated by calculating the relative mRNA expression level of each gene (mRNA expression level of each gene / GAPDH mRNA expression level), calculated as a ratio of the mRNA expression level of each gene in negative control dermal fibroblasts to the GAPDH mRNA expression level, which is an internal standard, and setting the relative mRNA expression level of each gene in dermal fibroblasts in which HOXA9 was knocked down, as 1. The test results are shown in Table 2.

[0049] [Table 2]

[0050] As shown in Table 2, knockdown of HOXA9 was confirmed in dermal fibroblasts. Meanwhile, the mRNA expression levels of the extracellular matrix-related factor genes COL1A1, COL3A1, COL5A1, ELN, and FBLN5 were all reduced by HOXA9 knockdown. Therefore, HOXA9 is thought to improve skin firmness and elasticity by positively regulating extracellular matrix production.

[0051] (Experimental Example 3) Effect of Kochia japonica extract on HOXA9 gene expression in dermal fibroblasts 5 × 10 abdominal dermal fibroblasts 4 The cells were seeded in 12-well plates at 1000 x g / well and cultured in DMEM culture medium containing 10% FBS at 37°C and 5% CO for 2 days. Subsequently, the cells were cultured for 1 day in a medium supplemented with Kochia sieboldii extract (Production Examples 1 to 3) to a final concentration of 10 μg / mL or 30 μg / mL. After the culture was completed, the HOXA9 mRNA expression level was measured using the method and primer set described in Experimental Example 1.

[0052] The HOXA9 mRNA expression level was evaluated by calculating the relative HOXA9 mRNA expression level (HOXA9 mRNA expression level / GAPDH mRNA expression level), which was calculated as a ratio of the HOXA9 mRNA expression level in untreated (control) dermal fibroblasts to the GAPDH mRNA expression level, which is an internal standard, and setting the relative HOXA9 mRNA expression level in dermal fibroblasts to which Kochia angustifolium extract had been added, relative to this. The test results are shown in Table 3.

[0053] [Table 3]

[0054] As shown in Table 3, the HOXA9 mRNA expression level in dermal fibroblasts was increased by the Kochia angustifolium extract. [Industrial Applicability]

[0055] The present invention can be used in the fields of manufacturing cosmetics, quasi-drugs, pharmaceuticals, functional foods, supplements and other foods and beverages that are primarily intended to improve and prevent body sagging and firmness.

Claims

1. A HOXA9 expression promoter containing an extract of Kochia japonica as an active ingredient.

2. The agent according to claim 1, wherein the promotion of HOXA9 expression is promotion of HOXA9 expression in dermal fibroblasts.

3. The agent according to claim 2, wherein the dermal fibroblasts are present in the skin of the body.

4. A composition for promoting HOXA9 expression, comprising the agent according to any one of claims 1 to 3.

5. The composition for promoting HOXA9 expression according to claim 4, wherein the composition is a cosmetic, a quasi-drug, a pharmaceutical, or a food or beverage.

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