Type 14 collagen production promoter

Abstract

To provide formulations effective for treating, ameliorating, and / or preventing skin or hair diseases or symptoms caused by morphological changes or weakening of dermal structures including perifollicular structures, by finding out a novel factor that enhances the expression of type 14 collagen, which is related to the promotion of collagen fiber formation.SOLUTION: Provided is an agent for promoting type 14 collagen production in dermal fibroblasts, characterized by containing, as an active ingredient, a truffle extract and / or an extract of panax ginseng that has been heat-dried after heat treatment with steam.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent for promoting the production of type XIV collagen in dermal fibroblasts.

Background Art

[0002] The skin is generally divided into three layers: the epidermis, the dermis, and the subcutaneous tissue. Dermal fibroblasts exist in the dermis layer, and collagen produced by dermal fibroblasts is known as an important component for maintaining the firmness and elasticity of the skin. Also, a decrease in collagen due to aging and inflammation has been cited as a cause of wrinkles and sagging. So far, 29 types of collagen have been found to exist in the human body. In the skin, it is present in the basement membrane, which is the junction between the epidermis and the dermis, and in the dermis, where it accounts for more than 70% of the total. In the extracellular matrix (ECM), the triple helix molecules of collagen further self-assemble to form supramolecular structures. Depending on its form, collagen is classified into fibril-forming collagen (types I, II, III, V, XI, etc.), network-forming collagen (types IV, VIII, X, etc.), FACIT-type collagen (types IX, XII, XIV, XVI, XIX, XX, etc.), transmembrane collagen (types XIII, XXII, XXIII, XXV, etc.), microfibril collagen (type VI), etc. Among the collagens in the dermis, type I collagen accounts for about 80%, type III collagen accounts for about 15%, and type V collagen forms thin collagen fibers by hybridizing with type I collagen and type III collagen. Also, type IV collagen, also called basement membrane collagen, forms a reticular structure. Types XIII and XXV are transmembrane collagens, with their C-terminal sides exposed extracellularly and involved in cell adhesion, etc. Type VI collagen forms tetramers and forms microfibrils in which short collagens are strung together. FACIT (Fibril Associated Collagen with Interrupted Triple helix)-type collagen exists in a state bound to fibril-forming collagen. Type 14 (XIV) collagen is this FACIT-type collagen and is thought to be involved in promoting the formation of collagen fibers, binding between collagen fibers, and binding between collagen fibers and other extracellular matrices by binding to fibril-forming collagen such as type I collagen in the dermis.The gene encoding type 14 collagen is represented by symbols such as COL14A1 and UND in humans, and it has been reported that dysfunction of this gene causes fibrotic abnormalities and biomechanical abnormalities (Non-Patent Document 1).

[0003] The growing hair follicles that form hair are about 2 to 6 mm in length, reaching from the hair pores on the surface of the epidermis to the deep part of the skin and penetrating the dermis and subcutaneous tissue. The outside of the hair follicle is wrapped by the basement membrane, but there are many unclear points about the connection mode between the outer hair follicle and the adjacent tissues. So far, it has been found that the basement membrane exists near the hair bulb of the hair follicle, but different from the connection part between the epidermis and dermis of the skin, hemidesmosomes and anchoring structures do not exist. This is thought to be due to the necessity of repeating growth and regression with the hair cycle, so that a firm connection structure is not formed between the hair follicle and the dermis (Non-Patent Document 2). On the other hand, on the epidermal side (upper part of the hair follicle) of the skin rather than the bulge area of the hair follicle whose structure is always maintained without being affected by the hair cycle, type 7 collagen which is a component of the anchoring plaque and laminin which is a basement membrane component are expressed, and a connection structure is formed between the hair follicle and the dermis, arrector pili muscle, etc. (Non-Patent Document 3). However, when the connection structure between the hair follicle and the dermis becomes unstable, the hair follicle is not fixed by the dermis, so the hair follicle deforms, and as a result, abnormalities occur in the shape of the newly generated hair, making the waves and curly hair more prominent.

[0004] Therefore, it can be expected that by activating components such as type 14 collagen that bind to fibrotic collagen and are involved in promoting the formation of collagen fibers, the connection structure between the hair follicle and the dermis and the uniformity of the collagen fibers around the hair follicle are improved, and the structure of the dermis is stabilized.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] In view of the above circumstances, the present invention aims to find a new factor that enhances the expression of type XIV collagen involved in promoting the formation of collagen fibers, and to provide a preparation effective for the treatment, improvement, and / or prevention of skin and hair diseases or conditions caused by morphological changes and fragility of dermal structures including those around hair follicles. [Means for Solving the Problems]

[0007] As a result of intensive research to solve the above problems, the inventors of the present invention have found that an extract of truffle, an extract of carrot after a predetermined pretreatment, and a mixture thereof have an effect of promoting the production of type XIV collagen in dermal fibroblasts, and have thus completed the present invention.

[0008] That is, the present invention includes the following inventions. (1) A promoter for promoting the production of type XIV collagen in dermal fibroblasts, characterized by containing, as an active ingredient, an extract of truffle and / or an extract of carrot that has been heat-dried after steam heating treatment. (2) The promoter for promoting the production of type XIV collagen according to (1), wherein the carrot is dried or raw carrot. (3) A composition for stabilizing dermal structure, containing the promoter for promoting the production of type XIV collagen according to (1) or (2). (4) The composition for stabilizing dermal structure according to (3), wherein the composition is a cosmetic, quasi-drug, or pharmaceutical.

Effects of the Invention

[0009] According to the present invention, a promoter for promoting the production of type XIV collagen is provided. Type XIV collagen binds to a fiber-forming collagen such as type I collagen in the dermis and is involved in promoting the formation of collagen fibers. Therefore, the promoter for promoting the production of type XIV collagen of the present invention is effective in improving and preventing wrinkles, sagging, loss of firmness and elasticity, hair undulation, and hair kinks caused by morphological changes and weakening of the dermal structure including the periphery of hair follicles due to aging and the like.

Modes for Carrying Out the Invention

[0010] Hereinafter, the present invention will be described in detail. 1. Promoter for Promoting the Production of Type XIV Collagen The agent for promoting type 14 collagen production in dermal fibroblasts of the present invention is characterized by containing, as active ingredients, an extract of truffle and / or an extract of ginseng that has been steam-heated and then heat-dried (hereinafter, may be referred to as "refined ginseng"). In the present invention, "promotion of type 14 collagen production" refers to promotion of type 14 collagen production at the living body level or culture level.

[0011] "Type 14 collagen" is a collagen classified as Fibril Associated Collagen with Interrupted Triple-helix (FACIT), and in humans it is represented by symbols such as COL14A1 and UND.

[0012] The truffles (scientific name: Tuber spp.) used in the present invention are ascomycetes belonging to the order Tuberales and family Tuberaceae, which grow underground in clumps and do not open to the outside. Many of the fruiting bodies have a strong fragrance and are dug up and eaten by animals such as squirrels and rabbits. Truffles are one of the three great delicacies in the world and are used in high-end French cuisine. The types of truffles used in the present invention are preferably white truffles (Tuber magnatum Pico) and black truffles (Tuber melanosporum Vitt).

[0013] In the present invention, the extract of truffles refers to an extract of the fruiting body or mycelium, preferably an extract of the fruiting body. For the extraction, the fruiting body or mycelium may be used as is, or may be processed by drying, crushing, shredding, etc.

[0014] The extraction method is not particularly limited, but it can be carried out by using water or hot water, or a mixed solvent of water and an organic solvent, and stirring or column extraction. Examples of the organic solvent include lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (such as acetone, methyl ethyl ketone, etc.), acetonitrile, esters (such as ethyl acetate, butyl acetate, etc.), hydrocarbons (such as hexane, heptane, liquid paraffin, etc.), and ethers (such as ethyl ether, tetrahydrofuran, propyl ether, etc.). Among them, polar solvents such as lower alcohols and liquid polyhydric alcohols are preferred, and water-soluble organic solvents such as ethanol, 1,3-butylene glycol, and propylene glycol are more preferred, and one or more of these can be used. Particularly preferred extraction solvents include water or a water-ethanol-based mixed polar solvent. The amount of the solvent used is not particularly limited. For example, it may be 10 times or more, preferably 20 times or more, based on the above-mentioned truffle (dry weight), but it is preferably 100 times or less for the convenience of operations when concentrating or isolating after extraction. Also, the extraction temperature and time depend on the type of solvent used. For example, it can be exemplified as 10 to 100 °C, preferably 30 to 90 °C, and 30 minutes to 24 hours, preferably 1 to 10 hours. Also, the extract may be used as the extracted solution as it is, but if necessary, within the range not affecting the effect, treatments such as concentration (concentration by an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, and ethanol precipitation may be carried out before use. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, and freeze drying, and used as a dried product.

[0015] The ginseng (scientific name: Panax ginseng C.A.Mey, alias: Korean ginseng, Dangsam, medicinal ginseng) used in the present invention is a perennial herb belonging to the genus Panax of the Araliaceae family, and is the original plant of the crude drug "carrot" (Japanese name: ginseng, scientific name: Ginseng Radix). The dried root of ginseng is used as a crude drug, and depending on the manufacturing method, it is roughly classified into "white ginseng" which has its skin peeled off and dried, and "red ginseng" which is steamed and dried with its skin attached.

[0016] In the present invention, any part of the plant body such as the leaves, stems, fruits, pericarp, flowers, flower buds, seeds, whole plants, roots, rhizomes, or the whole plant body, or a mixture thereof can be used for ginseng, but roots are preferred, and lateral roots of the roots are more preferred.

[0017] In the present invention, either dried ginseng or raw ginseng can be used for ginseng, but dried ginseng is preferred. In the case of dried ginseng, it is preferably dried until the moisture content is 20% or less, preferably 10% or less. The moisture content can be measured using methods such as loss on drying in the Japanese Pharmacopoeia. As the drying method, any method that is commonly used as a plant drying method and can bring the moisture content within the above range is not particularly limited, and examples include natural drying (air drying), sun drying, ventilation drying, hot air drying, spray drying, vacuum drying, vacuum drying, etc.

[0018] In the present invention, before extracting the above-mentioned dried carrot or raw carrot, a steam heat treatment and a heat drying treatment corresponding to the trimming treatment (a processing treatment of steaming and drying) performed during the processing of crude drugs are carried out. The steam heat treatment refers to a treatment in which hot water, saturated steam, superheated steam, reduced-pressure (vacuum) steam, etc. are used as a heat medium, and an object is heated in a high-humidity atmosphere, for example, an atmosphere with a humidity of 80% or more. The steam heating may be performed by bringing the heat medium into direct contact with the object, or the object may be indirectly heated through a heat exchanger. Also, the heating may be performed either under normal pressure or under pressure. As the conditions of the steam heat treatment, the temperature is preferably 70 to 180°C, more preferably 100 to 150°C. The time varies depending on the temperature, but is preferably 1 to 15 hours, more preferably 2 to 10 hours, and even more preferably 2 to 6 hours. These temperature and time conditions are merely illustrative and can be appropriately changed according to the mutual relationship between the temperature and the time. Further, the steam heat treatment in the present invention may be performed using a continuous or batch-type steamer (steaming machine), autoclave, or the like.

[0019] Since the carrot that has been subjected to the steam heat treatment contains moisture, a heat drying treatment is performed. As the temperature of the heat drying, 40 to 85°C is preferable, and 50 to 70°C is more preferable. As the drying method, ventilation drying, hot air drying, microwave drying, etc. can be used. The drying time (heating time) varies depending on the heating temperature, the moisture content of the carrot after the steam heat treatment, and the total amount to be dried, and cannot be specified, but is in the range of about 6 to 24 hours.

[0020] The carrot that has been subjected to the heat drying treatment after the steam heat treatment as described above is extracted. The extraction method may be carried out in the same manner as the extraction of the above-mentioned truffle. Regarding the type of extraction solvent, extraction temperature and time, and the treatment after extraction, the extraction method of the above-mentioned truffle may be followed.

[0021] In the present invention, either the extract of truffle or the extract of refined carrot may be used alone, but it is preferable to use both in combination because the effect of promoting the production of type 14 collagen in dermal fibroblasts is enhanced. When the extract of truffle and the extract of refined carrot are used in combination, the mixing ratio is not limited, but preferably 1:10 to 10:1, more preferably 1:5 to 5:1, still more preferably 1:2 to 2:1, and most preferably 1:1.

[0022] The above-mentioned extract of truffle and / or the extract of refined carrot has the effect of promoting the production of type 14 collagen in dermal fibroblasts. In the present invention, promoting the production of type 14 collagen means promoting the expression of the type 14 collagen gene and / or promoting the expression of the type 14 collagen protein by promoting the expression of the type 14 collagen gene. As described above, since type 14 collagen binds to fibrotic collagens such as type I collagen in the dermis and has the effect of promoting the formation of collagen fibers, the type 14 collagen production promoter of the present invention is effective for treating, improving, and / or preventing skin and hair diseases or symptoms caused by changes in the dermal structure including the area around hair follicles. Here, the changes in the dermal structure include morphological abnormalities, weakening, cavitation, etc. of the structure. Examples of skin and hair diseases and symptoms caused by changes in the dermal structure include, for skin, wrinkles, dullness, nasolabial folds, marionette lines, loss of firmness and elasticity, roughness, dullness, linear atrophoderma (cutaneous striae), solar elastosis, scleroderma, fibrosarcoma, xeroderma pigmentosum, cutaneous histiocytoma, etc., and for hair, waviness, kinky hair, frizzy hair, thin hair, split ends, loss of firmness and body, volume reduction, etc.

[0023] The content of the extract of truffle and / or the extract of refined carrot in the type 14 collagen production promoter according to the present invention is not particularly limited, but depending on the properties of the extract (extract solution, concentrate, or dried product), for example, it is preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight, based on the total amount of the drug.

[0024] 2. Composition for stabilizing dermal structure When the type 14 collagen production promoter according to the present invention is administered in vivo, it can be administered as it is, but it can be formulated into various compositions in the form of cosmetics, quasi-drugs, pharmaceuticals, etc. together with appropriate additives within a range that does not impair the effects of the present invention, and can be provided as a composition for stabilizing dermal structure. In particular, it is preferably in the form of a topical composition for skin (including hair compositions applied to the scalp and hair).

[0025] When formulating the type 14 collagen production promoter according to the present invention into cosmetics or quasi-drugs, the dosage form may be any of an aqueous solution system, solubilized system, emulsified system, powder system, powder dispersion system, oil solution system, gel system, ointment system, aerosol system, water-oil two-layer system, or water-oil-powder three-layer system, etc. Further, the cosmetics or quasi-drugs may be selected and appropriately formulated with various components, additives, bases, etc. that are usually used in skin external compositions together with the type 14 collagen production promoter, and can be manufactured according to methods known in the art. The form may be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of the components, additives, and base materials to be formulated into the skin external composition include diluents (purified water, ethanol, etc.), 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, cetyl octanoate, glyceryl trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidonecarboxylic acid, etc.), saccharides (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extract components, various surfactants, silicone oil, humectants, ultraviolet absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances, colorants, etc.

[0026] In addition, when it is used as a hair composition, as long as it does not adversely affect the effects of the present invention, components conventionally known as hair growth agents and hair nourishing agents may be included. For example, plant extract extracts such as assembly extract and citrus extract, vitamin B 6Examples include vitamins such as vitamin E and its derivatives, biotin, pantothenic acid and its derivatives, glycyrrhizic acid and its derivatives, nicotinic acid esters, amino acids such as serine and methionine, cephradine, capronium chloride, minoxidil, nicorandil, acetylcholine derivatives, cyclosporins, and female hormone agents such as estradiol, and mixtures thereof.

[0027] Examples of types of cosmetics and quasi-drugs include, for example, lotions, milky lotions, gels, beauty essences, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, bath agents, body lotions, body shampoos, hair shampoos, hair rinses, hair treatments, hair conditioners, scalp treatments, hair sprays, hair packs, hair essences, hair tonics, hair liquids, hair mousses, and the like.

[0028] When formulating the type 14 collagen production promoter according to the present invention into a pharmaceutical product, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various pharmaceutical preparations in a formulation form suitable for application to the affected area. As pharmacologically and pharmaceutically acceptable additives, depending on the dosage form and use, appropriately selected pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegrant aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizing aids, isotonic agents, pH adjusters, propellants, coloring agents, sweeteners, flavoring agents, fragrances, etc. can be added as appropriate, and it can be prepared into various pharmaceutical formulation forms that can be administered orally or parenterally, systemically or locally by various known methods. When the pharmaceutical product of the present invention is provided in the above-mentioned various forms, it can be manufactured by production methods commonly used by those skilled in the art, for example, the production methods shown in each article of the General Rules of Pharmaceutical Preparations [2] of the Japanese Pharmacopoeia.

[0029] The form of the pharmaceutical of the present invention is not particularly limited, and examples thereof include oral preparations such as tablets, dragees, capsules, troches, granules, powders, liquids, pills, emulsions, syrups, suspensions, elixirs, etc., injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories, ointments, lotions, sprays, transdermal absorption agents, transmucosal absorption agents, patches, etc. non-oral preparations. Further, it may be a dry product that is redissolved when used, and in the case of an injectable preparation, it is provided in the state of a unit dose ampule or a multi-dose container.

[0030] When the type 14 collagen production promoter according to the present invention is used as a pharmaceutical for treating, improving, and / or preventing the skin diseases and symptoms, a suitable form is a topical preparation, and examples thereof include ointments, creams, gels, liquids, patches (poultices, plasters), foams, sprays, aerosols, etc. An ointment refers to a homogeneous semi-solid topical preparation and includes oleaginous ointments, emulsion ointments, and water-soluble ointments. A gel refers to a topical preparation in which a water-insoluble component's water-containing compound is suspended in an aqueous liquid. A liquid refers to a liquid topical preparation and includes lotions, suspensions, emulsions, liniments, etc.

[0031] For oral administration preparations, 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 carboxymethyl cellulose, carboxymethyl cellulose calcium, starch, or hydroxypropyl cellulose; binders such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, gum arabic, or gelatin; lubricants such as magnesium stearate, calcium stearate, or talc; coating agents such as hydroxypropyl methylcellulose, sucrose, polyethylene glycol, or titanium oxide; bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat can be used, but are not limited thereto.

[0032] For parenteral administration preparations, solvents such as distilled water, physiological saline, ethanol, glycerin, propylene glycol, macrogol, alum solution, vegetable oil, etc.; tonicity agents such as glucose, sodium chloride, D-mannitol, etc.; pH adjusters such as inorganic acids, organic acids, inorganic bases or organic bases, etc. can be used, but are not limited thereto.

[0033] The pharmaceutical of the present invention functions as a prophylactic agent for suppressing the above skin diseases and symptoms and / or as a therapeutic agent for improving to a normal state. Since the active ingredient of the pharmaceutical of the present invention is derived from natural products, it is very safe and has no side effects. Therefore, when used as a medicine for treating, improving, and / or preventing the above-mentioned diseases, it can be orally or parenterally administered to mammals such as humans, mice, rats, rabbits, dogs, cats, etc. in a wide range of dosages.

[0034] The content of the type 14 collagen production promoter in the cosmetics, pharmaceuticals, and quasi-drugs of the present invention is not particularly limited, but in terms of the total weight of the preparation (composition), in terms of the dried product of the above extract, 0.001 to 30% by weight is preferable, and 0.01 to 10% by weight is more preferable. The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, general usage amount, efficacy and effect, etc. Also, regarding the method of adding the active ingredient in the formulation, it may be added in advance or during the manufacturing process, and may be appropriately selected in consideration of workability.

Example

[0035] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited thereto.

[0036] [Example 1] Production Example of Extracts of Truffle and Otanenjin Extracts of truffle and otanenjin were produced as follows.

[0037] (Production Example 1) Preparation of Hot Water Extract of Truffle 100 g of truffle powder was added to 1 L of purified water, extracted at 90 - 100 °C for 2 hours, filtered, the filtrate was concentrated, and freeze-dried to obtain 5.1 g of a hot water extract of truffle.

[0038] (Production Example 2) Preparation of a hot water extract of the root of raw Otaneninnjin (steam heating: 105 °C, heat drying: 50 °C) After harvesting, raw Otaneninnjin (moisture content 80%) was steamed at 105 °C for 8 hours and then dried at 50 °C (conforming to "Red Ginseng" in the Japanese Pharmacopoeia). 800 mL of purified water was added to 40 g of this dried Otaneninnjin, extracted at 95 - 100 °C for 2 hours, filtered, and the filtrate was concentrated to dryness to obtain 17.5 g of a hot water extract of the root of raw Otaneninnjin that had been steam-heated (105 °C).

[0039] (Production Example 3) Preparation of a hot water extract of the root of dried Otaneninnjin (steam heating: 105 °C, heat drying: 50 °C) The dried root of Otaneninnjin (moisture content 9%) was steamed at 105 °C for 8 hours and then dried at 50 °C. 800 mL of purified water was added to 40 g of this dried Otaneninnjin, extracted at 95 - 100 °C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried to obtain 18.7 g of a hot water extract of the root of dried Otaneninnjin that had been steam-heated (105 °C).

[0040] (Production Example 4) Preparation of a hot water extract of the root of dried Otaneninnjin (steam heating: 121 °C, heat drying: 60 °C) The dried root of Otaneninnjin (moisture content 7%) was steamed at 121 °C for 4 hours and then dried at 60 °C. 800 mL of purified water was added to 40 g of this dried Otaneninnjin, extracted at 95 - 100 °C for 2 hours, filtered, and the filtrate was concentrated and freeze-dried to obtain 16.2 g of a hot water extract of the root of dried Otaneninnjin that had been steam-heated (121 °C).

[0041] (Comparative Production Example 1) Preparation of a hot water extract of the root of raw Otaneninnjin (without steam heating treatment) Forty grams of raw carrots' roots after harvest, dried at 65°C, were added with 800 mL of purified water, extracted at 95 - 100°C for 2 hours, filtered, the filtrate was concentrated and freeze-dried to obtain 12.5 g of the hot water extract of raw carrots' roots.

[0042] [Example 2] Type 14 Collagen Production Promotion Test Using human-derived normal fibroblasts (manufactured by Kurabo Industries Ltd.), the effect of promoting the production of type 14 collagen by the extract of truffles and / or carrots that had been heat-treated with steam and then heat-dried was measured under the following conditions. Also, as a comparison of cell types, human-derived normal keratinocytes (manufactured by Kurabo Industries Ltd.), human dermal papilla cells, and human hair follicle stem cells were also tested simultaneously. For dermal papilla cells and hair follicle stem cells, hair follicles were collected from the subjects and cultured by the following method.

[0043] (Culture of Human Dermal Papilla Cells) Human hair was collected by plucking, and tissues containing the dermal papilla of the hair follicle tissue were collected using a scalpel, etc. After washing with PBS(-), the dermal papilla wrapped by the hair matrix was excised using forceps, etc. The excised dermal papilla was scratched on a culture plate, and then maintained with DMEM medium containing 10% FBS (manufactured by SIGMA) until confluent. The confluent cells were collected, seeded again on the same culture plate, and then the cells that had attached and were growing were used as dermal papilla cells in the following tests.

[0044] (Culture of Human Hair Follicle Stem Cells) Human hair was collected by plucking, and tissues containing the bulge region of the hair follicle tissue were collected using a scalpel, etc. After washing with PBS(-), trypsin (manufactured by BD Biosciences) treatment was performed. Then, using a cell strainer (manufactured by FALCON), the cells were isolated and collected. The collected cells were seeded on a culture plate and maintained with KG2 medium (manufactured by Kurabo) until confluent. The confluent cells were collected, seeded again on the same culture plate, and then the cells that had attached and were growing were used as hair follicle stem cells in the following tests.

[0045] Each of the above various cells (dermal fibroblasts, epidermal keratinocytes, dermal papilla cells, hair follicle stem cells) was seeded at 5 × 10 5 cells per well of a 12-well plate and cultured for 24 hours. Then, each extract obtained in the above Production Example was added so that the final concentration was 100 μg / ml. However, for the mixed extract of the truffle extract and the carrot extract, 50 μg / ml of each of the two extracts was added so that the final concentration of the mixed extract was 100 μg / ml. After adding the extract, the cells after 48 hours were collected, and RNA was extracted from the cells in each section using Trizol Reagent (manufactured by Invitrogen). Using a 2-STEP real-time PCR kit (manufactured by Applied Biosystems), the extracted RNA was reverse-transcribed into cDNA, and then real-time PCR (95°C: 15 seconds, 60°C: 30 seconds, 40 cycles) was performed using the following primer sets with ABI7300 (manufactured by Applied Biosystems) to confirm the expression of type 14 collagen. Other operations were carried out according to the defined methods.

[0046] Primer set for type 14 collagen: 5’-GATCAGTGGCGCGTCAAGT-3’ (SEQ ID NO: 1) 5’-GCTGGGAATCTGGTTGAGGAT-3’ (SEQ ID NO: 2) Primer set for GAPDH: 5’-TGCACCACCAACTGCTTAGC-3’ (SEQ ID NO: 3) 5’-TCTTCTGGGTGGCAGTGATG-3’ (SEQ ID NO: 4)

[0047] The expression of type XIV collagen was calculated as the ratio of the expression level of type XIV collagen mRNA without the addition of the extract to the expression level of GAPDH, which was used as an internal standard (relative expression level of type XIV collagen gene (expression level of type XIV collagen gene / expression level of GAPDH gene)), and the value was set to 1. In contrast, the value of the relative expression level of the type XIV collagen gene when the extract was added was calculated and evaluated. The results of these tests are shown in Table 1 below.

[0048]

Table 1

[0049] As shown in Table 1, compared with the extract of Otanenjinn without steam heating treatment (Comparative Production Example 1), the truffle extract (Production Example 1) and the Otanenjinn extract (Production Examples 2 to 4) that had been subjected to heat drying treatment after steam heating treatment showed an increase in the expression level of the type XIV collagen gene in human dermal fibroblasts, and a promoting effect on type XIV collagen production was recognized. In addition, the effect was higher when using Otanenjinn dried before steam heating treatment (Production Examples 3 and 4) than when using raw Otanenjinn (Production Example 2). Furthermore, when comparing Production Example 3 (steam heating temperature: 105°C) and Production Example 4 (steam heating temperature: 121°C), Production Example 4 with steam heating treatment at a higher temperature had a higher effect. Also, it was confirmed that when the truffle extract (Production Example 1) and the Otanenjinn extract (Production Examples 2 to 4) were used in combination, the effect was particularly enhanced, and the combination of Production Example 1 and Production Example 4 showed the highest effect. On the other hand, such an effect of promoting the expression of the type XIV collagen gene was not recognized or was low in cells other than fibroblasts (epidermal keratinocytes, dermal papilla cells, hair follicle stem cells).

Industrial Applicability

[0050] The present invention can be used in the field of manufacturing cosmetics, quasi-drugs, or pharmaceuticals for the purpose of treating, improving, and / or preventing skin and hair problems such as wrinkles, sagging, undulations, and kinky hair caused by changes in the dermal structure due to aging and the like.

Claims

1. A promoter for promoting the production of type 14 collagen in dermal fibroblasts, characterized by containing, as an active ingredient, a mixture of an extract of truffle and an extract of dried burdock root that has been subjected to steam heat treatment and then heat drying treatment.

2. A composition for improving hair waves or kinky hair caused by changes in the dermal structure, comprising the promoter for promoting the production of type 14 collagen according to Claim 1.

3. The composition for improving hair waves or kinky hair caused by changes in the dermal structure according to Claim 2, wherein the composition is a cosmetic, quasi-drug, or pharmaceutical.

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