Whitening agent

Abstract

To provide a novel skin-whitening agent.SOLUTION: A skin-whitening agent contains (A) rosmarinic acid and (B) luteolin glucuronide with the weight ratio of (A): (B)=1: 0.2-1.5. The agent preferably further contains tranexamic acid and / or nicotinamide.SELECTED DRAWING: None

Description

【Technical Field】 【0001】 The present invention relates to a novel whitening agent containing rosmarinic acid and luteolin glucuronide for use in humans and the like. 【Background Art】 【0002】 Due to air pollution and the destruction of the ozone layer, the amount of ultraviolet rays reaching the epidermis has been increasing year by year. Along with this, skin problems such as skin spots, freckles, and darkening caused by ultraviolet rays have been growing. Under this situation, various whitening agents have been developed to achieve fair and beautiful skin. Known whitening agents include ascorbic acid derivatives such as ascorbic acid, ascorbic acid phosphate esters, and ascorbic acid glucosides, glycosides of hydroquinones such as hydroquinone glucoside, tranexamic acid and tranexamic acid derivatives such as tranexamic acid esters (Patent Document 1, Patent Document 2). 【Prior Art Documents】 【Patent Documents】 【0003】 【Patent Document 1】 JP-A-2004-115381 【Patent Document 2】 JP-A-2021-80236 【Patent Document 3】 Patent No. 6786193 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0004】 Problems to be solved by the present invention include providing a novel whitening agent and the like. 【Means for Solving the Problems】 【0005】 Therefore, as a result of intensive studies by the inventor of the present invention, a composition containing rosmarinic acid and luteolin glucuronide was newly confirmed to have an inhibitory effect on melanin production, and thus the present invention was completed. 【0006】 The present invention includes the following items. [Item 1] A skin whitening agent in which the weight ratio of (A) rosmarinic acid and (B) luteoning glucronide is (A):(B) = 1:0.2 to 1.5. [Item 2] The whitening agent described in [Item 1], which contains tranexamic acid. [Item 3] A whitening agent according to [Item 1] or [Item 2], comprising nicotinamide. [Item 4] A composition according to any one of [Item 1] to [Item 3], in the form of a topical skin preparation. [Effects of the Invention] 【0007】 The present invention provides a novel skin whitening agent. [Modes for carrying out the invention] 【0008】 The following describes embodiments for carrying out the present invention. 【0009】 (Peppermint, rosmarinic acid, luteoning glucronide) Rosmarinic acid and luteoning glucronide used in the present invention are, for example, contained in peppermint extract (Patent Document 3). 【0010】 Peppermint (also known as European mint or black peppermint) is a plant belonging to the genus Mentha in the Lamiaceae family: Mentha piperita L. When producing peppermint extract, the roots, rhizomes, leaves, stems, flowers, whole plant, or mixtures thereof can be used as materials. However, since the active ingredients are thought to be more abundant in the leaves, it is considered preferable to use the leaves as the material. 【0011】 Peppermint extracts can be prepared, for example, by crushing and pressing the raw or dried material, or by crushing the raw or dried material and extracting it with a solvent. For example, peppermint extracts are prepared according to the following manufacturing example. The peppermint extracts used in the following examples were prepared according to the following manufacturing example. In the peppermint extract used in the following examples, rosmarinic acid was contained at 403.55 ppm and luteolin glucuronide was contained at 166.35 ppm, and the content ratio of (A) rosmarinic acid to (B) luteolin glucuronide was (A):(B) = 1:0.41 by weight ratio. 【0012】 The peppermint extract contained in the agent of the present invention is preferably contained at 0.1% or more, more preferably 0.2% or more, and still more preferably 0.5% or more in order to exhibit a desired effect (such as a whitening effect). Also, the peppermint extract contained in the agent of the present invention is preferably contained at 20% or less, more preferably 15% or less, and still more preferably 10% or less in consideration of, for example, desired toxicity. 【0013】 When the peppermint extract contained in the agent of the present invention is 1%, the results of calculating the contents of (A) rosmarinic acid and (B) luteolin glucuronide in the agent are shown in Table 1 below. 【0014】 【Table 1】 【0015】 When the peppermint extract contained in the agent of the present invention is 0.5%, the results of calculating the contents of (A) rosmarinic acid and (B) luteolin glucuronide in the agent are shown in Table 2 below. 【0016】 【Table 2】 【0017】 (Production Example of Peppermint Extract) Crush the leaves of peppermint to produce a crushed product. Immerse 100 g of this crushed product in 2 kg of a 50% ethanol solution. Conduct this immersion for 5 to 10 days in an environment of about 10°C to about 30°C. Fractionate the solution obtained through this immersion using a column (HP-20) to extract the fraction containing rosmarinic acid and luteolin glucuronide. Further purify this extracted fraction using a column (HP-20). The solution after purification preferably has “(A) rosmarinic acid and (B) luteolin glucuronide with a content ratio by weight of (A):(B) = 1:0.2 to 1.5 (when (A) is 1, (B) is 0.2 or more and 1.5 or less).” Note that the extract of peppermint used in the following examples has a content ratio by weight of (A) rosmarinic acid and (B) luteolin glucuronide of (A):(B) = 1:0.46. 【0018】 (Rosmarinic acid) Rosmarinic acid is a polyphenol and is soluble in ethanol. The rosmarinic acid contained in the agent of the present invention is preferably contained at 0.1 ppm or more, more preferably 0.2 ppm or more, and even more preferably 0.5 ppm or more in order to exhibit a desired effect (such as a whitening effect). Also, the rosmarinic acid contained in the agent of the present invention is preferably contained at 20 ppm or less, more preferably 15 ppm or less, and even more preferably 10 ppm or less in consideration of, for example, desired toxicity. 【0019】 (Luteolin glucuronide) Luteolin glucuronide is a flavonoid, such as luteolin 7-glucuronide and luteolin 3'-glucuronide. The luteolin glucuronide contained in the agent of the present invention is preferably present at a concentration of 0.08 ppm or more, more preferably 0.16 ppm or more, and even more preferably 0.40 ppm or more, in order to exhibit a desired effect (such as a skin whitening effect). The peppermint extract contained in the agent of the present invention is preferably present at a concentration of 16 ppm or less, more preferably 12 ppm or less, and even more preferably 8 ppm or less, taking into consideration the desired toxicity, for example. 【0020】 (Ratio of rosmarinic acid and luteoning luconide content) From the viewpoint of exhibiting activity, the agent of the present invention preferably has a weight ratio of (A) rosmarinic acid to (B) luteoning glucronide, where (A):(B) = 1:0.2~1.5 (when (A) is 1, (B) is 0.2 to 1.5), more preferably (A):(B) = 1:0.2~1.2 (when (A) is 1, (B) is 0.2 to 1.2), more preferably (A):(B) = 1:0.25~1.18 (when (A) is 1, (B) is 0.25 to 1.18), and even more preferably (A):(B) = 1:0.3~1.16 (when (A) is 1, (B) is 0.3 to 1.16). 【0021】 (Extraction solvent used in the production of peppermint extract) Examples of extraction solvents include water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, and other lower alcohols or aqueous lower alcohols, propylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,3,5-pentanetriol, glycerin, polyethylene glycol (molecular weight 100 to 100,000), and other polyhydric alcohols or aqueous lower alcohols. Examples of suitable solvents include various organic solvents such as aqueous polyhydric alcohols, acetone, ethyl acetate, diethyl ether, dimethyl ether, ethyl methyl ether, dioxane, acetonitrile, xylene, benzene, chloroform, carbon tetrachloride, phenol, and toluene, as well as one or more mixtures of acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) and alkalis (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) selected from those with appropriately adjusted normalities. However, ethanol is preferred to allow for the possibility of solvent substitution. 【0022】 (others) The peppermint extract used in this invention can be further purified after solvent extraction as appropriate. Purification operations include, for example, decomposition by adding acid (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or alkali (sodium hydroxide, calcium hydroxide, ammonia, etc.), fermentation or metabolic transformation by microorganisms, adsorption of components by ion exchange resin, activated carbon, diatomaceous earth, etc., fractionation using chromatography with various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.), filtration using filter paper, membrane filters, ultrafiltration membranes, etc., pressurization or depressurization, heating or cooling, drying, pH adjustment, deodorization, decolorization, and long-term static storage. It is also possible to arbitrarily select and combine these processes. 【0023】 (Tranexamic acid) The agent of the present invention may preferably further contain tranexamic acid to exhibit greater efficacy. Tranexamic acid is an abbreviation for trans-4-aminomethylcyclohexane-1-carboxylic acid. Tranexamic acids refer to tranexamic acid, tranexamic acid salts, tranexamic acid esters, tranexamic acid amides, and polymers of tranexamic acid. Examples of salts in tranexamic acid salts include metal salts such as sodium salts, potassium salts, and magnesium salts; and inorganic salts such as hydrochloride salts, phosphate salts, and sulfate salts. Examples of esters in tranexamic acid esters include vitamin esters such as vitamin A esters, vitamin E esters, and vitamin C esters, and alkyl esters. Examples of amides in tranexamic acid amides include methylamide. 【0024】 (Nicotinamide) The agent of the present invention may preferably further contain nicotinamide to enhance its effectiveness. Nicotinamide is an amide compound of nicotinic acid (vitamin B3, niacin), and is a derivative of nicotinic acid, also called nicotinamide or niacinamide. Nicotinamide is a water-soluble vitamin and one of the vitamin B group substances, and can be extracted from natural products (such as rice bran) or synthesized by known methods. Specifically, those listed in the 15th edition of the Japanese Pharmacopoeia 2008 can be used. 【0025】 (whitening) Skin whitening refers to the prevention and / or improvement of pigmentation, more specifically, to the prevention and / or improvement of pigmentation symptoms caused by increased melanin production, excessive accumulation, and abnormal deposition, such as age spots, dullness, freckles, sunburn, and darkening due to skin inflammation or irritation, as well as pigmentation symptoms caused by diseases that result in pigmentation, such as skin melanosis caused by drugs such as steroids. 【0026】 (Form of the drug) The preparations according to the present invention (such as topical skin preparations) are available in the following forms suitable for use: 1) pharmaceuticals, 2) quasi-drugs, and 3) topical or systemic skin preparations (for example, basic cosmetics such as lotions, emulsions, creams, ointments, oils, and packs; facial cleansers and skin cleansers such as solid soaps, liquid soaps, and hand washes; massage preparations; cleansing preparations; depilatory agents; hair removal agents; shaving preparations; aftershave lotions; pre-shave lotions; shaving creams; foundations; lipsticks; blushes; eyeshadows; eyelashes) 1) Makeup cosmetics such as inhalers and mascaras, perfumes, nail care products, nail enamels, nail enamel removers, poultices, plasters, tapes, sheets, adhesive patches, aerosols, etc. 2) Medicinal and / or cosmetic preparations applied to the scalp and hair (for example, shampoos, conditioners, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and nourishing products, poultices, plasters, tapes, sheets, aerosols, etc. 3) Bath additives used by adding them to bathwater 4) Others include deodorants and antiperspirants, antiperspirants, hygiene products, sanitary cotton, wet wipes, etc. 【0027】 (Components of the agent) Furthermore, such agents can be manufactured by optionally selecting and using the following examples of components and additives, as necessary, within the limits that do not impair the effects of the present invention. Note that tranexamic acid and nicotinamide are as described above. 【0028】 (1) Various oils and fats Avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange rafur oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peach oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japanese wax, coconut oil, beef tallow, lard, squalene, squalane, pristane, or hydrogenated versions of these oils (hydrogenated oils, etc.). 【0029】 (2) Waxes Beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montane wax, shellac wax, rice wax, etc. 【0030】 (3) Mineral oil Liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystalline wax, etc. 【0031】 (4) Fatty acids Natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acids; and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid. 【0032】 (5) Alcoholic beverages Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, and phenoxyethanol, and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol. 【0033】 (6) Polyhydric alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, treitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc. 【0034】 (7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc. 【0035】 (8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc. 【0036】 (9) Gum, sugars or water-soluble polymer compounds Acacia gum, benzoin gum, dammar gum, guaiac butter, Irish moss, karaya gum, tragacanth gum, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide have been added, low molecular weight chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salts, hya Rulonic acid or its salts, chondroitin sulfate or its salts, heparin, ethylcellulose, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, sodium carboxyethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylates, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their crosslinked polymers, carboxyvinyl polymers, polyethyleneimines, etc. 【0037】 (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkylamines, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium and silicon, fluorinated carbon surfactants), etc. 【0038】 (11) Various vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), Vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, Vitamin C group: vitamin C acid or its derivatives, Vitamin D group: ergocalcifer Vitamin D2 (Cholecalciferol), Vitamin D3 (Cholecalciferol), Dihydrotachisterol, Vitamin E group: Vitamin E or its derivatives, Ubiquinones, Vitamin K group: Phytonadione (Vitamin K1), Menaquinone (Vitamin K2), Menadione (Vitamin K3), Menadiol (Vitamin K4), Others, Essential fatty acids (Vitamin F), Carnitine, Ferulic acid, γ-Oryzanol, Orotic acid, Vitamin P group (Rutin, Eriocitrin, Hesperidin), Vitamin U, etc. 【0039】 (12) Various amino acids Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., or their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidone carboxylic acid. 【0040】 (13) Additives Depending on the type and form of product to be added, processing can be carried out using conventional methods (for example, crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc., by arbitrarily selecting and combining these processes), and the material can be arbitrarily selected from various materials and supplied. 【0041】 The solvent used for extraction should be selected considering the intended use, type, and subsequent processing of the product. However, it is generally preferable to use one or more solvents selected from water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, or other lower alcohols or aqueous lower alcohols, propylene glycol, 1,3-butylene glycol, glycerin, or other polyhydric alcohols or aqueous polyhydric alcohols, acetone, ethyl acetate, and other organic solvents. However, if the inclusion of organic solvents is undesirable depending on the application, water alone may be used, or ethanol, which is easily removed after extraction, may be used alone or in any mixture with water. Extraction by pressing may also be used. 【0042】 Furthermore, when additives derived from plant or animal raw materials are used in topical preparations or cosmetics for use on the whole body or topically, in addition to cosmetic effects such as protection of the skin and hair, moisturizing, improvement of feel and texture, imparting flexibility, alleviation of irritation, stress relief through fragrance, cell activation (prevention of cell aging), suppression of inflammation, improvement of skin and hair quality, prevention and improvement of rough skin, hair growth, hair nourishment, prevention of hair loss, imparting shine, cleansing effect, fatigue relief, improved blood circulation, and warming effect, effects such as fragrance, deodorization, thickening, preservative, and buffering can also be expected. 【0043】 Furthermore, by combining the various cosmetic and pharmaceutical effects of each known raw material, it is possible to enhance the effects targeted by this invention and create a product with multifunctional effects. 【0044】 The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % in the numerical values ​​indicating the amount of each component added means mass %. [Examples] 【0045】 The following describes embodiments of the present invention. The experimental materials used in the experiments described in the following embodiments are as follows. • B16 mouse melanoma cells (melanoma cells): B16 melanoma 4A5, RIKEN CELL BANK • Pre-culture medium for the cells: MEM medium containing 5% fetal bovine serum • MEM medium: E-MEM (051-07615), Fujifilm Wako Pure Chemical Corporation • Fetal bovine serum: Thermofisher Scientific • This culture medium: MEM medium containing 0.036% deforin • Theophylline (1,3-dimethylxanthine): Tokyo Chemical Industries, Ltd., CAS RN: 58-55-9, Product Code: T0179 • Peppermint extract: Contains 403.55 ppm rosmarinic acid and 166.35 ppm luteoning lucuronide, with a weight ratio of (A) rosmarinic acid to (B) luteoning lucuronide of (A):(B) = 1:0.41 Nicotinamide: Product code 000-54225, CAS RN TM 98-92-0, Kishida Chemical Co., Ltd. • Tranexamic acid: Japanese Pharmacopoeia Tranexamic Acid ·Arbutin:Product ID:A6804, LKT Laboratories INC 【0046】 • Normal human neonatal foreskin fibroblasts: Kurabo, KF-4009 • TNF-α: Fujifilm Wako Pure Chemical Industries, 203-15263 (Tumor Necrosis Factor α Human, Recombinant) D-MEM: Fujifilm Wako Pure Chemical Industries, 041-29775 • ELISA kit used for measuring MMP-1 levels (MMP-1 ELISA kit): Human MMP1 ELISA Kit (Abcam: ab100604) 【0047】 (Experiment 1: Melanin production inhibition test) By adding the sample to a specified cell type (the melanoma cell described above), we confirmed whether there was a change in melanin production in that cell type. B16 mouse melanoma cells were used as the cells to which the sample was added. 【0048】 (1) Pre-culture process of melanoma cells The melanoma cells described above were cultured for 24 hours in the aforementioned pre-culture medium (MEM medium containing 5% fetal bovine serum) under conditions of 5% CO2 and 37°C. 【0049】 (2) Main culture process with sample addition After pre-culturing of (1), 8.5 × 10 4 Melanoma cells were seeded at a cell-per-well rate into each well (wells containing the culture medium, 24-well plate). After seeding, the cells were cultured for 24 hours under conditions of 5% CO2 and 37°C. 【0050】 After this culturing, the culture medium in each well was replaced with fresh culture medium. The following samples, shown in Table 3, were added to the replaced medium (containing the cells). The addition of these samples was carried out to create the following groups. In Experiment 1, since the melanin production inhibitory effect of arbutin is known (Japanese Journal of Dermatology: 101(6), 609-613, 1991), a group to which arbutin was added was set up as a positive control example (Experiment Example 2). 【0051】 [Table 3] 【0052】 After the addition, the samples were cultured for 72 hours (3 days) under conditions of 5% CO2 and 37°C. After this 72-hour culture, each group was divided into two parts: one for use in Measurement 1 below, and the other for use in Measurement 2 below. 【0053】 (Measurement 1: Melanin amount measurement) The cultured sample used in Measurement 1 was recovered by trypsin treatment. The recovered cells were dissolved in a solution (containing 1N NaOH and 10% DMSO). The absorbance of the dissolved solution (absorbance for Test Examples 1 to 7) was measured at 420 nm. The measured value was defined as the melanin content. The measurement results are shown in Table 4. 【0054】 The values ​​(relative values) shown in Table 4 were calculated as follows. First, the absorbance of the measurement results for each group (n=3) was calculated. Next, for the mean values, the value of the control group for each group was set to 100, and the relative value of each group was calculated in comparison to the control group. The asterisk (*) in Table 2 indicates a significant difference (p<0.05) when comparing with Test Example 1 (value set to 100) using Dunnett's test. The † in Table 2 indicates a significant difference (p<0.05) when comparing Test Example 3 and Test Example 4 using Student's t-test, compared to Test Example 4. 【0055】 [Table 4] 【0056】 In test examples 3, 5, and 7, significant inhibition of melanin production was confirmed, similar to test example 2 (positive control). In a comparison between test example 3 and test example 4, significant inhibition of melanin production was confirmed in test example 3 compared to test example 4. 【0057】 (Measurement 2: Cell count measurement of melanoma cells using the WST method) The measurement was performed using Cell Counting Kit-8 (Dojindo). Kit-8 was added to the cultured sample to be used in Measurement 2. After adding the kit, the cells were cultured for 2 hours under conditions of 5% CO2 and 37°C. After the culture, the color development of each group was confirmed by measuring the absorbance at 450 nm according to the instructions for "Cell Counting Kit-8 (Dojindo)". This was performed for each group (n=4). Upon confirmation, no change in color development was observed in any group compared to the control group. This absence of change indicates that, as shown in Table 2, no cytotoxicity occurred in the cultured cells due to the addition of the sample. 【0058】 (Experiment 2: Melanin production inhibition test) By adding the sample (rosmarinic acid and / or luteoning lucnonide) to a specified cell (the melanoma cell described above), we confirmed whether there was a change in melanin production in the specified cell. B16 mouse melanoma cells were used as the cells to which the sample was added. 【0059】 (1) Pre-culture process of melanoma cells The melanoma cells described above were cultured for 24 hours in the aforementioned pre-culture medium (MEM medium containing 5% fetal bovine serum) under conditions of 5% CO2 and 37°C. 【0060】 (2) Main culture process with sample addition After pre-culturing of (1), 8.5 × 10 4 Melanoma cells were seeded at a cell-per-well rate into each well (wells containing the culture medium, 24-well plate). After seeding, the cells were cultured for 24 hours under conditions of 5% CO2 and 37°C. 【0061】 After this culturing, the culture medium in each well was replaced with fresh culture medium. The following samples, shown in Table 5, were added to the replaced medium (containing the cells). The addition of these samples was carried out to create the following groups. In Experiment 2, since arbutin is known to inhibit melanin production (Japanese Journal of Dermatology: 101(6), 609-613, 1991), a group to which arbutin was added was set up as a positive control example (Test Example 7). 【0062】 [Table 5] 【0063】 After the addition, the samples were cultured for 72 hours (3 days) under conditions of 5% CO2 and 37°C. After this 72-hour culture, each group was divided into two parts: one for use in Measurement 1 below, and the other for use in Measurement 2 below. 【0064】 (Measurement 2-1: Melanin content measurement) The cultured sample used in Measurement 1 was recovered by trypsin treatment. The recovered cells were dissolved in a solution (containing 1N NaOH and 10% DMSO). The absorbance of the dissolved solution (absorbance for Test Examples 1 to 7) was measured at 420 nm. The measured value was defined as the melanin content. The measurement results are shown in Table 4. 【0065】 The values ​​(relative values) shown in Table 6 were calculated as follows. First, the absorbance of each group (n=3) was calculated. Next, for the mean values, the value of the control group for each group was set to 100, and the relative value of each group was calculated in comparison to the control group. The asterisk (*) in Table 2 indicates a significant difference (p<0.05) when compared with Test Example 6 (value set to 100) in the Dunnett test. 【0066】 [Table 6] 【0067】 In Test Example 8, significant suppression of melanin production was confirmed. 【0068】 (Experiment 3: Melanin production inhibition test) By adding the sample (rosmarinic acid and / or luteoning lucnonide) to a specified cell (the melanoma cell described above), we confirmed whether there was a change in melanin production in the specified cell. B16 mouse melanoma cells were used as the cells to which the sample was added. 【0069】 (1) Pre-culture process of melanoma cells The melanoma cells described above were cultured for 24 hours in the aforementioned pre-culture medium (MEM medium containing 5% fetal bovine serum) under conditions of 5% CO2 and 37°C. 【0070】 (2) Main culture process with sample addition After pre-culturing of (1), 8.5 × 10 4 Melanoma cells were seeded at a cell-per-well rate into each well (wells containing the culture medium, 24-well plate). After seeding, the cells were cultured for 24 hours under conditions of 5% CO2 and 37°C. 【0071】 After this culturing, the culture medium in each well was replaced with fresh culture medium. The following samples, shown in Table 7, were added to the replaced medium (containing the cells). The addition of these samples was carried out to create the following groups. In this Experiment 3, since the melanin production inhibitory effect of arbutin is known (Japanese Journal of Dermatology: 101(6), 609-613, 1991), a group to which arbutin was added was set up as a positive control example (Test Example 10). 【0072】 [Table 7] 【0073】 After the addition, the samples were cultured for 72 hours (3 days) under conditions of 5% CO2 and 37°C. After this 72-hour culture, each group was divided into two parts: one for use in Measurement 1 below, and the other for use in Measurement 2 below. 【0074】 (Measurement 3-1: Melanin amount measurement) The cultured sample used in Measurement 1 was recovered by trypsin treatment. The recovered cells were dissolved in a solution (containing 1N NaOH and 10% DMSO). The absorbance of the dissolved solution (absorbance for Test Examples 1 to 7) was measured at 420 nm. The measured value was defined as the melanin content. The measurement results are shown in Table 8. 【0075】 The values ​​(relative values) shown in Table 8 were calculated as follows. First, the absorbance of each group (n=3) was calculated. Next, for the mean values, the value of the control group for each group was set to 100, and the relative value of each group was calculated in comparison to the control group. The asterisk (*) in Table 2 indicates a significant difference (p<0.05) when compared with test example 9 (value set to 100) in the Dunnett test. 【0076】 [Table 8] 【0077】 In Test Example 11, significant suppression of melanin production was confirmed. 【0078】 (Experiment 4: Evaluation of MMP-1 production) By stimulating specific cells (normal human neonatal foreskin fibroblasts) with the inflammatory cytokine (TNF-α) and then adding a sample (peppermint extract and nicotinamide, etc.) after stimulation, changes in the production of MMP-1 (matrix metalloproteinase) were observed in the specific cells. As described in Patent Document 3, it is known that in thinning of human skin, the degradation of the extracellular matrix by matrix metalloproteinases (hereinafter referred to as MMPs) is enhanced. Thinning of the skin is one of the typical phenomena of skin aging in which the epidermis and dermis thin with age. A high production of MMP-1 suggests that skin thinning is occurring. 【0079】 The experimental procedure performed is described below. 5 x 10 4 1 normal human epidermal keratinocyte was seeded in a 24-well plate and pre-cultured under specified conditions (5% CO2, 37°C) until it reached 75% confluence. 【0080】 After the initial culture, the medium was replaced with DMEM medium containing 0.25% FBS, and the following samples shown in Table 9 were added to the replaced medium (containing the cells). The addition of these samples was carried out to create the following groups. 【0081】 [Table 9] 【0082】 After adding the sample, incubation was carried out for 72 hours under specified conditions (5% CO2, 37°C). After the 72 hours of incubation, the supernatant of the culture medium was collected. The amount of MMP-1 produced in the collected supernatant was measured using an MMP-1 ELISA kit. 【0083】 Table 10 shows the results of measuring the production of MMP-1. In Table 10, the value for group 13 is set to 100.00, and the values ​​for the other groups are listed as relative values ​​compared to group 13. In Table 10, the asterisk (*) indicates a significant difference (p<0.05) when compared with the group from Test Example 13 (value set to 100.00) in the Dunnett test. In Table 10, the † symbol indicates a significant difference (p<0.05) between the group of Test Example 15 and the group of Test Example 16 compared to the group of Test Example 14, as determined by Dunnett's test. 【0084】 [Table 10] 【0085】 As shown in Table 10, in particular, in test examples 15 to 17, a further suppression of MMP-1 production in TNF-α-stimulated cells was confirmed compared to test example 14. 【0086】 Although embodiments of the present invention (including examples) have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and any design changes, etc., that do not depart from the spirit of the present invention are still included. [Industrial applicability] 【0087】 This invention can be used, for example, as an agent that exerts a whitening effect on the skin of humans and other animals.

Claims

[Claim 1] A skin whitening agent containing (A) rosmarinic acid, (B) luteolinic acid, and (C) tranexamic acid, wherein the weight ratio of (A) to (B) is (A):(B) = 1:0.2 to 1.

5. [Claim 2] A skin whitening agent containing (A) rosmarinic acid, (B) luteolinic acid, and (D) nicotinamide, wherein the weight ratio of (A) to (B) is (A):(B) = 1:0.2 to 1.

5. [Claim 3] The agent according to claim 2, wherein the whitening agent is an agent for whitening and for improving skin thinning.

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