JP7262319B2 - Melanin production inhibitor and method for producing melanin production inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a melanogenesis inhibitor and a method for producing a melanogenesis inhibitor.

2. Description of the Related Art As the standard of living in modern society increases and interest in beauty and health increases, there is an increasing demand for improvement of skin and improvement of lifestyle-related diseases through cosmetics, health foods, nutritional supplements, and the like. For these reasons, efforts are being made to screen safe plant materials that have wide-ranging improvement effects such as skin whitening, skin protection, skin aging prevention, suppression of blood sugar level increases, and reduction of blood lipids. there is

It has been reported that extracts of plants and the like are often used as substances that exhibit melanin production inhibitory action. For example, extracts of Tosendan, Souka, Seneciogracilis, and Cochrillo have inhibitory action on tyrosinase activity and whitening action. It is reported that it is effective for (Patent Document 1). In addition, it has been reported that cacao nibs, cacao mass, cocoa, and their extracts have melanin production inhibitory action and are effective in preventing and treating pigmentation such as age spots and freckles (Patent Document 2). As a skin protective effect, grape seed extract has been reported to reduce skin disorders caused by ultraviolet rays (Patent Document 3). It has been disclosed that ginseng has anti-arteriosclerosis, amelioration of lifestyle-related diseases such as immune function regulating action, skin whitening, and skin improvement effects such as skin aging inhibition (Patent Document 4).

JP 2010-195732 A Japanese Patent Application Laid-Open No. 2007-15943 JP 2008-88188 A JP 2016-164145 A

Patent Documents 1 and 2 disclose that it has the effect of suppressing melanin production, and Patent Document 3 discloses a component that protects the skin, and is incorporated in various products such as some foods, cosmetics, and pharmaceuticals. Some have been put into practical use. However, conventionally reported components are not always satisfactory in terms of melanogenesis inhibitory action, skin protective action and stability thereof. In addition, Patent Documents 1 to 3 only show melanin-suppressing effects and skin-improving effects, and do not provide wide-ranging applications such as lifestyle-related diseases. Patent Document 4 discloses that ginseng has a wide range of uses such as lifestyle-related diseases and skin improvement effects, but ginseng is very expensive and cannot be used casually.

The present invention has been made in view of the above problems, and has excellent melanogenesis inhibitory action, neutrophil elastase inhibitory action, intercellular adhesion protective action, and blood sugar level reduction by a small amount and a simple treatment or ingestion method. The purpose of the present invention is to provide an agent having a wide range of uses, such as action and blood lipid abnormality suppressing action. Furthermore, it aims at providing the manufacturing method of the said agent.

In order to solve the above-mentioned problems, the present inventors have made intensive studies, and as a result, Asteraceae Adenostemma or its extract has an inhibitory effect on melanin production and neutrophil elastase. We have found that it has inhibitory action, protective action against destruction of intercellular adhesion caused by exposure to ultraviolet light, exposure to blue light, etc., blood sugar level lowering action, and blood lipid abnormality inhibitory action, leading to the completion of the present invention. rice field.

That is, the present invention includes the following inventions.
[Invention 1]
A method for producing a melanin production inhibitor, which comprises extracting a leaf part of white radish and/or okadakon radish with water.
[Invention 2]
A melanin production inhibitor characterized by comprising, as an active ingredient, an aqueous extract of leaf parts of white radish and/or okadakon radish .

Since the melanin production inhibitor according to the present invention contains an active ingredient obtained from a plant of the genus Panthera, it has a melanin production inhibitory action without any safety problems. Such melanin production inhibitors are low cost, highly safe, and can be used for food and drink, pharmaceuticals, or quasi-drugs with few side effects.

The melanogenesis inhibitor according to the present invention can be produced by a simple and low-cost method.

FIG. 1 is a diagram visually showing the relationship between the concentrations of water extracts of white radish and okadakon and their melanin production inhibitory effects. FIG. 2 is a diagram showing the relationship between the concentrations of the aqueous extracts of white radish and okadakon and the effect of inhibiting melanin production in terms of absorbance. FIG. 3 is a diagram visually showing the effect of suppressing melanin production by different extraction methods of the extracts of white radish and okadakon. FIG. 4 is a diagram showing the melanin formation inhibitory effect of the extracts of white radish and okadakon by different extraction methods in terms of absorbance. FIG. 5 is a diagram showing melanin production inhibitory effects depending on the extraction method of the white radish extract. FIG. 6 is a diagram showing cytotoxicity depending on the extraction method of the white radish extract. FIG. 7 is a diagram showing cytotoxicity due to differences in the purification process of the white radish extract. FIG. 8 shows the thermal stability of the water extract of white radish. FIG. 9 is a diagram showing the evaluation of melanin production inhibitory effects by different purification methods of water extracts of white radish. FIG. 10 is a diagram showing the intercellular adhesion protective effect of the water extract of white radish in terms of electric resistance. FIG. 11 is a graph showing the neutrophil elastase inhibitory effect of water extracts of white radish and okadakon radish in terms of mRNA expression levels. FIG. 12 is a photograph showing the neutrophil elastase inhibitory effect of water extracts of white radish and okadakon radish by Western blotting. FIG. 13 is a graph showing the blood sugar level lowering effect of the water extract of white radish. FIG. 14 is a graph showing the cholesterol-lowering effect of water extract of white radish. FIG. 15 is a graph comparing the intercellular adhesion protective action between the water extract of radish and a hyaluronidase activity inhibitor. FIG. 16 is a diagram showing the melanogenesis inhibitory effect in leaves of radish. FIG. 17 is a graph showing the inhibitory effect of the leaf extract of white radish on melanin production by mice. FIG. 18 is a diagram comparing melanogenesis inhibitory effects using melanoma cells of white radish and amakusasida. FIG. 19 is a graph comparing melanogenesis inhibitory effects using melanoma cells of white radish and amakusasida. FIG. 20 is a graph comparing the effect of suppressing melanin production by mice of radish and fern.

The intercellular adhesion protective agent, melanin production inhibitor, blood sugar level reducing agent, neutrophil elastase inhibitor, and blood dyslipidemia inhibitor of the present invention contain a plant of the genus Adenostemma or an extract thereof as an active ingredient. do. Here, the extract also includes a purified product purified to a single component or a fraction close to a single component.

Plants belonging to the genus Rhinoceros radix are widely distributed in East Asia including Japan, Southeast Asia, India, Australia, etc., and about 10 species are known. In Japan, it is often found in Honshu (to the west of the Tohoku region), Shikoku, Kyushu, and Okinawa. In Japan, until now, the genus radish was considered to be a single species, but it has come to be divided into two varieties, one with a smooth achene and the other with a wart-like appearance.

The cultivar of the genus radish used in the present invention is not particularly limited as long as it is a plant belonging to the genus radish, but A. lavenia (L.) Kuntze var. L.) Kuntze var. madurense), A. lavenia var. tintrium, A. viscosum and the like.

From the viewpoint of exhibiting the effects of the present invention remarkably, it is preferable to use a radish and/or a radish as the genus plant. In Japan, the white radish is distributed from the Kanto region west of Honshu to Shikoku, Kyushu, and Okinawa, and inhabits wetlands in warm regions. Okadaikon lives in areas that are a little drier than the white radish, such as forest edges in mountains.

In the present invention, when a plant belonging to the genus Glycine is used as it is, the preparation method is not particularly limited. The radish genus plant may be obtained by pulverizing a raw radish genus plant and using the pulverized product as it is, or may be a dried product obtained by drying the whole plant or part of the genus radish plant, and cutting the dried product. It may be crushed or pulverized to prepare a powder. A dry product or powder of a plant belonging to the genus Panthera can be used, for example, by extracting it with water or hot water.

When preparing a plant belonging to the genus Glycine, each of the leaf part, stem part, root part, flower part and seed may be prepared separately, or two or more parts thereof may be combined. Preferable parts are the leaf part and the stem part, and more preferably the leaf part.

When using an extract of a radish genus plant as an intercellular adhesion protective agent, a melanin production inhibitor, a blood sugar level reducing agent, a neutrophil elastase inhibitor, and a blood dyslipidemia inhibitor, the extract of a radish genus plant is used It may be used as it is, or may be used after being diluted or concentrated as appropriate. When obtaining an extract of a plant belonging to the genus Panthera, the unprocessed plant body may be used as it is, or the plant may be refrigerated, frozen, or dried.

The part of the plant belonging to the genus Glycine used for extraction is not particularly limited, but includes leaves, stems, roots, flowers, and seeds, preferably leaves, stems, or a combination thereof, more preferably This is the leaf part.

Water, an organic solvent, or a water-containing organic solvent is used as a solvent for extracting a plant belonging to the genus Panthera. By using these solvents, a water extract, an organic solvent extract, or a water-containing organic solvent extract can be obtained from the plant of the genus Panthera. Examples of extraction solvents include water; lower alcohols such as methanol, ethanol and butyl alcohol; polyhydric alcohols such as butylene glycol, polyethylene glycol and glycerin; ketones such as acetone and methyl ethyl ketone; Esters and the like can be mentioned, but water, ethanol, or hydrous ethanol is preferred, and water or hydrous ethanol is more preferred. When ethanol is used, its concentration is not particularly limited. For example, it is used at a concentration of 20% or less, preferably 10% or less. Thereby, a highly safe extract can be obtained.

The method for extracting the genus radish is not particularly limited. After being immersed in an extraction solvent, it may be allowed to stand for a certain period of time at room temperature or under heat treatment conditions to obtain an extract of a plant of the genus Radix, or an unprocessed whole or part of a plant of the genus Radix, or a plant of the genus Radix. After crushing, pulverizing, etc. the whole plant or part of the dried product, etc., adding an extracting solvent and stirring, after processing for a certain period of time at room temperature or heat treatment conditions, the extract of the radish genus plant and the like.

The extraction conditions are not particularly limited, but a method of immersing a dried product of a plant belonging to the genus Rhinoceros radish in a solvent at 5 to 100° C., for example, can be employed. The extraction temperature is not particularly limited as long as an appropriate yield of the extract can be obtained. When water is used as the extraction solvent, the extraction temperature can be 5-100°C, preferably 20-70°C, more preferably 20-50°C. The extraction time is not particularly limited as long as an appropriate yield of extract can be obtained, but is, for example, 1 to 240 hours, preferably 1 to 48 hours, more preferably 2 to 12 hours. Extraction is usually carried out under normal pressure, but it can also be carried out under pressure. If it is under pressure, it is possible even at 120°C. The amount of the solvent added is not particularly limited as long as an appropriate yield of the extract can be obtained. is to add a part.

Impurities are easily removed by subjecting the obtained extract of a plant belonging to the genus of radish to activated charcoal treatment, acid treatment, etc., and an intercellular adhesion protective agent, a melanin production inhibitor, a blood sugar level lowering agent, and a neutrophil elastase inhibitor. , and blood dyslipidemia inhibitory effect can be purified, and safety can be improved.

The obtained extract of a plant belonging to the genus Panthera can be made into a purified product by further performing a purification treatment. Silica gel, liquid-liquid partition method, ion-exchange resin, adsorption chromatography, partition chromatography, or the like can be used for the purification treatment, and silica gel and ion-exchange resin are preferred.

The obtained extract of the plant belonging to the genus of radish can maintain stable activity even when subjected to heat treatment. Advantageous during sterilization.

[Use]
Structural abnormalities of keratinocytes that cause skin abnormalities are derived from the disruption of intercellular adhesion of keratinocytes, and melanogenesis that causes dark spots and freckles synthesizes dopaquinone from the essential amino acid tyrosine by tyrosinase, and the dopaquinone is oxidized and polymerized. generated by The radish genus plant or its extract of the present invention is effective as an intercellular adhesion protective agent that prevents the destruction of intercellular adhesion of keratinocytes due to exposure to ultraviolet rays such as sunlight and exposure to blue light from displays such as computers and smartphones. Furthermore, it has the effect of suppressing the overproduction of melanin in the melanocytes present in the lower part of the skin epidermis, and can be used for the prevention, improvement, and treatment of pigmentation such as rough skin, age spots, and freckles. . The radish genus plant or its extract of the present invention can be used not only for medicinal purposes but also for non-medicinal purposes, and is directed to patients with these diseases and healthy people who are aware of the prevention of these diseases. It can also be suitably used in food and drink.

The radish genus plant or its extract of the present invention has an effect as a neutrophil elastase inhibitor that suppresses the expression of neutrophil elastase, so it is an anti-wrinkle agent, an anti-chronic obstructive pulmonary disease agent (anti- COPD), anti-autoimmune disease agent, anti-ischemia reperfusion injury agent. Therefore, it can be used for prevention, amelioration, and treatment of chronic obstructive pulmonary disease (COPD), autoimmune diseases, and ischemia-reperfusion injury. The radish genus plant or its extract of the present invention can be used not only for medicinal purposes but also for non-medicinal purposes, and is directed to patients with these diseases and healthy people who are aware of the prevention of these diseases. It can also be suitably used in food and drink.

Neutrophil elastase digests and decomposes phagocytosed bacteria and foreign substances in neutrophils in a physiological state, and elastin, collagen (type III, type IV), fibronectin, and immunoglobulin outside neutrophils. , Decomposes blood coagulation factor XIII, etc., and plays a role in healing wounds by decomposing injured cells. However, pathological conditions and excessive release of neutrophil elastase can cause damage to their own tissues. Examples of diseases involving neutrophil elastase include skin wrinkles and serious diseases such as COPD, autoimmune diseases, and ischemia-reperfusion injury.

INDUSTRIAL APPLICABILITY The Panradius genus plant or its extract of the present invention has an excellent effect on lifestyle-related diseases, and can be used, for example, for the prevention, improvement and treatment of obesity. The Panradius plant or its extract of the present invention lowers blood glucose, and thus functions as a blood sugar level-lowering agent. Therefore, it can be used for prevention, improvement and treatment of diabetes. Furthermore, the radish genus plant or its extract of the present invention lowers blood cholesterol, and thus functions as a blood dyslipidemia inhibitor. Therefore, it can be used for prevention, amelioration, and treatment of abnormal blood lipids. The radish genus plant or its extract of the present invention can be used not only for medicinal purposes but also for non-medicinal purposes, and is directed to patients with these diseases and healthy people who are aware of the prevention of these diseases. It can also be suitably used in food and drink.

[Cosmetics]
Cosmetics contain the intercellular adhesion protective agent, melanogenesis inhibitor, and/or neutrophil elastase inhibitor of the present invention as active ingredients, and are provided in combination with pharmaceutically acceptable substrates and carriers. Excipients, coating agents, binders, extenders, disintegrants, lubricants, diluents, osmotic pressure adjusters, pH adjusters, emulsifiers, and dispersants are used in cosmetics to the extent that they are pharmaceutically acceptable. , Stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, coloring agents, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. can also

Cosmetics, specifically skin care cosmetics such as emulsions, creams, cleansers, packs, oil liquids, massage agents, serums, cleansers, deodorants, hand creams, lip balms; make-up bases, powders, liquids makeup cosmetics such as foundation, oily foundation, blusher, eye shadow, mascara, eyeliner, eyebrow, and lipstick; antiperspirant, sunscreen lotion, sunscreen cream, and other UV protection cosmetics; , including those used as quasi-drugs.

The mixing ratio of the intercellular adhesion protective agent, the melanogenesis inhibitor, and/or the neutrophil elastase inhibitor in the cosmetic is appropriately set according to the effective amount, the form of the cosmetic, etc. It is 0.001 to 10% by mass, preferably 0.001 to 1% by mass, more preferably 0.001 to 0.1% by mass, based on the total amount of the ingredients.

[Whitening cosmetics]
A whitening cosmetic contains the melanogenesis inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base material or carrier. Excipients, coating agents, binders, bulking agents, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifiers, dispersing agents, etc. Additives such as agents, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. can also

The mixing ratio of the melanin production inhibitor in the whitening cosmetic agent is appropriately set according to its effective amount, the form of the whitening cosmetic agent, and the like. %, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass.

[Skin protectant]
The skin protective agent contains the intercellular adhesion protective agent of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base material or carrier. Skin protection agents include excipients, coating agents, binders, bulking agents, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifiers, dispersing agents, to the extent that they are pharmaceutically acceptable. Additives such as agents, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. can also

The mixing ratio of the intercellular adhesion protective agent in the skin protective agent is appropriately set according to its effective amount, the form of the skin protective agent, and the like. % by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass.

[Bath additive]
The bath agent contains the intercellular adhesion protective agent, melanogenesis inhibitor, and/or neutrophil elastase inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base material or carrier. Bath additives include excipients, coating agents, binders, bulking agents, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifiers, and dispersing agents to the extent that they are pharmaceutically acceptable. , Stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, coloring agents, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. can also

The mixing ratio of the intercellular adhesion protective agent, the melanin production inhibitor, and/or the neutrophil elastase inhibitor in the bath agent is appropriately set according to the effective amount, the form of the bath agent, and the like. It is 0.001 to 20% by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass, relative to the total amount of the agent.

[Anti-wrinkle agent]
The anti-wrinkle agent contains the neutrophil elastase inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base material or carrier. Anti-wrinkle agents include excipients, coating agents, binders, bulking agents, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifying agents, dispersing agents, to the extent pharmaceutically acceptable. Additives such as agents, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. can also

The mixing ratio of the neutrophil elastase inhibitor in the anti-wrinkle agent is appropriately set according to its effective amount, the form of the anti-wrinkle agent, and the like. 20% by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass.

[Anti-chronic obstructive pulmonary disease agent]
An anti-chronic obstructive pulmonary disease agent contains the neutrophil elastase inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base or carrier. Anti-chronic obstructive pulmonary disease agents include excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure adjusters, and pH adjusters to the extent that they are pharmaceutically acceptable. , emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, additives such as anti-inflammatory agents can also be blended as appropriate.

The proportion of the neutrophil elastase inhibitor in the anti-chronic obstructive pulmonary disease agent is appropriately set according to its effective amount and the form of the anti-chronic obstructive pulmonary disease agent. It is 0.001 to 50% by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass, relative to the total amount of the disease agent.

[Anti-autoimmune disease agent]
An anti-autoimmune disease agent contains the neutrophil elastase inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base material or carrier. Excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure adjusters, pH adjusters, emulsifiers are used in anti-autoimmune disease agents to the extent that they are pharmaceutically acceptable. , Dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. It can also be blended.

The proportion of the neutrophil elastase inhibitor in the anti-autoimmune disease agent is appropriately set according to its effective amount and the form of the anti-autoimmune disease agent. is 0.001 to 50% by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass.

[Anti-ischemia-reperfusion injury agent]
An anti-ischemia-reperfusion injury agent contains the neutrophil elastase inhibitor of the present invention as an active ingredient, and is provided in combination with a pharmaceutically acceptable base or carrier. Anti-ischemia-reperfusion injury agents include excipients, coating agents, binders, bulking agents, disintegrants, lubricants, diluents, osmotic pressure adjusters, and pH adjusters to the extent that they are pharmaceutically acceptable. , emulsifiers, dispersants, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, additives such as anti-inflammatory agents can also be blended as appropriate.

The proportion of the neutrophil elastase inhibitor in the anti-ischemia-reperfusion injury agent is appropriately set according to its effective amount and the form of the anti-ischemia-reperfusion injury agent. It is 0.001 to 50% by mass, preferably 0.001 to 10% by mass, more preferably 0.001 to 0.1% by mass, based on the total amount of the inhibitor.

[Food and drink]
The intercellular adhesion protective agent and/or the melanogenesis inhibitor of the present invention can be blended as an active ingredient in food and drink. These foods and drinks can also be used as foods and drinks for skin protection and whitening effect. Moreover, these foods and drinks can also be provided as functional drinks or functional foods. Functional foods include foods for specified health uses, foods with nutrient function claims, foods with functional claims, foods for the elderly, health supplements (balanced nutritional foods, supplements), and the like.

The blood sugar level-lowering agent and/or the blood dyslipidemia inhibitor of the present invention can be blended as an active ingredient in food and drink. These food and drink products can also be used as food and drink products for improving obesity and improving lifestyle-related diseases. Moreover, these foods and drinks can also be provided as functional drinks or functional foods. Functional foods include foods for specified health uses, foods with nutrient function claims, foods with functional claims, foods for the elderly, health supplements (balanced nutritional foods, supplements), and the like.

Food and drink can contain the neutrophil elastase inhibitor of the present invention as an active ingredient. These foods and drinks can also be used as foods and drinks used for wrinkle improvement and skin improvement. Moreover, these foods and drinks can also be provided as functional drinks or functional foods. Functional foods include foods for specified health uses, foods with nutrient function claims, foods with functional claims, foods for the elderly, health supplements (balanced nutritional foods, supplements), and the like.

Foods include, for example, grains, potatoes, seafood, meats, eggs, oils and fats, milk, vegetables, beans, fruits, sugars, seaweeds, confectionery, seasonings, and cooked and processed foods. etc.

Processed foods are not particularly limited, but for example, processed marine products such as chikuwa and kamaboko; processed livestock products such as ham and sausage; confectionery such as cookies, biscuits, snacks, chocolate and cakes; soba, udon and raw noodles. , Chinese noodles, noodles such as pasta; Bread such as bread and sweet bread; Fermented processed foods such as natto and miso; Soybean foods such as tofu and okara; canned fruits and the like; dairy products such as butter, margarine, yogurt, cheese and milk; frozen dessert foods such as ice cream and sherbet;

The beverage is not particularly limited. Fruit drinks; carbonated drinks; coffee, coffee drinks, coffee-containing soft drinks, cocoa drinks, black tea, green tea, matcha, oolong tea, barley tea, roasted tea, and other beverages; vinegar drinks; soft drinks such as sports drinks; lactic acid beverages; lactic acid bacteria beverages; soybean beverages such as soymilk and modified soymilk; alcoholic beverages such as beer, sake, shochu, liqueur, and wine; nutritional beverages containing taurine, royal jelly, amino acids, vitamins, minerals, iron, etc. etc.

When applied to a food or drink containing at least one of the intercellular adhesion protective agent, melanogenesis inhibitor, blood sugar level-lowering agent, neutrophil elastase inhibitor, and blood dyslipidemia inhibitor of the present invention as an active ingredient is not particularly limited, but in terms of solid content, for humans and animals, it is generally 0.00001 to 1000 mg / kg body weight per day, preferably 0. 01 to 200 mg/kg body weight, more preferably 0.1 to 100 mg/kg body weight.

[Pharmaceuticals, quasi-drugs]
Pharmaceuticals and quasi-drugs can contain the intercellular adhesion protective agent and/or melanogenesis inhibitor of the present invention as an active ingredient. Pharmaceuticals and quasi-drugs can be formulated into appropriate forms and administered to humans or animals in any dosage form. The mode of administration is not particularly limited, and examples thereof include oral, transdermal, enteral, transmucosal, and injection.

Pharmaceuticals and quasi-drugs can contain a blood sugar level-lowering agent and/or blood dyslipidemia inhibitor as an active ingredient. Pharmaceuticals and quasi-drugs can be formulated into appropriate forms and administered to humans or animals in any dosage form. The mode of administration is not particularly limited, and examples thereof include oral, transdermal, enteral, transmucosal, and injection.

Pharmaceuticals and quasi-drugs can contain a neutrophil elastase inhibitor as an active ingredient. Pharmaceuticals and quasi-drugs can be formulated into appropriate forms and administered to humans or animals in any dosage form. The mode of administration is not particularly limited, and examples thereof include oral, transdermal, enteral, transmucosal, and injection.

Dosage forms for oral administration include, for example, pills, powders, tablets, granules, capsules, syrups, liquids and the like.

Parenteral administration includes, for example, intravenous injection, intramuscular injection, percutaneous absorption, inhalant, suppository, eye drops, nasal drops, and the like.

These various formulations can be produced by conventional methods using pharmaceutically acceptable carriers such as commonly used excipients, disintegrants, binders, lubricants, colorants and diluents.

Excipients include, for example, lactose, corn starch, glucose, sorbitol, calcium carbonate, kaolin, and crystalline cellulose.

Examples of disintegrants include starch, sodium alginate, carboxymethylcellulose, carboxymethylcellulose calcium, carmellose calcium, croscarmellose sodium, carboxymethylstarch sodium, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, and stearic acid monoglyceride. , lactose and the like.

Examples of binders include dimethylcellulose, glucose solution, starch solution, gelatin solution, bound cellulose, sucrose, D-mannitol, dextrin, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylstarch, ethylcellulose, shellac, and calcium phosphate. , polyvinylpyrrolidone, and the like.

Lubricants include, for example, talc, magnesium stearate, calcium stearate, colloidal silica, borax, polyethylene glycol and the like.

When applied to pharmaceuticals and quasi-drugs containing at least one of the intercellular adhesion protective agent, melanogenesis inhibitor, blood sugar level-lowering agent, neutrophil elastase inhibitor, and blood dyslipidemia inhibitor of the present invention is not particularly limited, but in terms of solid content, for humans and animals, it is generally 0.00001 to 1000 mg / kg body weight per day, preferably 0. 01 to 200 mg/kg body weight, more preferably 0.1 to 100 mg/kg body weight.

EXAMPLES Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

[Evaluation of melanin production inhibitory effect of radish plant extract]
(Method for extracting plants belonging to the genus Panthera)
The leaves of radish and okadakon, which are plants belonging to the genus Rhinoceros radish, were dried with hot air at 40° C. for 2 days using a ventilation/circulation type drying thermostat (manufactured by SANYO, Model No. MOV-1125). The dried leaves were pulverized using a desktop grinder (Tokyo Unicom Co., Ltd., model number T-351). 1 g of powdered dry leaves of each of the white radish and okadakon radish was immersed in 10 ml of water and extracted at 23° C. for 12 hours. Thereafter, filter filtration was performed to recover 8 ml of each extract of white radish and okadakon. Melanin production suppression was evaluated for each of the collected extracts of white radish and okadakon radish. In order to evaluate each extract, B16F10 melanoma cells, which are melanin-producing cells, were used, and the amount of melanin produced by the cultured B16F10 melanoma cells was evaluated by visual observation and absorbance.

(Culture of B16F10 melanoma cells)
In order to evaluate the amount of melanin production, B16F10 melanoma cells, which are melanin-producing cells, were cultured. A method for culturing B16F10 melanoma cells is shown below.
(1) In a 6-well dish, B16F10 melanoma cells (JCRB cell bank, cell registration number: JCRB0202), which are melanin producing cells, were seeded at 5×10 ⁴ cells (2 ml/well), After adding the culture medium, the cells were cultured at 37° C. under a 5% carbon dioxide stream for 1 day.
Pre-culture medium: DMEM High Glucose medium containing 10% FCS (fetal calf serum) (antibiotic MIX (manufactured by Wako Pure Chemical Industries, Ltd.) added to a final concentration of 1X) was used.
(2) After removing the pre-culture medium for the B16F10 melanoma cells cultured in (1), 2 ml of the B16F10 melanoma cell treatment medium was added and cultured for 72 hours.
(3) The B16F10 melanoma cells cultured in (2) were washed with phosphate buffered saline (PBS), collected in a 1.5 ml Eppendorf tube, and the color of the cells was visually compared.
Medium for treatment: Extracts of white radish and okadakon radish were added to the same medium as the preculture at 1000-fold dilution and 3000-fold dilution (volume ratio). Forskolin (FSK) was added to each test sample at 20 μM (final concentration) and DMSO at 0.2% (final concentration). FSK is a substance that induces signal induction and promotes melanin synthesis in the same manner as α-melanocyte-stimulating hormone (αMSH) in B16F10 melanoma cells.
Blank medium for treatment (-): No white radish or okadakon radish extract was added to the above treatment medium.
Control medium for treatment: No white radish extract, okadakon radish extract or FSK was added to the above treatment medium.

(measurement of melanin)
Melanin was extracted from each B16F10 melanoma cell collected. The method for extracting melanin is shown below.
(1) PBS was removed from each protein-matched test sample, 300 μl of 1N NaOH was added, and homogenized until no cell debris was visible.
(2) Each test sample was incubated at 45°C for 2 hours.
(3) 100 μl of methanol:chloroform (1:2) mixed solution was added to each test sample and stirred to extract melanin.
(4) Centrifugation was performed at 1200 rpm for 10 minutes, and the supernatant was recovered to obtain a melanin extract.
(5) 100 µl of the melanin extract was dispensed into a 96-well plate and the absorbance at 405 nm was measured.

FIG. 1 is a diagram visually showing the relationship between the concentration of water extracts of white radish and okadakon and the effect of suppressing melanin production, and FIG. It is a diagram indicated by . As shown in FIGS. 1 and 2, it was confirmed that melanin production was inhibited even when the extracts of white radish and okadakon were each diluted 3000-fold.

[Evaluation of extraction methods from plants belonging to the genus Rhinoceros radish]
1 g of powdered dried leaves of each of the radish radish and okadakon, which are plants of the genus Rhinoceros radish, are immersed in 10 ml of water: ethanol mixture (water: ethanol 0%, 80%, 90%, 100%) at 23 ° C. for 72 hours. extraction was performed. Thereafter, filter filtration was performed to recover 8 ml of each extract of white radish and okadakon. Melanin production suppression was evaluated for each of the collected extracts of white radish and okadakon radish. In order to evaluate each extract, B16F10 melanoma cells, which are melanin-producing cells, were used, and the amount of melanin produced by the cultured B16F10 melanoma cells was visually observed. B16F10 melanoma cells are cultured using the same method as the above method, and the extracts of white radish and okadakon are added at a 1000-fold dilution, and the amount of melanin produced by different extraction methods is visually observed and absorbance is measured. evaluated with
Blank medium for treatment (-): No white radish or okadakon radish extract was added to the above treatment medium.
Control medium for treatment: No white radish extract, okadakon radish extract or FSK was added to the above treatment medium.

The amount of melanin produced was measured by absorbance in the same manner as described in the section [Evaluation of suppression of melanin production by plant extract of the genus Pygmaea].

FIG. 3 is a diagram visually showing the effect of suppressing melanin production by different extraction methods of the extracts of white radish and okadakon, and FIG. It is a diagram showing. As shown in FIGS. 3 and 4, it was confirmed that the extract whose ethanol concentration was set to 20% or less was highly effective in suppressing melanin production even when diluted 1000-fold.

[Evaluation of extraction method from white radish]
1 g of dry powdered leaves of radish radish was immersed in 10 ml of water:ethanol mixed solution (water: ethanol 0%, 80%, 90%, 100%) and extracted at 23°C for 12 hours. After that, filter filtration was performed to recover 8 ml of an extract of white radish. The melanin production suppression was evaluated for the recovered extract of white radish. In order to evaluate each extract, B16F10 melanoma cells, which are melanin-producing cells, were used, and the amount of melanin produced by the cultured B16F10 melanoma cells was visually observed. The B16F10 melanoma cells are cultured using the same method as the above method, and the radish extract is added at 300-fold and 3000-fold dilutions, and the amount of melanin production suppression due to the difference in the extraction method can be visually observed. evaluated.
Blank treatment medium (-): No radish extract was added to the above treatment medium.
Control medium for treatment: No radish extract or FSK was added to the above treatment medium.

FIG. 5 is a diagram showing melanin production inhibitory effects depending on the extraction method of the white radish extract. As shown in FIG. 5, the extract with an ethanol concentration of 20% or less exhibited an inhibitory effect on melanin production even when diluted 3000-fold, and the extract with an ethanol concentration of 10% or less showed melanin production inhibitory effects. It was confirmed that the production suppression effect is even higher.

[Cytotoxicity test of extract of white radish]
A cytotoxicity test was performed using the radish extract obtained in the section [Evaluation of extraction method from radish]. B16F10 mouse melanoma cells were seeded on a 96-well plate at 1×10 ⁴ cells/well. As culture conditions, 100 μl of 10% FCS-containing DMEM High Glucose medium was used and cultured at 37° C. under 5% carbon dioxide aeration for 72 hours. After that, 10 μl of WST-8 reagent (cell counting kit, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and cultured for 30 minutes. Cell numbers were counted. The test substance is an extract obtained by extracting powdered dried leaves of white radish with 10 times the amount of water: ethanol mixed solution (water: ethanol 0%, 80%, 90%, 100%), 1/3000, 1/1000 , was added at a 1/300-fold dilution.

FIG. 6 is a diagram showing cytotoxicity depending on the extraction method of the white radish extract. As shown in the graph of FIG. 6, it was confirmed that the white radish extract extracted at an ethanol concentration of 10% or less has low cytotoxicity.

[Cytotoxicity test of extract and purified product of white radish]
A 1/10 amount of powdered activated carbon (manufactured by Kanto Kagaku Co., Ltd.) was added to the water extract of white radish, and the mixture was mixed by inversion at room temperature for 30 minutes. Thereafter, the mixture was centrifuged at 12,000 rpm, the supernatant was recovered, and impurities were removed with a 0.45 μm disk filter (activated carbon passing fraction). Subsequently, 1/10 amount of 1N HCl was added, heated at 90° C. for 30 minutes, centrifuged at 12000 rpm, and the supernatant was recovered. A 1/10 amount of 1N NaOH was added to the recovered product to neutralize it (acid-soluble fraction). These preparations were diluted to 1/300, added to the medium of B16F10 mouse melanoma cells, and subjected to cytotoxicity test in the same manner as in the section [Cytotoxicity test of radish extract].

FIG. 7 is a diagram showing cytotoxicity due to differences in the purification process of the white radish extract. As shown in the graph of FIG. 7, it was confirmed that the white radish extract became less cytotoxic as the activated charcoal treatment and the acid treatment were performed.

[Evaluation of thermal stability of radish plant extract]
The heat stability of the radish plant extract was evaluated by comparing the melanin production inhibitory effect between the radish radish plant extract heat-treated at 120 degrees for 20 minutes and the radish radish plant extract not heat-treated. The same method as described in the section [Evaluation of Melanin Production Inhibitory Effect of Plant Extract of the genus Radix] was used for the method for extracting the plant of the genus Radix and the method for culturing the B16F10 melanoma cells. The amount of melanin produced was visually observed.

FIG. 8 shows the thermal stability of the water extract of white radish. As shown in FIG. 8, the water extract of white radish heat-treated at 120 degrees for 20 minutes has almost the same activity as the extract without heat treatment, and has heat stability. It was shown that

[Evaluation of removal of impurities from a plant extract of the genus Rhinoceros radish]
Water extract of white radish, 1/10 amount of water-saturated activated carbon (manufactured by Kanto Chemical Co., Ltd.), 1/10 amount of water-saturated silica gel (manufactured by Kanto Chemical Co., Ltd.), 1/10 amount of water-saturated DEAE-Separose ( GE Healthcare) was added and mixed, and mixed by inversion at room temperature for 30 minutes. After that, it is centrifuged at 12,000 rpm, the supernatant (resin-passing fraction) is collected in a 1.5 mL tube, and the effect of removing impurities (pigment) contained in the water extract of white radish is determined visually (photography). bottom. Then, these preparations are diluted 1/1000, added to the medium of B16F10 mouse melanoma cells, and tested for melanin synthesis inhibitory activity in the same manner as in the section [Evaluation of melanin production inhibitory effect of plant extract of the genus Columbine]. carried out.

FIG. 9 is a diagram showing the evaluation of melanin production inhibitory effects by different purification methods of water extracts of white radish. As shown in FIG. 9, all of activated carbon, silica gel, and DEAE-Separose, which is an ion-exchange resin, were found to have the effect of removing impurities while maintaining the effect of suppressing melanin production. In particular, silica gel and ion-exchange resin were found to be highly effective in removing impurities.

[Protective effect of radish extract on human keratinocytes from blue light]
Human keratinocyte PSVK1 (JCRB cell bank, cell registration number: JCRB1093) 10 ⁵ (GIBOCO Epilife medium, manufactured by ThermoFisher Scientific) was seeded in 12-well Vitrigel (manufactured by Kanto Chemical Co., Ltd.) and cultured for 9 days (3 days medium change every 10 days). In order to examine the protective effect of the radish extract on human keratinocytes, the water extract of the radish was passed through activated carbon and treated with acid (1/1000 dilution, 0.03% DMSO added), a control (0.03% Only DMSO was added), and cultured overnight. Intercellular adhesion was determined by electrical resistance between the upper and lower layers of cells. Destruction of intercellular adhesion was evaluated by a decrease in resistance value, with the initial value of electrical resistance set to 100%. Electric resistance was measured using TC-749 (manufactured by Kanto Kagaku Co., Ltd.). As a method for destroying intercellular adhesion (cytotoxicity), blue light (Visi-Blue Transilluminator UVP, Low intensity, manufactured by UVP) was applied from a distance of 5 cm below the cell plate. In order to avoid the temperature rise due to the irradiation of blue light, the experiment was carried out by placing the irradiation device in a constant temperature incubator. Experiments were carried out using unirradiated cells as blanks (no light) (0.03% DMSO added).

FIG. 10 is a diagram showing the intercellular adhesion protective effect of the water extract of white radish in terms of electric resistance. As shown in FIG. 10, the electric resistance of the control cells decreased sharply 30 minutes after irradiation with blue light, while the electric resistance of the cells treated with the water extract of white radish was maintained. Therefore, it was confirmed that the radish extract has an effect of protecting the intercellular adhesion of keratinocytes, that is, a skin-protecting effect.

[Evaluation of neutrophil elastase inhibitory effect of radish plant extract]
(mRNA expression level test)
The neutrophil elastase-suppressing effect of the radish plant extract was evaluated by measuring the expression level of neutrophil elastase mRNA. 1×10 ⁵ human neutrophil-derived cells U937 (JCRB cell bank, cell registration number: JCRB9021) were cultured in a 6-well plate using 5% FCS-containing RPMI1640 medium (manufactured by Nacalai Tesque). After 48 hours of culture, water extracts of white radish and okadakon radish were added to a concentration of 1/1000 (control was not added). After 24 hours, the medium was collected in a 2 ml tube and centrifuged at 3000 rpm for 5 minutes to collect U937 cells. After collecting the cells, RNA was purified using FastGene ^™ RNA Purification Kit (manufactured by Nippon Genetics). 0.5 ug of the purified RNA was reverse transcribed with reverse transcriptase Revertra Ace (registered trademark) (manufactured by Toyobo) and subjected to quantitative PCR using THUNDERBIRD SYBR (registered trademark) qPCR Mix (manufactured by Toyobo). Quantitative PCR was performed using the MyiQ system (manufactured by BioRad). The amplification rate of neutrophil elastase was corrected with the amplification rate of 36B4 (internal standard). The primers used are shown below.

Neutrophil elastase primer hEla2 Forward: 5′-CCGTGCAGCGCATCTTCGA-3′
hEla2 Reverse: 5'-TGAGCTGGAGAATCACGATGT-3'
Primer for 36B4 h36B4 Forward: 5'- ccaactgttgcatcagtacc-3'
h36B4 Reverse: 5'-gccacaaaggcagatggatc-3'

(Western blotting)
(1) SDS sample buffer (0.05M Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 0.6% 2-mercaptoethanol) for each test sample. The neutrophil-derived U937 cells were lysed, dispensed onto a polyacrylamide gel so as to have the same amount of protein, and subjected to electrophoresis.
(2) After transferring proteins separated on a polyacrylamide gel to a PVDF membrane, the PVDF membrane was blocked with 1% BSA.
(3) To evaluate the expression level of human neutrophil elastase, a primary antibody reaction was carried out at 4° C. for 16 hours with a 1/2000 dilution of an anti-neutrophil elastase monoclonal antibody (manufactured by Santa Cruz). After washing, a secondary antibody reaction was performed with an anti-GAPDH polyclonal antibody (manufactured by Santa Cruz) diluted 1/5000.
(4) The signal intensity of human neutrophil elastase (about 30 kDa) was detected from the antigen on the PVDF membrane by chemiluminescence using an ECL western blotting detection system (manufactured by GE Healthcare).
All tests were performed with n=3.

FIG. 11 is a graph showing the neutrophil elastase inhibitory effect of water extracts of white radish and okadakon radish in terms of mRNA expression level. The vertical axis represents the expression efficiency of neutrophil elastase. FIG. 12 is a photograph showing the neutrophil elastase inhibitory effect of water extracts of white radish and okadakon radish by Western blotting. ELA2 is the neutrophil elastase protein and GAPDH is the protein used as an internal standard control. As shown in FIGS. 11 and 12, the samples to which the water extracts of white radish and okadakon were added clearly suppressed the expression of neutrophil elastase protein compared to the control to which no extract was added. ing. Therefore, it was confirmed that the extracts of white radish and okadakon have a neutrophil elastase inhibitory effect.

[Evaluation of Blood Cholesterol and Blood Cholesterol-Reducing Effect of the Plant Extract of the genus Rhinoceros radish]
The effect of reducing blood sugar levels was evaluated by subjecting C57BL/6J mice (male, 8 weeks old, N=8) to a glucose load. A water extract of radish produced by the method described in [Cytotoxicity test of purified extract of radish] was autoclaved at 120°C for 20 minutes and then filtered through a 0.45 µm filter. Mice were allowed to freely ingest this water extract of white radish for one week. The dosage was about 150 mg (dry weight)/animal/day. After fasting for 4 hours, mice were orally administered glucose (1.5 g/kg), and 0, 15, 30, and 60 minutes after glucose ingestion, about 1 μl of blood was collected from the tail vein of the mice. , G sensor (manufactured by ARKRAY) was used to measure the blood glucose level. Control mice were given RO water instead of the water extract of white radish and were subjected to a glucose tolerance test in a similar manner.

The effect of reducing blood cholesterol was evaluated using mice treated with the above method. 50 μl was bled from the tail vein of mice 120 minutes after glucose ingestion and serum was collected. Blood cholesterol concentration was measured by laboratory assay cholesterol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) using this serum as a sample.

FIG. 13 is a graph showing the blood sugar level lowering effect of the water extract of white radish. The vertical axis represents blood sugar level (mg/dl) and the horizontal axis represents time. It was confirmed that the water extract of white radish significantly suppressed the increase in blood glucose level 15 to 60 minutes after administration of glucose as compared with the control. This water extract of radish was evaluated for melanin production inhibitory effect using the test method described in [Evaluation of melanin production inhibition by plant extract of the genus Rhinoceros radish]. It has been confirmed that even a 1/3000 diluted white radish water extract significantly suppresses melanin synthesis (data not shown).

FIG. 14 is a graph showing the cholesterol-lowering effect of water extract of white radish. The vertical axis represents the blood cholesterol concentration. The water extract of white radish was confirmed to have a lowering effect on blood cholesterol compared to the control.

[Comparison of intercellular adhesion protective effect of white radish and hyaluronidase activity inhibitor]
Human keratinocyte PSVK1 (JCRB cell bank, cell registration number: JCRB1093) 10 ⁵ (GIBOCO Epilife medium, manufactured by ThermoFisher Scientific) was seeded in 12-well Vitrigel (manufactured by Kanto Chemical Co., Ltd.) and cultured for 9 days (3 days medium change every 10 days). A neomycin derivative G418 (manufactured by Nacalai) was used as a hyaluronidase activity inhibitor, and ONO-5046 (manufactured by Cayman) was used as a neutrophil elastase inhibitor.

The water extract of white radish was passed through activated charcoal and acid-treated (1/1000 dilution, 0.03% DMSO added) and control (0.03% DMSO only added) were cultured overnight. 20 μM of G418, which has a hyaluronidase activity inhibitory action, was added to the medium immediately before measurement, and 10 μM of ONO-5046 was added to the medium immediately before measurement. Intercellular adhesion was determined by electrical resistance between the upper and lower layers of cells. Destruction of intercellular adhesion was evaluated by a decrease in resistance value, with the initial value of electrical resistance set to 100%. Electric resistance was measured using TC-749 (manufactured by Kanto Kagaku Co., Ltd.). As a method for destroying intercellular adhesion (cytotoxicity), blue light (Visi-Blue Transilluminator UVP, Low intensity, manufactured by UVP) was applied from a distance of 5 cm below the cell plate. In order to avoid the temperature rise due to the irradiation of blue light, the experiment was carried out by placing the irradiation device in a constant temperature incubator. Experiments were carried out using unirradiated cells as blanks (no light) (0.03% DMSO added).

FIG. 15 is a graph comparing the intercellular adhesion protective action between the water extract of radish and a hyaluronidase activity inhibitor. From the test results of hyaluronidase's intercellular adhesion protective effect, it became clear that G418, which has hyaluronidase inhibitory activity, has no intercellular adhesion protective effect. One thing has been confirmed. In addition, the white radish extract was found to have a higher intercellular adhesion protective effect than the neutrophil elastase inhibitor.

[Melanin production inhibitory effect using melanoma cells in leaves of radish]
Whole dried radish (including leaves, stems, and some roots) and selected leaves of radish were pulverized and extracted with 20 times the amount of water. These extracts were added to the medium of B16F10 melanoma cells at a dilution of 1/3000 to 1/300, and melanin synthesis induction treatment (forskolin 10 μM) was performed. B16F10 melanoma cells were cultured according to the method described in the section (B16F10 melanoma cell culture).

FIG. 16 is a diagram showing the melanogenesis inhibitory effect in leaves of radish. As shown in FIG. 16, it was shown that melanin synthesis suppression was remarkably enhanced when the extract was prepared by sorting the leaves from the white radish.

[Suppressive effect of melanin production by mice of radish]
Whole dried radish (including leaves, stems and some roots) and selected leaves of radish were pulverized and extracted with 30 times the amount of water. These were sterilized at 120 degrees for 20 minutes. The extract was further diluted 3 times with water, and 3-week-old mice (C57BL/6J strain) were allowed to ingest the water ad libitum as drinking water for 2 weeks.

FIG. 17 is a graph showing the inhibitory effect of the leaf extract of white radish on melanin production by mice. As shown in FIG. 17 , the black radish leaf extract ingested mice had darker coat color and less pigmentation on the skin of the ears, whereas no change was observed with the whole plant extract of dried radish.

[Comparison of inhibitory effects on melanin production using melanoma cells of white radish and amakusasida]
Dried leaves of radish radish and dried leaves of Amakusa fern, which has a melanogenesis inhibitory action in other plants, were extracted with 20 times the amount of water, added to the medium of B16F10 melanoma cells, and melanin synthesis induction treatment (forskolin 10 μM) was performed. B16F10 melanoma cells were cultured according to the method described in the section (B16F10 melanoma cell culture).

FIG. 18 is a diagram comparing melanogenesis inhibitory effects using melanoma cells of white radish and amakusasida. FIG. 19 is a graph comparing melanogenesis inhibitory effects using melanoma cells of white radish and amakusasida. FIG. 19 quantifies the melanin production inhibitory effect, and expresses the relative amount with 1 being the sample without induction of melanin synthesis. As shown in FIGS. 18 and 19, it was confirmed that both extracts suppress melanin synthesis with similar effects.

[Comparison of effects of black radish and amakusashida in suppressing melanin production in mice]
The leaves of white radish and the leaves of Amakusa fern were pulverized and extracted with 30 times the amount of water. These were sterilized at 120 degrees for 20 minutes. The extract was further diluted 3 times with water, and 3-week-old mice (C57BL/6J strain) were allowed to ingest the water ad libitum as drinking water for 2 weeks.

FIG. 20 is a graph comparing the effect of suppressing melanin production by mice of radish and fern. As shown in FIG. 20, the black color of the fur of mice ingesting the dried leaf extract of white radish became lighter, and the pigmentation of the skin of the ears was reduced. No oral effect was observed in

The melanogenesis inhibitor according to the present invention can be used in the food industry, cosmetics industry, and pharmaceutical industry.

Claims (2)

A method for producing a melanin production inhibitor, which comprises extracting a leaf part of white radish and/or okadakon radish with water. A melanin production inhibitor characterized by comprising, as an active ingredient, an aqueous extract of leaf parts of white radish and/or okadakon radish .

Images