JP2006306863A - Melanin formation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bleaching agent exhibiting sustainable excellent melanin formation-inhibiting activities or bleaching activities; and to provide a cosmetic for bleaching, a food for bleaching and an oral composition. <P>SOLUTION: The melanin formation inhibitor contains one or more compounds selected from the group consisting of Hibiscus acids represented by formula (1) (wherein, R<SP>1</SP>and R<SP>2</SP>are each a hydrogen atom, a substituted or nonsubstituted alkyl group or a benzyl group) and salts thereof as active ingredients. The bleaching agent, the cosmetic for bleaching, the food for bleaching and the oral composition contain the melanin formation inhibitor as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a melanin production inhibitor containing hibiscus acids as an active ingredient, and a whitening agent, a whitening cosmetic, a whitening food and an oral composition containing the melanin production inhibitor.

  Conventionally, it is known that the deposition of pigments such as spots and freckles is caused by the production and accumulation of melanin in skin cells. It is also said that melanin is generated not only in the skin but also in the oral mucosa.

  The production process of melanin has not yet been fully elucidated, but the following process has become clear as one of its main production processes. That is, (1) active oxygen is generated in skin epidermal cells when the skin is exposed to ultraviolet rays, (2) cells are damaged by active oxygen, and various information transmitters are generated and released, (3) Various signal transmitters cause the promotion and activation of tyrosinase and produce melanin.

  Therefore, in order to suppress the production of melanin and keep the skin white, it is considered as effective means to eliminate active oxygen on the skin, suppress the production of tyrosinase, or inhibit tyrosinase activity. Yes.

  So far, active oxygen scavengers such as ascorbic acid and tyrosinase activity inhibitors such as kojic acid and arbutin have been proposed as active ingredients of whitening agents (see Patent Document 1, etc.). However, the substances proposed as the active ingredients of conventional whitening agents only act on a part of the melanin production process, and melanin is produced by other processes. It wasn't. Furthermore, the conventional whitening agent components are not satisfactory from the viewpoint of the sustained effect.

On the other hand, cosmetics and pharmaceuticals containing hibiscus acids are known. For example, Patent Document 2 describes a cosmetic containing 3-hydroxy-3,4-dicarboxy-1,4-butanolide or a derivative thereof. Patent Document 3 describes a glycosidase inhibitor and a therapeutic agent for diabetes containing hibiscus acids as active ingredients. However, the melanin production inhibitory action and the whitening action of hibiscus acids have not been known so far.
JP-A-1-85907 JP 2000-154134 A JP 2000-239164 A

  The present invention has an excellent melanin production inhibitory action, has a melanin production inhibitory action sustainability, and can be used safely. Melanin production inhibitor, whitening agent, whitening cosmetic, whitening food, and oral cavity The main purpose is to provide a composition.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have found that a compound having a specific structure has an excellent melanin production inhibitory effect, and further intensively studied, the present invention. It came to be completed.

  That is, the present invention relates to the following melanin production inhibitors, whitening agents, cosmetics, foods and oral compositions.

  Item 1: The following formula (1)

(However, R ¹ and R ² in the formula each represent a hydrogen atom, a substituted or unsubstituted alkyl group or a benzyl group.)
The melanin production inhibitor which uses as an active ingredient 1 or 2 or more types of compounds chosen from the group which consists of Hibiscus acids represented by these, and its salt.

Item 2: The melanin production inhibitor according to Item 1, wherein at least one of R ¹ and R ² of the hibiscus acids represented by Formula (1) is a substituted or unsubstituted alkyl group.

  Preferably, the following formula (1)

(In the formula, R ¹ and R ² each represent a hydrogen atom, a substituted or unsubstituted alkyl group or a benzyl group, and at least one of R ¹ and R ² is a substituted or unsubstituted alkyl group. A melanin production inhibitor comprising one or more compounds selected from the group consisting of hibiscus acids represented by the formula:

  Preferably, the melanin production inhibitor which uses as an active ingredient 1 or 2 or more types of compounds chosen from the group which consists of a biscus acid monoalkyl ester and a biscus acid dialkyl ester.

  More preferably, the melanin production inhibitor which uses as an active ingredient 1 or 2 or more types of compounds chosen from the group which consists of a biscus acid monomethyl ester, a hibiscus acid monoethyl ester, a hibiscus acid dimethyl ester, and a hibiscus acid diethyl ester.

  Item 3: A whitening agent comprising the melanin production inhibitor according to any one of Items 1 and 2 as an active ingredient.

  Item 4: A whitening cosmetic comprising the melanin production inhibitor according to any one of Items 1 and 2 as an active ingredient.

  Item 5: A whitening food containing the melanin production inhibitor according to any one of Items 1 and 2 as an active ingredient.

  Item 6: An oral composition containing the melanin production inhibitor according to any one of Items 1 and 2 as an active ingredient.

  Hereinafter, the present invention will be described in more detail.

Melanin production inhibitor The melanin production inhibitor of the present invention has the following formula (1):

(Wherein, R ¹ and R ² each represent a hydrogen atom, a substituted or unsubstituted alkyl group or a benzyl group) 1 or 2 selected from the group consisting of hibiscus acids and salts thereof The above compound is an active ingredient.

  The type of the alkyl group is not particularly limited as long as the effect of the present invention is achieved, and may be linear or branched.

  The number of carbon atoms of the alkyl group can be appropriately set within the range where the effects of the present invention are exerted, but is preferably about 1 to 6 carbon atoms, particularly about 1 to 3 carbon atoms.

  Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, an isobutyl group, and a hexyl group.

  The alkyl group or benzyl group may have a substituent. Although the kind of substituent can be suitably set in the range with the effect of this invention, for example, a C1-C4 alkyl group etc. are mentioned.

Specific examples of the hibiscus acids used in the present invention include, for example, hibiscus acids in which R ¹ and R ² are both hydrogen atoms, and hibiscus acids in which ^one of R ¹ and R ^{2 in} the formula is substituted. Monoesters, hibiscus diesters in which R ¹ and R ² in the formula are both substituted.

  Hibiscus acid monoesters include, for example, hibiscus monoalkyl esters such as hibiscus monomethyl ester and hibiscus monoethyl ester.

  Hibiscus acid diesters include, for example, biscus acid dialkyl esters such as bismuth acid dimethyl ester and hibiscus acid diethyl ester.

The salt of the hibiscus acid used in the present invention is a salt of the hibiscus acid, for example, when R ¹ and / or R ² is a hydrogen atom, they are substituted with a monovalent or divalent metal base. Alkali metal salts and alkaline earth metal salts are included.

Examples of the alkali metal salt include sodium salt and potassium salt. Examples of alkaline earth metal salts include calcium salts and magnesium salts. The salt also includes those forming a complex. For example, an example of the complex includes an alkaline earth metal complex formed by replacing R ¹ and / or R ² with calcium or magnesium.

  The means for producing hibiscus acids and salts thereof used in the present invention are not particularly limited, and may be obtained by chemical synthesis, or may be obtained by separation from a plant or its extract. Further, the ester or salt may be obtained by esterifying hibiscus acids obtained by separation from a plant or its extract according to a conventional method or reacting with various bases.

  The method for separating hibiscus acids from the plant body or its extract is not particularly limited, and a known method can be used as appropriate. For example, a plant containing hibiscus acid may be extracted with a suitable extraction solvent and then separated by means such as chromatography.

  Examples of plants containing hibiscus acids include plants belonging to the genus Fuyo.

  The plants belonging to the genus Fuso include, for example, the tropical annual Roselle (Hibiscus sabdariffa L.), the tropical shrub (Hibiscus rosa-sinensis L.), the Chinese origin (Hibiscus syriacus L.), and the African origin Kenaf (Hibiscus cannabinus L.), North American-grown fir (Hibiscus coccineus L.), Japanese-grown foal (Hibiscus hamabo), Chinese-grown bud (Hibiscus mutabilis L.), and the like. Among these, it is preferable to use roselle, bushouge, and mukugee, and in particular, roselle and bushuge are preferred.

  The extraction site of the plant body is not particularly limited, and examples thereof include the whole plant of the plant or a part of the plant such as a flower, a bud, a leaf, a stem, a branch, a branch tip, a root, a rhizome, and a seed. it can. Among these, Preferably, a flower and a leaf can be mentioned.

  Further, as the extraction raw material, a plant body may be used as it is, or a pulverized product thereof or a product obtained by drying, chopping, crushing, pressing, boiling or fermenting them may be used.

  Although the kind of solvent used for extraction can be suitably set, for example, water or various organic solvents, or a mixture of water and organic solvent can be mentioned. Examples of the organic solvent include methanol, ethanol, n-propanol, isopropanol, butanol, isobutanol, hexanol, propylene glycol, 1,3-butylene glycol, acetone, ethyl acetate, chloroform, pentane, hexane, heptane, hydrochloric acid and the like. A single or a combination of two or more is included. Among these, preferable extraction solvents include lower alcohols such as methanol, ethanol and isopropanol, or a mixture of water and lower alcohols.

  After extraction, various chromatographies such as dissolution, re-extraction, liquid separation, gradient, filtration, concentration, distillation, sublimation, crystallization, thin layer chromatography, column chromatography, gas chromatography, and high performance liquid chromatography were used. Various methods such as separation can be performed as appropriate, alone or in combination.

  Further, if necessary, moisture can be removed by applying a drying treatment such as reduced-pressure drying or freeze-drying to use as a dried product.

  The specific conditions for the extraction are not particularly limited, and can be appropriately set according to a conventional method. For example, a dried roselle flower is pulverized, about 10 parts by weight of 1.5% hydrochloric acid-ethanol aqueous solution is added to 1 part by weight of the extraction object, stirred at room temperature, solids are removed by filtration, A method for purification by HPLC can be mentioned.

  In addition, as the hibiscus acids, those obtained commercially can be used simply. The ester body can also be obtained by esterifying commercially available hibiscus acid according to a known method.

  The compound used as an active ingredient of the melanin production inhibitor of the present invention may be a single compound selected from the group consisting of the hibiscus acids or salts thereof, or a mixture of two or more compounds. Good.

  The mixture comprising two or more compounds may be obtained by mixing various hibiscus acids or salts thereof obtained by synthesis or isolation from plants, or a plant or extract thereof containing hibiscus acids. It may be a mixture or a fraction obtained from

  The melanin production inhibitor of the present invention contains one or two or more compounds selected from the group consisting of the hibiscus acids and salts thereof as an active ingredient, preferably from a biscus acid monoalkyl ester and a hibiscus acid dialkyl ester. One or two or more compounds selected from the group consisting of:

  More preferably, the active ingredient contains one or more compounds selected from the group consisting of hibiscus monomethyl ester, hibiscus monodimethyl ester, hibiscus dimethyl ester and hibiscus diethyl ester.

  The melanin production inhibitor of the present invention contains one or two or more compounds selected from the group consisting of the hibiscus acids and salts thereof as an active ingredient, and has a high and sustained tyrosinase activity inhibitory action and a high tyrosinase production inhibitory action. In addition, it has an effective and continuous melanin production inhibitory action.

Whitening Agent The whitening agent of the present invention contains the melanin production inhibitor of the present invention as an active ingredient.

  The whitening agent of the present invention may consist of the melanin production inhibitor itself, or contains other ingredients such as pharmaceutically or sanitary acceptable carriers or additives as an active ingredient. It may be.

  The type and blending amount of the carrier or additive can be appropriately selected and adjusted according to the dosage form of the whitening agent or the application target, as long as the effects of the present invention are not impaired.

  Further, the form of the whitening agent is not particularly limited, and may be arbitrarily selected from liquid, emulsion, cream, powder, granule, pill, ointment, etc. depending on the dosage form, form, use, etc. of the applied product. Can be prepared.

  Further, the whitening agent of the present invention can be an external preparation or an internal preparation.

  In formulating the whitening agent of the present invention as an external preparation, an inorganic pigment, an ultraviolet absorber, other whitening components, a tyrosinase activity inhibitor or a melanin dye reducing agent, a surfactant, a cell activator, an antibacterial agent, if necessary. Additives such as inflammatory agents, antibacterial agents, humectants, fragrances, and coloring agents may be added as appropriate.

  The type of external preparation to which the whitening agent of the present invention can be applied is not particularly limited. For example, external medicines and external quasi-drugs (skin external preparations, etc.), basic cosmetics (skin lotion, emulsion, cream, ointment, Lotion, oil, pack, etc.), makeup cosmetics (foundation, lipstick, blusher, mascara, etc.), other skin cosmetics (face wash, skin cleanser, massage agent, cleansing agent, etc.), oral composition, or Examples include bath preparations.

  When formulating the whitening agent of the present invention as an external preparation, the amount of the external preparation varies depending on the compounding target, dosage form, expected effect, etc., and cannot be uniformly defined, but is generally applied to the entire external preparation. On the other hand, it is 0.00001 to 10% by weight, preferably 0.00005 to 5% by weight, more preferably about 0.0001 to 3% by weight as hibiscus acids and salts thereof.

  In formulating the whitening agent of the present invention as an internal preparation, if necessary, a binder, a disintegrant, a lubricant, a wetting agent, a buffering agent, an excipient, an extender, a preservative, a fragrance, etc. Additives can be blended. Moreover, when formulating the whitening agent of the present invention as an internal preparation, other pharmaceutically active ingredients can be further blended.

  The type of internal preparation to which the whitening agent of the present invention can be applied is not particularly limited, and examples thereof include oral preparations such as powders, tablets, granules, capsules, and syrups.

  When formulating the whitening agent of the present invention as an internal preparation, the blending ratio of the internal preparation varies depending on the administration subject, dosage form, administration method, etc., and cannot be uniformly defined. The total amount of hibiscus acids and salts thereof is 0.00001 to 100% by weight, preferably 0.0001 to 50% by weight, and more preferably about 0.001 to 30% by weight.

  The whitening agent of the present invention contains a melanin production inhibitor containing as an active ingredient one or more compounds selected from the group consisting of the above hibiscus acids and salts thereof as an active ingredient, and is a high and sustained tyrosinase activity inhibitor. It has a continuous and excellent melanin production-inhibiting action including an action and a high tyrosinase production-inhibiting action, and has a continuous and excellent whitening effect.

Whitening Cosmetic The whitening cosmetic of the present invention contains the melanin production inhibitor of the present invention as an active ingredient.

  The type of cosmetic is not particularly limited and can be applied to various cosmetics. For example, various basic cosmetics such as creams, emulsions, lotions, packs, facial cleansers, patches, etc., makeups such as foundations, cheeks, white powders, various cosmetics for hair such as hair shampoos, hair nourishing agents, shampoos, rinses, etc. It can be applied to various cosmetics such as soaps, beauty nails, eau de cologne and bath salts.

  The form of the cosmetic is not particularly limited, and can be used in various forms such as a solution, an emulsion, an ointment, a sol, a gel, a powder, a spray, a sheet, and a film.

  In the cosmetics of the present invention, various components that are generally blended in cosmetics within the scope of the effects of the present invention, such as aqueous components, powder components, moisturizers, surfactants, preservatives, thickeners, An ultraviolet absorber, a fragrance | flavor, etc. can be mix | blended.

  The manufacturing method of the cosmetics of this invention is not specifically limited, According to a well-known method, it can manufacture suitably.

  The method of applying the cosmetic composition of the present invention is not particularly limited, however, for example, it may be applied to an easily or affected part such as a stain on a face, neck, arm or hand, a buckwheat or the like, an easily tanned part or a tanned part. An appropriate amount may be applied once to several times.

  The content ratio of the melanin production inhibitor in the cosmetic of the present invention can be appropriately set within the range in which the effect of the present invention can be exerted, but as a hibiscus acid and its salt, 0.00001 to 10% by weight relative to the entire cosmetic composition. Preferably, it is about 0.00005 to 5% by weight, more preferably about 0.0001 to 3% by weight.

  The whitening cosmetic composition of the present invention comprises, as an active ingredient, a melanin production inhibitor containing one or more compounds selected from the group consisting of the hibiscus acids and salts thereof as an active ingredient, and is a high and sustained tyrosinase. It has a continuous and excellent melanin production inhibitory action including an activity inhibitory action and a high tyrosinase production inhibitory action, and exhibits a continuous and excellent whitening action.

Whitening Food The whitening food of the present invention contains the melanin production inhibitor of the present invention as an active ingredient.

  The whitening food of the present invention can be used as, for example, health foods, food additives, functional foods, nutritional functional foods, health functional foods, and the like.

  The manufacturing method of the food for whitening is not specifically limited, According to a well-known method, it can manufacture suitably. For example, it can be obtained by mixing the whitening agent with various foods or food materials.

  The kind of food is not particularly limited, and can be appropriately set from various known foods and drinks. For example, it can be used as cookies, cereals, candy, tablets, jelly, marine products, seasonings, bread, soft drinks, carbonated drinks, mineral water, fruit juice drinks, teas, nutritional drinks, drinks and the like.

  Moreover, the foodstuff of this invention can be mix | blended with the other component used for normal food-drinks in the range which does not impair the effect of this invention. Examples of other components include various vitamins, minerals, animal and plant ingredients, excipients, sweeteners, flavoring agents, coloring agents, preservatives, disintegrating agents, lubricants, wetting agents, bulking agents, and flavoring agents. It is done.

  The content ratio of the melanin production inhibitor in the food of the present invention can be appropriately set within the range where the effects of the present invention can be exerted, but as the hibiscus acids and salts thereof, 0.00001 to 10% by weight, preferably Is about 0.00005 to 5% by weight, more preferably about 0.0001 to 3% by weight.

  The amount of intake when eating the whitening food of the present invention can be appropriately set according to the age, weight, symptoms, etc. of the application target, but is usually about 0.00001-50 mg as hibiscus acids and salts per day for an adult. The amount is preferably about 0.0001 to 20 mg, more preferably about 0.001 to 10 mg.

  The food for whitening of the present invention comprises a melanin production inhibitor containing as an active ingredient one or more compounds selected from the group consisting of the hibiscus acids and salts thereof as an active ingredient, and has a high and sustained tyrosinase activity. It has a continuous and excellent melanin production-inhibiting action including an inhibitory action and a high tyrosinase production-inhibiting action, and has a continuous and excellent whitening action.

Oral Composition The oral composition of the present invention contains the melanin production inhibitor of the present invention as an active ingredient.

  The type of oral composition is not particularly limited, and can be applied to various oral compositions.

  For example, it can be applied to various oral compositions such as toothpastes such as toothpaste, mouthwash, mouth rinse, oral paste, troches, gum massage cream and the like.

  The form of the oral composition is not particularly limited, and can be used in various forms such as a solution, emulsion, ointment, sol, gel, paste, tablet, powder, spray, sheet, film and the like.

  In the composition for oral cavity of the present invention, various components generally blended in the composition for oral cavity can be blended within the range where the effects of the present invention are exhibited. Examples of the various components include pressure-sensitive adhesives, refreshing agents, binders, sweeteners, flavoring agents, disintegrating agents, lubricants, coloring agents, sustained-release adjusting agents, surfactants, solubilizers, wetting agents, and pH adjusting agents. Etc.

  The manufacturing method of the composition for oral cavity of this invention is not specifically limited, According to a well-known method, it can manufacture suitably.

  The application method of the composition for oral cavity of the present invention is not particularly limited, and for example, an appropriate amount may be applied once to several times a day to the affected area or the entire oral cavity.

  The content ratio of the melanin production inhibitor in the oral composition of the present invention can be set as appropriate within the range in which the effects of the present invention can be exerted. However, as a hibiscus acid and a salt thereof relative to the entire oral composition, 0.00001 to 10 % By weight, preferably about 0.00005 to 5% by weight, more preferably about 0.0001 to 3% by weight.

  The oral composition of the present invention includes a melanin production inhibitor containing as an active ingredient one or more compounds selected from the group consisting of the hibiscus acids and salts thereof as an active ingredient, and is a high and sustained tyrosinase It has a continuous and excellent melanin production inhibitory action including an activity inhibitory action and a high tyrosinase production inhibitory action, removes pigments deposited on the gums, and exhibits an excellent whitening action.

  According to this invention, the melanin production inhibitor which uses as an active ingredient 1 type, or 2 or more types of compounds chosen from the group which consists of the hibiscus acid which has a specific structure, and its salt is provided.

  The compound serving as an active ingredient of the melanin production inhibitor of the present invention has an excellent melanin production inhibitory action including a high tyrosinase activity inhibitory action and a high tyrosinase production inhibitory action, and has an excellent whitening effect. . Further, the whitening effect is exhibited for a long time. Moreover, the compound is highly safe.

  The melanin production inhibitor of the present invention comprising such a compound as an active ingredient has an excellent whitening action and has a long-lasting whitening action, and is capable of producing melanin generated in skin cells, oral mucosa and the like. It is possible to effectively suppress the deposition of pigments such as spots and freckles. Furthermore, the melanin production inhibitor of the present invention can be used with peace of mind.

  The melanin production inhibitor can be used as an active ingredient in a whitening agent, a whitening cosmetic, a whitening food and an oral composition, and has an excellent whitening action according to the present invention. A whitening agent, a whitening cosmetic, a whitening food, and an oral composition that are durable and safe to use are also provided.

  The present invention having such characteristics can be suitably used in the cosmetics industry, the pharmaceutical industry, the food industry, and the like, in which interest in the whitening effect has been increasing in recent years.

  EXAMPLES Hereinafter, although an Example, a manufacture example, and a test example demonstrate this invention in detail, this invention is not limited to these.

Production Example 1 (Hibiscus acid)
800 g of dried Hibiscus sabdariffa L. flowers were pulverized by a mill, 8 L of 1.5% hydrochloric acid-methanol aqueous solution was added thereto, and the mixture was extracted for 3 hours with stirring at room temperature. Subsequently, the solid content was removed by filtration, and the filtrate was concentrated with an evaporator (45 ° C.). The filtrate was purified and concentrated by HP-20 (φ = 70 mm, h = 500 mm) column chromatography to obtain 118 g of a crude product.

  The obtained crude product was purified and concentrated several times by ODS (φ = 110 mm, h = 1200 mm) column chromatography, and then crystallized to obtain 34.8 g of hibiscus acid.

  When the substance obtained for confirmation was analyzed, the molecular weight according to the mass spectrum was 190. Proton NMR was as follows.

Proton NMR spectrum (400 MHz) (acetone-d6) δ (ppm): 5.35 (1H, S), 3.26 (1H, d, J = 17.2 Hz), 2.78 (1H, d, J = 17.2 Hz).
These values were consistent with the values for hibiscus acid reported by Boll et al. (Acta Chemica Scandinavica, 1969, Vol. 23, 286-293).

Production Example 2 (Hibiscus acid monoethyl ester)
100 g of dried Hibiscus sabdariffa L. flowers were pulverized with a mill, added with 500 mL of 30% ethanol aqueous solution, and extracted for 3 days with stirring at room temperature. Subsequently, ethyl acetate extraction was performed, and the obtained aqueous layer was further extracted with butanol, and then purified by silica gel chromatography (eluent: ethyl acetate-methanol solution) to obtain a fraction.

  When the obtained fraction was analyzed, the molecular weight according to the mass spectrum was 218. In proton NMR, several signal overlaps were observed, and the presence of several isomers was expected. Moreover, the compound which has the following signals considered to be one of them was recognized.

  Proton NMR spectrum (500 MHz) (CDCl3) δ (ppm): 5.23 (1H, S), 3.20 (1H, d, J = 17.4 Hz), 2.90 (1H, d, J = 17.4 Hz), 4.29 (2H, m ), 1.37 (3H, t, J = 7.4Hz).

  From the above analysis results and the fact that Hibiscus sabdariffa L. contains a large amount of hibiscus acid, it was speculated that the fraction contained bismuth acid monoethyl ester and its isomer.

  The structures of the hibiscus monoethyl ester and its isomer are presumed to be the following (a) and (b).

  Hereinafter, the obtained fraction is also referred to as “hibiscus acid monoethyl ester fraction”.

Production Example 3 (Dimethyl Hibiscus acid ester)
450 mg of hibiscus acid obtained in Production Example 1 was dissolved in a mixed solvent of 1.8 ml of methyl-t-butyl ether (MTBE) and 1.8 ml of methanol. The solution was stirred while cooling in an ice-water bath, and 3.5 ml of a trimethylsilyldiazomethane 2.0M diethyl ether solution was added little by little. The resulting precipitate was filtered, washed with MTBE, and dried under reduced pressure to obtain 300 mg of bismuth dimethyl ester.

  When the substance obtained for confirmation was analyzed, the molecular weight by mass spectrum was 218. Proton NMR was as follows.

  Proton NMR spectrum (400 MHz) (chloroform-d1) δ (ppm): 5.11 (1H, S), 3.08 (1H, d, J = 17.5 Hz), 2.88 (1H, d, J = 17.2 Hz), 3.80 (1H , S), 3.84 (3H, S), 3.96 (3H, S).

  These values were consistent with the values for hibiscus acid dimethyl ester reported by IBnusaud et al. (Tetrahedron, 2002, Vol. 58, 4887-4892).

Production Example 4 (Hibiscus Extract)
For use as a comparison, a hibiscus extract (extract) was produced by the following method.

100 g of dried Hibiscus sabdariffa L. flowers were pulverized with a mill, added with 500 mL of 30% ethanol aqueous solution, and extracted for 3 days with stirring at room temperature. Next, the solid content was removed by filtration, and the filtrate was concentrated under reduced pressure and then freeze-dried to obtain 15 g of Hibiscus sabdariffa L. extract.

Example 1 Evaluation of Tyrosinase Production Inhibitory Effect In order to evaluate the tyrosinase production inhibitory effect of hibiscus acid, Western blotting was performed as follows.

B16 melanoma cells derived from mice (Dainippon Pharmaceutical Co., Ltd., B16F0) were cultured in a 25 cm ² flask for 24 hours in an 8 ml culture solution, and then 5% by weight aqueous solutions of hibiscus acid obtained in Production Example 1 were respectively 12.5 and 25 50 or 100 ml was added and cultured for 72 hours.

  After completion of the culture, the cells were pulverized to extract proteins and subjected to electrophoresis. Next, the protein is transferred to a polyvinylidene difluoride membrane (PVDF membrane), reacted with a goat anti-tyrosinase antibody, further reacted with an alkaline phosphatase-labeled anti-goat antibody, and finally added with a chromogenic substrate, tyrosinase is added. Detected. For comparison, the same test was performed using a bismuth acid-free additive.

  The obtained results are shown in FIG. FIG. 1 shows a PVDF membrane image showing the expression level of tyrosinase in B16 melanoma cells cultured in the presence or absence of hibiscus acid.

  In the figure, lane I is added with no hibiscus acid solution, lane II is added with 100 μl of 5% hibiscus acid solution, lane III is added with 50 μl of 5% hibiscus acid solution, lane IV is added with 25 μl of 5% hibiscus acid solution, and lane V is 5%. The result of the sample added with 12.5 μl of hibiscus acid is shown.

  As is clear from the thickness of the band shown in FIG. 1, the tyrosinase expression was suppressed with the increase in the concentration of bismuth acid added in the culture with hibiscus acid added, as compared to the case without hibiscus acid added. It was confirmed that there was a tendency to

  As a result, it was revealed that hibiscus acid has a tyrosinase production inhibitory action.

Example 2 Evaluation of Tyrosinase Activity Inhibitory Effect (Hibiscus Acid)
In order to evaluate the tyrosinase activity inhibitory effect of hibiscus acid, the following test was conducted.

Mouse-derived B16 melanoma cells (D16 manufactured by Dainippon Pharmaceutical Co., Ltd., B16F0) were seeded in a 96-well microplate at 5 × 10 ⁴ cells / well and cultured overnight to fix the cells. Next, the culture solution on the culture plate was removed with an aspirator, and PBS (phosphate buffered saline) containing 1% Triton X-100 was added at 50 μl / well to disrupt the cell membrane structure to obtain a crude tyrosinase solution. . Next, a solution obtained by diluting the hibiscus acid obtained in Production Example 1 with PBS so that the final concentration of the hibiscus acid in the well is 0.3 to 2.5 mg / ml (also referred to as a sample added with hibiscus acid in the following equation 1). 50) / well was added. Thereafter, 50 μL of 0.1% dopa solution diluted with PBS was added to each well, immediately incubated at 37 ° C. for 4 hours, and then the absorbance at 405 nm was measured using a microplate reader.

  As a control, the following conditions were also measured in the same manner.

Based on the measured absorbance value, the tyrosinase activity inhibition rate (%) was calculated based on Equation 1 below.
・ Blank: B16 melanoma cell non-seeding: Hibiscus acid solution added ・ Hibiscus acid added sample: B16 melanoma cell seeded ・ Hibiscus acid solution added ・ Control 1: B16 melanoma cell seeded ・ Hibiscus acid solution added (PBS added instead)
・ Control 2: B16 melanoma cell non-seeding ・ Hibiscus acid solution not added (PBS added instead)

  For comparison, a test was conducted in the same manner as above except that the hibiscus (Hibiscus sabdariffa L.) extract obtained in Production Example 4 was used instead of hibiscus acid, and the tyrosinase activity inhibition rate (%) of the hibiscus extract was also determined. Calculated.

  The obtained results are shown in FIG. FIG. 2 illustrates the relationship between the concentration of hibiscus acid and hibiscus extract and the inhibition rate (%) of tyrosinase activity.

  From this result, it was confirmed that hibiscus acid has a higher tyrosinase activity inhibitory action than hibiscus extract, and that the effect is enhanced in a concentration-dependent manner.

Example 3 Evaluation of Tyrosinase Activity Inhibitory Effect (Hibiscus acid monoethyl ester)
In Example 2, except that the bismuth acid monoethyl ester fraction obtained in Production Example 2 was used instead of hibiscus acid, and the final concentration of hibiscus monoethyl ester was adjusted to 0.25 to 5 mg / ml. Evaluation was performed in the same manner as in Example 2.

  In addition, as in Example 2, for the purpose of comparison, a test was conducted in the same manner except that the hibiscus (Hibiscus sabdariffa L.) extract obtained in Production Example 4 was used instead of the bismuth acid monoethyl ester fraction. The tyrosinase activity inhibition rate (%) of the hibiscus extract was also calculated.

  The obtained results are shown in FIG. FIG. 3 shows the relationship between the concentration of hibiscus monoethyl ester fraction and hibiscus extract and the inhibition rate (%) of tyrosinase activity.

  From this result, it was confirmed that the hibiscus acid monoethyl ester fraction has a higher tyrosinase activity inhibitory action than the hibiscus extract, and that the effect is enhanced in a concentration-dependent manner.

Example 4 Evaluation of Tyrosinase Activity Inhibitory Effect (Hibiscus acid dimethyl ester)
In Example 2, except that the bismuth acid dimethylster fraction obtained in Production Example 3 was used instead of hibiscus acid, and the final concentration of bismuth acid dimethyl ester was adjusted to 0.25 to 5 mg / ml. Evaluation was performed in the same manner as in Example 2.

  Further, as in Example 2, for comparison, a test was conducted in the same manner except that the hibiscus (Hibiscus sabdariffa L.) extract obtained in Production Example 4 was used instead of hibiscus dimethyl ester, and tyrosinase of hibiscus extract was used. The activity inhibition rate (%) was also calculated.

  The obtained results are shown in FIG. FIG. 4 shows the relationship between the concentration of bismuth dimethyl ester and hibiscus extract and the inhibition rate (%) of tyrosinase activity.

  From this result, it was confirmed that bismuth acid dimethyl ester has a tyrosinase activity inhibitory action equivalent to hibiscus extract, and that the effect is enhanced in a concentration-dependent manner.

Example 5 Evaluation of tyrosinase activity inhibitory effect (Hibiscus acid)
In order to evaluate the persistence of the melanin production inhibitory effect of hibiscus acid, a persistence confirmation test of the tyrosinase activity inhibitory effect was performed according to the following method.

Mouse-derived B16 melanoma cells (D16 manufactured by Dainippon Pharmaceutical Co., Ltd., B16F0) were seeded in a 96-well microplate at 5 × 10 ⁴ cells / well and cultured overnight to fix the cells. Next, the culture solution on the culture plate was removed with an aspirator, and PBS (phosphate buffered saline) containing 1% Triton X-100 was added at 50 μl / well to destroy the cell membrane structure to obtain a crude tyrosinase solution. Next, 50 μl / well of a solution obtained by diluting the hibiscus acid obtained in Production Example 1 with PBS so that the final concentration of hibiscus acid in the well becomes 0.3 to 10 mg / mL was added (hereinafter referred to as a sample with hibiscus acid added). Also called.) Thereafter, 50 μL of 0.1% dopa solution diluted with PBS was added to each well and immediately incubated at 37 ° C.

  After 20 hours and 108 hours from the start of incubation, the absorbance at 405 nm was measured using a microplate reader.

  As a control, the following conditions were also measured in the same manner.

  Based on the measured absorbance value, the tyrosinase activity inhibition rate (%) was calculated based on Equation 2 below.

・ Blank: B16 melanoma cell non-seeding: Hibiscus acid solution added ・ Hibiscus acid added sample: B16 melanoma cell seeded ・ Hibiscus acid solution added ・ Control 1: B16 melanoma cell seeded ・ Hibiscus acid solution added (PBS added instead)
・ Control 2: B16 melanoma cell non-seeding ・ Hibiscus acid solution not added (PBS added instead)

For comparison, a similar test was performed except that arbutin was used instead of hibiscus acid, and the tyrosinase activity inhibition rate (%) of arbutin was calculated (hereinafter referred to as arbutin-added sample).

  The obtained results are shown in FIG. In FIG. 5, ◯ and ● represent the tyrosinase activity inhibition rate (%) of the sample added with hibiscus acid, and Δ and ▲ represent the tyrosinase activity inhibition rate (%) of the sample added with arbutin. The dotted line shows the measurement result after 20 hours, and the solid line shows the measurement result after 108 hours.

  As is apparent from FIG. 5, in the measurement results after 20 hours, hibiscus acid showed a higher tyrosinase inhibitory effect than arbutin. Further, in the measurement results after 108 hours, the sample added with hibiscus acid maintained its high tyrosinase inhibitory effect.

  In contrast, in the arbutin-added sample, a significant decrease in the inhibition rate of tyrosinase activity was observed after 108 hours compared to 20 hours later.

  From these results, it was suggested that hibiscus acid has a high and sustained tyrosinase activity inhibitory effect and can maintain a high whitening effect over a long period of time.

Example 6 Melanin production inhibitory effect (Hibiscus acid)
From the above results, it has been clarified that hibiscus acid has a tyrosinase production inhibitory action and a tyrosinase activity inhibitory action involved in the production of melanin. evaluated.

  The evaluation test was performed according to the following method.

Mouse-derived B16 melanoma cells (manufactured by Dainippon Pharmaceutical Co., Ltd., B16F0) were seeded in a 6-well plate at about 1 × 10 ⁵ cells / well and cultured overnight to fix the cells. Next, the solution obtained by diluting the hibiscus acid obtained in Production Example 1 with PBS so that the final concentration of hibiscus acid in the wells is 0.2 to 2 mg / ml (also referred to as the target sample in Formula 3 below). After culturing for 4 days, the cells were detached using a 0.25% trypsin solution, and the cells were collected by centrifugation. After visually confirming the color difference of the collected cells, the membrane structure of the cells was destroyed with 5N NaOH to elute melanin, and the absorbance at 405 nm was measured.

  In addition, as a control, the same measurement was performed for a system in which hibiscus acid was not added (hereinafter referred to as an additive-free sample in Equation 3 below).

  Furthermore, in order to correct errors during preparation of the solution, the amount of protein in the sample was measured using a quantification kit, corrected with the amount of unit protein, and the melanin production rate (%) was calculated based on Equation 3 below. .

  The obtained result is shown in FIG. FIG. 6 shows the melanin production rate (%) at each added concentration of hibiscus acid.

  As a result, it was confirmed that the addition of hibiscus acid significantly suppressed the production of melanin, and it was revealed that hibiscus acid is useful as a melanin production inhibitor.

Example 7 Melanin production inhibitory effect (Hibiscus acid monoethyl ester)
In Example 6, tests and evaluations were performed in the same manner as in Example 6 except that the bismuth acid monoethyl ester fraction obtained in Production Example 2 was used instead of hibiscus acid.

  The obtained results are shown in FIG. FIG. 7 shows the melanin production rate (%) at each addition concentration of the bismuth acid monoethyl ester fraction.

  As a result, it was confirmed that the addition of the bismuth acid monoethyl ester fraction significantly suppressed the production of melanin, and it was revealed that bismuth acid monoethyl ester is useful as a melanin production inhibitor. .

Example 8 Melanin production inhibitory effect (Hibiscus acid dimethyl ester)
In Example 6, tests and evaluations were performed in the same manner as in Example 6 except that the bismuth acid dimethyl ester obtained in Production Example 3 was used instead of hibiscus acid.

  The obtained result is shown in FIG. FIG. 8 shows the melanin production rate (%) at each addition concentration of hibiscus acid dimethyl ester.

  As a result, it was confirmed that the production of melanin was significantly suppressed by the addition of bismuth acid dimethyl ester, and it was revealed that bismuth acid dimethyl ester is useful as a melanin production inhibitor.

Test Example Since an optical isomer exists in hibiscus acid, it is considered that an extract obtained from a plant also contains an optical isomer of hibiscus acid. Of the optical isomers of hibiscus acid represented by the following formula (2), a garcinic acid represented by the following formula (3) is considered as a naturally occurring one.

  Therefore, in order to confirm whether the whitening action is characteristic of the three-dimensional structure of hibiscus acid, a test on melanin production inhibitory action was performed on hibiscus acid and garcinia acid according to the following procedure. In addition, cytotoxicity was also evaluated in order to confirm the safety of hibiscus acid.

  About the melanin production inhibitory effect, it tested as follows.

Mouse-derived B16 melanoma cells (manufactured by Dainippon Pharmaceutical Co., Ltd., B16F0) were seeded in a 6-well plate at about 1 × 10 ⁵ cells / well and cultured overnight to fix the cells. Next, a solution diluted with PBS so that the final concentration of hibiscus acid or garcinia acid in the well is 0.5 mg / ml (referred to as “target sample” in the following formula 4) is added, and cultured for 4 days. The cells were detached using 0.25% trypsin solution, and the cells were collected by centrifugation. After visually confirming the color difference of the collected cells, the membrane structure of the cells was destroyed with 5N NaOH to elute melanin, and the absorbance at 405 nm was measured.

  In addition, as a control, the same measurement was performed for a system in which hibiscus acid or garcinia acid was not added (hereinafter referred to as “non-added sample” in Equation 4 below).

  Furthermore, in order to correct the error at the time of preparing the solution, the amount of protein in the sample was measured using a quantification kit, corrected with the unit protein amount, and the melanin production rate (%) was calculated based on the following formula.

  Regarding cytotoxicity, the collected culture was stained with trypan blue, and the number of viable cells was measured with a hemocytometer.

The obtained result is shown in FIG. FIG. 9 shows the melanin production rate (%) and cell number of garcinia acid and hibiscus acid at a concentration of 0.5%. BK represents a blank (non-added sample).

  As a result, it was found that hibiscus acid has an inhibitory effect on melanin production and low cytotoxicity.

  On the other hand, it has become clear that garcinia acid has almost no melanin production inhibitory effect and high cytotoxicity.

  From this, it was suggested that the three-dimensional structure of hibiscus acid is important for the melanin production inhibitory effect.

Formulation Examples Hereinafter, the present invention will be described in more detail with reference to formulation examples of the present invention, but the present invention is not limited to these.

<Prescription Example 1> Lotion Toner lotion was prepared according to a conventional method by blending raw materials at the blending ratio shown in the following table.
(Raw material) (wt%)
Hibiscus acid 0.5
Ascorbic acid phosphate magnesium 3.0
Polypropylene glycol 2.0
Oxybenzone 3.0
P-Hydroxybenzoate ester 0.5
Citric acid appropriate amount perfume appropriate amount
Purified water balance
Total 100.0

<Prescription example 2> Cosmetic cream The raw materials were blended at the blending ratios shown in the following table, and a cosmetic cream was produced according to a conventional method.
(Raw material) (wt%)
Hibiscus acid monomethyl ester 0.5
Ascorbic acid monostearate 3.0
Cysteine 1.0
Stearyl alcohol 5.0
Stearic acid 8.0
Squalane 10.0
Self-emulsifying glyceryl monostearate 3.0
Polyoxyethylene cetyl ether (20E.0) 1.0
Propylene glycol 5.0
Potassium hydroxide 0.3
Perfume Appropriate amount Preservative Appropriate amount
Purified water balance
Total 100.0

<Prescription Example 3> Emulsion The raw materials were blended at the blending ratios shown in the following table, and a milky lotion was produced according to a conventional method.
(Raw material) (wt%)
Hibiscus acid monomethyl ester 1.0
Hibiscus acid monoethyl ester 1.0
Squalane 8.0
Vaseline 2.0
Beeslow 0.5
Sorbitan sesquioleate 0.8
Polyoxyethylene oleyl ether (20E.0) 1.2
Carboxyvinyl polymer 0.2
Propylene glycol 0.5
Potassium hydroxide 0.1
Ethanol 7.0
Perfume Appropriate amount Preservative Appropriate amount
Purified water balance
Total 100.0

<Prescription example 4> Bread The raw material was mix | blended with the mixture ratio shown to the following table | surface, and bread was manufactured in accordance with the conventional method.
(Raw material) (wt%)
Flour 51.0
Sugar 3.0
Salt 1.0
Hibiscus acid 0.5
Nonfat dry milk 1.0
Oils and fats 3.1
East Food 0.1
East 1.0
Purified water balance
Total 100.0

<Prescription Example 5> Tablets Raw materials were blended in the blending ratios shown in the following table, and tablets were produced according to a conventional method.
(Raw material) (wt%)
Hibiscus acid 5
Hibiscus acid monoethyl ester 5
Ascorbic acid phosphate magnesium 10
Placenta extract 10
Collagen 20
Crystalline cellulose 20
Dextrin 14
Glycerin fatty acid ester 6
Excipient 10
Total 100

<Prescription example 6> Drink agent The raw material was mix | blended with the mixture ratio shown to the following table | surface, and the drink agent was manufactured in accordance with the conventional method.
(Raw material) (wt%)
Hibiscus acid 0.5
Fructose glucose 20.0
Citric acid 0.50
Sodium citrate 0.10
Ascorbic acid 0.20
Perfume
Purified water balance
Total 100.0

<Prescription Example 7> Toothpaste A
The raw materials were blended at the blending ratios shown in the following table, and toothpastes were produced according to a conventional method.
(Raw material) (wt%)
Dicalcium phosphate 30.00
Glycerin 10.00
Sorbitol 20.00
Sodium carboxymethylcellulose 1.00
Sodium lauryl sulfate 1.50
Carrageenan 0.50
Saccharin sodium 0.10
Perfume 1.00
Sodium benzoate 0.30
Hibiscus acid 0.10
Purified water balance
Total 100.0

<Prescription Example 8> Toothpaste B
The raw materials were blended at the blending ratios shown in the following table, and toothpastes were produced according to a conventional method.
(Raw material) (wt%)
Sodium lauryl sulfate 0.80
Lauric acid diethanolamide 1.00
Propylene glycol 3.00
60% Sorbit liquid 35.00
Methylparaben 0.20
Butylparaben 0.01
Saccharin sodium 0.15
Gelatin 0.30
Fragrance 0.60
Sodium alginate 0.90
Sodium benzoate 0.10
Aluminum hydroxide 45.00
Sodium monofluorate 0.76
Hibiscus acid 0.05
Purified water balance
Total 100.0

<Prescription Example 9> Mouthwash
The raw materials were blended at the blending ratios shown in the following table, and a mouthwash was produced according to a conventional method.
(Raw material) (wt%)
Ethanol 10.00
Glycerin 5.00
Citric acid 0.01
Sodium citrate 0.10
Polyoxyethylene hydrogenated castor oil 0.50
Methyl paraoxybenzoate 0.10
Fragrance 0.20
Hibiscus acid 0.1
Purified water balance
Total 100.0

<Prescription Example 10> Oral Pasta Raw materials were blended at the blending ratios shown in the following table, and oral pasta was produced according to a conventional method.
(Raw material) (wt%)
Liquid paraffin 13.00
Cetanol 10.00
Glycerin 25.00
Sorbitan monopalmitate 0.60
Polyoxyethylene sorbitan monostearate 5.00
Sodium lauryl sulfate 0.10
Benzotonium chloride 0.10
Methyl salicylate 0.10
Saccharin 0.20
Fragrance 0.25
Hibiscus acid 0.50
Purified water balance
Total 100.0

2 is a PVDF image showing the expression level of tyrosinase in B16 melanoma cells cultured in the presence or absence of hibiscus acid in Example 1. FIG. In the figure, lane I is added with no hibiscus acid; lane II is added with 100 μl of 5% hibiscus acid solution; lane III is added with 50 μl of 5% hibiscus acid solution; lane IV is added with 25 μl of 5% hibiscus acid solution; It is a figure which shows the sample of 12.5 microliters of solution addition. In Example 2, it is a figure which shows the relationship between the addition density | concentration of a hibiscus acid or a hibiscus extract, and a tyrosinase activity inhibition rate (%). In Example 3, it is a figure which shows the relationship between the addition density | concentration of a hibiscus acid monoethyl ester fraction or a hibiscus extract, and tyrosinase activity inhibition rate (%). The relationship between the addition density | concentration of bismuth acid dimethyl ester or a hibiscus extract and the inhibition rate (%) of tyrosinase activity in Example 4 is shown. In Example 5, it is a figure which shows the tyrosinase activity inhibition rate (%) 20 hours after incubation of a hibiscus acid addition group and an arbutin addition group, and 108 hours. In Example 6, it is a figure which shows the relationship between the addition density | concentration of a hibiscus acid, and a melanin production rate (%). In Example 7, it is a figure which shows the relationship between the addition density | concentration of a hibiscus acid monoethyl ester fraction, and a melanin production rate (%). The relationship between the addition density | concentration of bismuth acid dimethyl ester and the melanin production rate (%) in Example 8 is shown. It is a figure which shows a cell number and a melanin production rate (%) when the density | concentration in a well of a hibiscus acid and a garcinia acid is 0.5 mg / ml in a test example.

Claims (6)

The following formula (1)

(Wherein, R ¹ and R ² each represent a hydrogen atom, a substituted or unsubstituted alkyl group or a benzyl group) 1 or 2 selected from the group consisting of hibiscus acids and salts thereof A melanin production inhibitor comprising the above compound as an active ingredient. The melanin production inhibitor according to claim 1, wherein at least one of R ¹ and R ² of the hibiscus acids represented by the formula (1) is a substituted or unsubstituted alkyl group. A whitening agent comprising the melanin production inhibitor according to claim 1 as an active ingredient. A whitening cosmetic comprising the melanin production inhibitor according to claim 1 as an active ingredient. A food for whitening comprising the melanin production inhibitor according to claim 1 as an active ingredient. The composition for oral cavity containing the melanin production inhibitor in any one of Claims 1-2 as an active ingredient.

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