JP2016098188A - Melanogenesis inhibitor, cosmetics, and method for producing melanogenesis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanogenesis inhibitor which can effectively prevent and treat pigmentation such as skin discoloration and freckles.SOLUTION: The present invention provides a melanogenesis inhibitor comprising a compound included in Pteris dispar Kunze extract, the compound illustrated by the formula, or a salt thereof as an active ingredient and a method for producing the same.SELECTED DRAWING: None

Description

  The present invention relates to a melanin production inhibitor, a cosmetic and a pharmaceutical composition using the melanin production inhibitor, and a method for producing the melanin production inhibitor.

  It is known that pigmentation such as stains and buckwheat is caused by overproduction of melanin in melanocytes (melanocytes) existing in the skin due to exposure to ultraviolet rays by sunlight, abnormal hormone balance, etc. ing. Melanin is synthesized by synthesizing dopaquinone from tyrosine, an essential amino acid, by the action of tyrosinase, and oxidizing and polymerizing this dopaquinone. Screening of melanin production inhibitors targeting this melanin synthesis pathway has been vigorously conducted.

  Many compounds using extracts of plants and the like have been reported as compounds exhibiting a melanin production inhibitory effect. For example, it has been reported that extracts of towsendan, soka, senesiogracilis, and cocriro have an inhibitory effect on tyrosinase activity and are effective for whitening (Patent Document 1). Moreover, it has been reported that cacao nibs, cacao mass, cocoa, and extracts thereof have an inhibitory action on melanin production and are effective in preventing and treating pigmentation such as spots and buckwheat (Patent Document 2).

  In order to stably exhibit the melanin production inhibitory effect, it is desired to identify an effective component of the melanin production inhibitory effect contained in the extract of plants or the like and use it under the optimum conditions of the component. However, there are few specified active ingredients that exert melanin production-suppressing effects contained in plants and the like, and zederones compounds isolated from gadgets (Curcuma zedoaria Roscoe) and (Patent Document 3), Jatoba (Hymenaea courbaril) Tannin-type substance isolated from (Patent Document 4) and the like have been reported.

JP 2010-195732 A JP 2007-155943 A Japanese Patent No. 55143939 Japanese Patent No. 3650245

  The compounds and plant extracts described in Patent Documents 1 to 4 are disclosed to have an action of suppressing melanin production, and are put into practical use by being blended in various products such as some foods, cosmetics, and pharmaceuticals. Some are. However, the melanin production inhibitory effect and its stability were not always satisfactory.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a melanin production inhibitor capable of effectively preventing and treating pigmentation such as spots and freckles. Moreover, it aims at providing the cosmetics and pharmaceutical composition using the said melanin production inhibitor. Furthermore, it aims at providing the manufacturing method of the said melanin production inhibitor.

That is, the present invention includes the following inventions.
[Invention 1]
The following formula (I):
[Wherein, R ¹ and R ² are each H, OH, or CH ₃ , or R ¹ and R ² are combined to form CH ₂ ;
R ³ and R ⁴ are H or OH, and at least one of them is OH;
R ⁵ is H or OH;
R ⁶ is H, glucose group, galactose group, xylose group, or mannose group. ]
The melanin production inhibitor which uses the compound shown by these, or its salt as an active ingredient.
[Invention 2]
R ¹ and R ² together are CH ₂ ,
R ³ is OH;
R ⁴ and R ⁵ are each H;
The melanin production inhibitor according to invention 1, wherein R ⁶ is H or a glucose group.
[Invention 3]
Cosmetics containing the melanin production inhibitor of invention 1 or 2.
[Invention 4]
A pharmaceutical composition comprising the melanin production inhibitor according to invention 1 or 2.
[Invention 5]
A method for producing the melanin production inhibitor according to invention 1 or 2,
A dried weed (Pteris dispar Kunze) is dipped in at least one solvent selected from the group consisting of methanol, ethanol, butylene glycol, methanol aqueous solution, ethanol aqueous solution, butylene glycol aqueous solution, hexane, and ethyl acetate. An extraction process for extracting
A purification step of obtaining the melanin production inhibitor using at least one treatment method selected from the group consisting of liquid-liquid distribution method, adsorption chromatography, and distribution chromatography,
The manufacturing method of the melanin production inhibitor containing this.

  The melanin production inhibitor of the present composition can provide an excellent effect in inhibiting the production of melanin, and by blending the isolated component, it is excellent in stability and has a high whitening effect. And pharmaceutical compositions can be provided. Moreover, the manufacturing method of the melanin production inhibitor of this structure can manufacture the said melanin production inhibitor by a simple method.

FIG. 1 is a diagram showing the melanin production-inhibiting effect of an extract of a prickly pear. FIG. 2 is a graph showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention. FIG. 3 is a diagram showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention. FIG. 4 is a diagram showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention. FIG. 5 is a graph showing the evaluation results of toxicity in the melanin production inhibitor according to the present invention.

  As a result of intensive studies, the present inventors have found that certain types of kaurene compounds have a melanin production inhibitory effect, and have completed the present invention. Kauren compounds are typical diterpenes, and are known to have antibacterial activity, antitumor activity, anti-inflammatory activity, etc. ,unknown.

The melanin production inhibitor according to the present invention comprises a compound represented by the following formula (I) or a salt thereof as an active ingredient.

In the formula, R ¹ and R ² are each H, OH, or CH ₃ , or R ¹ and R ² together are CH ₂ ,
R ³ and R ⁴ are H or OH, and at least one of them is OH;
R ⁵ is H or OH;
R ⁶ is H, glucose group, galactose group, xylose group, or mannose group.

Preferred compounds of the present invention include the following compounds.
Compound 1:

Compound 2:

Compound 3:

Compound 4:

Compound 5:

Compound 6:

  Of the compounds listed above, the more preferred compound is Compound 1 (11 beta-hydroxy-15-oxo-ent-kaura-16-ene-19-euacid: 11β-Hydroxy-15-oxo-ent-kaur-16 -En-19-oic Acid) and Compound 4 (Pterisic acid D), and a more preferred compound is Compound 1. Moreover, the glycoside of the glucose group of the said compounds 1-6, a galactose group, a xylose group, or a mannose group is also mentioned as a preferable example, and the glycoside of a glucose group is especially preferable.

  The melanin production inhibitor of the present invention can be efficiently extracted and purified from Pteris dispar Kunze. Amakusa fern belongs to the family Pteridaceae and is a fern plant widely distributed in Honshu, Shikoku, Kyushu, Okinawa, Taiwan, China, and the like west of Chiba. Although the site | part which uses amakusa fern is not specifically limited, A leaf is mainly used.

  The melanin production inhibitor of the present invention is extracted with an organic solvent from a pulverized product obtained by pulverizing a dried product of Amakashi fern (extraction step). Examples of the organic solvent used for extraction include 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; ethyl acetate and butyl acetate. Ester etc. are mentioned. The organic solvent may be water-containing organic solvent by adding water. Preferred organic solvents are methanol, ethanol, butylene glycol, hexane, and ethyl acetate, and preferred aqueous solvents are methanol aqueous solution, ethanol aqueous solution, and butylene glycol aqueous solution. More preferable organic solvents are ethanol and butylene glycol, and more preferable aqueous solvents are ethanol aqueous solution and butylene glycol aqueous solution. An organic solvent may be used individually by 1 type, and may be used in combination of 2 or more type. When preparing a water-containing organic solvent, the ratio of the organic solvent is preferably 30% by mass or more, more preferably 50 to 99% by mass, and particularly preferably 70 to 99% by mass.

  The method for extracting Amakusashida with an organic solvent is not particularly limited. For example, 1 to 50 parts by mass, preferably 5 to 20 parts by mass of the above organic solvent or water-containing organic solvent is added to 1 part by mass of the dried powder of Amakashida. Then, after extracting the extract of Amakusa fern while standing or stirring at an extraction temperature of 5 to 60 ° C., preferably 10 to 40 ° C. and an extraction time of 5 to 120 hours, preferably 8 to 72 hours, centrifugation is performed. Examples include a method of removing a solid content with a separator or the like. Since the obtained Amakashi fern extract contains a melanin production inhibitor at a relatively high concentration (0.2% by mass or more), it is also possible to use the Amakashi fern extract as it is as a melanin production inhibitor. .

  The obtained extract of Amakusashida can be purified by a liquid-liquid distribution method, adsorption chromatography, distribution chromatography, or the like (purification step). A styrene-divinylbenzene synthetic adsorbent (for example, HP-20, manufactured by Mitsubishi Chemical Corporation) can be suitably used as a carrier for adsorption chromatography, and silica gel can be suitably used as a carrier for distribution chromatography. The extract may be purified by one kind of extraction method, or may be purified by combining two or more extraction methods.

  The melanin production inhibitor of the present invention is provided in combination with various base materials and carriers acceptable as cosmetics. For cosmetics, if necessary, excipients, coating agents, binders, extenders, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifiers, dispersing agents, stabilizers, Additives such as antioxidants, surfactants, preservatives, ultraviolet absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents and the like can be appropriately blended.

  As cosmetics, specifically, skin care cosmetics such as milky lotion, cream, cleansing, pack, oil liquid, massage, beauty essence, cleaning agent, deodorant, hand cream, lip balm; makeup base, white powder, liquid Makeup cosmetics such as foundation, oily foundation, blusher, eye shadow, mascara, eyeliner, eyebrow, lipstick, etc .; antiperspirants, UV protection cosmetics such as sunscreen milk and sunscreen, and skin external preparations Also included are those used as quasi-drugs.

  The blending ratio of the melanin production inhibitor in the cosmetic is appropriately set according to the effective amount, the form of the cosmetic, and the like. For example, it is 0.001 to 10% by mass with respect to the total amount of the cosmetic. , Preferably it is 0.001-1 mass%, More preferably, it is 0.001-0.1 mass%.

  The pharmaceutical composition comprises the melanin production inhibitor of the present invention as an active ingredient and is provided in combination with a pharmaceutically acceptable base material or carrier. Pharmaceutical compositions include excipients, coating agents, binders, extenders, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH adjusting agents, emulsifiers, and dispersions within the pharmaceutically acceptable limits. Additives such as additives, stabilizers, antioxidants, surfactants, preservatives, UV absorbers, moisturizers, colorants, fragrances, thickeners, bactericides, cell activators, anti-inflammatory agents, etc. You can also.

  The pharmaceutical composition includes a pharmaceutical product or quasi-drug and contains the melanin production inhibitor of the present invention as an active ingredient. The pharmaceutical composition may be in a dosage form that is applied either orally or parenterally. Examples of the dosage form of the pharmaceutical composition administered by oral administration include pills, powders, tablets, granules, capsules, syrups, and liquids. Examples of the dosage form of a pharmaceutical composition administered parenterally include an external preparation, a transdermal preparation, a nasal preparation, a solution, a patch, and an external preparation for skin.

  The blending ratio of the melanin production inhibitor of the present invention in the pharmaceutical composition is appropriately set according to the effective amount, the dosage form of the pharmaceutical composition, the dosage form, etc., for example, relative to the total amount of the pharmaceutical composition And 0.001 to 10% by mass, preferably 0.001 to 1% by mass, and more preferably 0.001 to 0.1% by mass.

  About the melanin production inhibitor of this invention, the test which evaluates the effect | action which suppresses melanin production was implemented. The test results are shown below.

<Evaluation of Amakusa fern extract>
Amakusa fern was dried with warm air at 50 ° C. for 2 days using leaves / collected drying thermostats (manufactured by Isuzu Seisakusho, model number EPFH-343-2T). The dried leaves were pulverized using a table crusher (Tokyo Unicom Co., Ltd., model number T-351). 50 g of Amaxashi fern powder was immersed in 500 ml of butylene glycol, and extracted at 23 ° C. for 72 hours. Thereafter, filtration with a filter was performed to recover 450 ml of an agaricus extract. Melanogenesis suppression was evaluated about the collected Aplysia extract. In order to evaluate the Amakusa fern extract, B16 melanoma cells, which are melanin producing cells, were used, and the amount of melanin produced by the cultured B16 melanoma cells was visually observed.

(B16 melanoma cell culture)
In order to evaluate the amount of melanin produced, B16 melanoma cells, which are melanin producing cells, were cultured. A method for culturing B16 melanoma cells is shown below.
(1) B16 melanoma cells (JCRB cell bank, cell number: JCRB0202) which are melanin-producing cells are seeded in 6-well dishes so as to be 1 × 10 ⁴ cells (1 ml / well), and 1 ml of precultured After adding the medium, the cells were cultured at 37 ° C. in 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 preculture medium of B16 melanoma cells cultured in (1), 2 ml of a medium for treatment of B16 melanoma cells was added and cultured for 72 hours.
(3) The B16 melanoma cells cultured in (2) were washed with a phosphate buffer solution (PBS), collected in a 1.5 ml Eppendorf tube, and the cell colors were visually compared.
Treatment medium: Amaxashi fern extract was added to the same medium as the preculture so as to be diluted 300-fold and 1000-fold (volume ratio). For each test sample, forskolin (FSK) was added to a concentration of 20 μM (final concentration) and DMSO to 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) of B16 melanoma cells.
Control treatment medium: The amakashi fern extract was not added to the treatment medium.
Blank treatment medium: Amakashi fern extract and FSK were not added to the treatment medium.

  FIG. 1 is a diagram showing the melanin production-inhibiting effect of an amaranthus extract, and shows the relationship between the concentration of butylene glycol extract of the amakashida and the melanin production-inhibiting effect. Even when the Amakusa fern extract was diluted 1000 times, the melanin production inhibitory effect was confirmed.

<Extraction and Purification of Melanin Inhibitor from Amaxashi>
<Evaluation of Amakashi fern extract> 50 g of Amakashi fern powder obtained by the same method described in the section above is immersed in 500 ml of ethanol, extracted at 23 ° C. for 72 hours, and then centrifuged at 2000 rpm for 10 minutes. , 450 ml of the extract of Amaxashi fern was collected. The Amakusa fern extract was evaporated in an evaporator to evaporate ethanol and concentrated to 10 ml. Chloroform 500 ml was added to the concentrated solution, and left for 1 week to dissolve in chloroform to prepare a chloroform solution. The chloroform solution was filtered through a filter, and the filtrate was concentrated to 5 ml using an evaporator. Next, the concentrated solution was fractionated using a column packed with 20 g of silica gel (model number: 30721, manufactured by Nacalai Tesque, Inc.). 90 ml of concentrated chloroform solution was applied to a silica gel column, and methanol was 2.5% by volume, 5% by volume, 7.5% by volume, 10% by volume, 12.5% by volume, 15% by volume, and 17.5% by volume, respectively. Elution was successively performed using 90 ml of an eluent of methanol / chloroform adjusted to 20% by volume, and 19 fractions were obtained. When the melanin production inhibitory effect of each fraction was confirmed by the same method described in the section of (B16 melanoma cell culture), three fractions of fraction (16), fraction (17), and fraction (18) were obtained. There was a strong melanin production inhibitory activity. The three fractions were combined and dried, and 1 ml of chloroform was added. This chloroform additive was separated into a fraction dissolved in chloroform and a fraction not dissolved in chloroform. The melanin production inhibitory effect was confirmed in any fraction. The fraction not dissolved in chloroform was hydrolyzed with sulfuric acid (final concentration 1%) at 100 ° C. for 30 minutes. The hydrolyzate was analyzed under the following conditions using high performance liquid chromatography (HPLC) to recover the main peak. The recovered solution was dried under reduced pressure to obtain a purified product of Compound 1 (11 beta-hydroxy-15-oxo-ent-kaura-16-ene-19-euacid). The same compound 1 was obtained when the main peak of the fraction dissolved in chloroform was similarly collected using HPLC. About 10 mg of Compound 1 was obtained from 50 g of Amaxashi powder. Further, mass spectrometry confirmed that the chloroform-insoluble fraction before hydrolysis was a glucose glycoside of Compound 1.

Column: COSMOSIL (registered trademark) 5C ₁₈ -MS-II (manufactured by Nacalai Tesque)
Mobile phase: acetonitrile: water = 10: 90 to 60:40 (gradient elution)
Flow rate: 2 ml / min Detection wavelength: 245 nm

The structure was determined based on ¹ H-NMR and ¹³ C-NMR. The signals of ¹ H-NMR and ¹³ C-NMR spectra are shown below.
¹ H-NMR (CDCl ₃ + CD ₃ OD): 0.85 (3H, s), 1.14 (3H, s), 2.29 (1H, d, J = 12.0 Hz), 2.95 (1H , broadcasts), 3.93 (1H, d, J = 4.6 Hz), 5.16 (1H, s), 5.73 (1H, s)
¹³ C-NMR (CDCl ₃ + CD ₃ OD): 40.5 (C-1), 18.7 (C-2), 37.8 (C-3), 43.4 (C-4), 55. 8 (C-5), 19.8 (C-6), 33.7 (C-7), 50.6 (C-8), 62.8 (C-9), 38.8 (C-10) ), 65.7 (C-11), 65.7 (C-12), 36.8 (C-13), 36.4 (C-14), 210.7 (C-15), 150.3 (C-16), 112.7 (C-17), 28.8 (C-18), 180.4 (C-19), 15.4 (C-20)

<Evaluation of melanin production suppression using B16 melanoma cells>
As a test compound for inhibiting melanin production, Compound 1 purified from Amakashida, a glycoside glycoside of Compound 1 purified from Amakashida, Compound 4 (Pterisolic acid D: ChemFaces), Comparative Compound 1 (11 , 15-dihydroxy-ent-kaura-16-ene-19-euacid, manufactured by ChemFaces), and comparative compound 2 (pterislic acid A: manufactured by ChemFaces) were evaluated for inhibition of melanin production. In order to evaluate these test compounds, B16 melanoma cells, which are melanin producing cells, were used. B16 melanoma cells were cultured by the same method described in the section (B16 melanoma cell culture). Evaluation was performed by visual observation and quantitative analysis of the melanin production amount of each B16 melanoma cell after culture | cultivation about the B16 melanoma cell which added the test compound (final concentration: 10 micromol). Hereinafter, a method for quantifying melanin will be described.

(Quantification of melanin)
Melanin was extracted from each collected B16 melanoma cell. The extraction method of melanin is shown below.
(1) PBS was removed from each test sample in which the amount of protein was combined, 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) To each test sample, 100 μl of a mixed solution of methanol: chloroform (1: 2) was added and stirred to extract melanin.
(4) Centrifugation was performed at 1200 rpm for 10 minutes, and the supernatant was collected to obtain a melanin extract.
(5) 100 μl of melanin extract was dispensed into a 96-well plate, and the absorbance at 405 nm was measured.
(6) A calibration curve was created from the absorbance of the melanin standard solution, and the concentration of the melanin amount of each test sample was calculated. Melanin is dissolved in 1N NaOH aqueous solution so that the concentration of melanin is 0 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml. Prepared. 300 μl of melanin standard solution (n = 3) was added to a 1.5 ml Eppendorf tube, melanin was measured by the same method as in the above (3) to (5), and a calibration curve was prepared.
The melanin production inhibitory effect was measured by measuring the amount of protein in each test sample, and expressed as the amount of melanin produced (mg) per mg of protein.

  FIG. 2 is a graph showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention. Compared with the control, a very strong melanin production inhibitory effect was confirmed on the compound 1 and the glucose glycoside of compound 1 extracted from the scabbard. Moreover, the melanin production inhibitory effect was recognized also by the compound 4. On the other hand, among the compounds of the same kaurenes, the comparative compound 1 which does not have an oxygen double bond at the 15th carbon of the kaurene skeleton and the 9th and 11th carbons have a hydroxyl group. No comparative compound 2 was found to have a melanin production inhibitory effect.

<Concentration of melanin production inhibitor and melanin production inhibitory effect>
FIG. 3 is a diagram showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention, and shows the relationship between the concentration of the melanin production inhibitor according to the present invention and the melanin production inhibitory effect. Using the compound 1 and the compound 4 in which the melanin production inhibitory effect was recognized, the relationship between the concentration of the melanin production inhibitor and the melanin production inhibitory effect was evaluated. As an evaluation method, after culturing B16 melanoma cells by the same method described in the section of (B16 melanoma cell culture) using test samples in which the concentrations of compound 1 and compound 4 were adjusted to 3 μM, 10 μM, and 30 μM, respectively. Comparison was made by visual observation.

  From the photograph in FIG. 3, Compound 1 was found to have a sufficient melanin production inhibitory effect even at a low concentration of 3 μM. In addition, for compound 4, a melanin production inhibitory effect was observed at a concentration of 30 μM.

<Evaluation of melanin production suppression using a three-dimensional cultured skin model>
FIG. 4 is a diagram showing the melanin production inhibitory effect of the melanin production inhibitor according to the present invention, and shows the melanin production inhibitory effect using the three-dimensional cultured skin model of the melanin production inhibitor according to the present invention. Compound 1 was used to conduct a melanin production inhibition test using a three-dimensional cultured skin model. The melanin production inhibition test was performed using a commercially available three-dimensional cultured skin model (MEL-300 kit Asian donor: Kurashiki Boseki Co., Ltd.). According to the method of using the kit, a MEL-300 skin model cup is set in each well of a 6-well plate and warmed in a 37 ° C. incubator (EPI-100: SCF reaches a final concentration of 10 ng / mL when the medium is added) Aseptically, 5 mL each was put into a skin model cup. Compound 1 was added to the skin model cup at 300 μM, and the 6-well plate containing the skin model cup was placed in an incubator (37 ° C., 5% CO ₂ ) and cultured for 14 days. The medium was changed once every 3 days. The test sample to which the control and Compound 1 were added was irradiated with UV so as to be 30 mJ / cm ^2, and the blank was not irradiated with UV. After culturing, the blackness of each sample was measured to evaluate the melanin production inhibitory effect. The gradation was measured using ImageJ (free software), which is image processing software, and the blackness was compared by setting the blank to 1.0.

  From the photograph of FIG. 4, it was confirmed that Compound 1 had substantially the same value in the blackness and the blank not irradiated with UV, and the production of melanin was also suppressed in the three-dimensional cultured skin model.

<Toxicity assessment using a three-dimensional cultured skin model>
FIG. 5 is a graph showing the evaluation results of toxicity in the melanin production inhibitor according to the present invention. Using compound 1, a toxicity test of a melanin production inhibitor using a three-dimensional cultured skin model was performed. In the toxicity test of the melanin production inhibitor, the addition amount of Compound 1 was adjusted to 30 μM, 100 μM, and 300 μM, and the culture was performed by the same method as the test of the three-dimensional cultured skin model. After culturing the MEL-300 skin model, 1/10 volume of cytotoxicity test reagent was added directly to the medium and further incubated for 30 minutes. Thereafter, the culture medium was collected, and the optical density (OD) was measured at 450 nm using a microplate spectrophotometer (680 type, manufactured by Bio-Rad). The cytotoxicity test reagent was WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium. Salt] (Cell-Counting Kit-8 (registered trademark), manufactured by Dojindo Laboratories).

  From the graph of FIG. 5, it was confirmed that Compound 1 had substantially the same value as the blank, and did not exhibit toxicity in the three-dimensional cultured skin model even at a concentration of 300 μM. It has been confirmed that Compound 1 does not show toxicity even at a concentration of 900 μM in a three-dimensional cultured skin model (data not shown).

<Safety test>
A skin patch test was conducted as a safety test for humans. Purified Amakusashi Extract powder (Compound 1) is kneaded into white petrolatum (Japanese Pharmacopoeia, Wako Pure Chemical Industries, Ltd.) to 0.01%, 0.05%, and 0.1% by mass. A closed patch test was carried out using a dust chamber and a fin chamber (manufactured by Smart Practice Japan Co., Ltd.). 6 adult men and women with 2 sensitive skins, 2 slightly sensitive skins and 2 normal skins. The fin chamber coated with 5 test samples (2 blanks, 3 samples of Compound 1 with different concentrations) was peeled off 24 hours after being applied to the inner side of the upper arm, and the determination was made 1 hour and 24 hours later. It was.
The test sample is calculated by calculating the total points of 6 subjects, with 0 points for normal skin, 1 point for mild redness only, and 3 points for abnormalities such as redness and swelling. It was evaluated in 19 stages (0 to 18 points). Judgment was rejected with a total of 5 points or more. For blanks without Compound 1, the same test was repeated twice. The test results are shown below.

  Both the sample containing Compound 1 and the blank were acceptable, but it was found that the sample containing Compound 1 had lower total points than the blank, and better results were obtained. Therefore, it was suggested that the melanin production inhibitor of the present invention has no problem when used on human skin and has high safety, so that it can be applied to products such as cosmetics and pharmaceutical compositions.

  The melanin production inhibitor according to the present invention, a cosmetic and a pharmaceutical composition using the melanin production inhibitor, and a method for producing the melanin production inhibitor are used in, for example, the food field, the cosmetic field, and the pharmaceutical field. Can do.

That is, the present invention includes the following inventions.
[Invention 1]
The following formula (I):
[Wherein, R ¹ and R ² are each H, OH, or CH ₃ , or R ¹ and R ² are combined to form CH ₂ ;
R ³ and R ⁴ are H or OH, and at least one of them is OH;
R ⁵ is H or OH;
R ⁶ is H, glucose group, galactose group, xylose group, or mannose group. ]
The melanin production inhibitor which uses the compound shown by these, or its salt as an active ingredient.
[Invention 2]
R ¹ and R ² together are CH ₂ ,
R ³ is OH;
R ⁴ and R ⁵ are each H;
The melanin production inhibitor according to invention 1, wherein R ⁶ is H or a glucose group.
[Invention 3]
Cosmetics containing the melanin production inhibitor of invention 1 or 2.
[Invention 4]
A pharmaceutical composition comprising the melanin production inhibitor according to invention 1 or 2.
[Invention 5]
A method for producing the melanin production inhibitor according to invention 1 or 2,
A dried weed (Pteris dispar Kunze) is dipped in at least one solvent selected from the group consisting of methanol, ethanol, butylene glycol, methanol aqueous solution, ethanol aqueous solution, butylene glycol aqueous solution, hexane, and ethyl acetate. as extraction factory to extract the
Method of manufacturing including melanogenesis inhibitor a.
[Invention 6]
A purification step for obtaining the melanin production inhibitor from the amaranthus extract using at least one treatment method selected from the group consisting of a liquid-liquid distribution method, an adsorption chromatography, and a distribution chromatography.
The manufacturing method of the melanin production inhibitor of invention 5 which further contains these.

That is, the present invention includes the following inventions.
[Invention 1]
The following formula (I):
[Wherein, R ¹ and R ² are each H, OH, or CH ₃ , or R ¹ and R ² are combined to form CH ₂ ;
R ³ and R ⁴ are H or OH, and at least one of them is OH;
R ⁵ is H or OH;
R ⁶ is H, glucose group, galactose group, xylose group, or mannose group. ]
The melanin production inhibitor which uses the compound shown by these, or its salt as an active ingredient.
[Invention 2]
R ¹ and R ² together are CH ₂ ,
R ³ is OH;
R ⁴ and R ⁵ are each H;
The melanin production inhibitor according to invention 1, wherein R ⁶ is H or a glucose group.
[Invention 3]
Cosmetics containing the melanin production inhibitor of invention 1 or 2 .
[Invention 4]
A method for producing the melanin production inhibitor according to invention 1 or 2,
A dried weed (Pteris dispar Kunze) is dipped in at least one solvent selected from the group consisting of methanol, ethanol, butylene glycol, methanol aqueous solution, ethanol aqueous solution, butylene glycol aqueous solution, hexane, and ethyl acetate. The manufacturing method of the melanin production inhibitor which includes the extraction process which extracts.
[Invention 5]
Invention 4 further includes a purification step of obtaining the melanin production inhibitor from the amaranthus extract using at least one treatment method selected from the group consisting of liquid-liquid partition method, adsorption chromatography, and partition chromatography. The manufacturing method of the melanin production inhibitor of description.

Claims (5)

The following formula (I):
[Wherein, R ¹ and R ² are each H, OH, or CH ₃ , or R ¹ and R ² are combined to form CH ₂ ;
R ³ and R ⁴ are H or OH, and at least one of them is OH;
R ⁵ is H or OH;
R ⁶ is H, glucose group, galactose group, xylose group, or mannose group. ]
The melanin production inhibitor which uses the compound shown by these, or its salt as an active ingredient. R ¹ and R ² together are CH ₂ ,
R ³ is OH;
R ⁴ and R ⁵ are each H;
The melanin production inhibitor according to claim 1, wherein R ⁶ is H or a glucose group.   Cosmetics containing the melanin production inhibitor of Claim 1 or 2.   The pharmaceutical composition containing the melanin production inhibitor of Claim 1 or 2. A method for producing the melanin production inhibitor according to claim 1 or 2,
A dried weed (Pteris dispar Kunze) is dipped in at least one solvent selected from the group consisting of methanol, ethanol, butylene glycol, methanol aqueous solution, ethanol aqueous solution, butylene glycol aqueous solution, hexane, and ethyl acetate. An extraction process for extracting
A purification step of obtaining the melanin production inhibitor using at least one treatment method selected from the group consisting of liquid-liquid distribution method, adsorption chromatography, and distribution chromatography,
The manufacturing method of the melanin production inhibitor containing this.