JP7736460B2 - Composition for inhibiting melanin production, composition for promoting BMP4 expression, composition for inhibiting c-KIT expression, and skin-whitening cosmetic - Google Patents

Description

The present invention relates to a composition for inhibiting melanin production, a composition for promoting BMP4 expression in melanocytes, a composition for inhibiting c-KIT expression in melanocytes, and a whitening cosmetic.

Various technologies using extracts from plants of the genus Zanthoxylum in the Rutaceae family have been proposed. For example, Patent Document 1 describes a tyrosinase activity promoter containing Zanthoxylum extract as an active ingredient. Patent Document 2 discloses a slimming composition containing a blood circulation promoter and amino acids, and describes Zanthoxylum extract as the blood circulation promoter. Patent Document 3 discloses a slimming composition containing a thermogenic protein expression promoter, and describes a plant extract of the genus Zanthoxylum in the Rutaceae family as the thermogenic protein expression promoter. Patent Document 4 describes an anti-inflammatory composition containing Sichuan pepper extract, which is an extract extracted from the pericarp of the fruit of Zanthoxylum chinense in the genus Zanthoxylum in the Rutaceae family.

Japanese Patent Application Publication No. 11-79951 JP 2008-69134 A JP 2010-180165 A JP 2011-93880 A

Pigmentation of the skin, such as age spots and freckles, can occur, for example, when melanin produced in excess by melanocytes is deposited in the skin. To prevent or improve pigmentation, various components capable of suppressing melanin production have been proposed, but the development of new components is desired.
Therefore, a main object of the present invention is to provide a novel composition for inhibiting melanin production.

The inventors have discovered that an extract from the pericarp of Zanthoxylum japonicum (Zanthoxylum japonicum), a member of the Rutaceae family, has the effect of inhibiting melanin production. The inventors have also discovered that an extract from the pericarp of Zanthoxylum japonicum (Zanthoxylum japonicum), has the effect of promoting BMP4 expression in melanocytes and inhibiting c-KIT expression in melanocytes.

That is, the present invention provides a composition for inhibiting melanin production, which contains an extract of Zanthoxylum chinense pericarp as an active ingredient.
The present invention also provides a composition for promoting BMP4 expression in melanocytes, which contains an extract of Zanthoxylum chinense pericarp as an active ingredient.
The present invention also provides a composition for inhibiting c-KIT expression in melanocytes, which comprises an extract of Zanthoxylum chinense pericarp as an active ingredient.
The present invention also provides a whitening cosmetic composition containing any one of the above compositions.
In the whitening cosmetic, the content of the extract of Zanthoxylum chinense pericarp may be 3 μg/mL or more in terms of dry solid content.

The present invention provides a new composition for inhibiting melanin production. Note that the effects of the present invention are not limited to those described herein, and may be any of the effects described within this specification.

1 is a graph showing the amount of melanin when a pepper extract-containing composition is added. 1 is a graph showing the amount of melanin when a composition containing Japanese pepper extract is added. 1 is a graph showing the amount of BMP4 mRNA expression when pepper extract is added. 1 is a graph showing the protein amount of c-KIT when pepper extract was added.

Preferred embodiments for implementing the present invention are described below. The embodiments described below are representative embodiments of the present invention, and the scope of the present invention is not limited to these embodiments.

<1. Overview>

The present invention provides a composition for inhibiting melanin production, a composition for promoting BMP4 expression in melanocytes, and a composition for inhibiting c-KIT expression in melanocytes. All of these compositions contain an extract of Zanthoxylum chinense pericarp as an active ingredient. In other words, the extract of Zanthoxylum chinense pericarp has the effects of inhibiting melanin production, promoting BMP4 expression in melanocytes, and inhibiting c-KIT expression in melanocytes. The relationship between melanin production and BMP4 and c-KIT is explained below.

Melanin is produced in melanocytes. BMP4 (Bone Morphogenetic Protein 4) is known to suppress melanin production in melanocytes.

c-KIT is a receptor for SCF (Stem Cell Factor), a melanocyte activating factor. When c-KIT expression is promoted in melanocytes, the melanocytes become more susceptible to activation by SCF, thereby promoting melanin production. In this way, c-KIT is known to promote melanin production in melanocytes.

The extract of Zanthoxylum chinense pericarp used in the present invention is thought to inhibit melanin production by promoting the expression of BMP4 and suppressing the expression of c-KIT in melanocytes.

The composition of the present invention is described in detail below.

<2. Composition for inhibiting melanin production>

(1) Ingredients

(1-1) Zanthoxylum chinense peel extract

A composition for inhibiting melanin production according to one embodiment of the present invention contains an extract of Zanthoxylum bungeanum pericarp as an active ingredient. Zanthoxylum bungeanum is a deciduous shrub of the genus Zanthoxylum in the family Rutaceae, and is also known as Kasho (Flower Pepper).

In this embodiment, the extract of Zanthoxylum japonicum pericarp is obtained by an extraction process that includes contacting an extraction raw material containing Zanthoxylum japonicum pericarp with a solvent. The extraction raw material used in the extraction process may include raw materials other than Zanthoxylum japonicum pericarp. The extraction raw material may include, for example, parts of Zanthoxylum japonicum other than the pericarp (e.g., fruit, seeds, leaves, trunk, branches, roots, and stem). The extraction raw material preferably does not include raw materials other than Zanthoxylum japonicum pericarp. This allows for efficient extraction of active ingredients. As used herein, "free of raw materials other than Zanthoxylum japonicum pericarp" means that the extract does not include any raw materials other than Zanthoxylum japonicum pericarp, and that the extract is substantially free of raw materials other than Zanthoxylum japonicum pericarp. "Substantially free of raw materials other than Zanthoxylum japonicum pericarp" means that no extraction raw materials other than Zanthoxylum japonicum pericarp have been intentionally added.

The Zanthoxylum japonicum pericarp used in the above extraction process may be processed after being collected from the fruit of Zanthoxylum japonicum. The post-collection process may be, for example, at least one process selected from washing, drying, shredding, and crushing. The process may be carried out by a method known to those skilled in the art. From the perspective of ease of acquisition, the Zanthoxylum japonicum pericarp used in the above extraction process may be dried, i.e., may be a dried product.

The solvent used in the extraction process may be any solvent known to those skilled in the art. Examples of solvents used in the extraction process include water, alcohols, glycols, ketones, esters, ethers, and hydrocarbons. Examples of alcohols include ethanol, methanol, and propanol. Examples of glycols include ethylene glycol, diethylene glycol, butylene glycol, and propylene glycol. Examples of ketones include acetone and methyl ethyl ketone. Examples of esters include ethyl acetate, propyl acetate, and ethyl formate. Examples of ethers include diethyl ether and tetrahydrofuran. Examples of hydrocarbons include hexane and benzene. One or a combination of two or more of these solvents may be used in the extraction process. The solvent used in the extraction process is preferably an alcohol solution (a mixed solvent of water and alcohol) or alcohol, more preferably an ethanol solution (a mixed solvent of water and ethanol) or ethanol, and even more preferably an ethanol solution. When the solvent used in the extraction process is an ethanol solution, the ethanol concentration may be, for example, 50% by volume or more, preferably 70% by volume or more, more preferably 80% by volume or more, even more preferably 85% by volume or more, and particularly preferably 90% by volume or more, or 95% by volume or more.

The extraction process is carried out by contacting the extraction raw material containing the pericarp of Zanthoxylum japonicum with the solvent. This contact may be, for example, by immersing the extraction raw material containing the pericarp of Zanthoxylum japonicum in the solvent.

The extraction process is preferably a heat extraction, which involves immersing the extraction material containing Zanthoxylum chinense pericarp in a solvent and heating it. This heat extraction is suitable for extracting the active ingredients of the composition for inhibiting melanin production according to this embodiment. The lower limit of the extraction temperature (solvent temperature) in the heat extraction is preferably a temperature above room temperature (20°C) (i.e., above 20°C), more preferably 30°C or higher, even more preferably 40°C or higher, and particularly preferably 50°C or higher. The upper limit of the extraction temperature in the heat extraction is preferably below the boiling point of the solvent. A preferred range of the extraction temperature in the heat extraction may be a combination selected from the lower and upper limits described above. When the solvent used in the extraction process has a low boiling point (e.g., a boiling point below 100°C), the heat extraction is preferably a heat reflux extraction. Specifically, when the solvent used in the extraction process is, for example, an ethanol solution or ethanol, the heat extraction is preferably a heat reflux extraction. This allows for efficient extraction. The preferred lower and upper limits of the extraction temperature in heated reflux extraction may be the same as the preferred lower and upper limits of the extraction temperature in heated extraction described above. Furthermore, the preferred numerical range of the extraction temperature in heated reflux extraction may be a combination selected from the lower and upper limits.

The extraction process time (the time the extraction material is in contact with the solvent) may be adjusted as appropriate by those skilled in the art depending on factors such as the amount of Zanthoxylum japonicum peel contained in the extraction material and the type of solvent. In the case of heated extraction (including heated reflux extraction), the extraction time may be, for example, 30 minutes to 24 hours, 30 minutes to 15 hours, 30 minutes to 10 hours, 30 minutes to 5 hours, or 1 hour to 5 hours.

The active ingredient of the melanin production-inhibiting composition is dissolved from the Zanthoxylum japonicum peel into the solvent by the extraction process. The Zanthoxylum japonicum peel extract used in the melanin production-inhibiting composition may be the solvent itself (raw extract) into which the active ingredient is dissolved by the extraction process, or it may be obtained by subjecting the raw extract to further processing after the extraction process. The processing performed on the raw extract after the extraction process may be at least one processing selected from purification, concentration, dilution, drying, and solvent substitution. In other words, the Zanthoxylum japonicum peel extract used in the melanin production-inhibiting composition may be, for example, a purified product, concentrate, dilution, dried product, or solvent-substituted product of the Zanthoxylum japonicum peel extract. Processing after the extraction process may be performed using methods known to those skilled in the art. Furthermore, the purification process may include, for example, filtration, washing, bleaching, deodorization, and sterilization.

The extract of Zanthoxylum japonicum pericarp used in the composition for inhibiting melanin production may be, for example, obtained by redissolving the dried extract of Zanthoxylum japonicum pericarp in a solvent. The solvent used for redissolution may be the same as or different from the solvent used in the extraction process.

The extract of Zanthoxylum japonicum pericarp used in the above-mentioned composition for inhibiting melanin production is preferably one that has been subjected to at least a purification process after extraction, i.e., it is preferably a purified extract of Zanthoxylum japonicum pericarp.

(1-2) Content of Zanthoxylum chinense peel extract

In the melanin production-inhibiting composition of this embodiment, the lower limit of the content of Zanthoxylum japonicum pericarp extract may be, for example, 1 μg/mL or more, preferably 3 μg/mL or more, more preferably 6 μg/mL or more, even more preferably 10 μg/mL or more, and particularly preferably 15 μg/mL or more, 20 μg/mL or more, 25 μg/mL or more, 30 μg/mL or more, 40 μg/mL or more, or 50 μg/mL or more, calculated on a dry solids basis. Such a content may result in a composition with a higher melanin production-inhibiting effect. The upper limit of the content of Zanthoxylum japonicum pericarp extract may be adjusted as appropriate by those skilled in the art depending on the intended use and form of the melanin production-inhibiting composition. The upper limit of the content may be, for example, 10 mg/mL or less, 5 mg/mL or less, or 2 mg/mL or less, calculated on a dry solids basis. A preferred range of the content may be a combination selected from the lower and upper limits described above. As used herein, "dry solids equivalent" refers to converting the amount of extract from Zanthoxylum chinense peel into dry solids, which is the amount excluding water. In this specification, the dry solids are measured in accordance with the evaporation residue test method, a general test method defined in the Quasi-drug Ingredients Standards 2021.

(1-3) Ingredients other than Zanthoxylum chinense peel extract

The composition for inhibiting melanin production of this embodiment may be a composition consisting of an extract of Zanthoxylum chinense pericarp. In other words, the composition for inhibiting melanin production may be a composition that does not contain any components other than the extract of Zanthoxylum chinense pericarp.

The melanin production-inhibiting composition may contain ingredients other than an extract of Zanthoxylum chinense pericarp. These ingredients may be ingredients known in the art, such as bases, solvents (e.g., water, alcohols, etc.), oils, preservatives, emulsifiers, colorants, antiseptics, surfactants, antioxidants, moisturizers, UV absorbers, thickeners, powders, chelating agents, pH adjusters, and fragrances. These ingredients may be included in the melanin production-inhibiting composition to the extent that they do not impair the effects of the present invention.

(2) Dosage form

The dosage form of the composition for inhibiting melanin production of this embodiment may be appropriately selected by those skilled in the art depending on the application and purpose. The dosage form may be, for example, a liquid, emulsion, cream, solid, gel, paste, or powder.

(3) Manufacturing method

The composition for inhibiting melanin production of this embodiment is produced by using the extract of Zanthoxylum chinense pericarp described above. That is, the method for producing the composition for inhibiting melanin production includes using the extract of Zanthoxylum chinense pericarp described above. In addition to using the extract of Zanthoxylum chinense pericarp described above, the production method may also include steps known in the art.

The extract of Zanthoxylum chinense pericarp used in the above manufacturing method is obtained by the extraction process described above in "(1-1) Extract of Zanthoxylum chinense pericarp." That is, the details of the extraction process are as described above in "(1-1) Extract of Zanthoxylum chinense pericarp."

In a preferred embodiment, the extract of Zanthoxylum japonicum pericarp used in the above-mentioned production method may be obtained by heat extraction (particularly heat reflux extraction), in which an extraction raw material containing Zanthoxylum japonicum pericarp (particularly dried Zanthoxylum japonicum pericarp) is contacted (particularly immersed) with an ethanol solution or ethanol and heated. The ethanol concentration of the ethanol solution is preferably 70% by volume or more, more preferably 80% by volume or more, even more preferably 85% by volume or more, and particularly preferably 90% by volume or more, or even 95% by volume or more. The lower limit of the extraction temperature in the heat extraction (particularly heat reflux extraction) is preferably a temperature above room temperature (20°C) (i.e., above 20°C), more preferably 30°C or more, even more preferably 40°C or more, and particularly preferably 50°C or more. The upper limit of the extraction temperature in the heat extraction (particularly heat reflux extraction) is preferably below the boiling point of the solvent. A preferred numerical range for the extraction temperature may be a combination selected from these lower and upper limits.

In the above manufacturing method, the extract of Zanthoxylum chinense pericarp may be used in the amount described above in "(1-2) Content of extract of Zanthoxylum chinense pericarp." In other words, the details of the amount of extract of Zanthoxylum chinense pericarp used in the above manufacturing method are as described above in "(1-2) Content of extract of Zanthoxylum chinense pericarp."

(4) Purpose

The composition for inhibiting melanin production in this embodiment may be, for example, a cosmetic, beauty product, quasi-drug, pharmaceutical, or food or beverage product for inhibiting melanin production, and is preferably a cosmetic or beauty product for inhibiting melanin production. Note that, in this specification, "cosmetics" refers to cosmetics and quasi-drugs (e.g., medicated cosmetics).

The melanin production-inhibiting composition described above may be used as a raw material for inhibiting melanin production in, for example, cosmetics, beauty products, quasi-drugs, pharmaceuticals, or food and beverage products. That is, the melanin production-inhibiting composition may be, for example, a raw material for inhibiting melanin production in cosmetics, beauty products, quasi-drugs, pharmaceuticals, or food and beverage products, and is preferably a raw material for inhibiting melanin production in cosmetics or cosmetics. The content of Zanthoxylum chinense peel extract in these cosmetics, beauty products, quasi-drugs, pharmaceuticals, or food and beverage products may be adjusted, for example, within the numerical range described above in "(1-2) Content of Zanthoxylum chinense peel extract."

The melanin production-inhibiting composition described above may be a whitening composition because it has the effect of inhibiting melanin production. Therefore, the melanin production-inhibiting composition may be, for example, a whitening cosmetic, a whitening cosmetic product, a whitening quasi-drug, a whitening pharmaceutical product, or a whitening food or beverage, and is preferably a whitening cosmetic or whitening cosmetic product. Note that, as used herein, "whitening" refers to preventing and/or improving skin pigmentation (e.g., age spots, freckles, uneven skin tone, etc.) caused by excessive melanin production, or maintaining a uniform skin tone.

(5) Effect

The above-mentioned composition for inhibiting melanin production has the effect of inhibiting melanin production. This effect can be confirmed by a decrease in the amount of melanin produced when the composition for inhibiting melanin production according to this embodiment is used compared to when it is not used. The amount of melanin produced can be measured, for example, by the procedure shown in Example 1 described below.

<3. Composition for promoting BMP4 expression in melanocytes>

(1) Ingredients, dosage form, and manufacturing method

A composition for promoting BMP4 expression in melanocytes according to one embodiment of the present invention contains an extract of Zanthoxylum chinense pericarp as an active ingredient. The ingredients, dosage form, and manufacturing method of this composition for promoting BMP4 expression may be the same as those described in "(1) Ingredients," "(2) Dosage Form," and "(3) Manufacturing Method" in "2. Composition for Inhibiting Melanin Production" above.

(2) Purpose

The composition for promoting BMP4 expression in melanocytes may be, for example, a cosmetic, makeup product, quasi-drug, pharmaceutical, or food or beverage product for promoting BMP4 expression in melanocytes, and is preferably a cosmetic or makeup product for promoting BMP4 expression in melanocytes.

The above-mentioned composition for promoting BMP4 expression in melanocytes may be used as a raw material for promoting BMP4 expression in melanocytes in, for example, cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products. That is, the composition for promoting BMP4 expression in melanocytes may be, for example, a raw material for promoting BMP4 expression in melanocytes for cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products, and is preferably a raw material for promoting BMP4 expression in melanocytes for cosmetics or cosmetics. The content of Zanthoxylum chinense peel extract in these cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products may be adjusted, for example, within the numerical range described above in "(1-2) Content of Zanthoxylum chinense peel extract."

The composition for promoting BMP4 expression in melanocytes can suppress melanin production by promoting BMP4 expression. Therefore, the composition for promoting BMP4 expression in melanocytes may be a whitening composition. Furthermore, the composition for promoting BMP4 expression in melanocytes may be, for example, a whitening cosmetic, whitening cosmetics, whitening quasi-drug, whitening pharmaceutical, or whitening food or beverage, and is preferably a whitening cosmetic or whitening cosmetics.

(3) Effect

The composition for promoting BMP4 expression in melanocytes has the effect of promoting the expression of BMP4 in melanocytes. This effect can be confirmed by an increase in the expression level of BMP4 in melanocytes when the composition for promoting BMP4 expression in melanocytes according to this embodiment is used compared to when it is not used. The expression level of BMP4 can be the expression level of BMP4 protein or the expression level of the gene encoding BMP4. The expression level of the protein or gene can be measured by methods known in the art. For example, the expression level of the gene encoding BMP4 can be measured according to the procedure for measuring the expression level of BMP4 mRNA, as shown in Example 2 below.

<4. Composition for inhibiting c-KIT expression in melanocytes>

(1) Ingredients, dosage form, and manufacturing method

A composition for inhibiting c-KIT expression in melanocytes according to one embodiment of the present invention contains an extract of Zanthoxylum chinense pericarp as an active ingredient. The ingredients, dosage form, and manufacturing method of the composition for inhibiting c-KIT expression may be the same as those described in "(1) Ingredients," "(2) Dosage Form," and "(3) Manufacturing Method" in "2. Composition for inhibiting melanin production" above.

(2) Purpose

The composition for inhibiting c-KIT expression in melanocytes may be, for example, a cosmetic, makeup product, quasi-drug, pharmaceutical, or food or beverage product for inhibiting c-KIT expression in melanocytes, and is preferably a cosmetic or makeup product for inhibiting c-KIT expression in melanocytes.

The composition for inhibiting c-KIT expression in melanocytes may be used as a raw material for inhibiting c-KIT expression in, for example, cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products. That is, the composition for inhibiting c-KIT expression in melanocytes may be, for example, a raw material for inhibiting c-KIT expression in cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products, and is preferably a raw material for inhibiting c-KIT expression in cosmetics or cosmetics. The content of Zanthoxylum chinense peel extract in the cosmetics, quasi-drugs, pharmaceuticals, or food and beverage products may be adjusted, for example, within the numerical range described above in "(1-2) Content of Zanthoxylum chinense peel extract."

The composition for inhibiting c-KIT expression in melanocytes can inhibit melanin production through the inhibition of c-KIT expression. Therefore, the composition for inhibiting c-KIT expression in melanocytes may be a whitening composition. Furthermore, the composition for inhibiting c-KIT expression in melanocytes may be, for example, a whitening cosmetic, whitening cosmetics, whitening quasi-drug, whitening pharmaceutical, or whitening food or beverage, and is preferably a whitening cosmetic or whitening cosmetics.

(3) Effect

The composition for inhibiting c-KIT expression in melanocytes has the effect of inhibiting the expression of c-KIT in melanocytes. This effect can be confirmed by a decrease in the expression level of c-KIT in melanocytes when the composition for inhibiting c-KIT expression in melanocytes of this embodiment is used compared to when it is not used. The expression level of c-KIT may be the expression level of the c-KIT protein or the expression level of the gene encoding c-KIT. The expression level of the protein or gene can be measured by methods known in the art. For example, the expression level of the c-KIT protein can be measured according to the procedure shown in Example 3 below.

<5. Whitening cosmetics>

The above-mentioned compositions for inhibiting melanin production, compositions for promoting BMP4 expression in melanocytes, and compositions for inhibiting c-KIT expression in melanocytes are all suitable for use in whitening cosmetics. Therefore, the present invention also provides whitening cosmetics containing any one of the compositions for inhibiting melanin production, compositions for promoting BMP4 expression in melanocytes, and compositions for inhibiting c-KIT expression in melanocytes.

(1) Ingredients

A whitening cosmetic according to one embodiment of the present invention may be a cosmetic comprising a composition for inhibiting melanin production, a cosmetic comprising a composition for promoting BMP4 expression in melanocytes, or a cosmetic comprising a composition for inhibiting c-KIT expression in melanocytes. In other words, the whitening cosmetic may be a cosmetic containing no ingredients other than the composition for inhibiting melanin production, the composition for promoting BMP4 expression in melanocytes, and the composition for inhibiting c-KIT expression in melanocytes.

The whitening cosmetic composition of this embodiment may be a cosmetic composition containing ingredients other than the composition for inhibiting melanin production, the composition for promoting BMP4 expression in melanocytes, and the composition for inhibiting c-KIT expression in melanocytes. These ingredients may be ingredients known in the art. For example, the whitening cosmetic composition may contain, in addition to these compositions, one or more ingredients known to have melanin production inhibitory or whitening effects.

In the above-mentioned whitening cosmetic, the lower limit of the content of Zanthoxylum japonicum peel extract may be, for example, 1 μg/mL or more, preferably 3 μg/mL or more, more preferably 6 μg/mL or more, even more preferably 10 μg/mL or more, and particularly preferably 15 μg/mL or more, 20 μg/mL or more, 25 μg/mL or more, 30 μg/mL or more, 40 μg/mL or more, or 50 μg/mL or more, calculated on a dry solids basis. Such a content may result in a composition with a higher melanin production inhibitory effect. The upper limit of the content of Zanthoxylum japonicum peel extract may be adjusted as appropriate by those skilled in the art depending on the intended use and form of the above-mentioned whitening cosmetic. The upper limit of the content may be, for example, 10 mg/mL or less, 5 mg/mL or less, or 2 mg/mL or less, calculated on a dry solids basis. A preferred range of the content may be a combination selected from the above-mentioned lower and upper limits.

(2) Form

The form of the whitening cosmetic may be appropriately selected by those skilled in the art depending on the application and purpose. Such forms may include, for example, basic cosmetics such as face wash, lotion, emulsion, gel, cream, serum, cosmetic oil, and pack; makeup cosmetics such as makeup base, foundation, face powder, lipstick, lip gloss, lip balm, eye shadow, and eye cream; sun care products such as sunscreen cream, sunscreen lotion, and sunscreen spray; and body cosmetics such as body cream and body lotion.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

<Preparation of Zanthoxylum chinense peel extract>

A 95% by volume ethanol solution (ethanol:water = 95:5) was added to dried peel of Zanthoxylum japonicum (Zanthoxylum japonicum) of the Rutaceae family, and heated under reflux for 3 hours before cooling. The mixture was filtered through filter paper, and an equal volume of water was added to the filtrate. The mixture was then left to stand at 4°C and then filtered through a membrane filter. An equal volume of 1,3-butylene glycol was added to the filtrate, and the mixture was thoroughly stirred and again filtered through a membrane filter to prepare an extract of Zanthoxylum japonicum peel (hereinafter also referred to as "Zanthoxylum japonicum extract") with a final solids concentration of 0.4 w/v%. The membrane filter used in the above membrane filtration was PTFE 0.5 μm (manufactured by Advantec Co., Ltd.).

<Preparation of Zanthoxylum chinense extract>

Dried peel of Zanthoxylum piperitum (Zanthoxylum piperitum) of the Rutaceae family was extracted with absolute ethanol and then filtered to prepare a Zanthoxylum piperitum extract (hereinafter also referred to as "Zanthoxylum piperitum extract") with a final solids concentration of 2.0 w/v%.

<Example 1: Verification of the melanin production inhibitory effect in cultured cells>

B16 melanoma cells were seeded into 10% FBS-containing MEM medium (Nissui Pharmaceutical Co., Ltd.) in a six-well plate and cultured at 37°C in a carbon dioxide concentration of 5%. The following day, the pepper extract was diluted with a 50% ethanol solution (ethanol:water = 50:50) to prepare six pepper extract-containing compositions with different concentrations. Each pepper extract-containing composition was added and mixed to the medium so that the concentration of pepper extract in the medium was 0 μg/mL (control), 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, or 50 μg/mL. Two days after addition, the medium was replaced, and the above composition was added again at the same concentration. The following day, the medium was removed, and the cells were washed with phosphate buffer and then harvested. Sodium hydroxide solution was added to the harvested cells to dissolve melanin, and the amount of melanin in the cells was quantified by measuring absorbance. The effect of adding a composition containing pepper extract on melanin levels was confirmed by standardizing the total amount of melanin in cells by cell number.

As a comparative example, Japanese pepper extract was also diluted with a 50% by volume ethanol solution (ethanol:water = 50:50) to prepare three Japanese pepper extract-containing compositions with different concentrations of Japanese pepper extract. Each Japanese pepper extract-containing composition was added and mixed so that the concentration of Japanese pepper extract in the medium was 0 μg/mL (control), 3.125 μg/mL, or 6.25 μg/mL. Tests were then conducted using the same procedures as for the Japanese pepper extract-containing compositions described above. Note that when the concentration of Japanese pepper extract in the medium exceeded 6.25 μg/mL, a decrease in cell growth rate was observed. Therefore, when the concentration of Japanese pepper extract in the medium exceeded 6.25 μg/mL, melanin levels were not examined.

Figure 1 is a graph showing the amount of melanin when a pepper extract-containing composition was added. In Figure 1, the vertical axis shows the relative amount when the amount of melanin per cell in the control group is set to 1. As shown in Figure 1, the addition of the pepper extract-containing composition resulted in a concentration-dependent decrease in the amount of melanin. These results demonstrate that the pepper extract-containing composition is capable of suppressing melanin production.

Figure 2 is a graph showing the amount of melanin when a composition containing Japanese pepper extract was added. In Figure 2, the vertical axis shows the relative amount when the amount of melanin per cell in the control group is set to 1. As shown in Figure 2, the amount of melanin increased slightly with the addition of a composition containing Japanese pepper extract. These results demonstrate that a composition containing Japanese pepper extract is unable to suppress melanin production, but rather has the effect of promoting melanin production.

<Example 2: Verification of the effect of promoting BMP4 expression in human epidermal melanocytes>

Normal human epidermal melanocytes (Sigma) were seeded in a 6-well plate containing human epidermal melanocyte medium (Medium 254, Thermofisher) containing HMGS growth additive (Thermofisher). The cells were then cultured at 37°C in a 5% CO2 atmosphere. After 3 days, pepper extract was added to the medium at concentrations of 0 μg/mL (control), 100 μg/mL, or 200 μg/mL. After 24 hours, the cells (normal human epidermal melanocytes) were washed with phosphate buffer and then harvested. mRNA was extracted from the harvested cells using the RNeasy Plus Mini Kit (QIAGEN). Subsequently, human BMP4 mRNA expression levels were measured by quantitative real-time PCR using cDNA prepared using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (BioRad). The BMP4 forward primer (actgcaaccgttcagaggtc) and the BMP4 reverse primer (aaacttgctggaaaggctca) were used in the quantitative real-time PCR. The BMP4 mRNA expression levels were normalized to the β-actin mRNA level and calculated as a ratio to the control mRNA level.

Figure 3 is a graph showing the level of BMP4 mRNA expression when pepper extract was added. In Figure 3, the vertical axis shows the relative level when the mRNA expression level in the control group is set to 1. As shown in Figure 3, the addition of pepper extract increased the level of BMP4 mRNA expression in melanocytes. These results demonstrate that pepper extract can promote the expression of BMP4 in melanocytes.

<Example 3: Verification of the effect of suppressing c-KIT expression in human epidermal melanocytes>

Human epidermal melanocyte medium (Medium 254 medium, Thermofisher) containing HMGS growth additive (Thermofisher) was added to a 35 mm dish, and normal human epidermal melanocytes (Human Epidermal Melanocytes, Sigma) were seeded and cultured at 37°C in a carbon dioxide concentration of 5 vol%. After two days, pepper extract was added to the medium to achieve a concentration of 0 μg/mL (control), 100 μg/mL, or 200 μg/mL. After 24 hours, the cells were irradiated with 80 mJ/ ^cm2 of UVB. After 24 hours, the cells were washed with phosphate buffer and then collected. Proteins were collected from the cells, and the protein amount was analyzed using Western blotting. The primary antibody used was a c-KIT antibody (IBL), and the secondary antibody used was an Anti-Mouse secondary antibody (Protein Simple). The amount of c-KIT protein obtained was normalized to the amount of β-actin protein and calculated as a ratio to the amount of the target protein.

Figure 4 is a graph showing the amount of c-KIT protein when pepper extract was added. In Figure 4, the vertical axis shows the relative amount, with the protein amount in the control group set at 1. As shown in Figure 4, the addition of pepper extract reduced the amount of c-KIT protein in melanocytes. These results demonstrate that pepper extract can suppress the expression of c-KIT in melanocytes.

<Example 4: Production of lotion>

(Component) (%)
1. Glycerin 5.0
2. 1,3-butylene glycol 6.5
3. Polyoxyethylene (20E.O.) sorbitan monolaurate 1.2
4. Ethanol 12.0
5. Lactic acid 0.05
6. Sodium lactate 0.1
7. Ascorbic acid glucoside 2.0
8. Kasho extract 0.01
9. Methyl parahydroxybenzoate 0.1
10. Flavoring: appropriate amount 11. Remaining amount of purified water

(Manufacturing method)
A: Components 3, 4, 9, and 10 were mixed and dissolved.
B: Components 1, 2, 5 to 8 and 11 were mixed and dissolved.
C: A and B were mixed uniformly to obtain a lotion.

<Example 5: Preparation of oil-in-water emulsion>

(Component) (%)
1. Polyoxyethylene (6E.O.) sorbitan monostearate 1.0
2. Polyoxyethylene tetraoleate (60E.O.) sorbitol 0.5
3. Glyceryl monostearate 1.0
4. Stearic acid 0.5
5. Behenyl alcohol 0.5
6. Squalane 8.0
7. Arbutin 3.0
8. Ethanol 5.0
9. Kasho extract 0.05
10. Purified water remaining 11. Methyl parahydroxybenzoate 0.1
12. Carboxyvinyl polymer 0.2
13. Sodium hydroxide 0.1
14. Hyaluronic acid 0.1
15. Fragrance (appropriate amount)

(Manufacturing method)
A: Component 10 was heated and maintained at 70°C.
B: Components 1 to 6, 8 and 11 were mixed and heated to 70°C.
C: A was added to B and mixed to emulsify uniformly.
D: After cooling C, ingredients 7, 9, and 12 to 15 were added and mixed uniformly to obtain an oil-in-water emulsion.

<Example 6: Preparation of oil-in-water emulsion>

(Component) (%)
1. 1,3-butylene glycol 12.0
2. Tranexamic acid 4.0
3. Purified water remaining 4. Polyethylene glycol monostearate (40 E.O.) 0.5
5. Sorbitan sesquioleate 0.1
6. Hydrogenated lecithin 0.1
7. Cholesteryl hydroxystearate (Note 1) 3.0
8. Vaseline 2.0
9. α-olefin oligomer 5.0
10. Shea Butter 2.0
11. Dimethylpolysiloxane (10CS) 1.0
12. Ceramide 3 0.1
13. Astaxanthin 0.1
14. Tocopherol 0.01
15. Cetostearyl alcohol 2.0
16. Behenyl alcohol 1.0
17. Methyl parahydroxybenzoate 0.1
18. Acrylic acid/alkyl methacrylate copolymer (Note 2) 0.1
19. Xanthan gum 0.1
20. Sodium hydroxide 0.03
21. Kasho extract 0.1
22. Fragrance (Note 1) Salakos HS (manufactured by Nisshin Oillio Co., Ltd.)
(Note 2) Carbopol ULTREZ21 (manufactured by Lubrizol)

(Manufacturing method)
A: Components 1 to 3 were uniformly dissolved and mixed at 70°C.
B: Components 4 to 16 were uniformly dissolved and mixed at 80°C.
C: B was added to A and emulsified at 70°C.
D: Components 17 to 22 were added to C and mixed, then cooled to 40°C to obtain an oil-in-water emulsion.

<Example 7: Preparation of an oil-in-water emulsion serum>

(Component) (%)
1. 1,3-butylene glycol 5.0
2. Tranexamic acid 1.0
3. Tripropylene glycol 3.0
4. Purified water remaining amount 5. Polyoxyethylene sorbitan monooleate (20 E.O.) 0.1
6. Polyethylene glycol monostearate (55 E.O.) 0.25
7. Macadamia nut oil fatty acid phytosteryl (Note 3) 2.0
8. Light liquid isoparaffin (Note 4) 3.0
9. Dimethylpolysiloxane (6CS) 3.0
10. Cholesterol 0.1
11. Tocopherol 0.01
12. Cetostearyl alcohol 0.5
13. Methyl parahydroxybenzoate 0.1
14. Carbomer (Note 5) 0.15
15. (Sodium acrylate/sodium acryloyldimethyltaurate)
Copolymer (Note 6) 0.1
16. Sodium hydroxide 0.05
17. Ethanol 5.0
18. Collagen 0.1
19. Elastin 0.1
20. Hyaluronic acid 0.1
21. Kasho extract 0.1
22. Fragrance 0.05
(Note 3) PLANDOOL-MAS (manufactured by Nippon Fine Chemical Co., Ltd.)
(Note 4) Chloratum LES (manufactured by Croda)
(Note 5) CARBOPOL 980 (manufactured by LUBURIZOL)
(Note 6) SIMULGEL EG (SEPIC)

(Manufacturing method)
A: Components 1 to 4 were uniformly dissolved and mixed at 70°C.
B: Components 5 to 11 were uniformly dissolved and mixed at 80°C.
C: B was added to A and emulsified at 70°C.
D: Components 12 to 22 were added to C and mixed, then cooled to 40°C to obtain an oil-in-water emulsion beauty serum.

<Example 8: Production of Sheet-Type Pack Cosmetic>

(Component) (%)
1. Isostearic acid 0.05
2. Polyoxyethylene (60 mol) hydrogenated castor oil 0.3
3. 2-ethylhexyl methoxycinnamate 0.05
4. Glycerin 3.0
5. 1,3-butylene glycol 5.0
6. Ethanol 8.0
7. Sodium citrate 0.02
8. Citric acid 0.05
9. Dipotassium glycyrrhizinate 0.1
10. Phenoxyethanol 0.05
11. Niacinamide 4.0
12. 40% aqueous solution of pentasodium diethylenetriaminepentaacetate 0.15
13. Kasho extract 0.02
14. Remaining purified water

(Manufacturing method)
A: Components 1 to 6 were mixed and dissolved.
B: Components 7 to 14 were mixed and dissolved.
C: A was added to B and mixed.
D: C was impregnated into a nonwoven fabric to obtain a sheet-type pack cosmetic.

<Example 9: Preparation of Ointment>

(Component) (%)
1. Triethanolamine 2.0
2. Glycerin 8.0
3. Purified water remaining 4. Kasho extract 0.001
5. Cetyl alcohol 6.0
6. Vaseline 32.0
7. Tocopherol 0.01
8. Stearyl glycyrrhetinate 0.1
9. Stearic acid 18.0
10. Sorbitan sesquioleate 1.5

(Manufacturing method)
A. Components 5 to 10 were dissolved uniformly at 75°C.
B. Components 1 to 4 were dissolved uniformly at 75°C.
C. B was gradually added to A, emulsified at 75°C, and cooled to room temperature to obtain an ointment.

When the lotion, emulsion, serum, sheet pack cosmetic, and ointment produced in Examples 4 to 9 above were each applied to human skin, all were confirmed to be effective in preventing age spots and freckles. These results demonstrate that the composition containing the above-mentioned pepper extract has the effect of inhibiting melanin production, and that cosmetics containing this composition have a whitening effect.

Claims (2)

A composition for promoting BMP4 expression in melanocytes, containing an extract of Zanthoxylum chinense pericarp as the active ingredient. A composition for inhibiting c-KIT expression in melanocytes, containing an extract of Zanthoxylum chinense pericarp as the active ingredient.