JP7687568B2 - Composition for inhibiting melanin production, composition for whitening, and composition for inhibiting expression - Google Patents

Description

The present invention relates to a composition for inhibiting melanin production, a composition for whitening, and a composition for inhibiting its expression.

One of the causes of pigmentation, age spots, freckles, etc. due to sunburn etc. is the excessive production of melanin in melanocytes. Whitening compositions that inhibit melanin production are known, for example, as those disclosed in Patent Documents 1 and 2. Patent Document 1 discloses a whitening composition that inhibits the activity of tyrosinase, an enzyme involved in the production of melanin from tyrosine. Patent Document 2 discloses an external skin preparation that inhibits the expression of MC1R (melanocortin-1 receptor), a melanocyte-stimulating hormone receptor.

JP 2011-088856 A JP 2021-004215 A

In recent years, various studies have been conducted on the effects of microalgae. The present invention was made based on the new discovery, as a result of intensive research by the present researchers, that an extract of Nostoc verrucosum, an alga belonging to the Nostoc genus of the Nostoc family in the Cyanobacteria order Nostociales, exhibits an effect of suppressing melanin production and the expression of genes involved in melanin production.

The composition for inhibiting melanin production that solves the above problems contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.
The composition for inhibiting melanin production that solves the above problem is a composition for inhibiting melanin production that inhibits the production of melanin promoted by stimulation of melanocyte-stimulating hormone, and contains as an active ingredient an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.

The skin whitening composition that solves the above problems contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.
The expression-inhibiting composition that solves the above problem is an expression-inhibiting composition that inhibits tyrosinase gene expression, and contains as an active ingredient an extract of Nostoc vercosum extracted using an extraction solvent containing an organic solvent.

The expression-suppressing composition that solves the above problem is an expression-suppressing composition that suppresses the gene expression of MC1R, and contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.

The present invention provides novel compositions for inhibiting melanin production, whitening, and expression.

Graph showing the amount of melanin produced upon treatment with hexane extract in Test 1. Graph showing the amount of melanin produced upon treatment with methanol extract in Test 1. Graph showing tyrosinase mRNA expression levels upon treatment with hexane extract in Test 2. Graph showing MC1R mRNA expression levels upon treatment with hexane extract in Experiment 2. Graph showing the mRNA expression levels of tyrosinase and MC1R upon treatment with hexane extract in Experiment 3.

Hereinafter, one embodiment of the present invention will be described.
The composition of this embodiment (hereinafter referred to as the present composition) contains an extract of Nostoc vercosum as an active ingredient.

[Raw materials]
Nostoc verrucosum (Japanese name: Ashitsuki) is an alga of the Nostoc genus in the Nostoc family of the Nostocaceae order of the Cyanobacteria class. Nostoc verrucosum (hereinafter referred to as the above alga) may be a naturally occurring alga or an artificially cultured alga. From the viewpoints of stable supply and ease of quality maintenance, it is industrially preferable to use artificially cultured alga.

The extract contained in the present composition can be obtained by carrying out an extraction treatment using the algae as a raw material.
The algal cells as the raw material may be in the state as collected, crushed after collection, dried after collection, or crushed and dried after collection. From the viewpoint of efficiency in the extraction process, it is preferable to use crushed algal cells as the raw material.

[Extract]
Examples of the extraction solvent for extracting the extract from the algae include organic solvents and mixed solvents of organic solvents and water. Examples of the organic solvent include lower alcohols, hexane, ethyl acetate, dimethyl sulfoxide, acetonitrile, acetone, glycerin, and propylene glycol. Examples of the lower alcohol include alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propanol, isopropanol, and butanol.

As the organic solvent, a single type may be used, or a mixed solvent of multiple types may be used. When a mixed solvent of water and an organic solvent is used as the extraction solvent, the content of the organic solvent in the mixed solvent is preferably 50% by volume or more, more preferably 80% by volume or more. In addition, additives such as organic salts, inorganic salts, buffers, and emulsifiers may be dissolved in the extraction solvent.

The method for extracting the extract from the algae may be any known extraction method, such as cold extraction, room temperature extraction, or heated extraction. The extraction temperature may be appropriately set depending on the type of solvent, extraction efficiency, deterioration of the components, etc.

The extraction operation is carried out by immersing the algae as the raw material in the extraction solvent for a predetermined period of time. In this case, the concentration of the algae in the extraction solvent can be appropriately set depending on the type of extraction solvent, the extraction efficiency, the efficiency of the concentration process after extraction, etc. In such an extraction operation, in order to increase the extraction efficiency, further treatments such as refluxing, stirring, pressurization, and ultrasonic treatment may be carried out as necessary.

The extraction operation and solid-liquid separation operation may be performed once or multiple times on the same algae. When performing the extraction operation multiple times, the same extraction operation may be performed repeatedly, or different extraction operations may be performed in combination. For example, after performing the extraction operation using an organic solvent and the solid-liquid separation operation, the algae that has been subjected to these operations may be subjected to an extraction operation using another organic solvent. When performing a combination of different extraction operations, the extracts obtained by each extraction operation may be mixed and used as one extract, or may be used as separate, different extracts.

[Action and application field]
Ingestion of the present composition suppresses melanin production, and therefore the present composition can be used as a composition for inhibiting melanin production, which is intended to exert an inhibitory effect on melanin production, and as a composition for whitening, which is intended to exert a whitening effect based on the inhibition of melanin production.

In addition, by ingesting this composition, gene expression of tyrosinase, an enzyme involved in the production of melanin from tyrosine, is suppressed. Therefore, this composition can be used as an expression suppressing composition intended to exert an inhibitory effect on gene expression of tyrosinase. The above extract contained in the expression suppressing composition intended to exert an inhibitory effect on gene expression of tyrosinase is preferably a hexane extract extracted using an extraction solvent containing hexane. In this case, the effect of suppressing gene expression of tyrosinase is more pronounced.

Furthermore, by ingesting this composition, gene expression of MC1R (melanocortin-1 receptor) is suppressed. Therefore, this composition can be used as an expression-suppressing composition intended to exert an inhibitory effect on the expression of MC1R. MC1R is a receptor present on the cell membrane of melanocytes or melanoma. The above extract contained in the expression-suppressing composition intended to exert an inhibitory effect on the expression of the MC1R gene is preferably a hexane extract extracted using an extraction solvent containing hexane. In this case, the effect of suppressing the expression of the MC1R gene is more pronounced.

The mechanism by which melanin production is inhibited when the present composition is ingested can be assumed as follows: Melanin is produced in the body through the following steps 1 to 4.
Step 1: Exposure to ultraviolet rays and active oxygen.

Step 2: Melanocyte-stimulating hormone is secreted.
Step 3: Upon binding of melanocyte-stimulating hormone to MC1R, production of enzymes involved in melanin synthesis, such as tyrosinase, is promoted.

Step 4: Melanin is synthesized by enzymes.
When melanocyte-stimulating hormone is secreted in step 2, the composition inhibits the production of the enzyme in step 3. As a result, melanin synthesis in step 4 is inhibited. Therefore, the composition can be used as a melanin production inhibiting composition for inhibiting melanin production promoted by stimulation of melanocyte-stimulating hormone. Specifically, the composition can be selectively used for subjects in which melanocyte-stimulating hormone is secreted in step 2 and the concentration of melanocyte-stimulating hormone is increased or is likely to increase.

The melanin production inhibiting composition, skin whitening composition, and expression inhibiting composition of the present composition can each be applied in various fields such as food and beverages, pharmaceuticals, quasi-drugs, and cosmetics.
Examples of the food and drink include various beverages (drinks containing fruit or vegetable juice, soft drinks, mineral drinks, sports drinks, tea drinks, coffee, carbonated drinks, dairy products such as milk and yogurt, etc.), jelly foods (jelly, agar, jelly drinks, etc.), capsules (soft capsules, hard capsules), and various confectioneries. The food and drink may appropriately contain gelling agents such as pectin and carrageenan, sugars and sweeteners such as glucose, sucrose, fructose, lactose, stevia, aspartame, and sugar alcohols, food additives such as flavorings, oils and fats such as vegetable oils and animal oils and fats, etc. The uses of the food and drink are not particularly limited, and the food and drink may be used as so-called general foods, health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods with functional claims, and foods for the sick.

When used as a medicine, quasi-drug, or cosmetic, the method of administration is not particularly limited. Specific administration methods include, for example, administration by ingestion (oral ingestion), intravascular administration, enteral administration, transdermal administration, and intraperitoneal administration. Furthermore, when used as a medicine, quasi-drug, or cosmetic, the dosage form is not particularly limited. Specific dosage forms include, for example, powders, powders, granules, tablets, capsules, pills, suppositories, liquids, and injections. Furthermore, additives such as excipients, bases, emulsifiers, solvents, and stabilizers may be contained.

Next, the effects of the above embodiment will be described.
(1) The composition for inhibiting melanin production contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.

According to the above-mentioned configuration, the effect of suppressing melanin production can be obtained.
(2) The composition for inhibiting melanin production inhibits melanin production promoted by stimulation of melanocyte-stimulating hormone. The composition for inhibiting melanin production contains, as an active ingredient, an extract of Nostoc verrucosum extracted with an extraction solvent containing an organic solvent.

The above-mentioned configuration allows selective use in subjects who have secreted melanocyte-stimulating hormone and have seen an increase in the concentration of melanocyte-stimulating hormone or who are likely to experience an increase in said concentration.

(3) The whitening composition contains an extract of Nostoc vercosum extracted using an extraction solvent containing an organic solvent.
According to the above-mentioned composition, the production of melanin is suppressed, thereby achieving a skin whitening effect.

(4) A composition for inhibiting expression that inhibits the expression of a tyrosinase gene contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.
According to the above-mentioned composition, the effect of suppressing the expression of the tyrosinase gene can be obtained.

(5) A composition for suppressing expression of the MC1R gene contains, as an active ingredient, an extract of Nostoc vercosum extracted using an extraction solvent containing an organic solvent.
According to the above configuration, the effect of suppressing the expression of the MC1R gene can be obtained.

This embodiment can be modified as follows: This embodiment and the following modifications can be combined with each other to the extent that no technical contradiction occurs.
The composition for inhibiting melanin production, the composition for whitening, and the composition for inhibiting expression may contain other ingredients to the extent that the intended effects of each ingredient are not impaired.

- The amount and duration of intake of the composition for inhibiting melanin production, the composition for whitening, and the composition for inhibiting expression are not particularly limited and are determined appropriately taking into consideration the state of the body functions, age, sex, and other conditions of the person taking them.

- The melanin production-inhibiting composition, skin-whitening composition, and expression-inhibiting composition can be applied not only to humans, but also to feed, medicines, etc. for livestock and other captive animals.

Next, the technical ideas that can be understood from the above embodiment and modified examples will be described.
(i) A method for producing a composition for inhibiting melanin production, a composition for whitening, and a composition for inhibiting expression, comprising an extraction step of obtaining an extract from Nostoc vercosum using an extraction solvent containing an organic solvent.

[Explanation of impossible/impractical circumstances]
The description in the claims of this application of "an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent" corresponds to a case where "circumstances exist at the time of filing that make it impossible or nearly impractical to directly identify the substance by its structure or characteristics ('impossible or impractical circumstances')."

The components of Nostoc verrucosum contain a countless number of components that dissolve in extraction solvents, including organic solvents. The task of isolating active ingredients that have effects such as inhibiting melanin production from these countless components and analyzing their structures requires significantly excessive economic expenditure and time. Furthermore, when formulating a drug, it is common to include specific active ingredients in the form of extracts or crudely purified products rather than in an isolated and purified state. In particular, biological substances tend to be safer in their unrefined or crudely purified state compared to chemically synthesized substances, and therefore they are actively included in their unrefined or crudely purified state.

Therefore, in the world of patent acquisition, where technology is rapidly advancing and competition is fierce on an international scale, it is unreasonable to require an applicant to isolate an active ingredient that has an effect of inhibiting melanin production from an extract of Nostoc verrucosum and analyze its structure. Therefore, the description in the claims of this patent application of "an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent" corresponds to a case where "at the time of filing, there exist circumstances in which it is impossible or practically impractical to directly identify the substance by its structure or characteristics ('impossible/impractical circumstances')."

The above embodiment will be described in more detail below with reference to test examples, although the present invention is not limited thereto.
[Preparation of extract]
Hexane (2000 mL) was added to the dried powder (10 g) of Nostoc verrucosum and stirred at room temperature for 60 minutes. The liquid obtained by centrifugation and filtration was then concentrated under reduced pressure to obtain 16.3 mg of concentrate. The concentrate was dissolved in dimethyl sulfoxide to prepare a hexane extract with a solid concentration of 100 μg/mL.

Methanol (2000 mL) was added to the residue of Nostoc verrucosum, which was the solid matter recovered by filtration after the above-mentioned hexane extraction process, and the mixture was stirred at room temperature for 60 minutes. The liquid obtained by centrifugation and filtration was then concentrated under reduced pressure to obtain 999.7 mg of concentrate. The concentrate was dissolved in dimethyl sulfoxide to prepare a methanol extract with a solid concentration of 100 μg/mL.

Water (2000 mL) was added to the residue of Nostoc verrucosum, which was the solid matter recovered by filtration after the above-mentioned extraction with methanol, and the mixture was stirred at 90-95°C for 60 minutes. The liquid obtained by centrifugation and filtration was then concentrated under reduced pressure to obtain 2350 g of concentrate. The concentrate thus obtained was dissolved in ultrapure water to prepare a hot water extract with a solid matter concentration of 100 μg/mL.

[Preparation of medium]
Low glucose Dulbecco's modified Eagle's medium (DMEM: Sigma-Aldrich D6046) was supplemented with heat-inactivated fetal bovine serum to a final concentration of 5%, penicillin to a final concentration of 50 units/mL, and streptomycin to a final concentration of 50 μg/mL, and this was used as the medium for the tests described below.

[Test 1: Measurement of melanin production]
The effect of adding each extract on melanin production promoted by stimulation with α-MSH (melanocyte stimulating hormone) was evaluated. α-MSH is a physiologically active substance that functions as a ligand for MC1R present on the cell membrane of melanocytes or melanomas. α-MSH activates the intracellular signal transduction system and induces tyrosinase expression, ultimately inducing melanin production.

B16F1 cells (cultured mouse melanoma cells) were seeded at 1.0 x ¹⁰⁵ cells/60 mm dish and cultured in the above medium at 37°C and 5% _CO2 for 24 hours. After that, the medium was replaced and α-MSH was added to the medium to a final concentration of 0.10 nM, and a hexane extract or methanol extract was added to the medium.

The hexane extract was added to the medium to give a final concentration of 0-25 μL/mL. The methanol extract was added to the medium to give a final concentration of 0-20 μL/mL. An equal amount of dimethylsulfoxide was added to the control with a final concentration of 0 μL/mL. After culturing for 72 hours under conditions of 37°C and 5% _CO2 , the medium was removed and the cells were treated with trypsin/EDTA to obtain a cell suspension. The cell suspension was collected and the cell number was counted using a Fuchs-Rosenthal cell counting chamber.

Next, the cell suspension was centrifuged at 1000 rpm for 5 minutes, resuspended in PBS, and then centrifuged at 1000 rpm for 5 minutes to obtain a cell pellet. 2M NaOH was poured into the cell pellet so that 100 μL per 1×10 ⁶ cells was added, and the cell pellet was treated in a water bath at 100° C. for 20 minutes. After confirming that the melanin was completely dissolved, the absorbance at 405 nm was measured. The measured value was taken as the melanin content, and the relative value when each extract was added was calculated with the melanin content when only α-MSH was added being taken as 100%. The results are shown in the graphs of FIG. 1 and FIG. 2.

The graphs in Figures 1 and 2 show that the melanin production promoted by the stimulation of α-MSH was significantly suppressed by adding the hexane extract or methanol extract. In particular, the hexane extract was found to be highly effective in suppressing melanin production. In addition, although details of the test are omitted, no change in the amount of melanin produced was confirmed when the hot water extract was added instead of the hexane extract or methanol extract.

[Test 2: Measurement of expression levels of genes involved in melanin synthesis (72-hour treatment)]
The effect of adding each extract on the gene expression of tyrosinase and MC1R, which is promoted by stimulation with α-MSH, was evaluated.

B16F1 cells were seeded at 1.0 x ¹⁰⁵ cells/60 mm dish and cultured in the above medium at 37°C and 5% _CO2 for 24 hours. The medium was then replaced, and α-MSH was added to the medium at a final concentration of 0.10 nM, and the hexane extract was added to the medium at a final concentration of 0 to 25 μL/mL. An equal amount of dimethylsulfoxide was added to the control with a final concentration of 0 μL/mL.

After culturing for 72 hours under conditions of 37°C and 5% _CO2 , the medium was removed, the cells were lysed using an RNA extraction reagent (ISOGEN2, Nippon Gene Co., Ltd.), and total RNA was extracted. Then, cDNA was prepared using an RT-PCR kit (RNA PCR kit (AMV) Ver.3.0, Takara Bio Inc.). The obtained cDNA, primers, and PCR reagent (Thunderbird SYBR qPCR mix, TOYOBO Co., Ltd.) were mixed and a real-time PCR reaction was performed. The mRNA expression levels of tyrosinase, MC1R, and GAPDH as an internal standard were measured using the ^2-ΔΔCt method.

A real-time PCR system (Quant Studio 3, Applied Biosystems) was used for the real-time PCR reaction. The reaction conditions for the real-time PCR reaction were 95°C for 10 seconds and 60°C for 30 seconds per cycle, for a total of 40 cycles.

The measured tyrosinase mRNA expression level was corrected by the GAPDH mRNA expression level, and the tyrosinase mRNA expression level was calculated by setting the corrected value of the tyrosinase mRNA expression level when α-MSH was added and no hexane extract was added to "1". The results are shown in the graph in Figure 3. Similarly, the measured MC1R mRNA expression level was corrected by the GAPDH mRNA expression level, and the MC1R mRNA expression level was calculated by setting the corrected value of the MC1R mRNA expression level when α-MSH was added and no test solution was added to "1". The results are shown in the graph in Figure 4.

As shown in the graph in Figure 3, in the absence of α-MSH, no reduction in the tyrosinase mRNA expression level was observed with the addition of the hexane extract. On the other hand, in the presence of α-MSH, the expression level was significantly reduced with the addition of the hexane extract. As shown in the graph in Figure 4, in the absence and presence of α-MSH, the expression level of MC1R mRNA was significantly reduced with the addition of the hexane extract. In addition, although details of the test are omitted, a similar test was performed using a methanol extract instead of a hexane extract, and a reduction in the MC1R mRNA expression level with the addition of the hexane extract was confirmed.

[Test 3: Measurement of expression levels of genes involved in melanin synthesis (24-hour treatment)]
The expression levels of tyrosinase mRNA and MC1R mRNA were determined by carrying out a test similar to that of Test 2, except that the incubation time after the addition of α-MSH and the hexane extract in Test 2 was changed to 24 hours. The results are shown in the graph of FIG.

As shown in the graph in Figure 5, the expression level of tyrosinase mRNA was significantly decreased by the addition of the hexane extract in both the absence and presence of α-MSH. As shown in the graph in Figure 5, the expression level of MC1R mRNA was significantly decreased by the addition of the hexane extract in both the absence and presence of α-MSH.

The results of Tests 2 and 3 show that when the above extract of Nostoc verrucosum is added, gene expression of tyrosinase and MC1R is suppressed due to transcriptional suppression of tyrosinase and MC1R. Furthermore, it is found that as a result of the suppression of gene expression of tyrosinase and MC1R, melanin production is suppressed.

Claims (5)

A composition for inhibiting melanin production, comprising as an active ingredient an extract of Nostoc vercosum extracted using hexane . A composition for inhibiting melanin production, which inhibits melanin production promoted by the secretion of melanocyte-stimulating hormone,
A composition for inhibiting melanin production, comprising as an active ingredient an extract of Nostoc vercosum extracted using hexane . A skin-whitening composition containing the melanin production inhibiting composition according to claim 1 or 2. A composition for suppressing expression of a tyrosinase gene, comprising:
A composition for inhibiting expression, comprising as an active ingredient an extract of Nostoc verrucosum extracted using hexane . A composition for suppressing expression of MC1R gene, comprising:
A composition for inhibiting expression, comprising as an active ingredient an extract of Nostoc verrucosum extracted using hexane .