Composition for inhibiting melanin production, composition for whitening, and composition for suppressing expression

Abstract

To provide a novel melanogenesis inhibitory composition.SOLUTION: A melanogenesis inhibitory composition contains extract of Nostoc verrucosum, extracted using extraction solvents such as an organic solvent, as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for suppressing melanin production, a composition for whitening, and a composition for suppressing expression.

Background Art

[0002] Excessive production of melanin in melanocytes is one of the causes of pigmentation, freckles, and lentigines due to sunburn and the like. As a composition for whitening that suppresses melanin production, for example, those disclosed in Patent Document 1 and Patent Document 2 are known. Patent Document 1 discloses a composition for whitening that suppresses the activity of tyrosinase, an enzyme involved in the production of melanin from tyrosine. Patent Document 2 discloses an external preparation for skin that suppresses the expression of MC1R (melanocortin-1 receptor), a melanocyte-stimulating hormone receptor.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] In recent years, various studies have been conducted on the effects of microalgae. As a result of intensive research by the present inventors, an extract of Nostoc verrucosum, which is a thallus belonging to the genus Nostoc of the order Nostocales of the class Cyanophyceae, has been newly found to exhibit an action of suppressing melanin production and an action of suppressing the expression of genes involved in melanin production.

Means for Solving the Problems

[0005] The composition for suppressing 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 suppressing melanin production that solves the above problems is a composition for suppressing melanin production that suppresses the production of melanin promoted by the 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.

[0006] The composition for whitening 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 suppressing gene expression that solves the above problems is a composition for suppressing gene expression that suppresses the gene expression of tyrosinase, and contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent.

[0007] The composition for suppressing gene expression that solves the above problems is a composition for suppressing gene expression 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.

Effects of the Invention

[0008] According to the present invention, novel compositions for suppressing melanin production, whitening, and suppressing gene expression are provided.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

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Figure 5

Mode for Carrying Out the Invention

[0010] Hereinafter, an embodiment of the present invention will be described. The composition of this embodiment (hereinafter referred to as "this composition") contains an extract of Nostoc verrucosum as an active ingredient.

[0011] [Raw Materials] Nostoc verrucosum (Japanese name: Ashitsuki) is an algal body of the genus Nostoc in the order Nostocales of the class Cyanophyceae. Nostoc verrucosum (hereinafter referred to as "the above algal body") may be an algal body that grows naturally or an algal body that is artificially cultured. From the viewpoints of stable supply and easy quality retention, it is industrially preferable to use an artificially cultured algal body.

[0012] The above extract contained in this composition can be obtained by performing an extraction treatment using the above algal body as a raw material. The above algal body as a raw material may be in the state as collected, the state after being crushed after collection, the state after being dried after collection, or the state after being crushed and dried after collection. From the viewpoint of efficiency during the extraction treatment, it is preferable to use the crushed above algal body as a raw material.

[0013] [Extract] As the extraction solvent for extracting the extract from the above algal body, for example, an organic solvent and a mixed solvent of an organic solvent and water can be used. Examples of the above 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.

[0014] As the organic solvent, a single species may be used, or a mixed solvent of a plurality of species 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. Further, additives such as organic salts, inorganic salts, buffers, and emulsifiers may be dissolved in the extraction solvent.

[0015] As the extraction method for extracting the extract from the above algal thalli, any known extraction method such as cooling extraction, normal temperature extraction, and heating extraction may be used. Further, the extraction temperature can be appropriately set according to the type of solvent, extraction efficiency, degradation of components, etc.

[0016] The extraction operation is performed by immersing the above algal thalli as a raw material in the extraction solvent for a predetermined time. At this time, the concentration of the above algal thalli in the extraction solvent can be appropriately set according to the type of extraction solvent, extraction efficiency, efficiency of the concentration treatment after extraction, etc. In such an extraction operation, in order to enhance the extraction efficiency, treatments such as reflux treatment, stirring treatment, pressurization treatment, and ultrasonic treatment may be further performed as necessary.

[0017] The extraction operation and the solid-liquid separation operation may be performed only once on the same above algal thalli, or may be performed a plurality of times. Further, when the extraction operation is performed a plurality of times, the same extraction operation may be repeated, or different extraction operations may be combined. For example, after performing an extraction operation and a solid-liquid separation operation using an organic solvent, an extraction operation using another organic solvent may be performed on the above algal thalli on which those operations have been performed. When different extraction operations are combined, the extracts obtained by each extraction operation may be mixed and used as one extract, or may be used as separate different extracts.

[0018] [Function 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.

[0019] In addition, by taking this composition, the gene expression of tyrosinase, an enzyme involved in melanin production from tyrosine, is suppressed. Therefore, this composition can be used as an expression suppressing composition for the purpose of exerting an inhibitory effect on the gene expression of tyrosinase. In addition, the above-mentioned extract contained in the expression suppressing composition for the purpose of exerting an inhibitory effect on the gene expression of tyrosinase is preferably a hexane extract extracted using an extraction solvent containing hexane. In this case, the effect of suppressing the gene expression of tyrosinase is more remarkable.

[0020] 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 for the purpose of exerting 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 for the purpose of exerting 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.

[0021] 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.

[0022] 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.

[0023] Step 4: Melanin is synthesized by an enzyme. When the melanocyte-stimulating hormone in Step 2 is secreted, the present composition suppresses the production of the enzyme in Step 3. As a result, melanin synthesis in Step 4 is suppressed. Therefore, the present composition can be used as a composition for suppressing melanin production for suppressing the production of melanin promoted by the stimulation of melanocyte-stimulating hormone. Specifically, the present composition can be selectively used for an administration subject in which the melanocyte-stimulating hormone is secreted in Step 2 and an increase in the concentration of the melanocyte-stimulating hormone is observed or the concentration is likely to increase.

[0024] The composition for suppressing melanin production, the composition for whitening, and the composition for suppressing expression, each comprising the present composition, can be applied to each field such as foods and drinks, pharmaceuticals, quasi-drugs, cosmetics, and the like. Examples of the foods and drinks include various beverages (beverages containing fruit juice or vegetable juice, soft drinks, mineral drinks, sports drinks, tea beverages, coffee, carbonated beverages, dairy products such as milk and yogurt, etc.), jelly-like foods (jelly, agar, jelly-like beverages, etc.), capsules (soft capsules, hard capsules), and various confectioneries. The foods and drinks can appropriately contain gelling agents such as pectin and carrageenan, saccharides and sweeteners such as glucose, sucrose, fructose, lactose, stevia, aspartame, sugar alcohols, food additives such as fragrances, and oils and fats such as vegetable oils and animal fats. Further, the use of the foods and drinks is not particularly limited, and it can be applied as so-called general foods, health foods, functional foods, dietary supplements, supplements, foods for specified health use, foods with functional claims, and foods for patients.

[0025] When used as a pharmaceutical, quasi-drug, or cosmetic, the administration method is not particularly limited. Specific administration methods include, for example, administration by ingestion (oral intake), intravascular administration, enteral administration, transdermal administration, and intraperitoneal administration. Also, when used as a pharmaceutical, quasi-drug, or cosmetic, the dosage form is not particularly limited. Specific dosage forms include, for example, powders, fine powders, granules, tablets, capsules, pills, suppositories, liquids, and injections. Further, as additives, for example, excipients, bases, emulsifiers, solvents, stabilizers, etc. may be contained.

[0026] Next, the effects of the above-described embodiments will be explained. (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.

[0027] According to the above configuration, an effect of inhibiting melanin production can be obtained. (2) The composition for inhibiting melanin production inhibits the production of melanin promoted by the stimulation of melanocyte-stimulating hormone. 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.

[0028] According to the above configuration, it can be selectively used for administration subjects in which melanocyte-stimulating hormone is secreted and an increase in the concentration of melanocyte-stimulating hormone is observed or there is a high possibility that the concentration will increase.

[0029] (3) The composition for whitening contains an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent. According to the above configuration, by suppressing the production of melanin, a skin whitening effect can be obtained.

[0030] (4) The composition for expression suppression that suppresses the gene expression of tyrosinase contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent. According to the above configuration, an effect of suppressing the gene expression of tyrosinase can be obtained.

[0031] (5) The expression-suppressing composition for suppressing the expression of the MC1R gene contains, as an active ingredient, an extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent. According to the above configuration, an effect of suppressing the expression of the MC1R gene can be obtained.

[0032] Note that this embodiment can be implemented with the following modifications. This embodiment and the following modification examples can be implemented in combination with each other within a range where there is no technical contradiction. · The composition for suppressing melanin production, the composition for whitening, and the expression-suppressing composition may contain other components within a range that does not impair their respective intended effects.

[0033] · The intake amount and intake period of the composition for suppressing melanin production, the composition for whitening, and the expression-suppressing composition are not particularly limited and are appropriately determined in consideration of the physical function status, age, gender, and other conditions of the intake person.

[0034] · The composition for suppressing melanin production, the composition for whitening, and the expression-suppressing composition can be applied not only to humans but also to feeds, drugs, etc. for domestic animals such as livestock.

[0035] Next, the technical idea that can be grasped from the above embodiment and modification examples will be described. (A) A method for producing a composition for suppressing melanin production, a composition for whitening, and an expression-suppressing composition, the method having an extraction step of obtaining an extract from Nostoc verrucosum using an extraction solvent containing an organic solvent.

[0036] [Explanation of impossible / impractical matters] The description of "extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent" in the claims of the present application applies to the case where "at the time of filing, it is impossible to directly identify the substance by its structure or properties, or there are circumstances where it is not approximately practical ('impossible / impractical circumstances')".

[0037] The components of Nostoc verrucosum contain innumerable components that elute in an extraction solvent containing an organic solvent. Isolating the active ingredients having effects such as melanogenesis inhibition from these innumerable components and analyzing their structures requires extremely excessive economic expenditure and time. Also, in formulating drugs, it is generally practiced to include them in the state of extracts or their crude purified products, rather than in the state of isolating and purifying specific active ingredients. In particular, substances derived from organisms tend to have higher safety in the unpurified or crudely purified state compared to chemically synthesized substances, and they are actively included in the unpurified or crudely purified state.

[0038] Therefore, in the situation of obtaining patents with rapid technological progress and intense international competition, it is too harsh to require the applicant to isolate the active ingredients having effects such as melanogenesis inhibition from the extract of Nostoc verrucosum and analyze their structures. Thus, the description of "extract of Nostoc verrucosum extracted using an extraction solvent containing an organic solvent" in the claims of the present application applies to the case where "at the time of filing, it is impossible to directly identify the substance by its structure or properties, or there are circumstances where it is not approximately practical ('impossible / impractical circumstances')".

Example

[0039] Test examples are given below to further specifically explain the above embodiments. Note that the present invention is not limited to these. [Preparation of Extract] Hexane (2000 mL) was added to the dry powder of Nostoc vercosum (10 g), and the mixture was stirred at room temperature for 60 minutes. Then, the liquid obtained by centrifugation and filtration was concentrated under reduced pressure to obtain 16.3 mg of a concentrate. The obtained concentrate was dissolved in dimethyl sulfoxide to prepare a hexane extract with a solid content concentration of 100 μg / mL.

[0040] Methanol (2000 mL) was added to the residue of Nostoc vercosum, which was the solid content recovered by filtration after the extraction treatment with the above hexane, and the mixture was stirred at room temperature for 60 minutes. Then, the liquid obtained by centrifugation and filtration was concentrated under reduced pressure to obtain 999.7 mg of a concentrate. The obtained concentrate was dissolved in dimethyl sulfoxide to prepare a methanol extract with a solid content concentration of 100 μg / mL.

[0041] Water (2000 mL) was added to the residue of Nostoc vercosum, which was the solid content recovered by filtration after the extraction treatment with the above methanol, and the mixture was stirred at 90 - 95 °C for 60 minutes. Then, the liquid obtained by centrifugation and filtration was concentrated under reduced pressure to obtain 2350 g of a concentrate. The obtained concentrate was dissolved in ultrapure water to prepare a hot water extract with a solid content concentration of 100 μg / mL.

[0042] [Preparation of medium] Heat-inactivated fetal bovine serum was added to low-glucose Dulbecco's modified Eagle's medium (DMEM: D6046 manufactured by Sigma-Aldrich) 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.

[0043] [Test 1: Measurement of melanin production amount] Regarding melanogenesis promoted by the stimulation of α-MSH (melanocyte-stimulating hormone), the effects were evaluated when each extract was added. α-MSH is a bioactive substance that functions as a ligand for MC1R present on the cell membranes of melanocytes or melanoma. α-MSH activates the intracellular signal transduction system and induces melanogenesis ultimately by inducing tyrosinase expression.

[0044] B16F1 cells (mouse-derived melanoma cultured cells) were seeded at 1.0×10 5 cells / 60mm dish and cultured for 24 hours at 37°C under 5% CO 2 conditions using the above medium. Then, the medium was changed, and α-MSH was added to the medium to a final concentration of 0, 10 nM, and a hexane extract or a methanol extract was added to the medium.

[0045] The hexane extract was added to the medium to a final concentration of 0 - 25 μL / mL. The methanol extract was added to the medium to a final concentration of 0 - 20 μL / mL. For the control with a final concentration of 0 μL / mL, an equal volume of dimethyl sulfoxide was added. After culturing for 72 hours at 37°C under 5% CO 2 conditions, 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 count was measured using a Fuchs-Rosenthal cell counting chamber.

[0046] Next, the cell suspension was centrifuged at 1000 rpm for 5 minutes, resuspended in PBS, and then centrifuged again at 1000 rpm for 5 minutes to obtain a cell pellet. 1×10 62 M NaOH was poured into the cell pellet to reach 100 μL per cell, and the mixture was treated on a boiling 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 determined with the melanin content when only α-MSH was added set as 100%. The results are shown in the graphs of Fig. 1 and Fig. 2.

[0047] From the graphs of Fig. 1 and Fig. 2, it can be seen that the melanin production promoted by the stimulation of α-MSH was significantly suppressed by adding the hexane extract or the methanol extract. In particular, the hexane extract had a high effect of suppressing melanin production. Also, although the details of the test are omitted, when a hot water extract was added instead of the hexane extract and the methanol extract, no change in the amount of melanin production was confirmed.

[0048] [Test 2: Measurement of the expression level of genes involved in melanin synthesis (72-hour treatment)] Regarding the gene expressions of tyrosinase and MC1R promoted by the stimulation of α-MSH, the effects when each extract was added were evaluated.

[0049] B16F1 cells were seeded at 1.0×10 5 cells / 60 mm dish, and cultured for 24 hours at 37 °C under 5% CO 2 using the above medium. Then, the medium was changed, α-MSH was added to the medium to a final concentration of 0, 10 nM, and the hexane extract was added to the medium to a final concentration of 0 - 25 μL / mL. Note that an equal amount of dimethyl sulfoxide was added to the control with a final concentration of 0 μL / mL.

[0050] At 37 °C, 5% CO 2After culturing for 72 hours under the conditions, the medium was removed, and the cells were lysed using an RNA extraction reagent (ISOGEN2 manufactured by Nippon Gene Co., Ltd.) to extract total RNA. Then, cDNA was prepared using an RT-PCR kit (RNA PCR kit (AMV) Ver. 3.0 manufactured by Takara Bio Inc.). The obtained cDNA, primers, and PCR reagent (Thunderbird SYBR qPCR mix manufactured by TOYOBO Co., Ltd.) were mixed to perform a real-time PCR reaction. 2 -ΔΔCt Using the method, the mRNA expression levels of tyrosinase, MC1R, and GAPDH as an internal standard were measured.

[0051] For the real-time PCR reaction, a real-time PCR system (Quant Studio 3 manufactured by Applied Biosystems) was used. The reaction conditions for the real-time PCR reaction were 40 cycles with 10 seconds at 95°C and 30 seconds at 60°C as one cycle.

[0052] The measured mRNA expression level of tyrosinase was corrected with the mRNA expression level of GAPDH, and the mRNA expression level of tyrosinase was determined with the corrected value of the mRNA expression level of tyrosinase when α-MSH was added and no hexane extract was added set as "1". The results are shown in the graph of Figure 3. Similarly, the measured mRNA expression level of MC1R was corrected with the mRNA expression level of GAPDH, and the mRNA expression level of MC1R was determined with the corrected value of the mRNA expression level of MC1R when α-MSH was added and no test solution was added set as "1". The results are shown in the graph of Figure 4.

[0053] As shown in the graph of Figure 3, regarding the mRNA expression level of tyrosinase, in the absence of α-MSH, a decrease in the expression level due to the addition of the hexane extract could not be confirmed. On the other hand, in the presence of α-MSH, the expression level decreased significantly due to the addition of the hexane extract. As shown in the graph of Figure 4, regarding the mRNA expression level of MC1R, in both the absence and presence of α-MSH, the expression level decreased significantly due to the addition of the hexane extract. Also, although the details of the test are omitted, as a result of conducting the same test using a methanol extract instead of the hexane extract, a decrease in the mRNA expression level of MC1R due to the addition of the hexane extract could be confirmed.

[0054] [Test 3: Measurement of the expression level of genes involved in melanin synthesis (24-hour treatment)] By changing the culture time after the addition of α-MSH and the hexane extract in Test 2 to 24 hours and conducting the same test as in Test 2, the mRNA expression levels of tyrosinase and MC1R were determined. The results are shown in the graph of Figure 5.

[0055] As shown in the graph of Figure 5, regarding the mRNA expression level of tyrosinase, in both the absence and presence of α-MSH, the expression level decreased significantly due to the addition of the hexane extract. As shown in the graph of Figure 5, regarding the mRNA expression level of MC1R, in both the absence and presence of α-MSH, the expression level decreased significantly due to the addition of the hexane extract.

[0056] From the results of Test 2 and Test 3, it can be seen that when the above extract of Nostoque Vercosum is added, the gene expression of tyrosinase and MC1R is suppressed based on the transcriptional repression of tyrosinase and MC1R. And as a result of the suppression of the gene expression of tyrosinase and MC1R, it can be seen that melanin production is suppressed.

Claims

1. A composition for inhibiting melanin production, containing an extract of Nostoc verrucosum extracted using hexane as an active ingredient.

2. A composition for inhibiting melanin production that inhibits the production of melanin promoted by the secretion of melanocyte-stimulating hormone, A composition for inhibiting melanin production, containing an extract of Nostoc verrucosum extracted using hexane as an active ingredient.

3. A composition for whitening, containing the composition for inhibiting melanin production according to Claim 1 or Claim 2.

4. An expression-inhibiting composition that inhibits the gene expression of tyrosinase, An expression-inhibiting composition, containing an extract of Nostoc verrucosum extracted using hexane as an active ingredient.

5. An expression-inhibiting composition that inhibits the gene expression of MC1R, An expression-inhibiting composition, containing an extract of Nostoc verrucosum extracted using hexane as an active ingredient.

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