Composition

Abstract

To provide a novel composition for regulated expression.SOLUTION: A composition for regulated expression contains extract of Nostoc verrucosum as an active ingredient. The composition for regulated expression regulates the expression of at least one cell protection-related gene selected from the nrf-2 gene, sirt-1 gene, and MMP-1 gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention ,set Related to products. [Background technology]

[0002] In recent years, various studies have been conducted on the effects of microalgae. For example, Patent Document 1 discloses that components contained in the algae of the genus Nostoc, belonging to the order Nostoc, Cyanobacteria, exhibit an effect of promoting the expression of brain-derived neurotrophic factor. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2020-059684 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention was made based on the new discovery made as a result of intensive research by the present researchers that extracts of Nostoc verrucosum, an alga of the genus Nostoc in the family Nostocaceae of the order Nostociales of the class Cyanobacteria, have the effect of promoting or suppressing the expression of genes involved in cell protection. [Means for solving the problem]

[0005] To solve the above problems Ru group The product contains the nrf-2 gene, and sirt-1 inheritance Child? At least one selected from The release of Present To promote A composition containing an extract of Nostoc verrucosum as an active ingredient. The extract includes one or both of an alcohol extract extracted using an extraction solvent containing a lower alcohol and a hot water extract. .

[0006] above Record group One form of the composition promotes expression of the nrf-2 gene. It is a composition for . above Record group One form of the composition promotes the expression of the sirt-1 gene. It is a composition for Composition that solves the above problems suppresses MMP-1 gene expression The composition for the treatment of rhododendron amylase contains an extract of Nostoc verrucosum as an active ingredient, and the extract contains a hexane extract obtained by extracting with an extraction solvent containing hexane. . [Effects of the Invention]

[0008] According to the present invention, a novel Group A composition is provided. [Brief explanation of the drawings]

[0009] [Figure 1] Graph showing the mRNA expression level of the nrf-2 gene upon treatment with alcohol extract in Test 1. [Figure 2] Graph showing the mRNA expression level of the nrf-2 gene upon treatment with the hot water extract in Test 1. [Figure 3] Graph showing the mRNA expression level of the sirt-1 gene upon treatment with alcohol extract in Test 2. [Figure 4] Graph showing the mRNA expression level of the sirt-1 gene upon treatment with the hot water extract in Test 2. [Figure 5] Graph showing mRNA expression levels of MMP-1 gene upon treatment with hexane extract in Test 3. DETAILED DESCRIPTION OF THE INVENTION

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

[0011] [Raw materials] Nostoc verrucosum (Japanese name: Ashitsuki) is an alga of the genus Nostoc, family Nostocaceae, order Nostociales, class Cyanobacteria. Nostoc verrucosum (hereinafter referred to as the alga) may be a naturally occurring alga or an artificially cultivated alga. However, from an industrial perspective, it is preferable to use artificially cultivated alga because it allows for a stable supply and is easy to maintain quality.

[0012] 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 of 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.

[0013] [Extract] Examples of the extraction solvent used to extract the extract from the alga include water, 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.

[0014] The organic solvent may be a single type or a mixed solvent of multiple types. 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, for example, 25% by volume to 75% by volume, or 40% by volume to 60% by volume. In addition, additives such as organic salts, inorganic salts, buffers, and emulsifiers may be dissolved in the extraction solvent.

[0015] The extraction 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 can be appropriately set depending on the type of solvent, extraction efficiency, deterioration of components, etc.

[0016] The extraction is carried out by immersing the raw algae in an extraction solvent for a predetermined period of time. The concentration of the algae in the extraction solvent can be appropriately determined depending on the type of extraction solvent, extraction efficiency, efficiency of the concentration treatment after extraction, etc. In this extraction, further treatments such as refluxing, stirring, pressurization, and ultrasonic treatment may be carried out as necessary to increase the extraction efficiency.

[0017] After the extraction, a solid-liquid separation operation is performed to separate the extract from the algal residue. Known separation methods such as filtration and centrifugation can be used for the solid-liquid separation operation. The resulting extract may be concentrated or dried as needed.

[0018] The extraction operation and solid-liquid separation operation may be performed once or multiple times on the same algal cells. When the extraction operation is performed multiple times, the same extraction operation may be performed repeatedly, or different extraction operations may be performed in combination. For example, after performing an extraction operation using an organic solvent and a solid-liquid separation operation, the algal cells that have been subjected to these operations may be subjected to an extraction operation using water. When different extraction operations are performed in combination, the extracts obtained by each extraction operation may be mixed and used as a single extract, or may be used as separate, different extracts.

[0019] [Action and application field] Ingestion of this composition promotes or suppresses the expression of at least one cytoprotection-related gene selected from the nrf-2 gene, the sirt-1 gene, and the MMP-1 gene. Therefore, this composition can be used as an expression-regulating composition for the purpose of exerting an effect of regulating the expression of cytoprotection-related genes. Furthermore, this composition can be used as a cytoprotective composition for the purpose of exerting an effect of protecting cells by regulating the expression of cytoprotection-related genes.

[0020] Nrf-2 (NF-E2 related factor-2) is a transcription factor involved in promoting the transcription of antioxidant genes. Promoting the transcription of the nrf-2 gene is known to be effective in reducing carcinogenesis and lifestyle-related diseases. By ingesting this composition, the expression of the nrf-2 gene is promoted. Therefore, this composition can be used as an expression-regulating composition that promotes the expression of the nrf-2 gene and as a cell-protecting composition.

[0021] When the present composition is used as an expression-regulating composition that promotes the expression of the nrf-2 gene or as a cell-protecting composition, the extract preferably comprises an alcohol extract extracted using an extraction solvent containing a lower alcohol such as methanol. Furthermore, when the present composition is used as an expression-regulating composition or expression-promoting composition that promotes the expression of the nrf-2 gene, the extract preferably comprises a hot water extract extracted using hot water. In these cases, the effect of promoting the expression of the nrf-2 gene is more pronounced. The hot water extract is, for example, an extract extracted using hot water at 90°C or higher.

[0022] The sirt-1 (Sirtuin-1) gene is an anti-aging gene involved in the control of aging and lifespan. It is known that increased expression of the sirt-1 gene has the effect of extending lifespan, and that expression of the sirt-1 gene decreases with aging. Ingestion of this composition promotes expression of the sirt-1 gene. Therefore, this composition can be used as an expression-regulating composition that promotes expression of the sirt-1 gene and as a cell-protecting composition.

[0023] When the present composition is used as a composition for regulating expression of the sirt-1 gene and a composition for protecting cells, the extract preferably includes the alcohol extract. Furthermore, when the present composition is used as a composition for regulating expression of the sirt-1 gene and a composition for protecting cells, the extract preferably includes the hot water extract. In these cases, the effect of promoting the expression of the sirt-1 gene is more pronounced.

[0024] MMP-1 (Matrix metalloproteinase-1) is a proteolytic enzyme involved in the degradation of extracellular matrix, particularly collagen. MMP-1 is known to be involved in cancer metastasis, wrinkle formation, and sagging skin. Ingestion of this composition suppresses MMP-1 gene expression. Therefore, this composition can be used as an expression-regulating composition that suppresses MMP-1 gene expression and as a cell-protecting composition.

[0025] When the present composition is used as a composition for regulating expression of the MMP-1 gene or for protecting cells, the extract is preferably a hexane extract obtained using an extraction solvent containing hexane, which provides a more pronounced inhibitory effect on the expression of the MMP-1 gene.

[0026] [Application] The expression-regulating composition and cell-protecting composition comprising the present composition can each be applied in various fields such as food and beverages, pharmaceuticals, quasi-drugs, and cosmetics.

[0027] Examples of foods and beverages include various beverages (fruit or vegetable juice drinks, 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. Foods and beverages may 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, and fats and oils such as vegetable oils and animal oils and fats. The uses of foods and beverages are not particularly limited, and they can be used as so-called general foods, health foods, functional foods, dietary supplements, supplements, foods for specified health uses, foods with functional claims, and foods for patients.

[0028] When used as a pharmaceutical, quasi-drug, or cosmetic, the administration method is not particularly limited. Specific administration methods include, for example, oral administration, intravascular administration, enteral administration, transdermal administration, and intraperitoneal administration. Furthermore, when used as a pharmaceutical, quasi-drug, or cosmetic, the dosage form is not particularly limited. Specific dosage forms include, for example, powders, dusts, granules, tablets, capsules, pills, suppositories, liquids, and injections. Furthermore, additives such as excipients, bases, emulsifiers, solvents, and stabilizers may be contained.

[0029] Next, the effects of the above embodiment will be described. (1) The expression-regulating composition and the cell-protecting composition contain an extract of Nostoc verrucosum as an active ingredient.

[0030] The above-mentioned composition provides the effect of regulating the expression of at least one cytoprotective gene selected from the group consisting of nrf-2 gene, sirt-1 gene, and MMP-1 gene, and also provides the effect of protecting cells by regulating the expression of the cytoprotective gene.

[0031] (2) The extract of Nostoc verrucosum includes one or both of an alcohol extract extracted using an extraction solvent containing a lower alcohol and a hot water extract. According to the above-mentioned configuration, the effect of promoting the expression of the nrf-2 gene is remarkable. Also, according to the above-mentioned configuration, the effect of promoting the expression of the sirt-1 gene is remarkable.

[0032] (3) The extract of Nostoc verrucosum includes a hexane extract extracted using an extraction solvent containing hexane. According to the above-mentioned configuration, the effect of suppressing the expression of the MMP-1 gene can be significantly obtained.

[0033] This embodiment can be modified as follows: This embodiment and the following modifications can be combined and implemented within the scope of technical compatibility. The expression-regulating composition and the cell-protecting composition may contain other ingredients to the extent that their intended effects are not impaired.

[0034] The intake amount and intake period of the expression-regulating composition and cell-protecting composition are not particularly limited and may be determined appropriately taking into consideration the state of the recipient's physical functions, age, sex, and other conditions. The expression-regulating composition and cell-protecting composition can be applied not only to humans but also to feeds and medicines for domestic animals and other farm animals.

[0035] Next, the technical ideas that can be understood from the above-described embodiment and modified examples will be described. (i) A method for producing an expression-regulating composition and a cell-protecting composition, which comprises an extraction step of obtaining an extract from Nostoc verrucosum.

[0036] [Explanation of impossible / impractical circumstances] The description of "extract of Nostoc verrucosum" in the claims of this application falls under the case where "circumstances exist at the time of filing that make it impossible or practical to directly identify the substance by its structure or properties ('impossible or impractical circumstances')."

[0037] Nostoc verrucosum contains numerous components that dissolve in the extraction solvent. Isolating and structurally analyzing active ingredients that regulate the expression of cytoprotective genes from these numerous components requires significant financial and time expenditures. Furthermore, formulations typically contain specific active ingredients in the form of extracts or crudely purified products rather than in their isolated and purified form. In particular, biologically derived substances tend to be safer in their unpurified or crudely purified state than chemically synthesized substances, and therefore are actively used in these forms.

[0038] Therefore, in the context of rapid technological advances and fierce international competition in the patent acquisition arena, it is unreasonable to require the applicant to isolate an active ingredient from an extract of Nostoc verrucosum that has the effect of regulating the expression of cytoprotection-related genes and analyze its structure. Therefore, the description of "an extract of Nostoc verrucosum" in the claims of this patent application falls under the case where "circumstances exist that make it impossible or impractical at the time of filing to directly identify the product by its structure or properties ('impossible or impractical circumstances')." [Example]

[0039] The above embodiment will be described in more detail below with reference to test examples, although the present invention is not limited to these. [Extract preparation] Hexane (2000 mL) was added to dried powder (10 g) of Nostoc verrucosum and stirred at room temperature for 60 minutes. The resulting solution was then centrifuged and filtered, and 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.

[0040] Methanol (2000 mL) was added to the solid residue of Nostoc verrucosum recovered by filtration after the hexane extraction process described above, and the mixture was stirred at room temperature for 60 minutes. The resulting solution, obtained by centrifugation and filtration, was 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.

[0041] Water (2000 mL) was added to the residue of Nostoc verrucosum, the solid fraction recovered by filtration after the above-mentioned methanol extraction, 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 was then dissolved in ultrapure water to prepare a hot water extract with a solid concentration of 100 μg / mL.

[0042] [Preparation of medium] Heat-inactivated fetal bovine serum was added to a final concentration of 10%, penicillin to a final concentration of 50 units / mL, and streptomycin to a final concentration of 50 μg / mL to high-glucose Dulbecco's modified Eagle's medium (DMEM: D0822 manufactured by Sigma-Aldrich), and this was used as the medium for the tests described below.

[0043] [Test 1: Measurement of nrf-2 gene expression level] The effect of adding the above extracts on the expression of the nrf-2 gene was evaluated. Human keratinocyte-derived HaCaT cells were cultured at 2.0 × 10 5The cells were seeded at 1000 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 the alcohol extract or hot water extract was added to the medium. The alcohol extract was added to the medium to a final concentration of 0 to 20 μg / mL. An equal volume of dimethyl sulfoxide was added to the control for a final concentration of 0 μg / mL. The hot water extract was added to the medium to a final concentration of 0 to 200 μg / mL. An equal volume of ultrapure water was added to the control for a final concentration of 0 μg / mL.

[0044] After culturing for 48 hours at 37°C and 5% CO2, the medium was removed, and the cells were lysed using an RNA extraction reagent (ISOGEN2, Nippon Gene Co., Ltd.) to extract total RNA. cDNA was then prepared using an RT-PCR kit (RNA PCR kit (AMV) Ver. 3.0, Takara Bio Inc.). The resulting cDNA, primers, and PCR reagent (Thunderbird SYBR qPCR mix, Toyobo Co., Ltd.) were mixed and used for real-time PCR. -ΔΔCt Using this method, we measured the mRNA expression levels of the nrf-2 gene and the β-actin gene as an internal standard.

[0045] The real-time PCR reaction was carried out using a real-time PCR system (Quant Studio 3, manufactured by Applied Biosystems). The reaction conditions for the real-time PCR reaction were 95°C for 10 seconds and 60°C for 30 seconds as one cycle, for a total of 40 cycles.

[0046] The measured mRNA expression level of the nrf-2 gene was corrected by the mRNA expression level of the β-actin gene. The corrected value of the mRNA expression level of the nrf-2 gene without the extract was set to "1," and the mRNA expression level of the nrf-2 gene was calculated. The results are shown in the graphs in Figures 1 and 2.

[0047] As shown in Figure 1, the addition of the alcohol extract increased the expression level of the nrf-2 gene by approximately two-fold. Furthermore, as shown in Figure 2, the addition of the hot water extract increased the expression level of the nrf-2 gene by more than 1.5-fold. These results demonstrate that the addition of the alcohol extract or hot water extract promotes the expression of the nrf-2 gene. Furthermore, although details of the test are omitted, no change in the expression level of the nrf-2 gene was observed when the hexane extract was added.

[0048] [Test 2: Measurement of sirt-1 gene expression level] The effect of adding the above extracts on the expression of the sirt-1 gene was evaluated. The mRNA expression level of the sirt-1 gene was determined by performing a similar test to Test 1, except that the primers used in Test 1 were changed to primers corresponding to the sirt-1 gene. The results are shown in the graphs of Figures 3 and 4.

[0049] As shown in the graph in Figure 3, the addition of the alcohol extract or hot water extract increased the expression level of the sirt-1 gene by 1.5 times or more. These results indicate that the addition of the alcohol extract or hot water extract promotes the expression of the sirt-1 gene. Furthermore, although details of the test are omitted, no change in the expression level of the sirt-1 gene was observed when the hexane extract was added.

[0050] [Test 3: Measurement of MMP-1 gene expression level] The effect of adding the above extracts on the expression of MMP-1 gene was evaluated. The same experiment as in Experiment 1 was performed, except that the primers used in Experiment 1 were replaced with primers corresponding to the MMP-1 gene, and the mRNA expression level of the MMP-1 gene was determined. The results are shown in the graph in Figure 5. In Experiment 3, the hexane extract was added to the medium to a final concentration of 0 to 25 μg / mL. An equal amount of dimethyl sulfoxide was added to the control to achieve a final concentration of 0 μg / mL.

[0051] As shown in the graph in Figure 5, the addition of the hexane extract reduced the expression level of the MMP-1 gene to 10% or less. This result indicates that the addition of the hexane extract inhibits the expression of the MMP-1 gene. Furthermore, although details of the test are omitted, no change in the expression level of the MMP-1 gene was observed when the alcohol extract or hot water extract was added.

Claims

1. A composition for promoting the expression of at least one gene selected from the group consisting of nrf-2 gene and sirt-1 gene, Contains Nostoc verrucosum extract as the active ingredient, The extract is a composition comprising one or both of an alcohol extract extracted using an extraction solvent containing a lower alcohol and a hot water extract.

2. The composition of claim 1 for promoting expression of the nrf-2 gene.

3. The composition according to claim 1 or 2, for promoting the expression of the sirt-1 gene.

4. A composition for suppressing MMP-1 gene expression, comprising: Contains Nostoc verrucosum extract as the active ingredient, The extract is a composition comprising a hexane extract extracted using an extraction solvent containing hexane.

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