JP7725009B2 - composition - Google Patents

Description

The present invention relates to a composition .

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, part of the order Nostoc, of the class Cyanobacteria, exhibit the effect of promoting the expression of brain-derived neurotrophic factor.

Japanese Patent Application Laid-Open No. 2020-059684

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

The composition that solves the above problem is a composition for promoting the expression of at least one gene selected from the nrf-2 gene and the sirt-1 gene , and contains an extract of Nostoc verrucosum as an active ingredient , and the extract includes one or both of an alcohol extract extracted using an extraction solvent containing a lower alcohol and a hot water extract .

One embodiment of the composition is a composition for promoting the expression of the nrf-2 gene.
One embodiment of the composition is a composition for promoting the expression of the sirt-1 gene.
The composition that solves the above problem is a composition for suppressing the expression of the MMP-1 gene, and contains an extract of Nostoc verrucosum as an active ingredient, and the extract includes a hexane extract extracted using an extraction solvent containing hexane .

According to the present invention, a novel composition is provided.

Graph showing the mRNA expression level of the nrf-2 gene upon treatment with alcohol extract in Test 1. Graph showing the mRNA expression level of the nrf-2 gene upon treatment with hot water extract in Test 1. Graph showing the mRNA expression level of the sirt-1 gene upon treatment with alcohol extract in Test 2. Graph showing the mRNA expression level of the sirt-1 gene upon treatment with the hot water extract in Test 2. Graph showing mRNA expression levels of MMP-1 gene upon treatment with hexane extract in Test 3.

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.

[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 cultured alga. From the viewpoints of stable supply and ease of maintaining quality, 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 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.

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

A single organic solvent may be used, or a mixed solvent containing multiple organic solvents 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, for example, 25% to 75% by volume, or 40% to 60% by volume. In addition, additives such as organic salts, inorganic salts, buffers, and emulsifiers may be dissolved in the extraction solvent.

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 set appropriately depending on the type of solvent, extraction efficiency, deterioration of components, etc.

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

Then, after the extraction operation, a solid-liquid separation operation is performed to separate the extract liquid as an extract from the algae residue. Known separation methods such as filtration and centrifugation can be used as the solid-liquid separation method. The obtained extract liquid may be concentrated or dried as necessary.

The extraction operation and solid-liquid separation operation may be performed once or multiple times on the same algal cells. Furthermore, 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 an extraction operation using an organic solvent and a solid-liquid separation operation, the algal cells that have undergone 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.

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

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.

When the present composition is used as an expression-regulating composition that promotes the expression of the nrf-2 gene and 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.

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

When the present composition is used as an expression-regulating composition that promotes the expression of the sirt-1 gene and as a cell-protecting composition, the extract preferably includes the alcohol extract. Furthermore, when the present composition is used as an expression-regulating composition that promotes the expression of the sirt-1 gene and as a cell-protecting composition, 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.

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

When the present composition is used as an expression-regulating composition that suppresses the expression of the MMP-1 gene and as a cell-protecting composition, the extract is preferably a hexane extract obtained using an extraction solvent containing hexane. In this case, the effect of suppressing the expression of the MMP-1 gene is more pronounced.

[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.

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, nutritional supplements, dietary supplements, foods for specified health uses, foods with functional claims, and foods for the sick.

When used as a pharmaceutical, quasi-drug, or cosmetic, the method of administration 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 also not particularly limited. Specific dosage forms include, for example, powders, dustings, 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 regulating expression and the composition for protecting cells contain an extract of Nostoc verrucosum as an active ingredient.

The above configuration provides the effect of regulating the expression of at least one cell protection-related gene selected from the nrf-2 gene, sirt-1 gene, and MMP-1 gene. Furthermore, the effect of protecting cells is achieved by regulating the expression of the above cell protection-related gene.

(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.

(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.

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 components to the extent that the intended effects of each are not impaired.

The intake amount and intake period of the expression-regulating composition and cell-protecting composition are not particularly limited and are 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 feed, medicines, etc. for domestic animals and other captive animals.

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

[Explanation of impossible or impractical circumstances]
The description of "an 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')."

The constituents of Nostoc verrucosum contain numerous components that dissolve in the extraction solvent. Isolating the active ingredient that regulates the expression of cytoprotection-related genes from these numerous components and analyzing its structure requires a significantly large financial investment and time. Furthermore, when formulating a drug, it is common to include specific active ingredients in the form of an extract or a crudely purified product, rather than in an isolated and purified state. In particular, biologically derived substances tend to be safer in their unpurified or crudely purified state compared to chemically synthesized substances, and therefore are actively included in their unpurified or crudely purified state.

Therefore, in the context of rapid technological advances and fierce international competition in the patent acquisition arena, it would be unreasonable to require an applicant to isolate an active ingredient from an extract of Nostoc verrucosum that regulates 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 category of "circumstances where, at the time of filing, it is impossible or impractical to directly identify the substance by its structure or properties ('impossible or impractical circumstances')."

The above embodiment will be described in more detail below with reference to test examples, although the present invention is not limited to these.
[Preparation of extract]
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.

Methanol (2000 mL) was added to the Nostoc verrucosum residue, which was the solid fraction 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 resulting 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 Nostoc verrucosum residue, which was the solid fraction recovered by filtration after the above-mentioned methanol extraction process, 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 yield 2350 g of concentrate. The resulting concentrate was dissolved in ultrapure water to prepare a hot water extract with a solid concentration of 100 μg/mL.

[Preparation of medium]
Heat-inactivated fetal bovine serum was added to a high-glucose Dulbecco's modified Eagle's medium (DMEM: D0822 manufactured by Sigma-Aldrich) 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, and this was used as the medium for the tests described below.

[Test 1: Measurement of nrf-2 gene expression level]
The effect of adding the above extract on the expression of the nrf-2 gene was evaluated.
Human keratinocyte-derived HaCaT cells were seeded at 2.0 x ¹⁰ 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.

After culturing for 48 hours under conditions of 37°C and 5% _CO2 , the medium was removed, the cells were lysed using an RNA extraction reagent (ISOGEN2 manufactured by 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 manufactured by Takara Bio Inc.). The obtained cDNA, primers, and PCR reagent (Thunderbird SYBR qPCR mix manufactured by TOYOBO Co., Ltd.) were mixed and a real-time PCR reaction was performed. The mRNA expression levels of the nrf-2 gene and the β-actin gene as an internal standard were measured using the ^2-ΔΔCt method.

A real-time PCR system (Quant Studio 3, manufactured by 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, 60°C for 30 seconds, for a total of 40 cycles.

The measured mRNA expression level of the nrf-2 gene was corrected by the mRNA expression level of the β-actin gene. The mRNA expression level of the nrf-2 gene was then calculated by setting the corrected value of the mRNA expression level of the nrf-2 gene when no extract was added to "1." The results are shown in the graphs in Figures 1 and 2.

As shown in Figure 1, the addition of alcohol extract increased the expression level of the nrf-2 gene by approximately 2-fold. Furthermore, as shown in Figure 2, the addition of 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 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.

[Test 2: Measurement of expression level of sirt-1 gene]
The effect of adding the above extract on the expression of the sirt-1 gene was evaluated.
The mRNA expression level of the sirt-1 gene was determined by performing a test similar to that of 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.

As shown in the graph in Figure 3, the addition of alcohol extract or hot water extract increased the expression level of the sirt-1 gene by more than 1.5 times. These results demonstrate that the addition of 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.

[Test 3: Measurement of MMP-1 gene expression level]
The effect of adding the above extract on the expression of MMP-1 gene was evaluated.
The mRNA expression level of the MMP-1 gene was determined by performing a similar test to Test 1, except that the primers used in Test 1 were replaced with primers corresponding to the MMP-1 gene. The results are shown in the graph in Figure 5. In Test 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 a control with a final concentration of 0 μg/mL.

As shown in the graph in Figure 5, the addition of hexane extract reduced the expression level of the MMP-1 gene to less than 10%. These results demonstrate that the addition of hexane extract suppresses 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 alcohol extract or hot water extract was added.

Claims (4)

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