JP2022112464A - Gls1 gene-inhibiting cosmetic material compositions, gls1 gene inhibitors, cosmetic material compositions for senescent cell removal and senescent cell removing agents - Google Patents

Abstract

To provide GLS1 gene-inhibiting cosmetic material compositions, GLS1 gene-inhibiting food compositions, GLS1 gene inhibitors, cosmetic material compositions for removing senescent cells, food compositions for removing senescent cells, as well as senescent cell removing agents.SOLUTION: The disclosure provides GLS1 gene-inhibiting cosmetic material compositions, GLS1 gene-inhibiting food compositions and GLS1 gene inhibitors characterized by containing a Euglena-derived substance as an active ingredient for inhibiting GLS1 gene expression. The disclosure also provides cosmetic material compositions, food compositions and senescent cell removing agents for removing senescent cells in the living body characterized by containing a Euglena-derived substance as an effective ingredient.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cosmetic composition for inhibiting the GLS1 gene, a food composition for inhibiting the GLS1 gene, a GLS1 gene inhibitor, a cosmetic composition for removing senescent cells, a food composition for removing senescent cells, and a senescent cell removing agent.

From the viewpoint of improving QOL (Quality of life) in a super-aging society, technologies related to extension of healthy life expectancy and anti-aging are attracting attention. For the purpose of extending healthy life expectancy, extensive research has been conducted on cellular senescence and aging control mechanisms.

Patent Document 1 describes a senescent cell-removing agent containing a glutaminase inhibitor as an active ingredient, which is a drug for removing senescent cells in vivo. Patent Document 1 discloses that the glutaminase inhibitor should at least inhibit the activity of KGA (kidney-type glutaminase), and a nucleic acid such as siRNA or miRNA for knocking out the gene (GLS1) encoding KGA. It is also disclosed that it may be used as a glutaminase inhibitor.

WO2020/095971

With the background art described above, there is a demand for a technique for removing senescent cells. An object of the present invention is to provide a GLS1 gene-inhibiting cosmetic composition, a GLS1 gene-inhibiting food composition, a GLS1 gene inhibitor, a cosmetic composition for removing senescent cells, a food composition for removing senescent cells, and a senescent cell-removing agent. to provide.

The above-mentioned problems are solved by a cosmetic composition for inhibiting the GLS1 gene, which contains an Euglena-derived substance as an active ingredient and is used for inhibiting the expression of the GLS1 gene.
The above problems are solved by a food composition for inhibiting the GLS1 gene, which contains an Euglena-derived substance as an active ingredient and is used to inhibit the expression of the GLS1 gene.
The above problems are solved by GLS1 gene inhibition, which contains an Euglena-derived substance as an active ingredient and is used to inhibit the expression of the GLS1 gene.
The above problems are solved by a cosmetic composition for removing senescent cells, which contains an Euglena-derived substance as an active ingredient and is used for removing senescent cells in vivo.
The above problems are solved by a food composition for removing senescent cells, which contains an Euglena-derived substance as an active ingredient and is used for removing senescent cells in vivo.
The above-mentioned problems are solved by a senescent cell-removing agent that contains an Euglena-derived substance as an active ingredient and is used for removing senescent cells in vivo.
At this time, the Euglena-derived substance is preferably an Euglena extract.
At this time, the Euglena-derived substance is preferably an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena.

According to the present invention, it is possible to provide a GLS1 gene-inhibiting cosmetic composition, a GLS1 gene-inhibiting food composition, and a GLS1 gene inhibitor capable of inhibiting expression of the GLS1 gene. Further, according to the present invention, it is possible to provide a senescent cell-removing cosmetic composition, a senescent cell-removing food composition, and a senescent cell-removing agent capable of removing senescent cells.

1 is a graph showing the results of examining the effects of Euglena-derived substances on the GSL1 gene in human epidermal keratinocytes.

An embodiment of the present invention will be described below with reference to FIG. The present embodiment includes a GLS1 gene-inhibiting cosmetic composition, a GLS1 gene-inhibiting food composition, a GLS1 gene inhibitor, a senescent cell-removing cosmetic composition, and a senescent cell-removing food containing an Euglena-derived substance as an active ingredient. It relates to a composition and a senescent cell removing agent.

<Cellular senescence and senescent cells>
Normal human somatic cells have a limited number of divisions. In the present embodiment, "cell senescence" means changes in cells that progress toward this division limit. In addition, "senescent cells" refer to cells whose cell proliferation or cell cycle has irreversibly stopped. Whether or not a cell is a senescent cell can be determined based on the characteristics of cellular senescence. Specifically, senescent cells show increased expression levels of senescent markers compared to normal cells. Senescence markers include, but are not limited to, senescence-associated acid β-galactosidase, P53, P16INK4a, P21CIP1, and the like.

<GLS1 gene inhibitor>
The GLS1 gene inhibitor according to this embodiment contains an Euglena-derived substance as an active ingredient and is used to inhibit the expression of the GLS1 gene. “Inhibiting the expression of the GLS1 gene” includes not only expression at the transcription level (expression as mRNA) but also expression at the translation level (expression as protein).

(GLS1 gene)
The "GLS1 gene" is a gene related to glutamine metabolism, and is known to be a gene necessary for the survival of senescent cells. GLS1 (glutaminase 1) is a type of amidohydrolase enzyme that produces glutamic acid and ammonia from glutamine. Due to abnormalities in intracellular organelles, the inside of senescent cells tends to be acidic, and GLS1 works excessively to neutralize them to maintain the cells.

The expression of KGA, one of the GLS1 isoforms, is markedly increased in senescent cells, and there is also a positive correlation between KGA expression and age in human skin. From the results of examination of the effects of GLS1 inhibition on normal cells and senescent cells, it has been reported that senescent cells are selectively killed by inhibiting GLS1 (Patent Document 1).

Specifically, inhibition of senescent cell glutaminolysis (glutamine metabolism) induces selective cell death in senescent cells. Glutaminolysis is composed of several reaction steps, among which senescent cell-specific cell death is efficiently induced by inhibiting the reaction step that produces glutamic acid from glutamine.

<Senior cell removing agent>
The senescent cell removing agent according to the present embodiment contains an Euglena-derived substance as an active ingredient, and is used to remove senescent cells in vivo. Here, "senescent cell removal" means removing senescent cells from a tissue or organ or the like, or killing senescent cells. to selectively remove senescent cells from a cell population containing senescent cells.

The number of senescent cells increases with aging in various organs and tissues. Accumulation of senescent cells in aged tissue can lead to a variety of dysfunctions, diseases, and diminished regenerative capacity. Cells are induced to become senescent cells by being stressed. Senescent tissue, with an accumulation of senescent cells, is incapable of responding to the stresses that require proliferation, resulting in reduced health.

Since the Euglena-derived substance, which is the active ingredient of the senolytic agent according to the present embodiment, has the effect of inhibiting the expression of the GLS1 gene, it is possible to selectively kill senescent cells. Therefore, according to the agent for removing senescent cells according to the present embodiment, it is possible to suppress the accumulation of senescent cells and to suppress the deterioration of health.

(Regarding target senescent cells)
Senescent cells to be targeted are not particularly limited, but eukaryotic cells are exemplified. Examples of senescent cells include skin-related cells such as keratinocytes (keratinocytes), epithelial cells, fibroblasts, and dermal papilla cells. It may be a cell.

<Euglena>
In the present embodiment, the term "euglena" includes taxonomically classified microorganisms classified into the genus Euglena, variants thereof, variants thereof, and closely related species of the family Euglenaceae. Here, the genus Euglena is a group of eukaryotic organisms belonging to Excoverta, Euglenozoa, Euglenomycetes, Euglenoides, and Euglenaceae.

Species included in the genus Euglena specifically include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis. As Euglena, E. gracilis, particularly E. gracilis Z strain can be used, but in addition, Euglena gracilis Z strain mutant strain SM-ZK strain (chloroplast-deficient strain), variants of E. gracilis var. bacillaris, gene mutant strains such as chloroplast mutant strains of these species, and other Euglenoids such as Astasia longa.

Euglenoids are widely distributed in freshwater such as ponds and marshes, and may be used by separating from them, or any already isolated Euglenoids may be used. The genus Euglena includes all its variants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, and transformation.

(Euglenoid algae)
In the present embodiment, Euglenoid algae can be used as Euglena. Euglenoid cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are as Euglenoid alga bodies. Euglena living cells can be used as they are after being harvested from the culture vessel, but they are preferably washed with water or physiological saline. Alternatively, the Euglenoid alga body may be used in the form of a dispersion in a liquid such as water. In the present embodiment, it is preferable to use dry Euglena alga bodies (Euglena powder) obtained by freeze-drying or spray-drying living Euglena cells as Euglena alga bodies.

Furthermore, a mechanically processed product of alga bodies obtained by subjecting living Euglena cells to ultrasonic irradiation treatment or mechanical treatment such as homogenization may be used as the Euglena alga bodies. Alternatively, a dried mechanically processed product obtained by subjecting a mechanically processed product to a drying treatment may be used as the Euglenoid algal body.

(Euglena extract)
In the present embodiment, it is possible to use an Euglena extract (Euglena extract) as Euglena, and it is particularly preferable to use an Euglena aqueous solvent extract. In the present embodiment, "Euglena aqueous solvent extract" means an extract extracted from Euglena using an aqueous solvent. It is preferable to use a water extract, a hot water extract, an alcohol extract, or a glycol extract of Euglena extracted for several seconds to several tens of hours at ℃. The water used for extraction does not necessarily have to be distilled, pure, or ultrapure water, and may, for example, be tap water or contain impurities, but may contain components that interfere with the extraction of the active ingredient. no water is preferred.

In the present embodiment, "water extract" means an extract with water at 0 to 50°C (excluding 0°C). Here, "water" means water at 0 to 50°C (excluding 0°C). The temperature of the water is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient. Especially preferred is 10 to 30°C.

In this embodiment, "hot water extract" means an extract with water at a temperature higher than 50°C, and can also be called a "hot water extract". Here, “hot water” means water having a temperature higher than 50° C., and is a concept that includes “hot water”, including water in a boiling state. Moreover, it is not limited to hot water in a liquid state, but also includes hot water in a gaseous state and a supercritical state. The temperature of the hot water is not particularly limited as long as the active ingredient can be extracted sufficiently without affecting the active ingredient. °C and 100°C or less.

The pH of the water used for extraction is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient, preferably pH 4 to 10, more preferably pH 5 to 9. , particularly preferably pH 6-8.

In the present embodiment, as the aqueous solvent, one or more solvents that can sufficiently extract the active ingredient without affecting the active ingredient and that can be used for extraction are usually selected. may be used. Examples include, but are not limited to, water, alcohols, glycols, and the like. Alcohols include ethanol, methanol, n-propanol, isopropanol and the like. Glycols include 1,3-butylene glycol (BG) and propylene glycol. Other aqueous solvents include acetone and the like. These solvents may be used alone or as an aqueous solution, or may be used as a mixed solvent of any two or three or more.

The temperature of the aqueous solvent used for extraction is, for example, 0° C. or higher, and is not particularly limited as long as it does not affect the active ingredients. Aqueous solvents in a boiling or supercritical state can also be used, but it is preferred to use aqueous solvents at 5°C to 600°C, more preferably 10°C to 200°C. Therefore, the aqueous solvent for extraction includes an aqueous solvent in a boiling state or a supercritical state. The amount of the aqueous solvent used for extraction is preferably an amount that can sufficiently dissolve the water-soluble active ingredient contained in Euglena.

The extraction method is also not particularly limited, and for example, extraction can be performed by the method shown below, but the extraction method is not limited to this, and can be freely selected and used. For example, a dry powder of Euglena algae is immersed in an aqueous solvent for a predetermined time and then centrifuged or filtered, or a dry powder of Euglena algae is added to an aqueous solvent and shaken to uniformly disperse, followed by centrifugation or filtration. method, etc. Moreover, in order to promote extraction, it is also possible to heat the aqueous solvent after adding Euglena.

Euglena can be extracted with water by a conventional method as shown below, but not limited thereto. For example, the Euglena tissue and water are placed in a container and allowed to stand for a predetermined period of time with appropriate stirring or shaking, and the obtained extract can be used as a water extract as it is. Moreover, for example, the supernatant obtained by centrifuging such an extract can also be used as an aqueous extract. Also, such an extract or supernatant can be concentrated and dried to remove water and used as an aqueous extract. The water extraction may be carried out by adding a small amount of alcohol, preferably ethanol, to water in an amount of, for example, 10% by mass or less, in order to increase the extraction efficiency and shorten the extraction time. The extraction time for water extraction is not particularly limited as long as the active ingredient is extracted, and can be appropriately set in the range of several seconds to several tens of hours depending on the extraction temperature.

Extraction with hot water can be performed by a commonly used method as shown below, but is not limited thereto. After Euglena is introduced into a commonly used extractor together with water, it is extracted by heating. When boiling water or water in a supercritical state is used for extraction, it is necessary to use an extractor that can withstand the vapor pressure of water. The pressure during extraction can be set to 1 to 5000 atmospheres, preferably 60 to 400 atmospheres.

When performing extraction under high temperature and high pressure, if the extraction time is too long, the active ingredients may decompose or chemical reactions may occur. Therefore, when the extraction is performed under high temperature and high pressure, the extraction time is short, for example, preferably within 3 minutes, more preferably within 1 minute, and particularly preferably within 30 seconds.

The extracted Euglena extract can be used as it is as an active ingredient of the GLS1 gene inhibitor and the senescent cell-removing agent according to the present embodiment. Filtration, silica gel chromatography, reverse-phase or normal-phase high-performance liquid chromatography, etc.) can be used to fractionate and use highly active fractions. Alternatively, the Euglena extract or a fraction thereof can be concentrated and dried to remove the aqueous solvent, and this can be used as an aqueous solvent extract.

Alternatively, an extract extracted using BG may be used as the Euglena extract. The Euglena extract may be in the form of a complex with other cosmetic raw materials, such as permeation type collagen, permeation type elastin, permeation type hyaluronic acid, and hexapeptide-33 in a multi-layered capsule.

(Hydrolyzed euglena extract)
As the Euglena extract, a water-soluble component extracted by enzymatically decomposing algal cells belonging to the genus Euglena, specifically, a hydrolyzed Euglena extract obtained by enzymatically hydrolyzing and extracting Euglena powder (Euglena algae body) is used. can be Hydrolyzed Euglena extract can be prepared according to the method described in JP-A-2010-90065.

A method for preparing the hydrolyzed Euglena extract will be described below. Purified water is preferably added in an amount (weight) 100 times the amount (weight) of the dried Euglena, and pressure and heat treatment is performed. After that, a proteolytic enzyme is added to treat the algal body. After completion of the treatment, it is deactivated, for example, at 90° C., and the residue and water-soluble components are separated by centrifugation or filtration.

Specifically, the pressure and heat treatment conditions are preferably 100 to 150 ° C. using an autoclave, atmospheric pressure to 0.255 MPa, and heat and pressure treatment for 1 minute to 30 minutes. It is preferable to heat and pressurize at 0.14 MPa and 121° C. for 10 minutes. As the proteolytic enzyme, for example, pepsin, pancreatin, papain, and other commonly used enzymes having protease activity may be used alone or in combination. It is preferable to employ an endo-type peptidase to decompose into .

Pantidase MP and Alloase AP-10 manufactured by Yakult Pharmaceutical Co., Ltd. can also be used as commercially available proteases. The concentration of the enzyme added, the pH and reaction temperature of the reaction solution, and other conditions may be selected optimally for each enzymatic agent.

The water-soluble component thus obtained can be used as it is, but it can also be used after being subjected to purification operations such as fractionation, deodorization, decolorization, concentration, etc., as long as the effects of the present invention are not lost.

<Paramylon>
"Paramylon" is a porous polymer (β-1,3-glucan) in which about 700 glucoses are polymerized by β-1,3-bonds, and is a storage polysaccharide contained in the genus Euglena. is. Paramylon particles are flattened spheroidal particles and are formed by helically intertwined β-1,3-glucan chains.

Paramylon exists as granules in Euglena cells of all species and varieties, and the number, shape, and uniformity of the particles are characteristic depending on the species. Paramylon is composed only of glucose, and the average degree of polymerization of paramylon obtained from E. gracilis Z wild strain and chloroplast-deficient strain SM-ZK is about 700 in terms of glucose units. Paramylon is insoluble in water and hot water, but soluble in dilute alkali, strong acid, dimethyl sulfoxide, formaldehyde and formic acid. The average density of paramylon is 1.53 for E. gracilis Z and 1.63 for E. gracilis var. bacillaris SM-L1.

According to X-ray analysis using the powder pattern method, paramylon has a gentle helical structure in which three straight-chain β-1,3-glucans are twisted like a right-handed rope. Several of these glucan molecules aggregate to form paramylon granules. Paramylon granules have a very large crystal structure portion, accounting for about 90%, and are compounds with the highest crystal structure ratio among polysaccharides (Euglena, Physiology and Biochemistry, edited by Shozaburo Kitaoka, Gakkai Shuppan Center). The particle size distribution of paramylon (manufactured by Euglena Co., Ltd.) has a median diameter of 1.5 to 2.5 μm when measured with a laser diffraction/scattering particle size distribution analyzer.

Paramylon particles are isolated from cultured Euglena cells by any appropriate method and purified into fine particles, and are usually provided as powder. For example, paramylon particles can be (1) cultured Euglena cells in any appropriate medium, (2) separation of Euglena cells from the medium, (3) isolation of paramylon from the separated Euglena cells, ( 4) purification of the isolated paramylon, and optionally (5) cooling and subsequent lyophilization. Isolation of paramylon is performed, for example, using non-ionic or anionic detergents of the type that are largely biodegradable. Purification of paramylon occurs substantially simultaneously with isolation.

(Processed product of paramylon)
Processed products of paramylon include water-soluble paramylon obtained by chemically or physically treating paramylon by various known methods, sulfated paramylon, and paramylon derivatives.

Examples of processed products of paramylon include amorphous paramylon and emulsion paramylon. Amorphous paramylon is a substance obtained by amorphizing crystalline paramylon derived from Euglena. Amorphous paramylon has a relative crystallinity of 1 to 20% with respect to crystalline paramylon produced from Euglena by a known method. However, this relative crystallinity is obtained by the method described in JP-A-2011-184592.

That is, amorphous paramylon and paramylon were each pulverized with a pulverizer (ball mill MM400 manufactured by Retsh) for 5 minutes at a frequency of 20 times/second, and then analyzed using an X-ray diffractometer (H'PertPRO manufactured by Spectris). At a voltage of 45 KV and a tube current of 40 mA, scanning is performed in the range of 2θ from 5° to 30° to obtain diffraction peaks Pc and Pa near 2θ=20° of paramylon and amorphous paramylon. Using the values of Pc and Pa, the relative crystallinity of amorphous paramylon is calculated by the relative crystallinity of amorphous paramylon=Pa/Pc×100 (%).

Amorphous paramylon is prepared according to the method described in JP-A-2011-184592 by subjecting crystalline paramylon powder to alkali treatment, followed by neutralization with acid, followed by washing, water removal, and drying. Processed products of paramylon also include water-soluble paramylon obtained by chemically or physically treating paramylon by various known methods, sulfated paramylon, and paramylon derivatives.

"Emulsion paramylon" is a substance also called emulsion paramylon because its processing method and physical properties are similar to emulsions, and is obtained by adding water to paramylon according to the method described in JP-A-2016-199650. It is processed paramylon that is swollen by bonding with water four times or more, obtained by performing a collision treatment in which the fluid is ejected from a fine nozzle at an ultrahigh pressure to collide with an object to be collided.

Emulsion paramylon is a known physical property modification device that ejects a slurry obtained by adding a water-soluble solvent to a solid such as powder at an ultrahigh pressure from a fine-pore nozzle to collide with an object to be collided (for example, JP-A-2011-88108 It can be obtained by performing collision treatment one or more times at a nozzle pressure of 245 MPa at the time of jetting using a device described in Japanese Patent Application Laid-Open No. 6-47264.

Emulsion paramylon has a median diameter of 5 times or more that of paramylon and 7 μm or more when the particle size is measured with a laser diffraction/scattering particle size distribution analyzer, and the particles are separated from adjacent particles by an optical electron microscope. Adhesion was observed, and it swelled by binding with water four times or more that of paramylon.

Slurry, which is a mixture of raw material paramylon and water, is a free-flowing fluid. It has excellent stickiness and elasticity, and has a paste-like feel. The processed paramylon obtained is called emulsion paramylon in this specification because of its processing method and physical properties, but it is unknown whether or not it is emulsified. is.

<Application>
The GLS1 gene inhibitor and the senescent cell removing agent according to the present embodiment are configured as cosmetic compositions, food compositions such as health foods, and pharmaceutical compositions, and are used and ingested preventively to remove senescent cells. • Administered. Euglena-derived substances such as Euglena, Euglena extracts, and paramylon can be ingested as food and have no side effects, so they can be used, ingested, and administered continuously.

(Cosmetic composition)
The GLS1 gene inhibitor and senolytic agent according to the present embodiment can be suitably used in cosmetic compositions by utilizing the GLS1 gene inhibitory action and senolytic action. The cosmetic composition can be applied to any form of cosmetics. For example, skin care cosmetics such as cleansing agents, facial cleansers, lotions, lotions, milky lotions, creams, serums, packs, and scrubs, and makeup such as foundations, concealers, makeup bases, lipsticks, blushers, eye shadows, and eyeliners. It can be applied to cosmetics, sunscreen cosmetics, and the like.

In the cosmetic composition according to the present embodiment, in addition to the GLS1 gene inhibitor and the senescent cell removing agent according to the present embodiment, one or more components that can be used in ordinary cosmetic compositions are freely added. It is possible to select and blend. For example, base materials, preservatives, emulsifiers, coloring agents, preservatives, surfactants, ultraviolet absorbers, antioxidants, moisturizing agents, ultraviolet absorbers, fragrances, antiseptic antifungal agents, extender pigments, coloring pigments, alcohols, All additives that can normally be used in the cosmetic field, such as water, can be included.

In the cosmetic composition according to the present embodiment, the contents of the GLS1 gene inhibitor and the senescent cell removing agent are not particularly limited, and can be freely set according to the purpose.

(Food composition)
In the field of foods, the GLS1 gene inhibitor and senolytic agent of the present embodiment contain an effective amount of Euglena-derived substances that can effectively exhibit the GLS1 gene inhibitory action and the senescent cell removing action as a food material, and are blended in various foods. By doing so, it is possible to provide a food composition having the action. That is, the present invention can provide a food composition for foods labeled for removing senescent cells in the field of foods. Examples of the food composition include general foods, foods for specified health uses, foods with nutrient function claims, foods with function claims, foods for hospital patients, supplements, and the like. It can also be used as a food additive.

Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (lactic acid beverages, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powders Beverages (powdered juice, powdered soup, etc.), concentrated drinks, confectionery (candies, cookies, biscuits, gums, gummies, chewables, tablets, chocolate, etc.), breads, cereals and the like. In the case of food for specified health uses, food with nutrient function claims, food with function claims, etc., the shape may be capsule, troche, syrup, granule, powder, or the like.

Foods for Specified Health Uses are foods that contain functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Director-General of the Consumer Affairs Agency. be. In the present invention, it is a food that is marketed with a label indicating a specific health use related to removal of senescent cells.

A food with nutrient function claims is a food that is used to supplement nutritional ingredients (vitamins and minerals), and the functions of the nutritional ingredients are indicated. In order to be sold as a food with nutrient function claims, the amount of nutrients contained in the recommended daily intake must be within the specified upper and lower limits. also need to

Foods with function claims are foods labeled with functionalities based on scientific evidence under the responsibility of the business operator. Information on the grounds for safety and functionality was submitted to the Commissioner of the Consumer Affairs Agency before sales.

In the food composition according to the present embodiment, in addition to the Euglena-derived substance, it is possible to freely select and add one or more components that can be used in ordinary food compositions. For example, all additives that can be commonly used in the food field, such as various seasonings, preservatives, emulsifiers, stabilizers, fragrances, coloring agents, preservatives, and pH adjusters, can be included.

(Pharmaceutical composition)
In the field of medicine, the GLS1 gene inhibitor and senolytic agent of the present embodiment are used together with an amount of Euglena-derived substances that can effectively exhibit the GLS1 gene inhibitory action and senolytic action, together with a pharmaceutically acceptable carrier or additive. A pharmaceutical composition having the action is provided by blending the agents. The pharmaceutical composition may be a drug or a quasi-drug.

The pharmaceutical composition may be applied internally or topically. Therefore, the pharmaceutical composition is used in the form of an oral preparation, an injection such as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection and/or an intraperitoneal injection, a transmucosal application, a transdermal application, and the like. be able to. The dosage form of the pharmaceutical composition can be appropriately set depending on the application form. Semi-solid formulations such as ointments or gels are included.

The pharmaceutical composition according to this embodiment can contain one or more pharmaceutically acceptable additives by freely selecting them. For example, when the pharmaceutical composition according to this embodiment is applied to an oral formulation, for example, excipients, binders, disintegrants, surfactants, preservatives, coloring agents, flavoring agents, fragrances, stabilizers, preservatives All additives that can be commonly used in the field of pharmaceutical formulations, such as agents, antioxidants, etc., can be contained. In addition, a drug delivery system (DDS) can be used to prepare a sustained-release preparation or the like.

EXAMPLES The present invention will be specifically described below based on specific examples, but the present invention is not limited to these.

<Example 1: Hydrolyzed Euglena extract>
As the Euglena-derived substance, a hydrolyzed Euglena extract, which is a water-soluble component extracted by proteolytic decomposition of Euglena gracilis cells, was used. The hydrolyzed Euglena extract is obtained by adding 100 times the amount of purified water to 1 g of Euglena gracilis powder (Euglena algae, manufactured by Euglena Co., Ltd.), heating to a pressure of 0.1 MPa and a temperature of 12 ° C., and heating for 10 minutes. pressure treated. After that, 5% of the total amount of proteolytic enzyme of endo-type protease (for example, pantidase MP manufactured by Yakult Pharmaceutical Co., Ltd.) was added, and the mixture was reacted with stirring at 35° C. for 16 hours. After the reaction, the water-soluble component was fractionated by filtering using a filter paper. A sample obtained by sterilizing the water-soluble component with a 0.45 μm filter was used as 100% hydrolyzed Euglena extract undiluted solution.

<Example 2: Euglena extract>
Euglena extract was used as the Euglena-derived substance. The Euglena extract was prepared using Euglena gracilis powder (Euglena algal body, manufactured by Euglena Co., Ltd.) as a raw material and 1,3-butylene glycol (BG) as a solvent.

To 0.5 g of Euglena powder (Euglena gracilis, Lot. EU-16189, manufactured by Euglena Co., Ltd.), 1 ml of BG was added and shaken to uniformly disperse, followed by extraction at room temperature (25° C.) for 10 minutes. The supernatant obtained by centrifugation (3000 rpm, 3 minutes, 25° C.) was fractionated and filtered through a 0.45 μm sterile filter to prepare an Euglena extract.

<Test 1>
In Test 1, the influence of the hydrolyzed Euglena extract (Example 1) on the genes of skin epidermal cells was evaluated.

(Method of Test 1)
Normal human epidermal cells (3.0×10 4 cells/well) were seeded in a 12-well plate using HuMedia-KG2 medium. Next, the hydrolyzed Euglena extract was dissolved in HuMedia-KB2 medium (non-proliferation medium), and the medium was replaced. After replacing the medium, the cells were cultured for 24 hours, the culture supernatant was removed, and total RNA was isolated from the cells using RNAiso Plus, followed by purification using NucleoSpin (registered trademark) RNA. RNA library construction and sequencing was performed using the TruSeq Stranded mRNA Library Prep Kit and NovaSeq 6000 (Illumina Inc, USA). In this test (n=1), FPKM values were calculated, and genes with |logFC|>0.5 were defined as variable genes.

(Results of Test 1)
As a result of comprehensively analyzing gene changes in human epidermal keratinocytes caused by hydrolyzed Euglena extract using a next-generation sequencer, a sufficient number of reads could be secured for 14,586 genes out of a total of 57,643 genes. When 5% hydrolyzed Euglena extract was added, 1,145 genes (46.8%) decreased and 1,308 genes (53.2%) increased. The expression level of the GLS1 gene was 198.515 in the control and 127.003 when the hydrolyzed Euglena extract was added.

<Test 2>
In Test 2, the effects of the hydrolyzed Euglena extract (Example 1) and the Euglena extract (Example 2) on the longevity-related gene (GLS1) were evaluated.

(Method of Test 2)
Normal human epidermal cells (3.0×10 4 cells/well) were seeded in a 12-well plate using HuMedia-KG2 medium. Next, the hydrolyzed Euglena extract (Example 1) or Euglena extract (Example 2) was dissolved in HuMedia-KB2 medium (non-proliferation medium), and the medium was replaced. After replacing the medium, the cells were cultured for 24 hours, the culture supernatant was removed, and total RNA was isolated from the cells using RNAiso Plus. RNA was reverse transcribed to synthesize cDNA, and gene expression levels were evaluated by real-time PCR.

(Results of test 2)
FIG. 1 shows the results of examining the effects of Euglena-derived substances on the GSL1 gene in human epidermal keratinocytes. When the hydrolyzed Euglena extract (Example 1) was added, the expression level of the GLS1 gene was low. When the Euglena extract (Example 2) was added, the expression level of the GLS1 gene was significantly reduced.

The above results showed that the Euglena-derived substance inhibits the expression of the GLS1 gene in human epidermal keratinocytes. In other words, it was suggested that the use of the Euglena-derived substance as an active ingredient can inhibit the expression of the GLS1 gene and eliminate senescent cells in vivo.

The present invention relates to a GLS1 gene-inhibiting cosmetic composition, a GLS1 gene inhibitor, a senescent cell-removing cosmetic composition , and a senescent cell-removing agent.

With the background art described above, there is a demand for a technique for removing senescent cells. An object of the present invention is to provide a GLS1 gene-inhibiting cosmetic composition, a GLS1 gene inhibitor, a senescent cell-removing cosmetic composition , and a senescent cell-removing agent.

The object is to contain a Euglena-derived substance as an active ingredient, which is used to inhibit the expression of the GLS1 gene in the skin , and wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or an algae cell belonging to the genus Euglena as a protein enzyme. The problem is solved by a cosmetic composition for inhibiting the GLS1 gene , which is a water-soluble component that is decomposed and extracted and is applied transdermally .
The object is to contain a Euglena-derived substance as an active ingredient, which is used to inhibit the expression of the GLS1 gene in the skin , and wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or an algae cell belonging to the genus Euglena as a protein enzyme. It is resolved by inhibiting the GLS1 gene, which is characterized by being a water-soluble component that is decomposed and extracted, and is applied percutaneously .
The above-mentioned subject contains an Euglena-derived substance as an active ingredient, is used to remove skin aging cells, and the Euglena-derived substance is an aqueous solvent extract of Euglena or algae cells belonging to the genus Euglena that are proteolytically degraded. The problem is solved by a cosmetic composition for removing senescent cells , which is a water-soluble component that is extracted through the skin and is applied transdermally .
The above-mentioned subject contains an Euglena-derived substance as an active ingredient, is used to remove aging cells from the skin , and the Euglena-derived substance is an aqueous solvent extract of Euglena or algae cells belonging to the genus Euglena that are proteolytically decomposed. It is a water-soluble component that is extracted through the skin, and is solved by a senescent cell removing agent that is characterized by being applied transdermally .
At this time, the Euglena-derived substance is preferably an Euglena extract.
At this time, the Euglena-derived substance is preferably an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena.

According to the present invention, it is possible to provide a GLS1 gene-inhibiting cosmetic composition and a GLS1 gene inhibitor capable of inhibiting the expression of the GLS1 gene. Further, according to the present invention, it is possible to provide a senescent cell-removing cosmetic composition and a senescent cell-removing agent capable of removing senescent cells.

Claims (18)

A cosmetic composition for inhibiting the GLS1 gene, comprising a Euglena-derived substance as an active ingredient and used for inhibiting the expression of the GLS1 gene. 2. The cosmetic composition for inhibiting the GLS1 gene according to claim 1, wherein the Euglena-derived substance is an algal extract belonging to the genus Euglena. 2. The GLS1 gene-inhibiting cosmetic according to claim 1, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algae cells belonging to the genus Euglena. Composition. A food composition for inhibiting the GLS1 gene, which contains a Euglena-derived substance as an active ingredient and is used for inhibiting the expression of the GLS1 gene. 5. The food composition for inhibiting the GLS1 gene according to claim 4, wherein the Euglena-derived substance is an Euglena extract. 5. The food composition for inhibiting the GLS1 gene according to claim 4, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena. thing. A GLS1 gene inhibitor containing a Euglena-derived substance as an active ingredient and used to inhibit expression of the GLS1 gene. 8. The GLS1 gene inhibitor according to claim 7, wherein the Euglena-derived substance is an Euglena extract. 8. The GLS1 gene inhibitor according to claim 7, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena. A cosmetic composition for removing senescent cells, which contains an Euglena-derived substance as an active ingredient and is used for removing senescent cells in vivo. The cosmetic composition for removing senescent cells according to claim 10, wherein the Euglena-derived substance is an Euglena extract. 11. The cosmetic for removing senescent cells according to claim 10, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena. Composition. A food composition for removing senescent cells, which contains an Euglena-derived substance as an active ingredient and is used for removing senescent cells in vivo. 14. The food composition for removing senescent cells according to claim 13, wherein the Euglena-derived substance is an Euglena extract. 14. The food composition for removing senescent cells according to claim 13, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena. thing. A senescent cell removing agent containing a Euglena-derived substance as an active ingredient and used for removing senescent cells in vivo. 17. The agent for removing senescent cells according to claim 16, wherein the Euglena-derived substance is an Euglena extract. 17. The agent for removing senescent cells according to claim 16, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena.

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