JP2021112181A - Composition for inhibiting accumulation of senescent cells - Google Patents

Abstract

To provide a composition for inhibiting accumulation of senescent cells.SOLUTION: One or more substances selected from Bifidobacterium bacteria, cultures thereof, and treated products thereof are blended in a composition for inhibiting accumulation of senescent cells. The composition of the present invention can inhibit accumulation of senescent cells preferably by cell senescence inhibitory action or cell senescence apoptosis-inducing action.SELECTED DRAWING: Figure 1

Description

The present invention relates to a composition capable of suppressing the accumulation of senescent cells.

Cellular senescence is generally defined as a condition in which cells remain viable and have metabolic activity but lose their ability to proliferate. Cellular senescence is a variety of stimuli such as telomere shortening due to DNA terminal replication, DNA damage, changes in expression and activity of tumor suppressor genes and cancer genes, oxidative stress, inflammation, chemotherapeutic agents, exposure to UV irradiation and ionizing radiation. Alternatively, it is known that it can be caused by a factor (Non-Patent Document 1).

Various factors collectively called Senescence Associated Secretory Phenotype (SASP) are secreted from cells in which cell senescence is induced (senescent cells) to age surrounding cells, and SASP is a blood vessel. When transferred to the whole body via, it may promote cell senescence and carcinogenesis at the transfer destination, cause chronic inflammation, and migrate immune cells, resulting in impaired tissue function (non-patented). Document 1). Cellular senescence is thought to be induced in various tissues with aging, and its relationship with arteriosclerosis, diabetes, dementia, etc. has been suggested, and it is considered to be one of the factors that cause age-related diseases (). Non-Patent Document 2).
Due to these properties of senescent cells, there is increasing interest in preventing the accumulation of senescent cells (Senolytics), and senolytic removal of senescent cells is effective for some age-related diseases. This has been shown in animal experiments and human clinical studies (Non-Patent Document 2). Furthermore, in the mouse model, the longevity-prolonging effect of Senolytic has been shown, and the anti-aging effect at the individual level has been clarified (Non-Patent Document 3).

Eiji Hara “New Phase of Cellular Senescence” Experimental Medicine, 37 (11); 1728-1733, (2019) Paez-Ribes M, Gonzalez-Gualda E, Doherty GJ, Munoz-Espin D. EMBO Molecular Medicine, 11 (12), PMID: 31746100, (2019) Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong JA, Saltness R, Jeganathan KB, Verzosa GC, Pezeshki A et al, Nature, 530, 184-189 (2016)

An object of the present invention is to provide a composition for suppressing the accumulation of senescent cells.

As a result of diligent research to solve the above problems, the present inventors have found that bifidobacteria and their cultures or processed products have an action of suppressing the accumulation of senescent cells. The invention was completed.

That is, one aspect of the present invention is a composition for suppressing the accumulation of senescent cells, which comprises one or more selected from a bacterium belonging to the genus Bifidobacterium, a culture of the bacterium, and a treated product of the bacterium. be.
In this embodiment, the suppression of the accumulation of senescent cells may be due to the cell senescence inhibitory action.
Further, in this embodiment, the suppression of the accumulation of senescent cells may be due to the apoptosis-inducing action on the senescent cells.
Another aspect of the present invention is a cell anti-aging composition containing one or more selected from a bacterium of the genus Bifidobacterium, a culture of the bacterium, and a treated product of the bacterium.
In the present invention, the bacterium belonging to the genus Bifidobacterium is preferably selected from Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis.
The composition of the present invention is preferably a food or drink.
The composition of the present invention is preferably a pharmaceutical product.

According to the present invention, the accumulation of senescent cells can be suppressed by inhibiting cell senescence or by causing apoptotic cells to be apoptotic and eliminated. Therefore, it is possible to improve or prevent cell senescence and diseases and symptoms associated with or caused by SASP secreted from senescent cells.

The graph which shows the SA-β-Gal staining rate (average value) in the TIG-3 cell on the 2nd day after the induction of cell senescence. The graph which shows the relative expression level of the p16 gene in TIG-3 cells 3 days after the induction of cell senescence. The graph which shows the cytotoxicity (change rate of the cell number) of each sample with respect to TIG-3 cell which induced cell senescence. The graph which shows the cytotoxicity (cell number proliferation rate) of each sample with respect to the proliferating TIG-3 cell.

Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.

The composition of the present invention contains one or more selected from a bacterium belonging to the genus Bifidobacterium, a culture of the bacterium, and a processed product of the bacterium.

Bifidobacterium genus bacteria are not particularly limited, but Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis,
Reclassified as Bifidobacterium longum subspecies infantis), Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium catenuratum (Bifidobacterium catenulatum), Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium lactis, and Bifidobacterium pseudocatenulatum. (Bifidobacterium pseudolongum).
Of these, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis are more preferred.

More specifically, Bifidobacterium longum BB536 can be mentioned. Bifidobacterium Longum BB536 is a National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (〒292-0818).
2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Room 122), NIT on January 26, 2018
The accession number is EBP-02621, which is an international deposit based on the Budapest Treaty. Bifidobacterium longum subsp. Longum ATCC BAA-999 (number: ATCC BAA-999), which is the same bacterium as this, is A.
merican Type Culture Collection (ATCC; USA, 2)
0110, Virginia, Manassas, University Boulevard 10801 (108)
01 University Boulevard, Manassas, VA 2011
0, United States of America)) to ATCC BAA-9
It is available as 99 (see, for example, Japanese Patent Application Laid-Open No. 2012-223134).

More specifically, Bifidobacterium breve M-16V can be mentioned. Bifidobacterium Breve M-16V has been deposited with NPMD on January 26, 2018 under the accession number NITE BP-02622 under the Budapest Treaty. For example, "Bifidobacterium Breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd., which is available as a commercially available product, may be used.
More specifically, Bifidobacterium breve MCC1274 can also be mentioned. Bifidobacterium Breve MCC1274 was issued on August 25, 2009 by the Patent Organism Depositary Center (currently the Incorporated Administrative Agency Product Evaluation Technology Infrastructure Organization Patent Organism Depositary Center (IPOD)) (〒292). -0818 A deposit is made internationally based on the Budapest Treaty at the accession number FERM BP-11175 at Kazusakamatari Room 2-5-8 120, Kisarazu City, Chiba Prefecture.

More specific examples of Bifidobacterium infantis include Bifidobacterium longum subspecies infantis M-63. Bifidobacterium Longum Subspecies Infantis M-63 is 2018
It is synonymous with Bifidobacterium Infantis M-63, which was deposited internationally under the Budapest Treaty with accession number NITE BP-02623 on January 26, 2014.

The bacteria specified by the above-exemplified bacterial names are not limited to the strains themselves that have been deposited or registered with a predetermined institution under the bacterial names (hereinafter, also referred to as "deposited strains" for convenience of explanation). Substantially equivalent strains (also referred to as "derivative strains" or "derivative strains") are also included. That is, the stock itself is not limited to the stock itself deposited with the depositary institution with the deposit number, and the stock substantially equivalent thereto is also included. For each bacterium, "a strain substantially equivalent to the above-mentioned deposit strain" belongs to the same species as the above-mentioned deposit strain, and further, the similarity of the genome sequence with the above-mentioned deposit strain (Average Nucleotide Identity value) is preferable. A strain having 99.0% or more, more preferably 99.5% or more, still more preferably 100% identity, and preferably having the same mycological properties as the deposited strain. For each bacterium, a strain substantially equivalent to the above-mentioned deposit strain may be, for example, a derivative strain having the deposit strain as a parent strain. Derivative strains include strains bred from deposit stocks and strains naturally generated from deposit stocks. Examples of the breeding method include modification by a genetic engineering method and modification by mutation treatment. Mutation treatment includes irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutant agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, and methylmethanesulfonate. Be done. Examples of naturally occurring strains from the deposited strains include strains naturally occurring during the use of the deposited strains. Examples of such a strain include a mutant strain naturally generated by culturing a deposited strain (for example, subculture). The derivative strain may be constructed by one type of modification, or may be constructed by two or more types of modification.

As the bifidobacteria to be contained in the composition of the present invention, a commercially available product may be used, those obtained by appropriately producing the bacteria may be used, or the above-mentioned bifidobacteria may be cultured. You may use the one obtained by.
The culture method is not particularly limited as long as Bifidobacterium spp. Can grow. As the culturing method, for example, a method usually used for culturing Bifidobacterium spp. Can be used as it is or after being appropriately modified. The culture temperature may be, for example, 25 to 50 ° C., preferably 35 to 42 ° C. The culture can be preferably carried out under anaerobic conditions, and can be carried out, for example, while aerating an anaerobic gas such as carbon dioxide. In addition, the culture can also be carried out under microaerobic conditions such as liquid static culture. Culturing can be carried out, for example, until the Bifidobacterium spp. Grow to the desired degree.

The medium used for culturing is not particularly limited as long as Bifidobacterium spp. Can grow. As the medium, for example, a medium usually used for culturing bifidobacteria can be used as it is or after being appropriately modified. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolyzate, and waste sugar honey can be used depending on the assimilation property. .. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate and nitrates can be used. Further, as the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. In addition, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract may be used. In addition, as a medium usually used for culturing bifidobacteria, specifically, a fortified Clostridia medium (Reinforced Clostridial medium), an MRS medium (de Man, Rogosa, and Sharpe medium), and an mMRS medium (modified MRS medium). ), TOSP medium (TOS propionate medium), TOSP Mup
Examples thereof include a medium (TOS propionate mupirocin medium), a GAM (Gifu Anaerobic Medium) medium, and a YCFA (Yeast Extract-casein Hydrolysate Acid) medium.

The form of the bacterium belonging to the genus Bifidobacterium contained in the composition of the present invention may be any of a bacterial cell, a culture of the bacterium, and a treated product of the bacterium.
The bacterial cells are usually preferably contained in a form containing live bacterial cells, but are not particularly limited. The bacterial cell may be, for example, a live bacterial cell, a dead bacterial cell, or a mixture of a live bacterial cell and a dead bacterial cell.
As the bacterial culture, for example, the culture obtained by the culture may be used as it is, the culture may be diluted or concentrated and used, or the bacterial cells recovered from the culture may be used. Moreover, you may use culture supernatant or culture fraction as a culture. When the culture supernatant is used, for example, the supernatant of the culture solution obtained by culturing in GAM medium at 37 ° C. and 16 h can be preferably used.
As the processed product of bacteria, crushing, heating, freeze-drying, and dilutions, dried products or fractions thereof can be used for bacterial cells or cultures.

The composition of the present invention can suppress the accumulation of senescent cells.
As used herein, the term "senescent cell" means a cell in which cell senescence has been induced, which is a state in which a viable state is maintained and a state in which a metabolic activity is exhibited but a proliferative ability is lost.
As used herein, suppressing the accumulation of senescent cells includes inhibiting the induction of cellular senescence. That is, "suppression of accumulation of senescent cells" is to suppress the accumulation of senescent cells by inhibiting the transformation of normal cells into senescent cells and not increasing the number of senescent cells. As used herein, the term "normal cell" means a cell that maintains a viable state and has metabolic activity and proliferative ability.
Further, in the present specification, suppression of accumulation of senescent cells includes specific apoptosis of senescent cells. That is, "suppression of accumulation of senescent cells" also suppresses the accumulation of cells that have already become senescent cells by inducing and reducing apoptosis. The apoptosis-inducing action of the compositions of the present invention on senescent cells is usually undamaged on proliferating normal cells in which cell senescence has not been induced.

The composition of the present invention can inhibit the induction of normal cells to senescent cells as described above. In addition, the composition of the present invention can suppress the aging of surrounding cells through the secretion of SASP by apoptotic senescent cells.
Therefore, the action of the composition of the present invention can be rephrased as the anti-aging action of cells. Here, the anti-aging of cells may be to suppress cell aging.

The composition of the present invention is expected to improve or prevent diseases or symptoms associated with or caused by cellular senescence and SASP secreted from senescent cells by its action of suppressing the accumulation of senescent cells. Such diseases and symptoms include premature aging, cataracts, alopecia, cardiac hypertrophy, fatty liver, interstitial pneumonia, COPD, renal glomerulosclerosis, arteriosclerosis, arthritis, disc degeneration, sarcopenia, inflammatory bowel disease, diabetes, etc. Examples include metabolic syndrome, cancer, sarcoma, lymphoma, dementia, pituitary tumor, neurodegenerative disease and the like. In addition, the composition of the present invention is also expected to have effects such as alleviation of bone loss, atrophy of adipose tissue, improvement of cardiac function, recovery of respiratory function, muscle strengthening, improvement of motor function, extension of healthy life expectancy, and effect of extension of life expectancy. Will be done.

The content of the Bifidobacterium genus bacterium, the culture of the bacterium, and the processed product of the bacterium in the composition of the present invention is not particularly limited, but for example, the bacterial cell mass of the bacterium is 1.0 per 1 mL of the composition. It is preferably × 10 ⁴ cfu or more, and more preferably 2.0 × 10 ⁷ cfu or more. In addition, cfu refers to a colony forming unit. In the present specification, for example, it can be the value when cultivated at 38 ° C. in a solid medium containing 10% by mass of reduced skim milk powder. Further, when the cells are dead cells, cfu can be replaced with individual cells (cells).
When the culture supernatant is used as the culture of the bacteria, it is preferably 0.1 to 100% by mass, more preferably 1 to 90% by mass, and further preferably 10 to 80% by mass of the entire composition. Can be.
These may usually be in the range of content when distributed as an oral composition.

Another aspect of the present invention is the use of one or more selected from bifidobacteria, cultures of the bacteria, and treatments of the bacteria in the production of compositions for suppressing the accumulation of senescent cells. be.
Another aspect of the present invention is the use of one or more selected from Bifidobacterium spp., Cultures of said bacteria, and treated products of said bacteria in suppressing the accumulation of senescent cells.
Another aspect of the present invention is one or more selected from Bifidobacterium spp., Cultures of said bacteria, and treated products of said bacteria used for suppressing the accumulation of senescent cells.
Another aspect of the present invention is the suppression of the accumulation of senescent cells, which comprises administering to a subject one or more selected from a Bifidobacterium bacterium, a culture of the bacterium, and a treated product of the bacterium. How to do it.

Another aspect of the present invention is the use of one or more selected from bifidobacteria, cultures of the bacteria, and treatments of the bacteria in the production of cell anti-aging compositions. ..
Another aspect of the invention is the use of one or more selected from Bifidobacterium spp., Cultures of said bacteria, and treated products of said bacteria in anti-aging of cells.
Another aspect of the invention is one or more selected from Bifidobacterium spp., Cultures of said bacteria, and treated products of said bacteria used for anti-aging of cells.
Another aspect of the invention is a method of cell anti-aging comprising administering to a subject one or more selected from a Bifidobacterium bacterium, a culture of the bacterium, and a treated product of the bacterium. be.

The subject to which the composition of the present invention is administered (administrator) and the subject to be ingested (ingestor) are not particularly limited as long as they are animals, but are usually humans. In addition, it may be any of adults, children, infants, newborns (including low-weight babies), etc., but it is usually an adult. The gender is not particularly limited.

In the present specification, "administering one or more species selected from a bacterium of the genus Bifidobacterium, a culture of the bacterium, and a treated product of the bacterium to an animal" means "a bacterium of the genus Bifidobacterium". , One or more selected from the culture of the bacterium and the processed product of the bacterium, may be synonymous with "to feed the animal." Ingestion is usually voluntary (free ingestion), but may be compulsory (compulsory ingestion). That is, specifically, in the administration step, for example, one or more selected from a bacterium belonging to the genus Bifidobacterium, a culture of the bacterium, and a processed product of the bacterium are blended into food or drink or feed. It may be a step of supplying to the bacterium and thus allowing the subject to freely ingest it.

The ingestion (administration) timing of the composition of the present invention is not particularly limited, and can be appropriately selected according to the state of the ingestion (administration) target.

The ingestion (administration) amount of the composition of the present invention is appropriately selected depending on the age, gender, condition, other conditions, etc. of the ingestion (administration) subject.
The intake (administration) amount of the composition of the present invention is preferably in the range of 0.5 to 5 g / day for adults, for example, as the intake (administration) amount of the bacterial cells of the genus Bifidobacterium according to the present invention. The range of 1 to 4 g / day is more preferable, and 2 to 3 g / day is even more preferable. Alternatively, the intake (administration) amount of the culture supernatant of Bifidobacterium spp. Is preferably in the range of 0.5 to 5 g / day, more preferably 1 to 4 g / day, and 2 to 3 g in adults, for example. / Day is more preferred.
The composition of the present invention can be ingested (administered) once or in a plurality of times a day regardless of the amount and duration of ingestion (administration).

The ingestion (administration) period of the composition of the present invention is not particularly limited. In addition, there is no particular upper limit on the intake (administration) period, and continuous and long-term intake (administration) is possible.

The route of ingestion (administration) of the composition of the present invention may be oral or parenteral, but oral is preferable. In addition, parenteral ingestion (administration) includes transdermal, intravenous injection, rectal administration, inhalation and the like.

When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of food and drink.

The morphology and properties of the food and drink are not particularly limited as long as they can be ingested (administered) orally without impairing the effects of the present invention, and the bacterium of the genus Bifidobacterium, the culture of the bacterium, and the treated product of the bacterium are not particularly limited. It can be produced by a usual method using raw materials usually used for foods and drinks, except that one or two or more kinds selected from the above are contained.

Foods and drinks may be in the form of liquid, paste, gel solid, powder, etc., for example, nutritional supplements (supplements), tablets; liquid foods (nutrient foods for tube intake); bread, macaroni, spaghetti. , Mens, cake mix, fried flour, bread flour and other wheat flour products; instant noodles, cup noodles, retort / cooked foods, canned cooking, microwave foods, instant soups / stews, instant miso soups / soups, canned soups, freeze-dried foods , Other instant foods such as instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grain products); canned marine products, fish hams and sausages , Seafood paste products, fishery delicacies, boiled fish and other marine products; canned livestock and pastes, processed livestock products such as meat ham and sausage; , Prepared milk powder, cream, other dairy products such as dairy products; butter, margarines, vegetable oils and other fats and oils; soy sauce, miso, sauces, tomato processing seasonings, mirins, vinegars, etc. Ingredients: Combined seasonings / foods such as cooking mixes, curry bases, sauces, dressings, noodles, spices, and other complex seasonings; Frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; carbonated beverages, natural fruit juice , Fruit juice drinks, soft drinks with fruit juice, fruit meat drinks, fruit drinks with fruit grains, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, etc. Favorite beverages such as favorite beverages, baby foods, sprinkles, other commercially available foods such as Ochazuke paste; prepared powdered milk for childcare; enteral nutritional foods; functional foods (foods for specified health use, nutritionally functional foods), etc. ..

It can also be used as feed as one aspect of food and drink. Examples of the feed include pet food, livestock feed, fish feed and the like.
The form of the feed is not particularly limited, and in addition to one or more selected from Bifidobacterium spp., Cultures of the bacteria, and processed products of the bacteria, for example, corn, wheat, barley, rye, etc. Grains such as mylo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and flaxseed oil cake; bran such as bran, wheat bran, rice bran, and defatted rice bran; Animal feeds such as fish flour, defatted milk powder, casein, yellow grease, and corn; yeasts such as torula yeast and beer yeast; mineral feeds such as calcium tertiary phosphate and calcium carbonate; fats and oils; single amino acids; containing sugars, etc. It may be something to do.

When the composition of the present invention is in the form of a food or drink (including feed), it can be provided and sold as a food or drink indicating an application for suppressing the accumulation of aging cells and an application for anti-aging of cells. be.

Such "display" act includes all acts for informing the consumer of the use, and if it is an expression that can recall or analogize the use, the purpose of the display, the content of the display, etc. Regardless of the object or medium to be displayed, all of them fall under the act of "displaying" in the present invention.
Further, it is preferable that the "display" is performed by an expression that allows the consumer to directly recognize the above-mentioned use. Specifically, the act of transferring a product related to food and drink or the packaging of the product with the above-mentioned use, displaying it for delivery, transfer or delivery, and importing it, advertising on the product, price list or transaction documents Examples include the act of describing the above-mentioned use and displaying or distributing it, or describing the above-mentioned use in the information containing these and providing it by an electromagnetic (Internet, etc.) method.

On the other hand, as the display content, it is preferable that the display is approved by the government or the like (for example, a display obtained based on various systems established by the government and performed in a manner based on such approval). In addition, it is preferable to attach such display contents to packaging, containers, catalogs, pamphlets, promotional materials such as POPs at sales sites, and other documents.

In addition, "labeling" includes labeling as health foods, functional foods, enteric nutritional foods, special purpose foods, health functional foods, specified health foods, nutritional functional foods, functional foods, non-pharmaceutical products, etc. Can also be mentioned. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, a system related to foods for specified health use, nutritionally functional foods, or functional foods, or labeling approved by a system similar to these can be mentioned. Specifically, labeling as a food for specified health use, labeling as a conditional food for specified health use, labeling to the effect that it affects the structure and function of the body, labeling for reducing the risk of illness, and functionality based on scientific grounds. Indications, etc. can be mentioned, and more specifically, Cabinet Office Ordinance on permission for special use indications stipulated in the Health Promotion Act (Cabinet Office Ordinance No. 57, August 31, 2001) Labeling as a food for specified health use (particularly labeling for health use) and similar labeling specified in the above are typical examples.

Such indications include, for example, "do not accumulate aging cells", "for those who want to prevent cell aging", "for you who are losing bone", "for those who want to improve heart function", and "motor function". "For those who want to improve their healthy life expectancy", "For those who want to stay young forever", etc.

The composition of the present invention can also be in the form of a pharmaceutical product.
The route of ingestion (administration) of the drug may be oral or parenteral, but oral is preferable. In addition, parenteral ingestion (administration) includes transdermal, intravenous injection, rectal administration, inhalation and the like.
As the form of the drug, it can be appropriately formulated into a desired dosage form according to the ingestion (administration) method. For example, in the case of oral ingestion (administration), it can be formulated into a solid preparation such as a powder, a granule, a tablet or a capsule; a liquid preparation such as a solution, a syrup, a suspension or an emulsion. In the case of parenteral ingestion (administration), it can be formulated into a suppository, an ointment, an injection, or the like.
At the time of formulation, components such as excipients, pH adjusters, colorants, and flavoring agents that are usually used for formulation can be used. It is also possible to use other medicines such as other medicinal ingredients and components having a known or future-finding inhibitory effect on the accumulation of aging cells or an anti-aging effect on cells.
In addition, the formulation can be carried out by a known method as appropriate depending on the dosage form. At the time of formulation, a carrier usually used for formulation may be blended and formulated as appropriate. Examples of such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannit, and sorbit; starch derivatives such as corn starch, horse bell starch, α-starch, dextrin, and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, and the like. Cellulous derivatives such as hydroxypropylmethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium; gum arabic; dextran; purulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonic acid Carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate can be mentioned.

Examples of the binder include gelatin; polyvinylpyrrolidone; macrogol and the like in addition to the above-mentioned excipients.

Examples of the disintegrant include, in addition to the above-mentioned excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Examples of the lubricant include talc; stearic acid; metal stearic acid salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and silicic acid; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. Sodium carboxylic acid salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic acid anhydride and silicic acid hydrate; starch derivatives and the like. Be done.

Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic acid anhydride; and sorbic acid.

Examples of the flavoring agent include sweeteners, acidulants, and flavors.
Examples of the carrier used in the case of a liquid preparation for oral ingestion (administration) include a solvent such as water.

The timing of ingesting (administering) the drug of the present invention is not particularly limited, for example, before meals, after meals, between meals, and before bedtime.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of culture supernatant of Bifidobacterium genus Bifidobacterium Longum NITE BP-02621 (BB536) and Bifidobacterium Breve NITE BP-02622 (M-16V), respectively. Subcultured in MRS medium (Difco Lactobacillo MRS Broth, Becton, Dickinson and Company) and precultured. The preculture solution was inoculated into GAM medium (GAM bouillon "Nissui", manufactured by Nissui Pharmaceutical Co., Ltd.) at 5% (v / v) and cultured at 37 ° C. for 16 hours, respectively. Each culture solution was centrifuged at 4 ° C. and 8000 G for 10 minutes to collect the supernatant, and then the pH was adjusted to 7.0 ± 0.1 and the culture supernatant was prepared by passing through a 0.22 μm filter. The culture supernatant was stored at −30 ° C.
Hereinafter, the obtained "culture supernatant of Bifidobacterium longum NITE BP-02621 (BB536)" is also simply referred to as "culture supernatant of BB536". Further, the obtained "culture supernatant of Bifidobacterium Breve NITE BP-02622 (M-16V)" is also simply referred to as "culture supernatant of M-16V".

[Test Example 1] Examination of cell senescence inhibitory effect 6-well plate (Falcon cell culture 6-well multi-well plate, Corning)
Add 2 mL each of MEM medium (Minimum Essential Medium Eagle With Earle's salts, manufactured by SIGMA), 10% FBS (manufactured by Fetal Bovine Serum, manufactured by MP Bio), and 1% P / S (manufactured by Pen Strep, manufactured by gibco). Then, TIG-3 cells (1801781/07262017 / JCRB Cell Bank) having a PDL (Population Doubling Level) value of 25 to 50 were seeded so as to be ^{1 × 10 4 cells / well.} The culture supernatant of BB536 prepared in Example 1 was added thereto in an amount of 1% (v / v) or 0.1% (v / v), and the cells were cultured under the conditions of _{37 ° C., 5% and CO 2.}
Eighteen hours after the start of the culture, a final concentration of 3 μM doxorubicin (Doxo; doxorubicin hydrochloride, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added, and aging-inducing stimulation was performed for 1 hour. After the aging-inducing stimulation, the cells were washed twice with 2 mL of phosphate buffered saline (PBS pH 7.4 (1X), manufactured by gibco), and 2 mL MEM medium, 10% FBS, and 1% P / S were added. The MEM medium, FBS and P / S used were the same as those at the time of seeding of TIG-3 cells.
Then, 1% (v / v) or 0.1% (v / v) of the culture supernatant of BB536 was added again, and the cells were cultured for 2 to 3 days. Hereinafter, the obtained sample is referred to as "culture supernatant group of BB536".
A well was also provided to which the culture supernatant of BB536 was not added (Dox group). In addition, as a control for Dox, wells in which the culture supernatants of Dox and BB536 were not added and DMSO was added were also provided (DMSO group). In addition, as a positive control for the culture supernatant group of BB536, 1 μL of 1 MN-acetylcysteine (manufactured by Nacalai Tesque), which is a known antioxidant, was added instead of adding the culture supernatant of BB536.
As a negative control for the NAC group) and the culture supernatant group of BB536, wells (GAM group) to which GAM medium was added instead of adding the culture supernatant of BB536 were provided, respectively.
After culturing for 48 hours after the second addition of the culture supernatant of BB536, senescence-related acidic β-galactosidase (SA-β-Gal) staining was performed to specifically stain senescent cells. The cells stained with SA-β-Gal were photographed at three locations using a bright-field microscope (5x objective lens), and the number of cells was counted as senescent cells.
In addition, gene expression analysis of p16, which is a marker for senescent cells, was performed after culturing for 72 hours after the second addition of the culture supernatant of BB536.
The reverse transcription reaction was performed using PrimeScript RT Reagent Kit (manufactured by Takara Bio Inc.), and qPCR was performed using TB Green Premix Ex Taq II (manufactured by Takara Bio Inc.).

FIG. 1 shows the ratio of the number of SA-β-Gal-stained cells to the total number of seeded cells in each group (
Dyeing rate%) is shown. In the Dox group, 36% of SA-β-Gal positive cells are present, whereas BB is present.
In the culture supernatant group (1%) of 536, SA-β-Gal positive cells were 15%. The result of the culture supernatant group of BB536 was lower than the staining rate (23%) of SA-β-Gal positive cells in the NAC group, which is a positive control.
FIG. 2 shows the gene expression level of p16 (relative expression level with respect to the internal control GAPDH) in each group. The relative expression level was 0.0214 in the Dox group, whereas it was 0.0156 in the 1% culture supernatant administration group of BB536, which was significantly lower than that in the Dox group (Welch's t-test, p value). 0.0387).
From the above results, it was found that the culture supernatant of Bifidobacterium spp. Has an inhibitory effect on cell senescence.

[Test Example 2] Examination of apoptosis-inducing action on senescent cells As for Test Example 2, the same sample as Test Example 1 was used unless otherwise specified.
Add 2 mL each of MEM medium, 10% FBS, and P / S to a dish with a grid (μ-Dish 35 mm High grid-500, manufactured by Nippon Genetics) to add TIG-3 cells with a PDL value of 25 to 50. The seeds were sown so as to be 1 × 10 ^{4 pieces / dish.} The next day, a final concentration of 3 μM doxorubicin was added, and aging-inducing stimulation was performed for 1 hour. 2 mL of PB after aging-induced stimulation
The cells were washed twice with S, MEM medium, 10% FBS, and 2 mL of P / S were added and cultured. The MEM medium, FBS, and P / S used were the same as those used for TIG-3 cell seeding.
After 72 hours, 5 dishes were selected and the number of cells was counted using a bright-field microscope (5x objective lens) (first time). Then, 1%, 0.1%, or 0.01% of the culture supernatant of M-16V prepared in Example 1 was added to the cells. After an additional 96 hours (4 days), the culture supernatant of M-16V was additionally administered at 1%, 0.1%, or 0.01% and cultured. That is, the culture supernatant of M-16V was administered twice in a total amount of 2%, 0.2%, or 0.02%. The obtained sample is referred to as "M-16V culture supernatant group".
In addition, a well to which the culture supernatant of M-16V was not added was also provided (Dox group).
As a positive control for the M-16V culture supernatant addition group, instead of adding the M-16V culture supernatant, a dish (Dasa) to which a known apoptosis-inducing substance dasatinib (manufactured by Funakoshi) was added at a final concentration of 300 μM. As a negative control for the group) and the M-16V culture supernatant group, a dish (GAM group) to which GAM medium was added instead of adding the M-16V culture supernatant was also provided.
After 192 hours (8 days) of culturing from the time of the second addition of the culture supernatant of M-16V, the number of cells was counted at the same place on the dish as at the time of the first count of the number of cells. The ratio of the number of cells in the second time to the number of cells in the first time was calculated, and the rate of change in the number of cells before and after culturing was determined.

The results are shown in FIG. In the Dox group, 97% of the cells were alive, whereas in the M-16V culture supernatant 2% administration group, only 58% were alive, and a significant decrease in senescent cells was observed (Student's t-test). , P value 0.0480).
From the above results, it was found that the culture supernatant of Bifidobacterium spp. Has an apoptosis-inducing effect on senescent cells.

[Test Example 3] Examination of Cytotoxicity to Normal Cells For Test Example 3, the same sample as Test Example 1 was used unless otherwise specified.
Add 1 mL each of MEM medium, 10% FBS, and P / S to a 24-well plate (Falcon cell culture 24-well multi-well plate, manufactured by Corning), and the PDL value is 25.
TIG-3 cells of ~ 50 were ^{seeded at 5 × 10 3} cells / well. After 24 hours, 1% (v / v), 0.5% (v / v), or 0.1% (v / v) of the culture supernatant of M-16V prepared in Example 1 was added to the cells. Incubated for 3 or 6 days. Cell Counting after culturing for 3 or 6 days
Kit-8 (manufactured by Dojin Chemical Co., Ltd.) was added at 100 μL / well and cultured for 3 hours. Hereinafter, these samples are referred to as "M-16V culture supernatant group".
Wells (Dasa group) to which dasatinib was added at a final concentration of 300 μM instead of M-16V culture supernatant, wells (GAM group) to which GAM medium was added instead of M-16V culture supernatant, M-16V Wells (No treatment group) to which the culture supernatant was not added were also provided.
Then, using a microplate reader (SH-9000, manufactured by Corona Electric Co., Ltd.), the absorbance (OD _{450 ) of each group sample was measured at 450 nm, and the ratio of OD 450} after culturing for 3 days and after culturing for 6 days was calculated. Then, the cell proliferation rate was used.

The results are shown in FIG. In the No treatment group, the cell proliferation rate was 2.86, whereas in the culture supernatant group of M-16V, the cell proliferation rate was about 2.76 to 2.82 in any amount of sample added. rice field. The cell proliferation rate was 3.13 in the GAM 1% administration group and 0.94 in the Dasa group.
From the above results, it was found that the culture supernatant of M-16V did not show cytotoxicity to normal cells.

Claims (7)

A composition for suppressing the accumulation of senescent cells, which comprises one or more selected from a bacterium belonging to the genus Bifidobacterium, a culture of the bacterium, and a processed product of the bacterium. The composition according to claim 1, wherein the accumulation of senescent cells is suppressed by the cell senescence inhibitory action. The composition according to claim 1, wherein the accumulation of senescent cells is suppressed by an apoptosis-inducing action on senescent cells. A composition for anti-aging of cells, which comprises one or more selected from a bacterium of the genus Bifidobacterium, a culture of the bacterium, and a processed product of the bacterium. The composition according to any one of claims 1 to 4, wherein the Bifidobacterium bacterium is selected from Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis. thing. The composition according to any one of claims 1 to 5, which is a food or drink. The composition according to any one of claims 1 to 5, which is a pharmaceutical product.

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