Composition for removing senescent cells and use thereof

Abstract

To provide a composition capable of removing senescent cells.SOLUTION: A composition of the present disclosure contains sphingosines.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present disclosure relates to a composition for removing senolytic cells and uses thereof. [Background technology]

[0002] Senescent cells refer to cells whose sustained proliferation has decreased or ceased compared to normal cells. Senescent cells have been shown to secrete inflammatory cytokines, chemokines, growth factors, etc. This phenomenon is called the senescence-associated secretory phenotype (SASP), and has been suggested to be related to the onset of aging-related diseases (Patent Document 1).

[0003] In recent years, the development of drugs that can selectively remove senescent cells is expected to lead to the establishment of new methodologies for extending healthy lifespan and treating age-related diseases. Known examples of such drugs include drugs containing glutaminase inhibitors as active ingredients (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 095971 Summary of the Invention [Problem to be solved by the invention]

[0005] Development of drugs that can selectively eliminate senescent cells has only recently begun and is not yet fully advanced.

[0006] Therefore, an object of the present disclosure is to provide a composition capable of removing senescent cells and uses thereof. [Means for solving the problem]

[0007] To achieve the above object, the composition for removing senescent cells of the present disclosure contains sphingosines.

[0008] The composition for improving cognitive function of the present disclosure contains a sphingosine.

[0009] The composition for improving learning function of the present disclosure contains a sphingosine.

[0010] The composition for improving motor function of the present disclosure contains a sphingosine. [Effects of the Invention]

[0011] According to the present disclosure, senescent cells can be removed. The composition for removing senescent cells of the present disclosure can be suitably used to improve, for example, cognitive function, learning function, or motor function. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the relationship between the number of days that NHDFs were passaged and maintained and PDL. [Figure 2] FIG. 2(A) is a micrograph of NHDFs at P10 (10th passage), and FIG. 2(B) is a micrograph of NHDFs at P15 (15th passage). [Figure 3] FIG. 3 is a graph showing relative gene expression levels in replicative senescent NHDF model cells. [Figure 4] FIG. 4 is a graph showing the relationship between each doxorubicin concentration and the survival rate of NHDF. [Figure 5] FIG. 5 is a graph showing the relative gene expression levels in doxorubicin-induced senescent NHDF model cells. [Figure 6] Figure 6 is a graph showing the relationship between the concentration of each test compound and the viability of NHDFs. Figures 6(A) to (C) show the results for sphingosine, phytosphingosine, and sphingadienine, respectively. [Figure 7] FIG. 7 is a graph showing relative gene expression levels in replicative senescent NHDF model cells. [Figure 8] FIG. 8 is a graph showing the relative gene expression levels in doxorubicin-induced senescent NHDF model cells. [Figure 9] FIG. 9 is a photomicrograph showing live, dead, and senescent cells in replicative senescent NHDF model cells. [Figure 10] FIG. 10 is a photomicrograph showing live, dead, and senescent cells in doxorubicin-senescent NHDF model cells. [Figure 11] FIG. 11(A) is a graph showing changes in body weight of doxorubicin-induced aging model mice, and FIG. 11(B) is a graph showing changes in food intake of the same model mice. [Figure 12] FIG. 12 is a graph showing changes in transepidermal water loss in doxorubicin-induced aging model mice. [Figure 13-1] Figure 13 is a graph showing wrinkle formation in a doxorubicin-induced aging model mouse, in which (A) to (D) show the total volume fraction, wrinkle area fraction, wrinkle volume fraction, and maximum wrinkle depth, respectively. [Figure 13-2] Figure 13 is a graph showing wrinkle formation in a doxorubicin-induced aging model mouse. Figures 13(E) to (G) show the maximum wrinkle width, average wrinkle depth, and number of wrinkles, respectively. [Figure 14] Figure 14 is a graph showing the results of a rotarod test on doxorubicin-induced aging model mice. Figure 14(A) shows the test results from days 18 to 20 after the start of the test, and Figure 14(B) shows the test results from days 32 to 34. [Figure 15] FIG. 15 is a graph showing the relative gene expression levels in the dorsal skin of doxorubicin-induced aging model mice. [Figure 16] FIG. 16 is a graph showing the amount of ceramide in the epidermis of doxorubicin-induced aging model mice. [Figure 17] FIG. 17 is a graph showing the results of a novel object recognition test in doxorubicin-induced aging model mice. [Figure 18]FIG. 18 is a graph showing the results of a novel object recognition test in doxorubicin-induced aging model mice using phytosphingosine. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Definition> As used herein, "sphingosines" refer to components that constitute sphingolipids, and are long-chain aliphatic amines containing two or three hydroxyl groups at their terminals.

[0014] As used herein, the term "cell" refers to any cell, and particularly includes cells derived from animals. Examples of such animals include humans and non-human animals other than humans. Examples of such non-human animals include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs, as well as birds.

[0015] As used herein, the term "senescent cells" refers to cells whose sustained proliferation has decreased or stopped compared to normal cells. Senescent cells are generally known to have irreversible proliferation arrest in the G1 phase, and express p16, which inhibits cell cycle progression by suppressing genes that promote cell cycle progression. INK4a (Cyclin-dependent kinase inhibitor 2A (CDKN2A), hereafter referred to as "p16"), p21 CIP1 It is known that senescent cells are formed in association with increased expression of cyclin-dependent kinase inhibitor 1 (CDKN1A), hereinafter referred to as "p21." The senescent cells can be identified, for example, using a senescence-related gene as a marker. The senescent cells can be prepared, for example, by dividing proliferative cells (normal cells, for example, human skin fibroblasts) 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more times.

[0016] As used herein, "senescence-cell-associated gene" refers to a gene whose expression is increased or suppressed in senescent cells compared to normal cells. Examples of the senescence-cell-associated gene include p16, p21, MMP1, GLS1, and IL1B. p16 is a gene encoding a protein that controls cell cycle progression and specifically inhibits CDK4 and CDK6 kinases. An example of human p16 is the gene encoding the protein registered in UniProt under accession number P42771. p21 is known as a cyclin-dependent kinase inhibitor and is a gene encoding a protein known to control cell cycle progression in the G1 phase by binding to and inhibiting cyclin-CDK2 or cyclin-CDK1 complexes. An example of human p21 is the gene encoding the protein registered in UniProt under accession number P38936. MMP1 (Matrix metalloproteinase-1) is a gene encoding a protease known to degrade type I collagen protein. An example of human MMP1 is a gene encoding a protein registered in UniProt under accession number P03956. GLS1 (Glutaminase kidney isoform, mitochondrial) is a type of amidohydrolase enzyme and is a gene encoding a protein known to hydrolyze glutamine to glutamic acid. An example of human GLS1 is a gene encoding a protein registered in UniProt under accession number O94925. IL1B (Interleukin 1 beta) is a gene encoding an inflammatory cytokine. An example of human IL1B is a gene encoding a protein registered in UniProt under accession number P01584. It is known that the senescent cells express higher levels of p16, p21, MMP1, GLS1, and / or IL1B than the normal cells.

[0017] As used herein, the term "food composition" refers to a substance that can be ingested by animals, particularly humans, such as dairy products, oils and fats, fruits, vegetables, seaweed, nuts and seeds, confectioneries, grains, beans, meat, seafood, eggs, sweeteners, spices, and beverages (e.g., tea, milk, fruit juice, and alcoholic beverages).

[0018] As used herein, the term "cosmetic composition" refers to a composition used as a cosmetic. Examples of such cosmetics include cosmetics for use on the skin, bath additives, and fragrances. Examples of such cosmetics include cleansers such as soap, synthetic toilet soap, liquid body wash (body soap), and facial cleanser; cleansing cream, cleansing lotion, lotion, emulsion, serum, lotion; face packs such as liquid face packs and paste face packs; face powders such as face powder, water face powder, and kneaded face powder; face powder, foundation, lipstick, and blush; eye cosmetics such as eyeliner and eye shadow; sunscreen cosmetics, tanning cosmetics, and hair removal cosmetics; and shaving cosmetics such as shaving lotion and aftershave lotion.

[0019] As used herein, "cognitive function" refers to intellectual functions such as memory, understanding, and judgment.

[0020] In this specification, the term "learning function" refers to a function that memorizes input information, executed operations, etc., and optimizes the processing to perform more appropriate processing based on that memory.

[0021] As used herein, "motor function" refers to the ability to move the body.

[0022] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequences using appropriate sequence conversion software, etc.

[0023] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0024] <Composition or agent for removing senescent cells> In one aspect, the present disclosure provides a composition or agent for removing senolytic cells. The composition or agent for removing senolytic cells of the present disclosure comprises a sphingosine.

[0025] As a result of extensive research, the present inventors have found, using senescent model cells, that sphingosines can reduce the viability of the senescent model cells compared to normal cells, leading to the establishment of the present disclosure. Therefore, the composition or agent for removing senescent cells of the present disclosure can be used to remove, for example, senescent cells.

[0026] The sphingosines may be derived from animals or plants. Examples of the sphingosines include sphingosine, phytosphingosine, sphinganine, sphingadienine, and sphingenin. In the sphingosines, the long-chain aliphatic group may be saturated or unsaturated. In the latter case, the long-chain aliphatic group includes a geometric isomer of the unsaturated aliphatic group. Specific examples of the sphingadienine include its geometric isomers, such as those in which the double bonds at the 4th and 8th positions are cis:cis, cis:trans, trans:cis, or trans:trans. An example of the geometric isomer of sphingadienine is trans-4-cis-8-sphingadienine. Examples of the geometric isomer of sphingenine include 4-hydroxy-cis-8-sphingenin. The number of carbon atoms in the long-chain aliphatic group is, for example, 12 to 20, and more preferably 18. Specific examples of the sphingosines include animal-derived sphingosines such as C12-sphingosine, C14-sphingosine, C16-sphingosine, C18-sphingosine (C18-sphingosine, (d18:1)), sphinganine (d18:0), and phytosphingosine (t18:0); trans-4-cis-8-sphingadienine; Examples of sphingosines include plant-derived sphingosines such as 4-hydroxy-cis-8-sphingenin (t18:1), 8-sphingenin (d18:1), and C20-sphingosine (d20:1), with C18-sphingosine being preferred because it can promote the removal of senescent cells. The sphingosines may be, for example, at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienin, sphinganine, and sphingenin.

[0027] The sphingosines may be, for example, isomers. Examples of the isomers include tautomers or stereoisomers. Examples of the tautomers or stereoisomers include all theoretically possible tautomers or stereoisomers. The sphingosines may be, for example, salts of the sphingosines or solvates such as hydrates of the salts.

[0028] The salt of the sphingosine is not particularly limited and may be, for example, a pharmaceutically acceptable salt. The sphingosine may form, for example, an acid addition salt or a salt with a base depending on the type of sphingosine. The pharmaceutically acceptable salt is not particularly limited and may, for example, be an alkali metal salt such as a sodium salt or a potassium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt; an ammonium salt; an aliphatic amine salt such as a trimethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an ethanolamine salt, a diethanolamine salt, or a triethanolamine salt; an aralkylamine salt such as N,N-dibenzylethylenediamine; a heterocyclic aromatic amine salt such as a pyridine salt, a picoline salt, a quinoline salt, or an isoquinoline salt; a quaternary ammonium salt such as a tetramethylammonium salt, a tetraethylammonium salt, a benzyltrimethylammonium salt, a benzyltributylammonium salt, a methyltrioctylammonium salt, or a tetrabutylammonium salt; an arginine salt, a lysine salt, an aspartate salt, or a glutamic acid salt. inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, and perchlorate; aliphatic organic acid or aromatic organic acid salts such as acetate, propionate, succinate, glycolate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate, hydroxymaleate, pyruvate, phenylacetate, benzoate, 4-aminobenzoate, anthranilate, 4-hydroxybenzoate, salicylate, 4-aminosalicylate, pamoate, gluconate, and nicotinate; sulfonates such as methanesulfonate, isethionate, ethanesulfonate, benzenesulfonate, halobenzenesulfonate, p-toluenesulfonate, toluenesulfonate, naphthalenesulfonate, sulfanilate, and cyclohexylsulfamate; and the like.

[0029] The sphingosines may be derived from sphingolipids, for example. The sphingolipid refers to a lipid containing the sphingosines. The sphingolipid may include, for example, ceramide. The ceramide refers to a compound in which the sphingosines and a fatty acid are amide-bonded. The ceramide may be, for example, a complex sphingolipid in which a polar group is bonded to the hydroxyl group at the 1-position. Examples of the sphingolipid include a sphingophospholipid in which phosphate choline is bonded to ceramide as the polar group, and a sphingoglycolipid in which a sugar is bonded to ceramide as the polar group. Examples of the sphingophospholipid include sphingomyelin. Examples of the sphingoglycolipid include glucosylceramide, galactosylceramide, ganglioside, and the like. It is known that when the sphingolipid is orally administered, the sphingolipid is broken down in the gastrointestinal lumen into polar groups, sphingosines, fatty acids, etc., and each of these is then absorbed into the body through gastrointestinal epithelial cells (Sugawara T, Biochemistry, Vol. 92, No. 5, 649-657 (2020); Sugawara T, J. Agric. Food Chem., 70, 31, 9597-9609 (2022)). Since the sphingolipid can produce the sphingosines during digestion, in the present disclosure, the sphingolipid may be used as the sphingosines.

[0030] The composition for removing senescent cells of the present disclosure can remove senescent cells, for example, by applying it to a subject. The conditions for use (administration conditions) of the composition for removing senescent cells of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, and the like can be appropriately set depending on, for example, the type of subject.

[0031] The composition for removing senolytic cells of the present disclosure may be used, for example, in vivo or in vitro.

[0032] The subject to which the composition for removing senescent cells of the present disclosure is administered is not particularly limited. When the composition for removing senescent cells of the present disclosure is used in vivo, the subject to which the composition is administered can be, for example, a human or a non-human animal other than a human. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs, as well as birds. When the composition for removing senescent cells of the present disclosure is used in vitro, the subject to which the composition is administered can be, for example, a cell, tissue, or organ. Examples of the cell include cells collected from a living organism or cultured cells, and examples of the tissue or organ include tissue (biological tissue) or organ collected from a living organism.

[0033] In the composition for removing senescent cells of the present disclosure, the amount of the sphingosines may be within a range that exhibits the effect of removing senescent cells, i.e., an effective amount. Examples of the amount of the sphingosines include 0.000001 to 30% by weight, 0.000001 to 20% by weight, 0.000001 to 10% by weight, 0.000001 to 1% by weight, 0.000001 to 0.1% by weight, 0.00001 to 0.1% by weight, 0.0001 to 0.1% by weight, 0.001 to 0.1% by weight, and 0.01 to 0.1% by weight. The upper limit of the amount may be, for example, 1%, 10%, 20%, or 30% by weight.

[0034] The composition for removing senescent cells of the present disclosure can be administered orally or parenterally. Examples of parenteral administration include transdermal administration and application (contact) to the skin.

[0035] The dosage form of the composition for removing senescent cells of the present disclosure is not particularly limited and can be determined appropriately depending on, for example, the administration form. Examples of the dosage form include liquid and solid. When the administration form is oral administration, examples of the dosage form include tablets, pills, capsules, granules, powders, liquids, and creams. When the composition for removing senescent cells is administered transdermally, the dosage form is preferably, for example, a lotion, emulsion, or cream.

[0036] The composition for removing senescent cells of the present disclosure may contain, for example, additives as needed. When used as a composition, the additives preferably include pharmaceutically acceptable additives, pharmaceutically acceptable carriers, additives that can be added to cosmetic compositions, or carriers that can be added to cosmetic compositions. The additives or carriers are not particularly limited, and examples include base raw materials, excipients, colorants, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, and flavoring agents such as fragrances. In the present disclosure, the amount of the additives or carriers added is not particularly limited as long as they do not interfere with the function of sphingosines and the like.

[0037] In the senescent cell removal composition of the present disclosure, "removal of senescent cells" refers to, for example, reducing the viability of senescent cells, killing senescent cells, or removing senescent cells from tissues or organs. The removal of senescent cells can be evaluated, for example, by measuring the viability of senescent cells when a test substance is contacted with the senescent cells, as described in Example 3 below. Specifically, the removal of senescent cells by the test substance can be evaluated, for example, based on the viability of senescent cells in the absence of the test substance and the viability of senescent cells in the presence of the test substance. In the evaluation, the test substance can be evaluated as removing senescent cells if, for example, the viability of senescent cells in the presence of the test substance is 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, based on the viability of senescent cells in the absence of the test substance (100%).

[0038] The senescent cells are not particularly limited, and examples thereof include epithelial cells such as epidermal keratinocytes; endothelial cells such as vascular endothelial cells; fibroblasts such as dermal fibroblasts; muscle cells such as vascular smooth muscle cells and cardiac progenitor cells; glial cells such as astrocytes, oligodendrocytes, and microglia; and senescent leukocytes such as lymphocytes.

[0039] The senescent cells are not particularly limited, and examples thereof include senescent cells in tissues or organs such as muscle tissue, adipose tissue, skin tissue, cartilage tissue, tendon tissue, ligament tissue, interstitial parenchyma, vascular tissue, nervous system tissue, circulatory system tissue, respiratory system tissue, digestive system tissue, metabolic system tissue, lymphatic system tissue, bone marrow tissue, and blood.

[0040] The composition for removing senescent cells of the present disclosure can be suitably used, for example, as a food composition or a cosmetic composition. The composition for removing senescent cells of the present disclosure can, for example, improve the moisturizing effect of skin by removing senescent cells from the skin. Therefore, the composition for removing senescent cells of the present disclosure can also be said to be a composition for use in a method for improving the moisturizing effect of skin by removing senescent cells from the skin.

[0041] <Composition or agent for improving cognitive function> In another aspect, the present disclosure discloses a composition or agent for improving cognitive function. The composition or agent for improving cognitive function of the present disclosure comprises a sphingosine. The composition or agent for improving cognitive function of the present disclosure can improve, for example, cognitive function.

[0042] For the sphingosines, for example, the explanation of the sphingosines in the composition or agent for removing senescent cells can be used.

[0043] The cognitive function improving composition of the present disclosure can improve cognitive function, for example, by administering it to a subject. The conditions for use (administration conditions) of the cognitive function improving composition of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on, for example, the type of subject to be administered.

[0044] The composition for improving cognitive function of the present disclosure can be used, for example, in vivo.

[0045] In the cognitive function improving composition of the present disclosure, the amount of the sphingosines may be within a range that exhibits a cognitive function improving effect, i.e., an effective amount. Examples of the amount of the sphingosines include 0.000001 to 30% by weight, 0.000001 to 20% by weight, 0.000001 to 10% by weight, 0.000001 to 1% by weight, 0.000001 to 0.1% by weight, 0.00001 to 0.1% by weight, 0.0001 to 0.1% by weight, 0.001 to 0.1% by weight, and 0.01 to 0.1% by weight. The upper limit of the amount may be, for example, 1% by weight, 10% by weight, 20% by weight, or 30% by weight.

[0046] The administration target, administration form, dosage form, and additives of the composition for improving cognitive function of the present disclosure can be determined, for example, from the descriptions of the administration target, administration form, dosage form, and additives of the composition or agent for removing senescent cells described above.

[0047] In the cognitive function-improving composition of the present disclosure, "improvement of cognitive function" refers to improvement in functions such as memory, comprehension, and / or judgment. The improvement in cognitive function can be evaluated, for example, by a rotarod test in accordance with Example 8 described below. The rotarod test can measure, for example, the subject's coordinated movement and motor learning, thereby enabling the subject's cognitive function, learning function, and motor function to be evaluated. Specifically, the improvement in cognitive function caused by the test substance can be evaluated based on the measurement results of a subject (e.g., a mouse) not administered with the test substance and a subject administered with the test substance. In the evaluation, for example, if the measured values ​​of coordination and motor learning of a subject administered with the test substance improve by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, or 60% or more based on the measured values ​​of coordination and motor learning of a subject not administered with the test substance, the test substance can be evaluated as improving cognitive function.

[0048] The improvement in cognitive function can also be evaluated by a novel object recognition test, for example, according to Example 11 described below. In the novel object recognition test, for example, a subject is first made to memorize two identical objects, and then, after a certain period of time, one object is replaced with another novel object (novel object), and the approach time to each object (time spent in contact with the object) is measured. The cognitive function of the subject can be evaluated, for example, by calculating the ratio (novel object exploration ratio) (%) of the approach time to the novel object to the total approach time, which is the sum of the approach times to each object, and using the novel object exploration ratio as an index. Specifically, the improvement in cognitive function due to the test substance can be evaluated, for example, based on the measurement results of a subject (e.g., a mouse) not administered with the test substance and a subject administered with the test substance. In the evaluation, for example, if the novel object exploration rate of a subject administered with the test substance improves by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, or 60% or more based on the novel object exploration rate of a subject not administered with the test substance, the test substance can be evaluated as improving cognitive function.

[0049] The cognitive function improving composition of the present disclosure can be suitably used, for example, as a food composition or a cosmetic composition.

[0050] <Composition or agent for improving learning function> In another aspect, the present disclosure discloses a composition or agent for improving learning function. The composition or agent for improving learning function of the present disclosure comprises a sphingosine. The composition or agent for improving learning function of the present disclosure can improve, for example, learning function.

[0051] For the sphingosines, for example, the explanation of the sphingosines in the composition or agent for removing senescent cells can be used.

[0052] The composition for improving learning function of the present disclosure can improve learning function, for example, by being used on a subject to be administered. The conditions for use (administration conditions) of the composition for improving learning function of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on, for example, the type of subject to be administered.

[0053] The composition for improving learning function of the present disclosure can be used, for example, in vivo.

[0054] In the composition for improving learning function of the present disclosure, the amount of the sphingosine compound may be within a range that exhibits the effect of improving learning function, i.e., an effective amount. Examples of the amount of the sphingosine compound include 0.000001 to 30% by weight, 0.000001 to 20% by weight, 0.000001 to 10% by weight, 0.000001 to 1% by weight, 0.000001 to 0.1% by weight, 0.00001 to 0.1% by weight, 0.0001 to 0.1% by weight, 0.001 to 0.1% by weight, and 0.01 to 0.1% by weight. The upper limit of the amount may be, for example, 1% by weight, 10% by weight, 20% by weight, or 30% by weight.

[0055] The administration target, administration form, dosage form, and additives of the composition for improving learning function of the present disclosure can be determined, for example, from the descriptions of the administration target, administration form, dosage form, and additives of the composition or agent for removing senescent cells described above.

[0056] In the composition for improving learning function of the present disclosure, for example, improvement in learning function means improvement in the function of memorizing input information, executed operations, etc., and optimizing the function to perform more appropriate processing based on that memorization. The method for evaluating the improvement in learning function can be, for example, by replacing "improvement of cognitive function" with "improvement of learning function" in the above-mentioned description of the evaluation method for the composition for improving cognitive function.

[0057] The composition for improving learning function of the present disclosure can be suitably used, for example, as a food composition or a cosmetic composition.

[0058] <Composition or agent for improving motor function> In another aspect, the present disclosure discloses a composition or agent for improving motor function. The composition or agent for improving motor function of the present disclosure comprises a sphingosine. The composition or agent for improving motor function of the present disclosure can improve, for example, motor function.

[0059] For the sphingosines, for example, the explanation of the sphingosines in the composition or agent for removing senescent cells can be used.

[0060] The composition for improving motor function of the present disclosure can improve motor function, for example, by administering it to a subject. The conditions for use (administration conditions) of the composition for improving motor function of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on, for example, the type of subject to be administered.

[0061] The composition for improving motor function of the present disclosure can be used, for example, in vivo.

[0062] In the motor function improving composition of the present disclosure, the amount of the sphingosines may be within a range that exhibits a motor function improving effect, i.e., an effective amount. Examples of the amount of the sphingosines include 0.000001 to 30% by weight, 0.000001 to 20% by weight, 0.000001 to 10% by weight, 0.000001 to 1% by weight, 0.000001 to 0.1% by weight, 0.00001 to 0.1% by weight, 0.0001 to 0.1% by weight, 0.001 to 0.1% by weight, and 0.01 to 0.1% by weight. The upper limit of the amount may be, for example, 1% by weight, 10% by weight, 20% by weight, or 30% by weight.

[0063] The administration target, administration form, dosage form, and additives of the composition for improving motor function of the present disclosure can be determined, for example, from the descriptions of the administration target, administration form, dosage form, and additives of the composition or agent for removing senescent cells described above.

[0064] In the composition for improving motor function of the present disclosure, for example, improvement in motor function means improvement in the function of moving the body. The method for evaluating the improvement in motor function can be, for example, the same as the above-mentioned description of the evaluation method for the composition for improving cognitive function, except that "improvement in cognitive function" is replaced with "improvement in motor function."

[0065] The composition for improving motor function of the present disclosure can be suitably used, for example, as a food composition or a cosmetic composition.

[0066] <Method for removing senescent cells> In another aspect, the present disclosure discloses a method for removing senescent cells. The method for removing senescent cells of the present disclosure uses the senolytic agent and / or composition for removing senescent cells of the present disclosure. The method for removing senescent cells of the present disclosure can remove, for example, senescent cells.

[0067] The method for removing senescent cells of the present disclosure includes a step of applying the senolytic agent and / or composition for removing senescent cells of the present disclosure to a subject. The use may be, for example, by contact with the skin or the like, or by administration.

[0068] In the senescent cell removal method of the present disclosure, the using step may be carried out, for example, in vitro or in vivo. The subject (administration subject) and administration conditions of the senescent cell removal method of the present disclosure can be determined by reference to the explanation of the administration subject and administration conditions for the senolytic agent and / or composition for senescent cell removal of the present disclosure.

[0069] <Cognitive function improvement method> In another aspect, the present disclosure discloses a method for improving cognitive function. The cognitive function improving method of the present disclosure uses the cognitive function improving agent and / or composition for improving cognitive function of the present disclosure. According to the cognitive function improving method of the present disclosure, for example, cognitive function can be improved.

[0070] The cognitive function improving method of the present disclosure includes a step of administering the cognitive function improving agent and / or composition of the present disclosure to a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0071] In the cognitive function improving method of the present disclosure, the using step can be performed, for example, in vivo. The subject (administration subject) and administration conditions of the cognitive function improving method of the present disclosure can refer to, for example, the explanation of the administration subject and administration conditions for the senolytic agent and / or composition for removing senescent cells of the present disclosure.

[0072] <Method for improving learning function> In another aspect, the present disclosure discloses a method for improving learning function. The method for improving learning function of the present disclosure uses the agent for improving learning function and / or the composition for improving learning function of the present disclosure. According to the method for improving learning function of the present disclosure, for example, learning function can be improved.

[0073] The method for improving learning function according to the present disclosure includes a step of administering to a subject the agent for improving learning function and / or the composition for improving learning function according to the present disclosure. The use may be, for example, by contact with the skin or the like or by administration.

[0074] In the method for improving learning function of the present disclosure, the using step can be performed, for example, in vivo. The subject (administration subject) and administration conditions of the method for improving learning function of the present disclosure can be determined, for example, from the explanation of the administration subject and administration conditions of the senolytic agent and / or composition for removing senescent cells of the present disclosure.

[0075] <How to improve motor function> In another aspect, the present disclosure discloses a method for improving motor function. The method for improving motor function of the present disclosure uses the motor function improving agent and / or composition for improving motor function of the present disclosure. According to the method for improving motor function of the present disclosure, for example, motor function can be improved.

[0076] The method for improving motor function according to the present disclosure includes a step of administering the motor function improving agent and / or composition for improving motor function according to the present disclosure to a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0077] In the motor function improving method of the present disclosure, the using step can be performed, for example, in vivo. The subject (administration subject) and administration conditions of the motor function improving method of the present disclosure can refer to, for example, the explanation of the administration subject and administration conditions for the senolytic agent and / or composition for removing senescent cells of the present disclosure.

[0078] <Use> In another aspect, the present disclosure is directed to the use of a senolytic composition or senolytic agent of the present disclosure, for example, for use in removing senescent cells. The present disclosure is directed to the use of a senolytic composition or senolytic agent of the present disclosure, for example, for manufacturing a senolytic composition or senolytic agent.

[0079] In another aspect, the present disclosure relates to the use of the composition or agent for improving cognitive function of the present disclosure, for example, for use in improving cognitive function. The present disclosure relates to the use of the composition or agent for improving cognitive function of the present disclosure, for example, for producing the composition or agent for improving cognitive function.

[0080] In another aspect, the present disclosure relates to use of the composition or agent for improving learning function of the present disclosure, for example, for use in improving learning function. The present disclosure relates to use of the composition or agent for improving learning function of the present disclosure, for example, for producing a composition or agent for improving learning function.

[0081] In another aspect, the present disclosure relates to use of the composition for improving motor function or the agent for improving motor function of the present disclosure, for example, for use in improving motor function. The present disclosure relates to use of the composition for improving motor function or the agent for improving motor function of the present disclosure, for example, for producing a composition for improving motor function or the agent for improving motor function. [Example]

[0082] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to the attached protocols. In the following description, "mol / l" may also be abbreviated as "M."

[0083] [Example 1] By repeatedly expanding and passage-cultivating normal human dermal fibroblasts (NHDFs), we generated replicative senescent NHDF model cells, and confirmed that the expression of senescence-related genes was increased in the resulting cells.

[0084] (1) Pretreatment of NHDF A basal medium was prepared by adding 31.25 mL of low-serum liquid medium for dermal fibroblast proliferation (FibroLife S2 Comp Kit, Kurabo) to 480 mL of liquid medium for dermal fibroblasts (FibroLife BM, Kurabo). Normal human neonatal foreskin dermal fibroblasts (frozen NHDF (NB), Kurabo) were cultured in this basal medium at 37°C and 5% CO for 24 hours.

[0085] (2) Preparation of replicative senescent NHDF model cells The cultured NHDFs were serially passaged to generate replicative senescent NHDF model cells. Before reaching confluence, NHDFs were treated with trypsin (32778-34, Nacalai Tesque) to release them from the dish, and then passaged and maintained. The population doubling level (PDL) at each passage was calculated using the following formula (1): where C is the cell number, and n is the passage number. PDL n+1 =PDL n +3.32(logC n+1 -logCn ) ···(1)

[0086] The number of NHDF cells was measured by treating the NHDF with trypsin to release them from the dish, suspending them in the basal medium, and then counting them using a hemocytometer (8100204, Hirschmann). The NHDF cells were also observed using an optical microscope (TMS-F MFA20100, Nikon). The results are shown in Figures 1 and 2.

[0087] Figure 1 is a graph showing the relationship between the number of days NHDF were passaged and maintained and PDL. In Figure 1, the horizontal axis represents the number of days NHDF were passaged and maintained, and the vertical axis represents PDL. As shown in Figure 1, PDL increased with the number of days NHDF were passaged and maintained, but from around day 25, the graph gradually showed a gentle slope, reaching a plateau at P15 (the 15th passage). Therefore, it was found that sustained cell proliferation of NHDF had almost ceased at P15 (the 15th passage).

[0088] Figure 2 shows micrographs showing the appearance of NHDF at each passage. Figure 2(A) is a micrograph of NHDF at P10 (10th passage), and Figure 2(B) is a micrograph of NHDF at P15 (15th passage). The bars in the figure indicate a length of 50 μm. As shown in Figure 2, it was confirmed that the cell density of NHDF at P15 (15th passage) was reduced compared to the appearance of NHDF at P10 (10th passage).

[0089] (3) Measurement of gene expression levels in replicative senescent NHDF model cells NHDFs were cultured to obtain replicative senescent NHDF model cells. Total RNA was extracted from the NHDF model cells, and cDNA was synthesized from the total RNA. The resulting cDNA was then used to measure gene expression levels in the NHDFs by quantitative real-time PCR. Each step is described below.

[0090] (3-1) Cultivation of replicative senescent NHDF model cells The NHDF model cells at P16 (the 16th passage) were treated with trypsin to release them from the dish and suspended in the basal medium. Then, the number of the NHDF model cells was counted using the hemocytometer, and the cell count was 1.0 × 10 5 The NHDF model cells were suspended in the basal medium to give a concentration of 5.0 × 10 cells / mL. The resulting suspension was placed in each well of a 24-well plate. 4 After the addition, the cells were cultured at 37°C and 5% CO for 24 hours, and then the basal medium in each well was replaced with 500 μL of fresh basal medium, followed by culture under the same conditions for 48 hours.

[0091] (3-2) Total RNA extraction from replicative senescent NHDF model cells The NHDF model cells obtained in Example 1 (3-1) were washed with D-PBS(-) and then recovered by adding 500 μL of Sepasol®-RNA I Super G (Nacalai Tesque). Next, 100 μL of chloroform was added to the recovered NHDF model cells, and the cells were incubated at room temperature (approximately 25°C, the same applies below) for 3 minutes, followed by centrifugation at 10,800 rpm and 4°C for 15 minutes. After centrifugation, 120 μL of the supernatant was transferred to a new tube, and 250 μL of 2-propanol was added. The cells were then allowed to stand at room temperature for 10 minutes. After standing, the cells were centrifuged at 10,800 rpm and 4°C for 10 minutes. The supernatant after centrifugation was removed, and 500 μL of 75% ethanol was added to the resulting precipitate and stirred using a vortex mixer. The resulting solution was centrifuged at 8,500 rpm and 4°C for 5 minutes. After the centrifugation, the ethanol was removed and 500 μL of 75% ethanol was added to obtain an RNA solution, which was stored at −80° C. until use.

[0092] (3-3) cDNA synthesis The RNA solution obtained in Example 1 (3-2) was centrifuged at 8,500 rpm and 4°C for 5 minutes, after which the ethanol in the supernatant was removed and the tube was dried at room temperature for 10 minutes with the opening facing downward. After drying, 30 μL of DEPC-treated water was added to the tube to prepare an RNA solution, which was then incubated at 55°C for 10 minutes. The RNA concentration of the RNA solution was determined by measuring the absorbance at 260 nm using a NANODROP LITE spectrophotometer (Thermo Fisher Scientific). 3 μL of the RNA solution was transferred to another tube, diluted with DEPC-treated water based on the concentration of the RNA solution obtained from the measurement, incubated at 65°C for 5 minutes, and then cooled on ice. cDNA was synthesized from the RNA solution using ReverTra Ace® qPCR RT Master Mix (Toyobo Co., Ltd.). The resulting cDNA solution was diluted 30-fold with DEPC-treated water and stored at −30° C. until use in quantitative real-time PCR.

[0093] (3-4) Quantitative real-time PCR MilliQ water was added to the dried powder of each primer, and a portion of the stock solution was prepared at 100 μM. This was then diluted 20-fold with MilliQ water. 1 μL of the diluted forward primer solution, 1 μL of the diluted reverse primer solution, 5 μL of SYBR Green, and 3 μL of the cDNA solution obtained in Example 1 (3-3) were dispensed into each well of a 96-well multiplate (Bio-Rad) and mixed carefully to avoid foaming. PCR was performed using a real-time PCR device (CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad)). The PCR reaction conditions were 95°C for 3 minutes, followed by 50 cycles of 95°C for 15 seconds and 60°C for 30 seconds. Finally, the temperature was raised from 60°C to 95°C for 10 seconds, and melting curve analysis was performed. From the obtained Ct values, the expression level of each gene was calculated relative to the expression level of the endogenous control gene (β-actin gene). The control was calculated in the same manner, except that young cells (normal cells) at passage numbers 3 to 6 (P3 to P6) were used. The results are shown in Figure 3.

[0094] Primer set for P16 gene Forward primer 5'-ACCAGAGGCAGTAACCATGC-3' (SEQ ID NO: 1) Reverse primer 5'-CCTGTAGGACCTTCGGTGAC-3' (SEQ ID NO: 2) Primer set for the P21 gene Forward primer 5'-TAGCAGCGGAACAAGGAG-3' (SEQ ID NO: 3) Reverse primer 5'-AAACGGGAACCAGGACAC-3' (SEQ ID NO: 4) Primer set for MMP1 gene Forward primer 5'-ACGAATTTGCCGACAGAGAT-3' (SEQ ID NO: 5) Reverse primer 5'-GTCCTTGGGGTATCCGTGTA-3' (SEQ ID NO: 6) Primer set for the GLS1 gene Forward primer 5'-CTGGAAGCCTGCAAAGTAAAC-3' (SEQ ID NO: 7) Reverse primer 5'-TGAGGTGTGTACTGGACTTGG-3' (SEQ ID NO: 8)

[0095] Figure 3 is a graph showing the relative gene expression levels in replicative senescent NHDF model cells. In Figure 3, the horizontal axis shows each gene (p16, p21, MMP1, and GLS1) in replicative senescent NHDF model cells (rep. senescent), and the vertical axis shows the relative expression level of each gene. p16, p21, MMP1, and GLS1 are senescence-related genes known to be highly expressed in senescent cells. It was confirmed that replicative senescent NHDF model cells showed higher expression levels of p16, p21, MMP1, and GLS1 than young (normal) cells at passages 3 to 6 (P3 to P6).

[0096] These results suggest that replicative senescent NHDF model cells, like senescent cells, exhibit a near-certainty in sustained cell proliferation and high expression of senescence-related genes. Therefore, we found that replicative senescent NHDF model cells can be generated by continuous subculture.

[0097] [Example 2] By administering doxorubicin to NHDFs, we created doxorubicin-induced senescent NHDF model cells, and confirmed that the expression of senescence-related genes was increased in the resulting cells.

[0098] (1) Pretreatment of normal human skin fibroblasts As in Example 1, normal human neonatal foreskin dermal fibroblasts (frozen NHDF (NB), manufactured by Kurabo) were cultured in the basal medium at 37° C. and 5% CO 2 for 24 hours.

[0099] (2) Measurement of cell viability at each doxorubicin concentration To determine the concentration of doxorubicin to administer to NHDFs, we measured the cell viability of NHDFs at each doxorubicin concentration using the MTT assay. Doxorubicin is known to act at the G2 / M phase of the cell cycle to arrest cell proliferation and induce cellular senescence.

[0100] MTT reagent (23547-76, Nacalai Tesque) was dissolved in the basal medium to prepare a 5 mg / mL MTT stock solution. The cultured NHDFs were seeded (2,500 cells / well) into a 96-well plate containing the basal medium and cultured at 37°C and 5% CO for 24 hours. Then, 0.1, 0.25, 1, 5, or 10 μM doxorubicin hydrochloride (040-21521, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the basal medium and cultured at 37°C and 5% CO for 72 hours. The basal medium was then removed from the 96-well plate, 80 μL of fresh basal medium was added, followed by 20 μL of the MTT stock solution. The plate was then cultured at 37°C and 5% CO for 35 minutes. After the culture, the basal medium was removed, and 100 μL of DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to dissolve the NHDFs. The resulting lysate was stirred for 10 minutes using a microplate mixer, and the absorbance at 570 nm (A570) and 650 nm (A650) was measured using a microplate reader (Molecular Devices). The cell viability was calculated based on the following formula (2). As a control, an equal volume of MilliQ water was added to the basal medium instead of doxorubicin hydrochloride. The results are shown in Figure 4. Cell viability = {Sample (A570-A650) / Control (A570-A650)} × 100 (2)

[0101] Figure 4 is a graph showing the relationship between each doxorubicin concentration and NHDF viability. In Figure 4, the horizontal axis represents each doxorubicin concentration, and the vertical axis represents NHDF viability at each doxorubicin concentration. As shown in Figure 4, as the doxorubicin concentration increased, NHDF viability decreased, with a sharp decrease observed between 0.25 μM and 1 μM. Therefore, from the perspective of NHDF viability, it was found that doxorubicin at a concentration of 0.25 μM is appropriate. In the subsequent studies, doxorubicin-induced senescent NHDF model cells prepared at a concentration of 0.25 μM doxorubicin were used.

[0102] (3) Preparation of doxorubicin-induced senescent NHDF model cells Young cells (normal cells) from passages 3 to 6 (P3 to P6) were seeded into a 24-well plate containing the basal medium and cultured for 24 hours at 37°C and 5% CO2. The basal medium was then replaced with basal medium containing 0.25 μM doxorubicin hydrochloride, and the cells were cultured for 72 hours at 37°C and 5% CO2. In this way, doxorubicin-induced senescent NHDF model cells were prepared.

[0103] (4) Measurement of gene expression levels in doxorubicin-induced senescent NHDF model cells The relative expression levels of p16, p21, MMP1, and GLS1 in the doxorubicin-senescent NHDF model cells were calculated in the same manner as in Example 1 (3-2) to (3-4), except that the doxorubicin-senescent NHDF model cells of Example 2 (3) were used instead of the replicative senescent NHDF model cells of Example 1. Calculations were performed in the same manner, except that young cells (normal cells) at passages 3 to 6 (P3 to P6) were used as controls. These results are shown in Figure 5.

[0104] Figure 5 is a graph showing the relative gene expression levels in doxorubicin-induced senescent NHDF model cells. In Figure 5, the horizontal axis shows each gene (p16, p21, MMP1, and GLS1) in replicative senescent NHDF model cells (Dox. senescent), and the vertical axis shows the relative expression level of each gene. As shown in Figure 5, it was confirmed that doxorubicin-induced NHDF model cells showed higher expression levels of p16, p21, MMP1, and GLS1 than young (normal) cells at passages 3 to 6 (P3 to P6).

[0105] These results indicate that doxorubicin-induced senescent NHDF model cells, like senescent cells, highly express genes related to senescence. Therefore, it was found that doxorubicin-induced senescent NHDF model cells can be generated by administering doxorubicin to NHDFs.

[0106] [Example 3] Using replicative senescent NHDF model cells and doxorubicin-induced senescent NHDF model cells, we confirmed that sphingosines can eliminate senescent cells.

[0107] The senescent cell elimination function of each test compound was evaluated by measuring the viability of NHDFs at each test compound concentration using an MTT assay. Replicative senescent NHDF model cells, prepared by the same method as in Example 1 (3-1), were seeded (2,500 cells / well) into a 96-well plate containing the basal medium and cultured for 24 hours at 37°C and 5% CO2. The doxorubicin-senescent NHDF model cells were prepared in the same manner as in Example 2 (3), except that they were cultured for 24 hours in the presence of 2.5 μM doxorubicin hydrochloride. The basal medium of the replicative senescent NHDF model cells (replicative) and doxorubicin-senescent NHDF model cells (doxorubicin) was then replaced with basal medium containing a given concentration (1, 5, 7.5, or 10 μM) of the test compound (sphingosine, phytosphingosine, or sphingadienine), and the cells were cultured for 48 hours at 37°C and 5% CO2. Next, the cultured NHDF model cells were subjected to an MTT assay in the same manner as in Example 2(2). Young cells (normal cells) from passages 3 to 6 (P3 to P6) were used as controls. For negative controls, an equal volume of MilliQ water was added to the basal medium instead of each test compound. The results are shown in Figure 6.

[0108] Figure 6 is a graph showing the relationship between the concentration of each test compound and the viability of NHDF. In Figure 6, the horizontal axis represents the concentration of each test compound, and the vertical axis represents the viability of NHDF at each test compound concentration. Figures 6(A) to 6(C) show the results for sphingosine, phytosphingosine, and sphingadienine, respectively. As shown in Figure 6, sphingosine, phytosphingosine, and sphingadienine reduced the viability of senescent cells at all concentrations and in senescent NHDF model cells.

[0109] These results suggest that sphingosines, such as sphingosine, phytosphingosine, and sphingadienine, can selectively eliminate senescent cells in replicative senescent NHDF model cells and doxorubicin-induced senescent NHDF model cells. Phytosphingosine, in particular, was highly effective in eliminating senescent cells.

[0110] [Example 4] The effects of sphingosine and phytosphingosine on gene expression levels in replicative senescent NHDF model cells and doxorubicin-induced senescent NHDF model cells were evaluated.

[0111] Replicative senescent NHDF model cells prepared by a method similar to that described in Example 1(3-1) were seeded (2,500 cells / well) into a 24-well plate containing the basal medium and cultured for 24 hours at 37°C in 5% CO. Doxorubicin-senescent NHDF model cells were prepared in the same manner as in Example 2(3), except that they were cultured for 24 hours in the presence of 2.5 μM doxorubicin hydrochloride. The basal medium of the replicative senescent NHDF model cells and doxorubicin-senescent NHDF model cells was then replaced with basal medium containing 5 μM sphingosine or phytosphingosine (sphingosine-added group and phytosphingosine-added group, respectively), and the cells were cultured for 48 hours at 37°C in 5% CO. The relative expression levels of p16, p21, and IL1B in replicative senescent NHDF model cells and doxorubicin-induced senescent NHDF model cells were calculated in the same manner as in Example 1 (3-2) to (3-4), except that the cultured NHDF model cells were used instead of the replicative senescent NHDF model cells of Example 1 and the following primer sets were used. Calculations were performed in the same manner as above, except that young (normal) cells at passages 3 to 6 (P3 to P6) were used as controls. Furthermore, calculations were performed as a negative control using the basal medium to which an equal amount of MilliQ water was added in place of sphingosine and phytosphingosine. These results are shown in Figures 7 and 8. Primer set for the IL1B gene Forward primer 5'-TTCGACACATGGGATAACGAGG-3' (SEQ ID NO: 9) Reverse primer 5'-TTTTTGCTGTGAGTCCCGGAG-3' (SEQ ID NO: 10)

[0112] Figure 7 is a graph showing the relative gene expression levels in replicative senescent NHDF model cells. In Figure 7, the horizontal axis shows each gene (p16, p21, and IL1B) in replicative senescent NHDF model cells (rep. senescent), and the vertical axis shows the relative expression level of each gene. p16, p21, and IL1B are senescence-related genes known to be highly expressed in senescent cells. It was confirmed that the sphingosine-supplemented group and the phytosphingosine-supplemented group showed lower expression levels of p16, p21, and IL1B than the control group.

[0113] Next, Figure 8 is a graph showing the relative gene expression levels in doxorubicin-induced senescent NHDF model cells. In Figure 8, the horizontal axis shows each gene (p16, p21, and GLS1) in doxorubicin-induced senescent NHDF model cells (rep. senescent), and the vertical axis shows the relative expression level of each gene. It was confirmed that the sphingosine-added group and the phytosphingosine-added group showed lower expression levels than the control for all of the p16, p21, and GLS1 genes.

[0114] These results suggest that sphingosine and phytosphingosine can suppress the expression of senescence-related genes in both replicative senescent NHDF model cells and doxorubicin-induced senescent NHDF model cells by eliminating senescent cells.

[0115] [Example 5] Cell staining confirmed that sphingosine and phytosphingosine could selectively eliminate senescent cells in replicative senescent NHDF model cells and doxorubicin-senescent NHDF model cells.

[0116] Replicative senescent NHDF model cells prepared by a method similar to that described in Example 1(3-1) were seeded (2,500 cells / well) into a 24-well plate containing the basal medium and cultured for 24 hours at 37°C in 5% CO. The doxorubicin-senescent NHDF model cells were prepared in the same manner as described in Example 2(3), except that they were cultured for 24 hours in the presence of 2.5 μM doxorubicin hydrochloride. The basal medium of the replicative senescent NHDF model cells and doxorubicin-senescent NHDF model cells was then replaced with basal medium containing 5 μM sphingosine or phytosphingosine (sphingosine-added group or phytosphingosine-added group), and the cells were cultured for 48 hours at 37°C in 5% CO. After the culture, the NHDFs were washed with 500 μL of D-PBS(-) and incubated with 1 μM Cellstain®-Calcein-AM (Fujifilm Wako Pure Chemical Industries, Ltd.) solution or Cellstain®-PI (Fujifilm Wako Pure Chemical Industries, Ltd.) solution at 37°C for 15 minutes to stain live and dead cells. After the staining, the stained NHDFs in D-PBS(-) were observed using a fluorescence microscope (BZ-9000, KEYENCE Corporation).

[0117] After the observation, the NHDFs were washed with 500 μL of D-PBS(-) and then fixed using a Cellular Sensation Kit (OZ Biosciences). SA-β-gal staining was performed at 37°C for 18 hours. The resulting NHDFs were observed using an optical microscope (TMS-F MFA20100, Nikon). For the negative control, an equal volume of MilliQ water was added to the basal medium instead of sphingosine and phytosphingosine. These results are shown in Figures 9 and 10.

[0118] Figure 9 shows micrographs of live, dead, and senescent cells in replicative senescence NHDF model cells. In Figure 9, each photograph shows, from left to right, a calcein-AM fluorescent image, a PI fluorescent image, a SA-β-gal stained image (4x magnification), and a SA-β-gal stained image (20x magnification). In Figure 9, each photograph shows, from top to bottom, a control, a sphingosine-treated group (So-5 μM), and a phytosphingosine-treated group (P-5 μM). In Figure 9, calcein-AM-positive cells indicate live cells, PI-positive cells indicate dead cells, and SA-β-gal-positive cells indicate senescent cells. As shown in Figure 9, the sphingosine-treated group (So-5 μM) and the phytosphingosine-treated group (P-5 μM) showed an increase in the number of dead cells and a decrease in the number of senescent cells compared to the control. This confirms that sphingosine and phytosphingosine induce cell death in senescent cells.

[0119] Next, Figure 10 shows micrographs showing live, dead, and senescent cells in doxorubicin-induced NHDF model cells. In Figure 10, each photograph shows, from left to right, a Calcein-AM fluorescent image, a PI fluorescent image, and an SA-β-gal staining image. Also, in Figure 10, each photograph shows, from top to bottom, a control, a sphingosine-added group (So-5 μM), and a phytosphingosine-added group (P-5 μM). In Figure 10, Calcein-AM-positive cells indicate live cells, PI-positive cells indicate dead cells, and SA-β-gal-positive cells indicate senescent cells. As shown in Figure 10, similar to Figure 9, the sphingosine-added group (So-5 μM) and the phytosphingosine-added group (P-5 μM) showed an increase in the number of dead cells and a decrease in the number of senescent cells compared to the control. This confirms that sphingosine and phytosphingosine induce cell death in senescent cells.

[0120] From the above, it was found that senescent cells are selectively removed by sphingosine and phytosphingosine.

[0121] [Example 6] The effects of phytosphingosine on body weight and food intake were evaluated in doxorubicin-induced aging model mice.

[0122] Twenty-four C57BL / 6J mice (female, 6 weeks old) (Shimizu Experimental Materials) were purchased and housed in a breeding facility set at 24°C with a 12-hour light-dark cycle. From the age of 7 weeks, the mice were divided into three groups: a normal group (non-aging model mice) fed with AIN-93G (food, Shimizu Experimental Materials), a control group (doxorubicin-induced aging model mice) fed with AIN-93G, a 0.04% phytosphingosine-fed group (doxorubicin-induced aging model mice) fed with AIN-93G containing 0.04% phytosphingosine, and a 0.1% phytosphingosine-fed group (doxorubicin-induced aging model mice) fed with AIN-93G containing 0.1% phytosphingosine. Each group consisted of six mice (n = 6).

[0123] The control group, 0.04% phytosphingosine-fed group, and 0.1% phytosphingosine-fed group were intraperitoneally administered 5 mg / kg of doxorubicin twice (days 1 and 7) during the first week of the experimental feeding to create doxorubicin-induced aging model mice. The control group, 0.04% phytosphingosine-fed group, and 0.1% phytosphingosine-fed group were fed AIN-93G ad libitum from day 8 to day 35 of the experimental feeding. The control group received AIN-93G and water, the 0.04% phytosphingosine-fed group received AIN-93G containing 0.04% phytosphingosine and water, and the 0.1% phytosphingosine-fed group received AIN-93G containing 0.1% phytosphingosine and water. Body weight and food intake were measured. These results are shown in FIG.

[0124] On the 33rd day of the experimental feeding, the backs of the mice were shaved under isoflurane anesthesia, and on the 34th day, noninvasive measurements of the dorsal skin and collection of dorsal skin replicas were performed as described below. On the 36th day of the experimental feeding, the mice were fasted overnight and dissected under isoflurane inhalation anesthesia. The dorsal skin of the mice was collected, washed with saline, weighed, immediately frozen in liquid nitrogen, and stored at -80°C. A portion of the collected dorsal skin was added to RNAlater™ Stabilization Solution (Thermo Fisher Scientific) for genetic analysis, immersed overnight at 4°C, and then stored at -80°C.

[0125] Figure 11(A) is a graph showing changes in body weight of doxorubicin-induced aging model mice, and Figure 11(B) is a graph showing changes in food intake of the same model mice. In Figure 11(A), the horizontal axis shows the number of days of experimental feeding, and the vertical axis shows the body weight of the doxorubicin-induced aging model mice on each day. In Figure 11(B), the horizontal axis shows the number of days of experimental feeding, and the vertical axis shows the food intake of each group on each day. As shown in Figure 11(A), body weight increased in all groups as the number of days of experimental feeding increased. Furthermore, the 0.04% phytosphingosine-fed group and the 0.1% phytosphingosine-fed group showed faster weight gain than the control group. As shown in Figure 11(B), there was no difference in food intake between the groups.

[0126] From the above, it was found that the weight gain of doxorubicin-induced NHDF model mice was restored by the intake of phytosphingosine, just like the normal group (non-aged model mice).

[0127] [Example 7] The effects of phytosphingosine on the dorsal skin of doxorubicin-induced aging model mice were evaluated.

[0128] (1) Non-invasive measurement of dorsal skin For each group of mice prepared in Example 6, the transepidermal water loss (TEWL) of the dorsal skin of the mice was measured using a measurement probe (TEWAMETER, MPA580, manufactured by Courage+Khazaka) on days 0 and 34 of the test feeding. The results are shown in Figure 12.

[0129] Figure 12 is a graph showing changes in transepidermal water loss in the doxorubicin-induced aging model mice during the experimental feeding. In Figure 12, the horizontal axis represents the number of days during the experimental feeding, and the vertical axis represents transepidermal water loss. As shown in Figure 12, the control group had an increased transepidermal water loss on day 34 compared to the normal group, indicating a decline in skin barrier function with aging. In contrast, the 0.04% phytosphingosine-fed group and the 0.1% phytosphingosine-fed group had the same transepidermal water loss as the normal group even on day 34, completely suppressing the decline in skin barrier function associated with aging.

[0130] (2) Collection of dorsal skin replicas and three-dimensional image analysis On day 34 of the experimental period, dorsal skin replicas were prepared from the mice using a transmission replica preparation kit (ASB-01, manufactured by AsahiBioMed). The prepared dorsal skin replicas were analyzed for wrinkles using a reflection replica analysis system (ASA-03RXD, manufactured by AsahiBioMed). The wrinkle analysis involved quantifying the total volume fraction, wrinkle area fraction, wrinkle volume fraction, maximum wrinkle depth, maximum wrinkle width, average wrinkle depth, and number of wrinkles. The results are shown in Figure 13.

[0131] Figure 13 is a graph showing wrinkle formation in the doxorubicin-induced aging model mice in the experimental breeding study. In Figures 13(A) to 13(G), the horizontal axis indicates the type of each group, and the vertical axis indicates the total volume fraction, wrinkle area fraction, wrinkle volume fraction, maximum wrinkle depth, maximum wrinkle width, average wrinkle depth, and number of wrinkles, respectively. As shown in Figures 13(A) to 13(G), in all wrinkle analyses, the control group showed increased wrinkle severity compared to the normal group. In contrast, the 0.04% phytosphingosine-fed group and the 0.1% phytosphingosine-fed group showed similar results to the normal group in all wrinkle analyses, completely suppressing the increase in wrinkle severity due to aging.

[0132] From the above, it was found that oral intake of phytosphingosine can suppress the decline in skin barrier function and wrinkle formation in doxorubicin-induced aging model mice.

[0133] [Example 8] Using the rotarod test, we confirmed that phytosphingosine can improve cognitive, learning, and motor functions in doxorubicin-induced aging model mice.

[0134] For each group of mice prepared in Example 6, a rotarod test was performed using a rotarod device (Columbus Instruments) on days 18 and 32 after the start of the test. Specifically, in the test, mice were placed on a rod whose rotation speed changed from 0 rpm to 40 rpm over a 4-minute period, and the time until the mouse fell was measured. The test was performed three times for each mouse. A second and third test was performed 24 and 48 hours after the first test, and the time was measured in the same way. The rod was disinfected with 1% sodium hypochlorite and 70% ethanol after each test. The results are shown in Figure 14.

[0135] Figure 14 is a graph showing the results of the rotarod test of the doxorubicin-induced aging model mice in the experimental feeding. Figure 14(A) shows the test results from days 18 to 20 after the start of the experimental feeding, and Figure 14(B) shows the test results from days 32 to 34. In Figure 14, the horizontal axis represents the number of days, and the vertical axis represents the time (seconds) from when the mouse stepped onto the rod until it fell off. As shown in Figure 14(A), the normal group and the 0.1% phytosphingosine-fed group spent more time on the rod over the course of the experiment than the control group. Furthermore, as shown in Figure 14(B), the normal group and the 0.1% phytosphingosine-fed group spent longer time on the rod than the control group in all experiments.

[0136] These results demonstrate that oral administration of phytosphingosine can improve cognitive, learning, and motor functions in doxorubicin-induced aging model mice.

[0137] [Example 9] The effect of phytosphingosine on gene expression levels in the skin of doxorubicin-induced aging model mice was evaluated.

[0138] A portion of the dorsal skin from each mouse group preserved in Example 6 was collected and collected in a tube containing 800 μL of Sepasol®-RNA I Super G (Nacalai Tesque). The skin sections were finely cut with scissors in Sepasol®-RNA I Super G (Nacalai Tesque) on ice and then disrupted using a handheld homogenizer (T10 basic ULTRA-TURRAX, IKA). 200 μL of Sepasol®-RNA I Super G (Nacalai Tesque) was added to the resulting disrupted material, mixed by inversion, and allowed to stand at room temperature for 5 minutes. After this standing, 200 μL of chloroform was added, mixed by inversion, allowed to stand at room temperature for 3 minutes, and centrifuged at 10,800 rpm at 4°C for 15 minutes. 480 μL of the supernatant was transferred to another tube, 1 mL of 2-propanol was added, and the mixture was mixed by inversion. After standing at room temperature for 10 minutes, the mixture was centrifuged at 10,800 rpm and 4°C for 10 minutes. After the centrifugation, the supernatant was removed, 500 μL of 75% ethanol was added, and the mixture was stirred using a vortex mixer. The mixture was then centrifuged at 8,500 rpm and 4°C for 5 minutes. After removing the ethanol, 500 μL of 75% ethanol was added, and the resulting RNA solution was stored at -80°C.

[0139] The relative expression levels of p21 and Il6 were calculated in the same manner as in Example 1 (3-3) and (3-4), except that the obtained RNA solution and the following primer sets were used. The results are shown in Figure 15. p21 and Il6 are senescence-related genes and are known to be highly expressed in senescent cells. Primer set for p21 gene Forward primer 5'-CCTGGTGATGTCCGACCTG-3' (SEQ ID NO: 11) Reverse primer 5'-CCATGAGCGCATCGCAATC-3' (SEQ ID NO: 12) Primer set for Il6 gene Forward primer 5'-CCGGAGAGGAGACTTCACAG-3' (SEQ ID NO: 13) Reverse primer 5'-TTCTGCAAGTGCATCATCGT-3' (SEQ ID NO: 14)

[0140] Figure 15 is a graph showing the relative gene expression levels in the dorsal skin of the doxorubicin-induced aging model mice in the experimental breeding study. In Figure 15, the horizontal axis indicates the type of gene, and the vertical axis indicates the relative expression level of each gene. As shown in Figure 15, the expression levels of p21 and IL6 were increased in the control group, indicating the induction of aging. In contrast, the 0.04% phytosphingosine-fed group and the 0.1% phytosphingosine-fed group showed reduced expression levels of p21 and IL6 compared to the control group, which were equivalent to those of the normal group.

[0141] These findings demonstrate that phytosphingosine can suppress the expression of aging-related genes in the skin of doxorubicin-induced NHDF model mice.

[0142] [Example 10] We confirmed that phytosphingosine increases the amount of ceramide in the epidermis of doxorubicin-induced aging model mice.

[0143] The dorsal skin of each mouse group preserved in Example 6 was excised at two locations (diameter 8 mm, total area 1.0 cm) using a biopsy trephine (BP-80F, Kai Medical Co., Ltd.). 2The resulting skin was immersed in 1 mL of 3.8% ammonium thiocyanate-containing D-PBS(-) solution and incubated for 40 minutes to separate the epidermis from the dermis. The epidermis was washed twice with D-PBS(-) and then lipids were extracted in chloroform / methanol (2:1, v / v) overnight at 4°C. The extracted lipids were dried under nitrogen, and the resulting dried product was subjected to a weak alkaline treatment with 1 mL of methanol (containing 0.4 M KOH) at 38°C for 2 hours to remove glycerolipids. The removed lipids were recovered using the Folch method, loaded onto a Sep-Pak, and chloroform / methanol (9:1, v / v) was run through to obtain a ceramide fraction. After extracting the epidermal lipids in this manner, the epidermal residue was again immersed in 95% methanol (containing 1 M KOH) and incubated overnight at 4°C to extract lipids. The lipids were collected using the Folch method to extract ω-hydroxyceramide ([POS]ceramide) bound to the stratum corneum via ester bonds. These collected lipids were dried under nitrogen and used as analytical samples.

[0144] The analytical sample was redissolved in 1 mL of methanol, and 5 μL of the solution was subjected to quantitative analysis of ceramide using a QTRAP system MRM. The quantitative analysis was performed under the following analytical and gradient conditions. This allowed for the analysis of the sphingoid base and fatty acids that constitute ceramide. The results are shown in FIG. 16. The abbreviations for the sphingoid base and fatty acid are as follows. The types of ceramide are listed below in the order of the abbreviation for the fatty acid followed by the abbreviation for the sphingoid base. Abbreviation for sphingoid base DS: Dihydrosphingosine S: Sphingosine P: Phytosphingosine H: 6-hydroxysphingosine SD: 4,14-sphingadiene Fatty acid abbreviations N: Nonhydroxy fatty acid A: α-hydroxy fatty acid O: ω-hydroxy fatty acids EO: Ester ω-hydroxy fatty acid

[0145] <Analysis conditions> Detector: AB SCIEX QTRAP 5500 (manufactured by AB SCIEX) Probe: ESI Ion Spray Voltage: 5500 V GS1: 50 psi GS2: 50 psi Curtain Gas: 20 psi Interface Heater: 350℃ Declustering Potential: 85 V Entrance Potential: 10 V Collision Energy: 40 V ·Collision Cell Exit Potential: 12 V Column: TSKgel ODS-100Z, 3 μm, 2.0 × 50 mm (Tosoh Corporation) Column temperature: 40℃ Pump: LC-20AD (Shimadzu Corporation) ·Flow rate: 0.2 mL / min ·Mobile phase: (A) MilliQ water (containing 2 mM ammonium acetate) (B) Methanol (containing 1 mM ammonium acetate)

[0146] <Gradient conditions> Time(minutes) 0 15 30 35 40 A concentration (%) 10 1 1 10 10 B concentration (%) 90 99 99 90 90

[0147] Figure 16 is a graph showing the amount of ceramide in the epidermis of doxorubicin-induced aging model mice. In Figure 16, the horizontal axis indicates the type of each ceramide in the epidermis, and the vertical axis indicates the amount of each ceramide in the epidermis. As shown in Figure 16, the 0.04% phytosphingosine-fed group and the 0.1% phytosphingosine-fed group showed increased amounts of ceramide in the epidermis, particularly for NSD, NP, and AP, compared to the control group.

[0148] These findings suggest that phytosphingosine can increase the amount of ceramide in the epidermis and improve the skin's moisturizing function by removing senescent cells in the skin of doxorubicin-induced NHDF model mice.

[0149] [Example 11] Using a novel object recognition test, we confirmed that phytosphingosine can improve cognitive function in doxorubicin-induced aging model mice.

[0150] (1) Creation of a doxorubicin-induced aging model mouse As in Example 6, C57BL / 6J mice (female, 6 weeks old) were housed in a breeding facility set at 24°C with a 12-hour light-dark cycle. From the time the mice reached 7 weeks of age, they were pre-housed in individual cages for 7 days with free access to AIN-93G and water (distilled water).

[0151] After the preliminary breeding, the mice were divided into three groups: a normal group (non-aging model mice (Nor)) that received intraperitoneal administration of saline; test group 1 (doxorubicin-induced aging model mice (Exp1)) that received intraperitoneal administration of doxorubicin a total of two times; and test group 2 (doxorubicin-induced aging model mice (Exp2)) that received intraperitoneal administration of doxorubicin a total of three times. The normal group received intraperitoneal administration of saline on days 0, 7, and 14 from the start of the test breeding. Test group 1 received intraperitoneal administration of 5 mg / kg doxorubicin on days 0 and 7 from the start of the test breeding (twice in total), and were designated as doxorubicin-induced aging model mice. Test group 2 received intraperitoneal administration of 5 mg / kg doxorubicin on days 0, 7, and 14 from the start of the test breeding (three times in total), and were designated as doxorubicin-induced aging model mice. Each group consisted of 3 to 4 animals.

[0152] (2) Novel object recognition test in doxorubicin-induced aging model mice On the 20th day after the start of the experimental breeding (the day before the novel object recognition test), the mice in the normal group, test group 1, and test group 2 were placed in a square open field (50 cm × 50 cm × 50 cm) for 5 minutes to acclimate to the square open field environment. On the 21st day after the start of the experimental breeding (24 hours after acclimatization), the mice in the normal group, test group 1, and test group 2 were placed in the square open field with two identical objects and allowed to explore freely for 10 minutes before being returned to individual cages. After 1 hour of exploration, the mice in the normal group, test group 1, and test group 2 were placed in the square open field with one of the identical objects replaced with a novel object of a different shape, and their approach time to each object (time spent in contact with the object) was measured for 10 minutes. The ratio of approach time to the novel object to the total approach time (novel object exploration ratio) (%) was calculated, and the cognitive function of the doxorubicin-induced aging model mice was evaluated using the novel object exploration ratio as an index. Mice are known to be interested in novel objects they have never seen before, and when they find a novel object, they will approach and explore it. Therefore, a high novel object exploration ratio (long approach time to the novel object) can be evaluated as having high cognitive function, and conversely, a low novel object exploration ratio (short approach time to the novel object) can be evaluated as having low cognitive function. These results are shown in Figure 17.

[0153] Figure 17 is a graph showing the results of a novel object recognition test in doxorubicin-induced aging model mice. In Figure 17, the horizontal axis represents the normal group (Nor), test group 1 (Exp1), and test group 2 (Exp2), and the vertical axis represents the novel object exploration rate. As shown in Figure 17, it was confirmed that the novel object exploration rate was reduced in test group 1 and test group 2 compared to the normal group. Therefore, it was found that cognitive function was impaired in doxorubicin-induced aging model mice. Furthermore, when test group 1 and test group 2 were compared, there was almost no difference in the novel object exploration rate between the two groups. Therefore, it was found that intraperitoneal administration of doxorubicin a total of two times was sufficient to evaluate cognitive function. Furthermore, test group 1 was used in the following studies.

[0154] (3) Novel object recognition test using phytosphingosine in doxorubicin-induced aging model mice C57BL / 6J mice were pre-fed as described in Example 11(1) above and divided into a control group (doxorubicin-induced aging model mice (con)) receiving AIN-93G and a 0.04% phytosphingosine-fed group (doxorubicin-induced aging model mice (phs0.04%)) receiving AIN-93G containing 0.04% phytosphingosine. Each group received 5 mg / kg of doxorubicin intraperitoneally on days 0 and 7 from the start of the experimental feeding (twice in total) to create doxorubicin-induced aging model mice. During the experimental feeding period, the control group was fed AIN-93G and water, while the 0.04% phytosphingosine-fed group was fed AIN-93G containing 0.04% phytosphingosine and water ad libitum. Each group consisted of six mice (n=6).

[0155] As in Example 11(2), cognitive function of each group was evaluated by a novel object recognition test on day 21 after the start of the test feeding. The results are shown in FIG.

[0156] Figure 18 is a graph showing the results of a novel object recognition test in doxorubicin-induced aging model mice using phytosphingosine. In Figure 18, the horizontal axis represents the control group (con) and the 0.04% phytosphingosine-fed group (phs0.04%), and the vertical axis represents the novel object exploration rate. As shown in Figure 18, it was confirmed that the novel object exploration rate was increased in the 0.04% phytosphingosine-fed group compared to the control group. Therefore, it was found that phytosphingosine can improve the cognitive function of doxorubicin-induced aging model mice.

[0157] From the above, it was found that oral administration of phytosphingosine can improve cognitive function in doxorubicin-induced aging model mice.

[0158] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0159] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Composition for removing senescent cells> (Appendix 1) A composition for removing senescent cells, comprising sphingosines. (Appendix 2) The composition for removing senescent cells according to Appendix 1, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine. (Appendix 3) A composition for removing senescent cells according to Appendix 1 or 2, which is a food composition. (Appendix 4) The composition for removing senescent cells according to Appendix 1 or 2, which is a cosmetic composition. (Appendix 5) 5. The composition for removing senescent cells according to claim 4, wherein the content of the sphingosines is 0.000001 to 0.1% by weight. <Senocyte removal agent> (Appendix 6) A senolytic agent comprising a composition for removing senolytic cells according to any one of Appendices 1 to 5. <Composition for improving cognitive function> (Appendix 7) A composition for improving cognitive function, comprising sphingosines. (Appendix 8) 8. The composition for improving cognitive function according to claim 7, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphinganine, and sphingenine. (Appendix 9) A composition for improving cognitive function according to Appendix 7 or 8, which is a food composition. <Cognitive function improver> (Appendix 10) A cognitive function improving agent comprising a composition for improving cognitive function described in any one of Appendices 7 to 9. <Composition for improving learning function> (Appendix 11) A composition for improving learning function, comprising sphingosines. (Appendix 12) 12. The composition for improving learning function according to claim 11, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine. (Appendix 13) A composition for improving learning function described in Appendix 11 or 12, which is a food composition. <Learning function improver> (Appendix 14) A learning function improving agent comprising a composition for improving learning function described in any one of Appendices 11 to 13. <Composition for improving motor function> (Appendix 15) A composition for improving motor function, comprising a sphingosine. (Appendix 16) 16. The composition for improving motor function according to claim 15, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine. (Appendix 17) A composition for improving motor function according to Appendix 15 or 16, which is a food composition. <Motor function improving agent> (Appendix 18) A motor function improving agent comprising a composition for improving motor function described in any one of Appendices 15 to 17. <Method for removing senescent cells> (Appendix 19) A method for removing senescent cells using sphingosines. <Cognitive function improvement method> (Appendix 20) A method for improving cognitive function using sphingosines. <Method for improving learning function> (Appendix 21) A method for improving learning function using sphingosines. <How to improve motor function> (Appendix 22) A method for improving motor function using sphingosines. (Appendix 23) 23. The method of any one of claims 19 to 22, comprising administering the sphingosine to a subject. (Appendix 24) 24. The method of any one of claims 19 to 23, for use in vitro or in vivo. <Use> (Appendix 25) Use of a composition for removing senescent cells described in any one of Appendixes 1 to 5 or a senolytic agent for removing senescent cells described in Appendix 6 for use in removing senescent cells. (Appendix 26) Use of a composition for improving cognitive function described in any one of Appendixes 7 to 9 or a cognitive function improving agent described in Appendix 10 for use in improving cognitive function. (Appendix 27) Use of a composition for improving learning function described in any one of Appendixes 11 to 13 or an agent for improving learning function described in Appendix 14 for use in improving learning function. (Appendix 28) Use of a composition for improving motor function described in any one of Appendixes 15 to 17 or a motor function improving agent described in Appendix 18 for use in improving motor function. [Industrial Applicability]

[0160] As described above, according to the present disclosure, senescent cells can be removed, and therefore the present disclosure is extremely useful, for example, in the field of anti-aging.

Claims

1. A composition for removing senescent cells, comprising sphingosines.

2. 2. The composition for removing senescent cells according to claim 1, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine.

3. The composition for removing senescent cells according to claim 1 or 2, which is a food composition.

4. The composition for removing senescent cells according to claim 1 or 2, which is a cosmetic composition.

5. 5. The composition for removing senescent cells according to claim 4, wherein the content of the sphingosines is 0.000001 to 0.1% by weight.

6. A composition for improving cognitive function, comprising sphingosines.

7. 7. The composition for improving cognitive function according to claim 6, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine.

8. The composition for improving cognitive function according to claim 6 or 7, which is a food composition.

9. A composition for improving learning function, comprising sphingosines.

10. 10. The composition for improving learning function according to claim 9, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine.

11. The composition for improving learning function according to claim 9 or 10, which is a food composition.

12. A composition for improving motor function, comprising a sphingosine.

13. The composition for improving motor function according to claim 12, wherein the sphingosine is at least one selected from the group consisting of sphingosine, phytosphingosine, sphingadienine, sphinganine, and sphingenine.

14. The composition for improving motor function according to claim 12 or 13, which is a food composition.

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