Senescent cell remover

Abstract

To provide a senescent cell remover which is derived from a food product.SOLUTION: One aspect of the present invention relates to a senescent cell remover that contains a treated product of blueberry as an active ingredient. Another aspect of the present invention relates to a food product for protecting or improving one or more diseases, symptoms or disorders where a senescent cell participates, in an object having or the possibility of having the diseases, symptoms or disorders, contains a treated product of blueberry as an active ingredient, wherein the diseases, symptoms or disorders are selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatocholangitis, pulmonary fibrosis, Alzheimer disease, Parkinson disease, type-1 diabetes, type-2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, non-alcoholic fatty liver disease, immune function decrease, cachexia and weakness.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a senescent cell removing agent. In particular, one aspect of the present invention relates to a senescent cell removing agent containing a processed blueberry as an active ingredient. [Background technology]

[0002] When DNA damage that is difficult to repair occurs in cells that compose the body, the cells irreversibly stop proliferation and enter a state of aging. It is known that cellular aging can be caused by factors such as DNA damage, telomere shortening, mitochondrial dysfunction, activation of cancer genes, and / or oxidative stress (Non-Patent Document 1). When senescent cells accumulate in the body, they ultimately lead to the aging of the individual, causing various diseases (aging-related diseases) in the individual.

[0003] Since senescent cells themselves do not proliferate, cellular senescence can be an important cancer suppression mechanism. On the other hand, cellular senescence is characterized by the senescence-associated secretory phenomenon (SASP), which synthesizes and secretes large amounts of inflammation-inducing proteins. For this reason, it is known that cellular senescence can be a cause of aging-related diseases, including the induction of cancer in surrounding cells. Several drugs that have the effect of removing senescent cells have been developed, and it has been demonstrated at the mouse level that the healthy lifespan can be extended by removing senescent cells from the body using these drugs (Non-Patent Documents 2 and 3).

[0004] In addition to the development of drugs with senescent cell removal effects, procyanidin C1 (PCC1) has been reported as a compound contained in grape seeds that has the effect of removing senescent cells (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4892690 [Patent Document 2] JP 2022-168636 A [Non-patent literature]

[0006] [Non-Patent Document 1] Kudlova, N., J. B. De Sanctis & M. Hajduch, 2022, Cellular Senescence: Molecular Targets, Biomarkers, and Senolytic Drugs. Int J Mol Sci, p. 23 [Non-Patent Document 2] Musi, N., J. M. Valentine, K. R. Sickora, E. Baeuerle, C. S. Thompson, Q. Shen & M. E. Orr, 2018, Tau protein aggregation is associated with cellular senescence in the brain. Aging Cell, Vol. 17, e12840 [Non-Patent Document 3] Schafer, M. J., T. A. White, K. Iijima, A. J. Haak, G. Ligresti, E. J. Atkinson, A. L. Oberg, J. Birch, H. Salmonowicz, Y. Zhu, D. L. Mazula, R. W. Brooks, H. Fuhrmann-Stroissnigg, T. Pirtskhalava, Y. S. Prakash, T. Tchkonia, P. D. Robbins, M. C. Aubry, J. F. Passos, J. L. Kirkland, D. J. Tschumperlin, H. Kita & N. K. LeBrasseur, 2017, Cellular senescence mediates fibrotic pulmonary disease. Nat Commun, Vol. 8, p. 14532 [Non-Patent Document 4] Xu, Q., Q. Fu, Z. Li, H. Liu, Y. Wang, X. Lin, R. He, X. Zhang, Z. Ju, J. Campisi, JL Kirkland & Y. Sun, 2021, The flavonoid procyanidin C1 has senotherapeutic activity and increases lifespan in mice. Nat Metab, Volume 3, p. 1706-1726 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, the development of drugs with senescent cell removal effects is underway. However, under current laws in Japan, aging itself is not considered a disease. For this reason, it is difficult to implement drugs with senescent cell removal effects in society. On the other hand, if food-derived components with senescent cell removal effects can be found from foods that are widely consumed, it may be possible to prevent or improve aging-related diseases through the intake of the foods. Such foods or food-derived components can be continuously applied not only to subjects with aging-related diseases, but also to healthy subjects without aging-related diseases.

[0008] Therefore, an object of the present invention is to provide a food-derived senescent cell removing agent. [Means for solving the problem]

[0009] The present inventors have investigated various means for solving the above problems. They have found that processed blueberries have an effect of removing senescent cells. They have completed the present invention based on the above findings.

[0010] That is, the present invention includes the following aspects and embodiments. (Embodiment 1) An agent for removing senescent cells, comprising a processed product of blueberries as an active ingredient. (Embodiment 2) The senolytic agent according to embodiment 1 for preventing, treating, or ameliorating one or more diseases, symptoms, or disorders in which senescent cells are involved. (Embodiment 3) The senescent cell removing agent according to embodiment 2, wherein the one or more diseases, symptoms, or disorders involving senescent cells are selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatic cholangitis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, non-alcoholic fatty liver disease, impaired immune function, cachexia, and frailty. (Embodiment 4) The senescent cell removing agent according to any one of embodiments 1 to 3, wherein the processed blueberry product is a processed blueberry stem or leaf product. (Embodiment 5) The senescent cell removing agent according to any one of embodiments 1 to 4, wherein the processed blueberry product is one or more products or fractions selected from the group consisting of blueberry pulverization, squeezed juice, solvent extract, and separated fractions thereof. (Embodiment 6) A food product for preventing or ameliorating one or more diseases, symptoms, or disorders involving senescent cells in a subject having or likely to have the disease, symptom, or disorder selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatic cholangitis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, non-alcoholic fatty liver disease, impaired immune function, cachexia, and frailty, comprising a processed blueberry as an active ingredient. (Embodiment 7) The food according to embodiment 6, wherein the processed blueberry is a processed blueberry stem or leaf. (Embodiment 8) The food according to embodiment 6 or 7, wherein the processed blueberry product is one or more products or fractions selected from the group consisting of blueberry crush, juice, and solvent extract, and isolated fractions thereof. Effect of the Invention

[0011] The present invention makes it possible to provide a food-derived senescent cell removing agent. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 is a graph showing the results of evaluating the viability of WI-38 senescent cells and WI-38 non-senescent cells to which blueberry leaf extract was added in II-1 using the MTT assay. In the figure, the black bars indicate the viability of WI-38 non-senescent cells, and the shaded bars indicate the viability of WI-38 senescent cells (mean ± SD, n = 4 to 5). The horizontal axis indicates the concentration of blueberry leaf extract added (μg / mL), and the vertical axis indicates the cell viability (%). [Diagram 2] FIG. 2 is a graph showing the results of evaluating the viability of WI-38 senescent cells to which 5-(2,6-difluorophenoxy)-3-[[3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid (QVD-O-Ph) was added using the MTT assay in II-2. In the figure, the bars indicate the viability of WI-38 senescent cells to which no QVD-O-Ph was added or to which a given concentration of QVD-O-Ph was added (mean ± SD, n = 3). The horizontal axis indicates the concentration of QVD-O-Ph added (μM), and the vertical axis indicates the cell viability (%). [Diagram 3] Figure 3 is a graph showing the results of evaluating the viability of WI-38 senescent cells to which QVD-O-Ph and blueberry leaf extract were added in II-2 using an MTT assay. In the figure, the black bars show the viability when blueberry leaf extract was added to WI-38 senescent cells without QVD-O-Ph, and the gray bars show the viability when blueberry leaf extract was additionally added to WI-38 senescent cells to which QVD-O-Ph was added (mean ± SD, n = 3 to 4). The vertical axis shows the cell viability (%). [Figure 4] Figure 4 is a graph showing the results of evaluating the mitochondrial membrane potential of WI-38 senescent cells to which blueberry leaf extract was added using the JC-1 assay in II-3. In the figure, the vertical axis shows the JC-1 fluorescence intensity ratio (red fluorescence / green fluorescence) (mean ± SD, n = 3). [Diagram 5]FIG. 5 is a graph showing the results of comparing the cytotoxic activity of PCC1 described in the document by Xu et al. (Non-Patent Document 4) with the cytotoxic activity of proanthocyanidin (PAC) contained in blueberry leaf extract in II-4. In the figure, A shows the relationship between the concentration of PCC1 added to WI-38 senescent cells and the viability of WI-38 senescent cells. In the figure, the horizontal axis is the PCC1 concentration (μg / mL), and the vertical axis shows the viability of WI-38 senescent cells without addition or with addition of PCC1 (mean ± SD, n = 3). B shows the relationship between the concentration of PAC added to WI-38 senescent cells and the viability of WI-38 senescent cells. In the figure, the horizontal axis is the PAC concentration (ng / mL), and the vertical axis shows the viability of WI-38 senescent cells without addition or with addition of PAC (mean ± SD, n = 5). [Figure 6] Figure 6 is a graph showing the results of evaluating the viability of WI-38 senescent cells and WI-38 non-senescent cells to which each fraction of blueberry leaf extract and each fraction of blueberry stem extract were added using the MTT assay in II-5. In the figure, the black bars indicate the viability of WI-38 non-senescent cells, and the gray bars indicate the viability of WI-38 senescent cells (mean ± SD, n = 2 to 3). The vertical axis indicates the cell viability (%). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [1: Senescent cell remover] The present inventors have found that processed blueberries have an effect of removing senescent cells. Therefore, one aspect of the present invention relates to an agent for removing senescent cells comprising processed blueberries as an active ingredient.

[0014] In each aspect of the present invention, "cellular senescence" and "senescent cells" refer to a state in which cells irreversibly stop proliferating due to factors such as DNA damage, telomere shortening, mitochondrial dysfunction, oncogene activation, and oxidative stress, and to cells in such a state, respectively. Cellular senescence is known to cause various diseases (aging-related diseases) in individuals. In addition, in each aspect of the present invention, "elimination of senescent cells" refers to injury and death of senescent cells. Elimination of senescent cells refers to, for example, injury and death of senescent cells caused by apoptosis, particularly injury and death of senescent cells caused by caspase-dependent apoptosis.

[0015] The processed blueberry product, which is the active ingredient of the senescent cell removing agent of this embodiment, has selective cytotoxic activity against senescent cells, while having no particular effect on normal cells. Furthermore, the cytotoxic activity of the processed blueberry product is caused by caspase-dependent apoptosis. Therefore, the senescent cell removing agent of this embodiment can remove senescent cells through apoptosis, particularly caspase-dependent apoptosis. Furthermore, the senescent cell removing agent of this embodiment can prevent, treat, or ameliorate aging-related diseases.

[0016] In each embodiment of the present invention, the senescent cell elimination effect can be evaluated based on, for example, but not limited to, the cytotoxic activity against senescent cells induced under certain conditions. In addition, in each embodiment of the present invention, the mechanism of action of the senescent cell elimination effect can be evaluated based on, for example, but not limited to, the suppression or recovery effect of cytotoxic activity by co-addition with a pan-caspase inhibitor having an apoptosis inhibitory effect (e.g., 5-(2,6-difluorophenoxy)-3-[[3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid, QVD-O-OPh), or the change in mitochondrial membrane potential, which is an index of apoptosis.

[0017] The senescent cell removing agent of this embodiment is preferably used to prevent, treat or improve one or more diseases, symptoms or disorders involving senescent cells. The one or more diseases, symptoms or disorders involving senescent cells are preferably selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatic cholangitis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, nonalcoholic fatty liver disease, immunological impairment, cachexia and frailty, and more preferably frailty. All of the diseases, symptoms or disorders exemplified above are known to be aging-related diseases related to cell aging. Therefore, by administering the senescent cell removing agent of this embodiment to a subject who may have the above-exemplified diseases, symptoms or disorders in the future, such as a healthy human, the disease, symptoms or disorders can be prevented through the senescent cell removing effect of the blueberry processing product. Furthermore, by administering the senescent cell-removing agent of this embodiment to a subject, such as a human patient, having one of the diseases, symptoms, or disorders exemplified above, the disease, symptoms, or disorders can be treated or ameliorated via the senescent cell-removing effect of the blueberry processed product.

[0018] As used herein, "prevention" means substantially preventing or partially reducing the occurrence (onset or manifestation) of the disease, symptom, or disorder in a subject, such as a healthy human, who may have the disease, symptom, or disorder exemplified above in the future. In addition, as used herein, "treatment" means suppressing (e.g., suppressing progression), alleviating, repairing, and / or curing a disease, symptom, or disorder that has occurred (onset or manifested) in a subject, such as a human patient, who has the disease, symptom, or disorder exemplified above. Furthermore, as used herein, "improvement" means partially or completely suppressing (e.g., suppressing progression), alleviating, and / or repairing a disease, symptom, or disorder that has occurred (onset or manifested) in a subject, such as a human patient, who has the disease, symptom, or disorder exemplified above.

[0019] Blueberries are deciduous or evergreen, shrub or semi-tree-like fruit trees native to America, classified in the Ericaceae family, Vaccinium genus, Cyanococcus section (Vander Kloet, 1988; Tamada, 1996). Blueberries consist of about six species, but the three most important species in orchard gardening are (1) highbush blueberry (Vaccinium corymbosum. L), (2) rabbiteye blueberry (V. virgatum Aiton), and (3) lowbush blueberry (V. angustifolium Michaux and V. myrtilloides Aition). These blueberry species were developed from wild species by the United States Department of Agriculture or state universities since the beginning of the 20th century. The direction of breeding was aimed at making blueberries easy to cultivate and capable of producing large, sweet, high-quality fruits. Today, blueberries are cultivated all over the world. In recent years, uses for parts of the plant other than the fruit (for example, food uses) have also been developed, such as using blueberry leaves as tea.

[0020] In each aspect of the present invention, the type, origin, place of origin, etc. of blueberries used as an active ingredient are not particularly limited. Blueberries of any type, origin, and place of origin, including the species exemplified above, can be used as an active ingredient.

[0021] In each embodiment of the present invention, the processed product of blueberries used as an active ingredient may be a processed product of any part of the plant, but is usually a processed product of any part other than the fruit, for example, a processed product of stems, leaves, flowers, or roots, and particularly a processed product of stems or leaves. The above-exemplified parts of the blueberry plant may be used alone or in combination of one or more. As shown in the following examples, the processed product of blueberries has a senescent cell removing effect. Therefore, by administering the senescent cell removing agent of this embodiment to a subject having or possibly having the above-exemplified disease, symptom, or disorder, for example, a healthy human or human patient, the disease, symptom, or disorder can be prevented, treated, or ameliorated through the senescent cell removing effect of the processed product of blueberries. In addition, the senescent cell removing agent of this embodiment can provide a means for preventing, treating, or ameliorating the above-exemplified disease, symptom, or disorder using a processed product of blueberries, which is a food that is widely consumed.

[0022] In each embodiment of the present invention, blueberries used as an active ingredient may be used in its original form, but are usually used in the form of a processed product. In each embodiment of the present invention, the "processed product" refers to a product or fraction that has been processed or separated by physical or chemical means. Examples of means for obtaining the processed product include, but are not limited to, crushing, squeezing, drying, solvent extraction, filtration, centrifugation, adsorption, recrystallization, distillation, and various types of chromatography. Each of the above means may be combined and / or repeated multiple times under the same or different conditions as desired.

[0023] The crushing can be carried out using a crushing machine such as a mill or blender commonly used in the art. For example, crushed blueberries can be obtained by crushing a part of a blueberry, preferably a stem or a leaf, either as it is or after drying. The crushed product may be dried if desired.

[0024] Drying can be performed by any means and under any conditions as long as the senescent cell removing effect of the processed blueberry product is not impaired. Drying can be performed by means of, for example, vacuum freeze drying, hot air drying, far infrared drying, reduced pressure drying, microwave reduced pressure drying, superheated steam drying, etc. Since there is little change in the components of the processed product, it is preferable to dry blueberries or a processed product thereof (e.g., a pulverized product) by vacuum freeze drying.

[0025] Solvent extraction can be carried out using, but is not limited to, water or an organic solvent such as a lower alcohol (e.g., an alcohol having 1 to 6 carbon atoms such as methanol, ethanol, or 2-propanol (isopropyl alcohol)), a higher alcohol (e.g., an alcohol having 7 or more carbon atoms such as 1-heptanol or 1-octanol), acetone, acetonitrile, diethyl ether, or ethyl acetate. The solvent used for solvent extraction of blueberries or a processed product thereof (e.g., crushed product or squeezed juice) is preferably water or a lower alcohol, more preferably water. In addition, the solvent extraction may be carried out under either room temperature or heated conditions. Particularly preferably, the solvent extract of blueberries can be obtained by extracting blueberries or a processed product thereof (e.g., crushed product or squeezed juice) with hot water. The solvent extract may be dried as desired.

[0026] Chromatography can be carried out using various chromatographic supports commonly used in the art, such as adsorption, normal or reversed phase partition, or gel filtration.

[0027] The processed product of blueberries is preferably one or more products or fractions selected from the group consisting of crushed products, squeezed juice, and solvent extracts of the parts of blueberry plants (e.g., stems or leaves) exemplified above, and separated fractions thereof, more preferably a solvent extract of the crushed products or a separated fraction of the solvent extract, and even more preferably a hot water extract of the crushed products or a chromatographically separated fraction of the hot water extract. Particularly preferably, the processed product of blueberries is a fraction obtained by adding a hot water extract of crushed products of the parts of blueberry plants (e.g., stems or leaves) exemplified above to Sephadex LH-20 column chromatography and eluting with an eluent of 60% acetone aqueous solution. By administering the senescent cell removing agent of this embodiment to a subject having or possibly having the disease, symptom, or disorder exemplified above, such as a healthy human or human patient, the disease, symptom, or disorder can be prevented, treated, or ameliorated through the senescent cell removing effect of the processed product of blueberries. Furthermore, the senescent cell removing agent of this embodiment can provide a means for preventing, treating, or ameliorating the diseases, symptoms, or disorders exemplified above, using processed blueberries, a food that is widely consumed.

[0028] In a particularly preferred embodiment, a hot water extract of the ground product of the above-exemplified blueberry plant part (e.g., stem or leaf) is added to Sephadex LH-20 column chromatography, and the fraction eluted with an eluent of 60% acetone aqueous solution usually contains proanthocyanidin (hereinafter also referred to as "PAC"). The PAC contained in the fraction is, for example, a main constituent unit of two types of polymers (A type and B type) with different bonding modes represented by the following formula, in which a flavan-3-ol skeleton is polymerized, and further contains a phenylpropanoid unit such as cinchonaine I (Patent Publication No. 4892690 (Patent Document 1) and JP-A-2022-168636 (Patent Document 2)).

[0029] [ka] TIFF2025005056000002.tif135162

[0030] In the formula, R 1 and R 3 is H or OH, R 2 is OH and R 4 is H or a monovalent group (for example, a lower hydrocarbon group such as a C1-C6 alkyl), and the repeat number n of the extension unit is an integer of 2 or more.

[0031] In this embodiment, it is preferable that the PAC contained in the fraction contains a cinchonaine I unit as a terminal unit of the polymer, contains a B-type bond as an extension unit that is the intermediate portion of the polymer, and has an average degree of polymerization of 6 or more, for example, 10 or more. By containing a PAC having the above characteristics, the processed blueberry product can exhibit a particularly high senescent cell removal effect.

[0032] Due to the above characteristics, the processed blueberry product can exhibit a senescent cell removing effect, and through the senescent cell removing effect, the processed blueberry product can prevent, treat, or ameliorate the above-mentioned diseases, symptoms, or disorders in subjects who have or may have such diseases, symptoms, or disorders, such as healthy humans or human patients.

[0033] [2: Food use] Since processed blueberries have been used in food applications, they are foods with sufficient dietary experience. For this reason, processed blueberries have been established to be safe and low-toxic. Therefore, another aspect of the present invention relates to a food containing processed blueberries as an active ingredient for preventing or improving one or more diseases, symptoms, or disorders associated with senescent cells in a subject who has or may have the disease, symptom, or disorder. In the food of this aspect, the one or more diseases, symptoms, or disorders associated with senescent cells are preferably the same as the one or more diseases, symptoms, or disorders associated with senescent cells exemplified above for the senescent cell removing agent of one aspect of the present invention. The food of this aspect can prevent or improve the disease, symptom, or disorder exemplified above in a subject who has or may have the disease, symptom, or disorder, such as a healthy human or human patient, through a senescent cell inhibitory effect without substantially affecting the health of the subject who ingests the food.

[0034] In the food of this embodiment, the processed blueberry used as an active ingredient may be used alone or in combination with one or more edible ingredients. The food of this embodiment may be formulated into various dosage forms commonly used in the art according to the desired method of ingestion. Therefore, the food of this embodiment may also be provided in the form of a food composition containing the processed blueberry and one or more edible ingredients. In this embodiment, the one or more edible ingredients may include preservatives, stabilizers, dispersants, leavening agents, surfactants, oily liquids, buffers, antioxidants, sweeteners, flavorings, colorants, pigments, etc.

[0035] The food product of this embodiment may be used as it is as a food product, or may be mixed with other foods or food components and used as a food ingredient.

[0036] The food of this embodiment can be used as, for example, a dietary supplement, a functional food, or a food for specified health uses. The form of the food of this embodiment may be, for example, a normal food or beverage, or a dietary supplement such as a supplement. Examples of the form of the dietary supplement include tablets, pills, powders, capsules, soft capsules, microcapsules, elixirs, liquids, syrups, slurries, and suspensions. The tablets may be formulated as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, orally disintegrating tablets (OD tablets) or film-coated tablets, or as double- or multi-layered tablets, as desired. The food of this embodiment can be formulated using the same ingredients as the pharmaceutical of one embodiment of the present invention described below.

[0037] When a subject, particularly a human, ingests the food of this embodiment, the processed blueberry product, which is an active ingredient, is ingested by the subject in a manner and dosage (e.g., intake amount and intake route) that is effective and safe for food. For example, when a subject, particularly a human, ingests the food of this embodiment, the intake amount of the processed blueberry product used as an active ingredient is usually in the range of 1 to 1000 mg / kg body weight / day, for example, in the range of 10 to 100 mg / kg body weight / day.

[0038] [3:Medicinal uses] As explained above, processed blueberries can exert a senescent cell removing effect. Therefore, another aspect of the present invention relates to a medicament for preventing or treating one or more diseases, symptoms, or disorders involving senescent cells in a subject who has or may have the disease, symptom, or disorder, comprising a processed blueberry as an active ingredient. The medicament of this aspect can prevent or treat one or more diseases, symptoms, or disorders involving senescent cells in a subject who has or may have the disease, symptom, or disorder through the senescent cell removing effect.

[0039] In the senolytic agent and medicament of this embodiment, the processed blueberry used as an active ingredient may be used alone or in combination with one or more pharma- ceutically acceptable ingredients. The senolytic agent and medicament of this embodiment may be formulated into various dosage forms commonly used in the art, depending on the desired administration method. Thus, the senolytic agent and medicament of this embodiment may also be provided in the form of a pharmaceutical composition containing the processed blueberry and one or more pharma- ceutically acceptable carriers. In this embodiment, the pharmaceutical composition may contain, in addition to the above-mentioned ingredients, one or more pharma- ceutically acceptable additives such as medium (e.g., a solvent such as sterilized water or a solution such as saline), excipient, binder, vehicle, solubilizer, preservative, stabilizer, disintegrant, disintegration inhibitor, swelling agent, lubricant, surfactant, emulsifier, oily liquid (e.g., vegetable oil), suspending agent, buffer, soothing agent, antioxidant, sweetener, and flavoring agent.

[0040] The dosage form of the senescent cell removing agent and medicament of this embodiment is not particularly limited, and may be a preparation for use in parenteral administration, a preparation for use in transmucosal (e.g., nasal, sublingual, or oral mucosa), transdermal, transanal (enema), or transvaginal administration, or a preparation for use in oral administration. Furthermore, the dosage form of the senescent cell removing agent and medicament of this embodiment may be a preparation in a unit dose form or a preparation in a multiple dose form. Examples of preparations for use in parenteral administration include injections such as sterile solutions or suspensions with water or other pharma- ceutically acceptable liquids. Examples of additives that can be mixed with the injection include, but are not limited to, vehicles such as isotonic solutions containing physiological saline, glucose, or other auxiliary agents (e.g., D-sorbitol, D-mannitol, or sodium chloride), solubilizing agents such as alcohols (e.g., ethanol or benzyl alcohol), esters (e.g., benzyl benzoate), and polyalcohols (e.g., propylene glycol or polyethylene glycol), nonionic surfactants such as polysorbate 80 or polyoxyethylene hydrogenated castor oil, oily liquids such as sesame oil or soybean oil, buffers such as phosphate buffer or sodium acetate buffer, pain-relieving agents such as benzalkonium chloride or procaine hydrochloride, stabilizers such as human serum albumin or polyethylene glycol, preservatives, and antioxidants. The prepared injection is usually filled into a suitable container (e.g., a vial or an ampoule) and stored under an appropriate environment until use.

[0041] Formulations for oral administration include, but are not limited to, tablets, pills, powders, capsules, soft capsules, microcapsules, elixirs, liquids, syrups, slurries, suspensions, etc. Tablets may be formulated as sugar-coated tablets or gelatin-coated tablets, enteric-coated tablets, orally disintegrating tablets (OD tablets) or film-coated tablets, or as double or multi-layer tablets, if desired.

[0042] Ingredients that can be mixed into tablets or capsules include, but are not limited to, binders such as water, ethanol, propanol, simple syrup, glucose solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, gelatin, corn starch, tragacanth gum, or gum arabic; excipients such as crystalline cellulose, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, or silicic acid; dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, and the like. Examples of the disintegrating agent include ester, sodium lauryl sulfate, monoglyceride stearic acid, starch, or lactose; disintegration inhibitors such as white sugar, stearic cocoa butter, or hydrogenated oil; absorption promoters such as quaternary ammonium salts or sodium lauryl sulfate; moisturizing agents such as glycerin or starch; adsorbents such as starch, lactose, kaolin, bentonite, or colloidal silicic acid; lubricants such as purified talc, stearates (e.g., magnesium stearate), boric acid powder, or polyethylene glycol; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, saffron oil, or cherry. When the preparation is a capsule, it may further contain a liquid carrier such as oil or fat.

[0043] The senolytic agent and medicament of this embodiment can also be used in combination with one or more other pharmaceutical agents useful as pharmaceuticals. In this case, the senolytic agent and medicament of this embodiment may be provided in the form of a single pharmaceutical agent containing a processed product of blueberries and one or more other pharmaceutical agents, or may be provided in the form of a pharmaceutical combination or kit containing multiple preparations in which the processed product of blueberries and one or more other pharmaceutical agents are separately formulated. In the case of a pharmaceutical combination or kit form, each preparation can be administered simultaneously or separately (e.g., consecutively).

[0044] The medicine of this embodiment can be used to prevent or treat one or more diseases, symptoms, or disorders associated with senescent cells in a subject having or potentially having such a disease, symptom, or disorder, such as a healthy human or human patient, via the senescent cell-removing effect of a processed product of blueberries, in the same manner as the senescent cell-removing agent of one embodiment of the present invention described above. In the medicine of this embodiment, the one or more diseases, symptoms, or disorders associated with senescent cells are preferably the same as the one or more diseases, symptoms, or disorders associated with senescent cells exemplified above for the senescent cell-removing agent of one embodiment of the present invention. By using the medicine of this embodiment for the prevention or treatment of the disease, symptom, or disorder in a subject having or potentially having such a disease, symptom, or disorder, such as a healthy human or human patient, the disease, symptom, or disorder can be prevented or treated via the senescent cell-removing effect of a processed product of blueberries.

[0045] The processed blueberry used as the active ingredient of the senescent cell removing agent and medicine of this embodiment is derived from a blueberry plant. Therefore, the processed blueberry product is safe and low-toxicity. Therefore, the senescent cell inhibition and medicine of this embodiment can be applied to various subjects requiring prevention or treatment of the diseases, symptoms, or disorders exemplified above. The subject is preferably a human or non-human mammalian subject or patient (e.g., a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, Mandrill, or chimpanzee), more preferably a healthy human or human patient. By administering the senescent cell removing agent and medicine of this embodiment to the subject, the disease, symptoms, or disorders in the subject can be prevented or treated through the senescent cell removing effect of the processed blueberry product.

[0046] When the senolytic agent and medicament of this embodiment are administered to a subject, particularly a healthy human or human patient, the exact dosage and administration should be ultimately determined by the attending physician as a therapeutically effective dosage and administration, taking into consideration many factors such as the subject's age, sex, symptoms to be prevented or treated, the exact state (e.g., severity) of the disease and / or disorder, and the administration route. Therefore, in the senolytic agent and medicament of this embodiment, the processed product of blueberries, which is an active ingredient, is administered to the subject at a therapeutically effective dosage and administration (e.g., dosage and administration route). For example, when the senolytic agent and medicament of this embodiment are administered to a subject, particularly a human patient, the dosage of the processed product of blueberries used as an active ingredient is usually in the range of 1 to 1000 mg / kg body weight / day, for example, in the range of 10 to 100 mg / kg body weight / day.

[0047] The senolytic agent and medicament of this embodiment may be administered by any administration route. For example, the senolytic agent and medicament of this embodiment are preferably administered by routes such as intravenous administration, enema administration, subcutaneous administration, intramuscular administration, oral administration, or intraperitoneal administration, and more preferably orally. By using the senolytic agent or medicament of this embodiment for oral administration, the senolytic agent or medicament can be conveniently prescribed to a subject, for example, a healthy human or human patient.

[0048] The processed product of blueberries can be used to prevent or treat the above-mentioned senescent cell-associated diseases, symptoms, or disorders in subjects who have or may have the above-mentioned senescent cell-associated diseases, symptoms, or disorders, such as healthy humans or human patients, through the senescent cell removing effect. Therefore, another aspect of the present invention relates to a preventive or therapeutic agent for one or more diseases, symptoms, or disorders in which senescent cells are associated, comprising a processed product of blueberries as an active ingredient. The preventive or therapeutic agent of this aspect has the same characteristics as the senescent cell removing agent and medicine of this aspect described above. In addition, the preventive or therapeutic agent of this aspect can be used in the same manner and dosage as the senescent cell removing agent and medicine of this aspect described above. In the preventive or therapeutic agent of this aspect, the one or more diseases, symptoms, or disorders in which senescent cells are associated are preferably the same as the one or more diseases, symptoms, or disorders in which senescent cells are associated, exemplified above for the senescent cell removing agent of one aspect of the present invention. By administering an effective amount of a processed blueberry product to a subject having or likely to have any of the above-mentioned diseases, symptoms, or disorders, the above-mentioned diseases, symptoms, or disorders can be prevented or treated through the senescent cell removal effect.

[0049] Another aspect of the present invention is a method for preventing or treating one or more diseases, symptoms, or disorders involving senescent cells, comprising administering an effective amount of a processed product of blueberries to a subject having or possibly having one or more diseases, symptoms, or disorders involving senescent cells, such as a healthy human or human patient, to exert a senescent cell removal effect. The processed product of blueberries administered in the method of this aspect has the same characteristics as the active ingredients of the senolytic agent and medicament of this aspect described above. The method of this aspect can be carried out in the same manner and dosage as the senolytic agent and medicament of this aspect described above. In the method of this aspect, the one or more diseases, symptoms, or disorders involving senescent cells are preferably the same as the one or more diseases, symptoms, or disorders involving senescent cells exemplified above for the senolytic agent of one aspect of the present invention. By administering an effective amount of a processed product of blueberries to a subject having or possibly having one or more diseases, symptoms, or disorders involving senescent cells, such as a healthy human or human patient, the diseases, symptoms, or disorders exemplified above can be prevented or treated through the senescent cell removal effect.

[0050] Another aspect of the present invention is a processed product of blueberry for use in preventing or treating one or more diseases, symptoms or disorders involving senescent cells in a subject having or likely to have one or more diseases, symptoms or disorders involving senescent cells, such as a healthy human or a human patient, through the senescent cell-removing effect. Still another aspect of the present invention is the use of a processed product of blueberry in the manufacture of a medicament for preventing or treating one or more diseases, symptoms or disorders involving senescent cells in a subject having or likely to have one or more diseases, symptoms or disorders involving senescent cells, such as a healthy human or a human patient, through the senescent cell-removing effect. Still another aspect of the present invention is the use of a processed product of blueberry for preventing or treating one or more diseases, symptoms or disorders involving senescent cells in a subject having or likely to have one or more diseases, symptoms or disorders involving senescent cells, such as a healthy human or a human patient, through the senescent cell-removing effect. Compounds and the like of this aspect have the same characteristics as the active ingredients of the senescent cell-removing agent and the medicament of this aspect described above. Also, compounds and the like of this aspect can be used in the same usage and dosage as the senescent cell-removing agent and the medicament of this aspect described above. In compounds and the like of this aspect, it is preferable that one or more diseases, symptoms or disorders involving senescent cells are the same as one or more diseases, symptoms or disorders involving senescent cells exemplified above with respect to the senescent cell-removing agent of one aspect of the present invention. By administering an effective amount of the processed product of blueberry to a subject having or likely to have the diseases, symptoms or disorders exemplified above, such as a healthy human or a human patient, the diseases, symptoms or disorders exemplified above can be prevented or treated through the senescent cell-removing effect.

Example

[0051] Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[0052] <I: Materials and methods> [I-1: Preparation of processed product of blueberry] According to the procedures described in JP 2022-92576 A and JP 2022-168636 A, a hot water extract of the stems and leaves of freeze-dried blueberries (rabbit eye blueberry species), and each hot water extract was separated by Sephadex LH-20 column chromatography to obtain a separated fraction. The hot water extract of the blueberry stem was passed through a Sephadex LH-20 biocolumn, and the obtained fraction was designated as Fr. 0 (pass-through). Next, the eluate was passed through the biocolumn in the order of distilled water, 20% aqueous methanol solution, 40% aqueous methanol solution, 60% aqueous methanol solution, 80% aqueous methanol solution, 100% methanol, 40% aqueous acetone solution, 60% aqueous acetone solution, and 100% acetone to obtain fractions (Fr.) 1 to 9. In addition, the hot water extract of blueberry leaves was passed through a Sephadex LH-20 biocolumn, and the obtained fraction was designated as Fr. 0 (pass-through). Next, the eluates were passed through the biocolumn in the order of distilled water, 20% aqueous methanol solution, 40% aqueous methanol solution, 60% aqueous methanol solution, 80% aqueous methanol solution, 100% methanol, and 60% aqueous acetone solution, to obtain fractions (Fr.) 1 to 7. Each of the obtained fractions was freeze-dried when the eluate was only water, or after the organic solvent was removed by distillation using an evaporator when the eluate contained an organic solvent.

[0053] [I-2: Structural analysis of active ingredients contained in processed blueberries] It is known that blueberry stem extracts and leaf extracts contain proanthocyanidins (PACs) (Patent Publication No. 4892690 and JP 2022-168636 A). The content, constitutional units, bond type and average degree of polymerization of PACs contained in Fr. 3, 4, 5, 6, 7 and 8 of the blueberry stem extract prepared in I-1 and Fr. 4, 5, 6 and 7 of the leaf extract were determined according to the procedures described in Patent Publication No. 4892690 and JP 2022-168636 A. The PACs contained in each fraction of the blueberry stem extract and leaf extract are mainly composed of two types of polymers (A type and B type) with different bond types represented by the above chemical formula, in which the flavan-3-ol skeleton is polymerized.

[0054] [I-3: Cell culture] Human fetal lung fibroblasts, WI-38, were obtained from Riken (Lot 6, Tsukuba, Japan). WI-38 were maintained by culturing in Dulbecco's Modified Eagle's Medium (DMEM) low glucose medium containing 10% fetal bovine serum (FBS) under conditions of 5% CO2 and 37°C.

[0055] [I-4: Induction of senescent cells] In the case of WI-38, the cells were exposed to 80 mM H2O2 for 6 days, and then cultured for about 7 to 10 days to induce them to become senescent cells.

[0056] [I-5: Evaluation of cytotoxic activity] To evaluate the cytotoxic activity, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. 50 μL of 2x concentration of sample and DMEM low glucose medium containing 20% ​​FBS were added to the designated wells of a 96-well plate. Then, 1.0 × 10 550 μL of the cell suspension at 1000 cells / mL was added to each of the designated wells and pipetted. 50 μL of the above medium was added to the blank wells instead of the cell suspension. Culture was carried out for 72 hours. According to the protocol of Dojindo Laboratories, after 72 hours of culture, the medium in each well of the 96-well plate was replaced with DMEM low glucose medium containing 10% FBS. 10 μL of MTT solution was added to each well, and the color reaction was carried out in an incubator for 4 hours. After that, the supernatant was carefully collected from each well and 200 μL of PBS was carefully added. After washing, PBS was carefully aspirated from each well and dried. After sufficient drying, 200 μL of 0.04 mol / L HCl / IPA was added and the plate was shaken at 700 rpm for 10 minutes on a plate shaker. After that, after confirming that the mixture was sufficiently stirred, the absorbance of each well was measured at a wavelength of 570 nm. The survival rate (%) was calculated as a percentage of the absorbance of the wells to which each sample was added relative to the absorbance of the control wells (to which DMSO was added) based on the average absorbance measured in each assay.

[0057] [I-6: Evaluation of apoptosis inhibitory activity by pan-caspase inhibitors] The pan-caspase inhibitor 5-(2,6-difluorophenoxy)-3-[[3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid (QVD-O-OPh) acts on caspases 1, 3, 8 and 9 and inhibits their activity. Expression and activation of caspases lead to the induction of apoptosis. Therefore, apoptosis can be inhibited by adding the pan-caspase inhibitor QVD-O-OPh. Before the addition of the sample, QVD-O-Ph was added to the cells in advance and cultured for 4 hours. Then, the sample was additionally added (Liu et al. 2018, Wang et al. 2016, He et al. 2020a, Li et al. 2019, Xu et al. 2021, He et al. 2020b). After incubation for a given period of time, the cytotoxic activity was evaluated according to the procedure in I-5.

[0058] [I-7: Evaluation of apoptosis-inducing activity by JC-1 assay] The fluorescent dye JC-1 forms aggregates in normal cells with a high membrane potential (Δψm), but exists as monomers in apoptotic cells with a low membrane potential (Δψm). Also, because it has the property of changing fluorescence depending on the aggregability of the probe, it exhibits red fluorescence (excitation wavelength: 560 nm / emission wavelength: 590 nm) in aggregates and green fluorescence (excitation wavelength: 485 nm / emission wavelength: 535 nm) in monomers, respectively. Therefore, apoptosis-inducing activity can be evaluated based on the JC-1 fluorescence intensity ratio (red fluorescence / green fluorescence) in the JC-1 assay. The overall procedure was carried out according to the protocol prepared by Cayman Chemical Company, the vendor of the JC-1 assay kit.

[0059] [I-8: Statistical analysis] The results of each test were shown as mean ± standard deviation (SD). Student's t-test was used for comparison between two groups. Also, for multiple comparisons, one-way ANOVA with Dunnett's test or two-way ANOVA with Dunnett's test using Dunnett's test, or one-way ANOVA with Tukey's test using Tukey's test was used. All tests were statistically analyzed using Graphpad Prism Version 9.5.0.

[0060] <II: Results> [II-1: Cytotoxic activity of blueberry processed products] Blueberry leaf extract was prepared according to the procedure of I-1. WI-38 senescent cells and WI-38 non-senescent cells were prepared according to the procedures of I-3 and I-4. 15 μg / mL or 25 μg / mL of blueberry leaf extract was added to WI-38 senescent cells or WI-38 non-senescent cells. After 72 hours of culture, the viability of WI-38 senescent cells and WI-38 non-senescent cells was evaluated using the MTT assay according to the procedure of I-5. The results are shown in Figure 1. In the figure, the black bars indicate the viability of WI-38 non-senescent cells, and the shaded bars indicate the viability of WI-38 senescent cells (mean ± SD, n = 4 to 5). The horizontal axis indicates the concentration of blueberry leaf extract added (μg / mL), and the vertical axis indicates the cell viability (%). p values ​​were determined using two-way analysis of variance with Dunnett's test or t-test. ns: p>0.05, ***:p<0.001, ****:p<0.0001.

[0061] As shown in Figure 1, when blueberry leaf extract was added to non-senescent WI-38 cells, no significant change in viability was observed. In contrast, when blueberry leaf extract was added to senescent WI-38 cells, the viability decreased in a concentration-dependent manner. Furthermore, the viability of senescent WI-38 cells was significantly decreased compared to that of non-senescent WI-38 cells to which the same concentration of blueberry leaf extract had been added. These results demonstrated that blueberry leaf extract exhibits selective cytotoxic activity against senescent cells.

[0062] [II-2: Relationship between cytotoxicity and caspase activity of blueberry processed products] 5, 10, 15 or 20 μM of QVD-O-Ph was added to WI-38 senescent cells. After 72 h of culture, the viability of WI-38 senescent cells was evaluated using the MTT assay according to the procedure in I-5. The results are shown in Figure 2. In the figure, the bars indicate the viability of WI-38 senescent cells without addition or with the addition of a given concentration of QVD-O-Ph (mean ± SD, n = 3). The horizontal axis indicates the concentration of QVD-O-Ph added (μM), and the vertical axis indicates the cell viability (%). p values ​​were determined using two-way analysis of variance with Dunnett's test. ns: p>0.05.

[0063] As shown in FIG. 2, when QVD-O-Ph was added to WI-38 senescent cells, no significant change in viability was observed.

[0064] Next, we confirmed whether the cytotoxic activity of blueberry leaf extract against senescent cells was due to apoptosis. 20 μM QVD-O-Ph was added to WI-38 senescent cells. After 4 hours of incubation, 25 μg / mL of blueberry leaf extract was additionally added to the cells. After 24 hours of incubation, the viability of WI-38 senescent cells was evaluated using the MTT assay according to the procedure in I-5. The results are shown in Figure 3. In the figure, the black bars indicate the viability of WI-38 senescent cells without QVD-O-Ph when blueberry leaf extract was added, and the gray bars indicate the viability of WI-38 senescent cells with QVD-O-Ph when blueberry leaf extract was added (mean ± SD, n = 3-4). The vertical axis indicates the cell viability (%). p values ​​were determined using Student's t-test. ns: p>0.05, **: p<0.01.

[0065] As shown in Figure 3, when blueberry leaf extract was added alone to WI-38 senescent cells without QVD-O-Ph, the viability decreased (black bars). In contrast, when blueberry leaf extract was added to WI-38 senescent cells with QVD-O-Ph, no decrease in viability was observed. This result demonstrated that the cytotoxic activity of blueberry leaf extract against senescent cells is caspase-dependent.

[0066] [II-3: Relationship between cytotoxic activity of blueberry processed products and mitochondrial membrane potential] 25 μg / mL of blueberry leaf extract was added to WI-38 senescent cells. After 24 hours of culture, mitochondrial membrane potential was evaluated using the JC-1 assay according to the procedure in I-7. The results are shown in Figure 4. In the figure, the vertical axis indicates the JC-1 fluorescence intensity ratio (red fluorescence / green fluorescence) (mean ± SD, n = 3). p values ​​were determined using Student's t-test. *: p<0.05.

[0067] As shown in Figure 4, when blueberry leaf extract was added to WI-38 senescent cells, the JC-1 fluorescence intensity ratio decreased. This result indicates that the addition of blueberry leaf extract reduced normal cells with high membrane potential in WI-38 senescent cells and increased apoptotic cells with low membrane potential. The results of II-2 and II-3 strongly suggest that the cytotoxic activity of blueberry leaf extract against senescent cells is due to apoptosis.

[0068] [II-4: Comparison of the cytotoxic activity of procyanidins from the prior art with that of processed blueberries] Xu et al. (Non-Patent Document 4) describes that procyanidin C1 (PCC1) contained in grape seeds has a removal effect on WI-38 senescent cells. In contrast, Japanese Patent Publication No. 4892690 (Patent Document 1) and Japanese Patent Publication No. 2022-168636 (Patent Document 2) describe that blueberry leaf extract contains proanthocyanidin (PAC). Therefore, the cytotoxic activity of PCC1 described in the Xu et al. document was compared with the cytotoxic activity of PAC contained in blueberry leaf extract. The results are shown in FIG. 5. In the figure, A shows the relationship between the concentration of PCC1 added to WI-38 senescent cells and the viability of WI-38 senescent cells. In the figure, the horizontal axis is the PCC1 concentration (μg / mL), and the vertical axis shows the viability of WI-38 senescent cells without addition or with addition of PCC1 (mean ± SD, n = 3). The p value was determined using Student's t-test. *: p<0.05, ***: p<0.001, ****: p<0.0001. B shows the relationship between the concentration of PAC added to WI-38 senescent cells and the viability of WI-38 senescent cells. In the figure, the horizontal axis is the PAC concentration (ng / mL), and the vertical axis shows the viability of WI-38 senescent cells without or with PAC added (mean ± SD, n = 5). p values ​​were determined using Student's t-test. **: p<0.01, ****: p<0.0001. The graph shown in A was created by converting the concentration of PCC1 added, expressed as a molar concentration, to a μg / mL concentration using the molecular weight of PCC1, based on the results showing the relationship between the concentration of PCC1 added to WI-38 senescent cells and the viability of WI-38 senescent cells (culture time: 72 hours) described in the literature by Xu et al. The graph shown in B was prepared by converting the added concentrations of blueberry leaf extract shown in FIG. 1 into ng / mL concentrations of PAC contained in blueberry leaf extract based on the content of PAC contained in the blueberry leaf extract determined according to the procedure described in I-2.

[0069] As shown in Figure 5A, the IC of PCC1 on the cytotoxic activity of WI-38 senescent cells 50In contrast, as shown in Figure 5B, the IC value of PAC on the cytotoxic activity of WI-38 senescent cells was 65–87 μg / mL. 50 The IC value of PCC1 on the cytotoxic activity of WI-38 senescent cells was 675 ng / mL. Figure 5A shows the test results for the isolated compound PCC1, whereas Figure 5B shows the test results for the crude extract, blueberry leaf extract, converted into PAC concentration. Although a simple comparison is difficult, the IC value of PCC1 on the cytotoxic activity of WI-38 senescent cells is 50 Value and IC of PAC 50 The difference between the values ​​is about 100-fold or more. Therefore, this result demonstrates that the PAC contained in the blueberry leaf extract has a significantly higher senescent cell removal effect than the PCC1 of the prior art.

[0070] [II-5: Comparison of cytotoxic activity of processed blueberries] The content and average degree of polymerization of PAC in Fr. 4, 5, 6, and 7 of blueberry leaf extract and Fr. 3, 4, 5, 6, 7, and 8 of blueberry stem extract were determined according to the procedure described in I-2. Based on the determined PAC content, Fr. 4, 5, 6, and 7 of blueberry leaf extract and Fr. 3, 4, 5, 6, 7, and 8 of blueberry stem extract containing 1000 ng of PAC were prepared. Each fraction of blueberry leaf extract and each fraction of blueberry stem extract prepared were added to WI-38 senescent cells or WI-38 non-senescent cells. After 72 hours of culture, the viability of WI-38 senescent cells and WI-38 non-senescent cells was evaluated using the MTT assay according to the procedure described in I-5. The results are shown in FIG. 6. In the figure, the black bars indicate the viability of WI-38 non-senescent cells, and the gray bars indicate the viability of WI-38 senescent cells (mean ± SD, n = 2-3). The vertical axis indicates cell viability (%). p values ​​were determined using Student's t-test. ns: p>0.05, *: p<0.05, **: p<0.01. Table 1 also shows the average degree of polymerization of PAC contained in Fr. 4, 5, 6, and 7 of blueberry leaf extract and Fr. 3, 4, 5, 6, 7, and 8 of blueberry stem extract.

[0071] [Table 1]

[0072] As shown in Figure 6 and Table 1, when blueberry leaf extract Fr. 7 and stem extract Fr. 7 and 8, which contain PACs with a high average degree of polymerization, were added to WI-38 senescent cells, a significant decrease in viability was confirmed. These results suggest that the higher the average degree of polymerization of PACs, the greater the effect of removing senescent cells.

[0073] The present invention is not limited to the above-described embodiments, but includes various modified examples. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the configurations described. In addition, it is possible to add, delete, and / or replace part of the configuration of each embodiment with other configurations.

Claims

1. An aging cell remover containing processed blueberries as an active ingredient.

2. The senolytic agent according to claim 1, for preventing, treating or ameliorating one or more diseases, symptoms or disorders in which senescent cells are involved.

3. The senescent cell removing agent according to claim 2, wherein the one or more diseases, symptoms, or disorders involving senescent cells are selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatic cholangitis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, non-alcoholic fatty liver disease, impaired immune function, cachexia, and frailty.

4. The senescent cell removing agent according to claim 1, wherein the processed blueberry product is a processed blueberry stem or leaf product.

5. 2. The senescent cell removing agent according to claim 1, wherein the processed blueberry product is one or more products or fractions selected from the group consisting of blueberry crushed material, squeezed juice, solvent extract, and isolated fractions thereof.

6. A food product containing processed blueberries as an active ingredient, for preventing or ameliorating one or more diseases, symptoms or disorders involving senescent cells in a subject having or likely to have the disease, symptom or disorder selected from the group consisting of osteoarthritis, osteoporosis, sarcopenia, hepatic cholangitis, pulmonary fibrosis, Alzheimer's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, atherosclerosis, heart failure, cancer, renal dysfunction, non-alcoholic fatty liver disease, decreased immune function, cachexia and frailty.

7. 7. The food according to claim 6, wherein the processed blueberry product is a processed blueberry stem or leaf.

8. 7. The food according to claim 6, wherein the processed blueberry product is one or more products or fractions selected from the group consisting of blueberry crush, juice, and solvent extract, and isolated fractions thereof.

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