Senolytic agent

Abstract

The object of the present invention is to provide a preparation and composition that is effective for removing senescent cells and can be ingested in the food and beverage sector. [Solution] Provided is a senescent cell removing agent containing an effective amount of at least one selected from the group consisting of zygosperm flower, zygosperm bark, and extracts thereof.

Description

[Technical field]

[0001] The present invention relates to an agent for eliminating senescent cells. More specifically, the present invention relates to an agent for eliminating senescent cells, which is characterized by selectively eliminating senescent cells relative to normal cells. [Background technology]

[0002] Currently, developed countries are becoming increasingly aging societies and average life expectancy is increasing, but the gap between this and healthy life expectancy is becoming an issue. Healthy life expectancy is a health indicator proposed by the World Health Organization (WHO) and refers to the period during which one can live an independent and healthy life, both physically and mentally. The difference between healthy life expectancy and average life expectancy, which is the "unhealthy period" during which daily life is restricted, is said to be about 9 years for Japanese men and about 12 years for Japanese women.

[0003] From the perspective of measures to deal with the super-aging society, research and development of technologies to extend healthy lifespan is being conducted, and attention is being paid to cellular senescence and the removal of senescent cells. Senescent cells are cells in which cell division has irreversibly stopped, and they exist not only in elderly people but also in young people and newborns.

[0004] It has become clear that cellular senescence is induced by DNA damage caused by stress such as ultraviolet rays and active oxygen, shortening of telomeres, etc. It has also been suggested that various diseases may be caused by inflammatory proteins secreted by senescent cells, so there is a strong demand for technology to control cellular senescence in order to suppress the induction of senescent cells and to remove the senescent cells that have been created.

[0005] For example, Patent Document 1 reports that sodium-glucose cotransporter 2 (SGLT2) inhibitors have an effect of removing senescent cells. Specifically, it has been confirmed that the SGLT2 inhibitor canagliflozin has an effect of removing senescent cells in obese model mice, arteriosclerosis model mice, progeria model mice, and the like. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2022-101541 A Summary of the Invention [Problem to be solved by the invention]

[0007] However, the removal of senescent cells is itself difficult, and there are few reported techniques for selectively removing senescent cells. In addition, there are no reports of ingredients that are effective in removing senescent cells and can be ingested from plant resources in the fields of food and beverages.

[0008] Therefore, an object of the present invention is to provide a preparation and composition that is effective for removing senescent cells and can be ingested in the food and beverage sector. [Means for solving the problem]

[0009] In order to achieve the above object, the present inventors conducted extensive research and discovered that Albizia julibrissin, which is commonly consumed in the food industry, has the effect of removing senescent cells, thereby completing the present invention.

[0010] That is, the present invention provides the formulations and compositions described below.

[0011] Section 1. An agent for removing senescent cells comprising an effective amount of at least one member selected from the group consisting of uniflora flowers, uniflora bark, and extracts thereof.

[0012] Section 2. Item 1. The senescent cell removing agent according to Item 1, which is labeled with at least one or more indications selected from the group consisting of "reducing senescent cells," "removing senescent cells," "reducing the proportion of senescent cells," and "promoting the reduction of senescent cells."

[0013] Section 3. Item 3. The agent for removing senescent cells according to Item 1 or 2, wherein the removal of senescent cells is characterized by selectively removing senescent cells relative to normal cells.

[0014] Section 4. Item 4. The agent for removing senescent cells according to any one of Items 1 to 3, characterized in that the ratio (A / B) of (A) the 50% lethal concentration (LC50) in normal cells to (B) the 50% lethal concentration in senescent cells is greater than 1.1.

[0015] Section 5. Item 5. The senescent cell removing agent according to any one of items 1 to 4, which is a food product, a cosmetic product, or a pharmaceutical product.

[0016] Section 6. A food composition for reducing senescent cells, removing senescent cells, decreasing the proportion of senescent cells, or promoting the reduction of senescent cells, comprising an effective amount of at least one member selected from the group consisting of uniflora flowers, uniflora bark, and extracts thereof.

[0017] Section 7. Use of at least one member selected from the group consisting of hydrangea flower, hydrangea bark, and extracts thereof to manufacture a composition for reducing senescent cells, removing senescent cells, decreasing the proportion of senescent cells, or promoting the reduction of senescent cells.

[0018] Section 8. Item 8. The use of at least one selected from the group consisting of uniflora flowers, uniflora bark, and extracts thereof according to Item 7, wherein the composition is a food, cosmetic, or pharmaceutical composition. Effect of the Invention

[0019] According to the present invention, it is possible to effectively remove senescent cells by using Albizia julibrissin, which can also be ingested in the food and beverage sector. [Brief description of the drawings]

[0020] [Figure 1]FIG. 1 is a graph showing the results of senescent cell removing ability in Test Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] [Senocyte removal agent] In one embodiment, the senolytic agent of the present invention contains Albizia julibrissin.

[0022] (Albizia julibrissin) The Japanese bush clover is a deciduous tree of the Fabaceae subfamily. The Japanese bush clover is also called the Silk Tree, and its name comes from the fact that it closes its leaflets and hangs down during the night.

[0023] The Japanese dogwood is distributed in Honshu, Shikoku, Kyushu, and other parts of Japan, and also grows wild in Iran, Afghanistan, China, the Korean Peninsula, and other areas, so it can be easily obtained from these areas.

[0024] In addition, the flowers and skin of the jasmine are dried in the sun and used as a herbal medicine, and they are also brewed and drunk to relieve stress.

[0025] The part of the Synthetic Musk to be used is not particularly limited, and may be the whole plant, or may be leaves, fruits, flowers, branches, trunks, roots, seeds, or a mixture of at least two or more of them. In one embodiment, the part of the Synthetic Musk to be used preferably includes flowers and / or bark, and more preferably includes flowers and bark.

[0026] The hyacinth may be used fresh or dried (e.g., freeze-dried). When extracting from the hyacinth, it may be processed, for example, into a crushed or powdered state, from the viewpoint of improving the extraction efficiency.

[0027] The compound may be, for example, crushed or pulverized in the form of chips, powder, granules, or paste, and among these, it is preferable to use the powder.

[0028] The method for processing the plant body into a crushed or pulverized product can be a known method and is not particularly limited. For example, the plant body can be crushed or pulverized by any method commonly used by those skilled in the art using equipment or tools such as a crusher, mill, blender, mixer, and stone mill.

[0029] Before being crushed or pulverized, the plant body may be dried. The drying method may be any known method, and is not particularly limited, but examples thereof include freeze-drying, drying under reduced pressure, air drying, and heat drying.

[0030] When using an extract of the rhododendron, the method is not particularly limited, and it can be extracted by a normal extraction method used for extracting plant materials. The extraction method is not limited, but examples thereof include solvent extraction, steam distillation, squeezing (direct, high temperature, or low temperature), and supercritical extraction. In addition, a combination of these extraction methods may be used, such as a method of crushing and then solvent extraction.

[0031] The extraction solvent is not particularly limited as long as it is a solvent that is commonly used for extracting plant materials. The extraction solvent may be, for example, an organic solvent, an aqueous solvent, or a mixture of an organic solvent and an aqueous solvent, but is preferably an aqueous solvent or a mixture of an organic solvent and an aqueous solvent, and more preferably an aqueous solvent.

[0032] Examples of the organic solvent include alcohol, acetone, benzene, ester, ethyl acetate, hexane, chloroform, and diethyl ether, with alcohol being preferred. The alcohol is not particularly limited as long as it is a common solvent used in the extraction of plant raw materials, and examples of the alcohol include monohydric alcohols having 1 to 5 carbon atoms, such as ethanol, methanol, propyl alcohol, isopropyl alcohol, and butyl alcohol, and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, dipropylene glycol, and glycerin.

[0033] The aqueous solvent is not particularly limited as long as it contains water, and examples thereof include water, physiological saline, and buffer solutions. Examples of the buffer solution include phosphate buffer, sodium phosphate buffer, sodium carbonate buffer, citrate buffer, citrate phosphate buffer, acetate buffer, and Tris buffer. Preferred aqueous solvents are water and / or physiological saline. The pH of the aqueous solvent is not particularly limited.

[0034] Of these extraction solvents, from the viewpoint of significantly achieving the effects of the present invention, at least one selected from the group consisting of water, ethanol, 1,3-butylene glycol, and propylene glycol is preferred, water and / or ethanol is more preferred, and water or aqueous ethanol is even more preferred.

[0035] When a mixture of an organic solvent and an aqueous solvent is used as the extraction solvent, the content of the aqueous solvent in the total amount of the extraction solvent is not particularly limited, but may be, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more.

[0036] The extraction temperature is not particularly limited, but examples include -50°C to 150°C, 0°C to 100°C, 5°C to 95°C, 10°C to 95°C, 15°C to 95°C, 30°C to 90°C, 50°C to 90°C, 70°C to 90°C, and 80°C to 90°C.

[0037] From the viewpoint of improving the extraction efficiency, it is preferable to carry out the extraction while stirring or shaking. The extraction time can be appropriately determined depending on the part to be used, such as leaves, fruits, flowers, branches, trunks, roots, seeds, etc. The extraction time can also be appropriately determined depending on the state of the hyacinth, that is, whether it is raw or dried, or whether it is crushed or pulverized, or the processed state thereof. The extraction time is usually 1 minute to 72 hours, preferably 10 minutes to 48 hours, and most preferably 30 minutes to 2 hours.

[0038] As an example, the extract of the sycamore tree refers to an extract obtained by immersing and extracting the flowers and / or bark of the sycamore tree, or a crushed product thereof, in water and / or an organic solvent and filtering the residue, or the extract from which the solvent has been removed, or a fine powder of these, or the extract or the product from which the solvent has been removed, dissolved, dispersed, or diluted using an appropriate solvent, and it is also possible to use a commercially available product. As an example of another extraction method, the extract of the sycamore tree can be extracted after processing the flowers and / or bark of the sycamore tree, such as steaming. In addition, the leaves and branches associated with the flowers and / or bark of the sycamore tree may be removed, or they may be used as they are without being removed.

[0039] The content of the plant body of the Sympetrum japonica or an extract thereof is appropriately adjusted depending on the type and amount of other ingredients, the dosage form, etc., and is not limited, and may be, for example, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, etc. In addition, the content of the plant body of the Sympetrum japonica or an extract thereof may be, for example, 100% by mass or less, and may be 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, etc., based on the total amount of the composition. The content of the Hemerocallis arbutus plant or its extract relative to the total amount of the composition may be, for example, 0.01 to 100 mass%, 0.1 to 100 mass%, 1 to 100 mass%, 5 to 100 mass%, 10 to 100 mass%, 20 to 100 mass%, 30 to 100 mass%, 40 to 100 mass%, 50 to 100 mass%, 0.01 to 90 mass%, 0.1 to 90 mass%, 1 to 90 mass%, 5 to 90 mass%, %, 10-90% by mass, 20-90% by mass, 30-90% by mass, 40-90% by mass, 50-90% by mass, 0.01-80% by mass, 0.1-80% by mass, 1-80% by mass, 5-80% by mass %, 10-80% by mass, 20-80% by mass, 30-80% by mass, 40-80% by mass, 50-80% by mass, 0.01-70% by mass, 0.1-70% by mass, 1-70% by mass, 5-70% by mass %, 10-70% by mass, 20-70% by mass, 30-70% by mass, 40-70% by mass, 50-70% by mass, 0.01-60% by mass, 0.1-60% by mass, 1-60% by mass, 5-60% by mass %, 10-60% by mass, 20-60% by mass, 30-60% by mass, 40-60% by mass, 0.01-50% by mass, 0.1-50% by mass, 1-50% by mass, 5-50% by mass, 10-50% by mass %, 20-50 mass%, 30-50 mass%, 0.01-40 mass%, 0.1-40 mass%, 1-40 mass%, 5-40 mass%, 10-40 mass%, 20-40 mass%, 0.01-30 Examples include mass%, 0.1 to 30 mass%, 1 to 30 mass%, 5 to 30 mass%, 10 to 30 mass%, 0.01 to 20 mass%, 0.1 to 20 mass%, 1 to 20 mass%, 5 to 20 mass%, and the like.

[0040] [Application] The senescent cell removing agent of the present invention is preferably used to effectively remove senescent cells, and preferably to selectively remove senescent cells from normal cells. In this specification, senescent cells refer to cells that have lost the ability to proliferate through cell division. When DNA is irreparably damaged by stress such as ultraviolet rays, becoming a senescent cell may be said to be a defense mechanism that stops cell division to prevent cancer. However, it is known that if senescent cells remain in the body without being eliminated, they secrete inflammatory proteins and the like, causing chronic inflammation. In addition, in this specification, normal cells that can be contrasted with senescent cells refer to cells that have not lost the ability to proliferate through cell division.

[0041] Senescent cells have been identified as markers of increased p16 gene and β-galactosidase activity, and it is becoming clear that age-related changes can be suppressed by removing senescent cells from aging individuals. For example, in nonalcoholic steatohepatitis (NASH), senescent cells accumulate and inflammation can occur, but it has been reported that removing senescent cells leads to improvement of inflammation.

[0042] Even if there are anti-inflammatory ingredients for treating inflammation caused by the accumulation of senescent cells as symptomatic treatment, simply suppressing inflammation cannot be a fundamental solution as long as senescent cells accumulate. In addition, various ingredients have been screened for anti-aging, but there are not enough reports of ingredients that can suppress the generation of senescent cells. Furthermore, there are almost no reports of technologies that can distinguish senescent cells that have been generated and accumulated from normal cells and selectively remove only senescent cells.

[0043] The accumulation of senescent cells does not necessarily occur only in the elderly or sick; it can also occur in young people and newborns. Therefore, rather than relying on pharmaceuticals to solve the problem, there is a need for technology that can selectively remove senescent cells using ingredients that can be ingested on a daily basis. However, there have been few research reports from this perspective.

[0044] In the examples (test examples) described later, etoposide, an inducer of cellular senescence, was administered to human liver cancer-derived HepG2 cells to obtain senescent cells, and the effects of at least one selected from the group consisting of euonymus flower, euonymus bark, and extracts thereof, together with quercetin, which is known to have the ability to remove senescent cells, were then confirmed.

[0045] As a result, it was confirmed that, compared to quercetin, at least one member selected from the group consisting of zygosperm flower, zygosperm bark, and extracts thereof, has a significantly greater ability to selectively remove senescent cells.

[0046] Therefore, at least one selected from the group consisting of uniflora flower, uniflora bark, and extracts thereof can be suitably used as an agent for selectively removing senescent cells, which can effectively remove senescent cells, and preferably selectively remove senescent cells relative to normal cells.

[0047] The ability to selectively remove senescent cells relative to normal cells (senescent cell removal ability) can be calculated by determining the ratio (A / B) of (A) the 50% lethal concentration in normal cells to (B) the 50% lethal concentration in senescent cells, as in the Examples described below. The ability to remove senescent cells is not limited as long as it achieves the effects of the present invention, but for example, the ratio (A / B) is preferably greater than 1.1, and may be 1.2 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, 6 or more, 7 or more, 8 or more, 9 or more, etc.

[0048] The formulation and composition of the present invention can be added to or mixed with food and beverages, cosmetics, medicines, quasi-drugs, pet food, and feed. Alternatively, they can be used as they are as medicines, quasi-drugs, food and beverages, pet food, and feed. The formulation and composition of the present invention can be used as food and beverages that explicitly or implicitly state or imply that their functional function is the removal of senescent cells, i.e., health foods, functional foods, foods for sick people, and foods for specified health uses. Even if the functional function is not explicitly or implicitly stated, they can be used as so-called doctor's supplements recommended or presented by doctors in internal medicine, orthopedics, veterinary hospitals, and the like in hospitals and / or clinics. When the functional function is explicitly stated, at least one or more types of indications selected from the group consisting of "reducing senescent cells," "removing senescent cells," "reducing the proportion of senescent cells," and "promoting the reduction of senescent cells" may be used, but are not limited thereto. In addition, the effects attributable to the functionality may be displayed as at least one or more of the following selected from the group consisting of "improvement of blood liver function enzyme levels", "maintenance of cognitive function", "maintenance of muscle strength", "maintenance of walking ability", "maintenance of muscle mass, muscle strength and muscle endurance required for independent daily living", "maintenance of agility", "reduction of fasting blood glucose level", "moderating rise in blood glucose level after meals", "reduction of blood triglycerides", "maintenance of vascular flexibility that decreases with age", "maintenance of kidney filtration function", "reduction of temporary facial and leg swelling", "reducing urination frequency", "suppression of dizziness", "maintenance of bone health", "maintenance of bone density", "reduction of knee discomfort", "support of flexibility and mobility of knee joints", "improvement of skin moisturizing ability", "maintenance of skin elasticity", "regeneration and hair growth", "regulation of stomach condition", "improvement of visual function", "reduction of temporary physical and mental fatigue" and "reduction of biological age".

[0049] When the above functionality is not clearly stated, it is also possible to use the formulation and composition of the present invention by displaying, for example, "for those who are concerned about senescent cells, those who wish to remove senescent cells, those who wish to inhibit or reduce senescent cells, those who wish to reduce the proportion of senescent cells, those who are concerned about their liver function or kidney function values, those who feel a decline in muscle strength, those who are concerned about blood sugar levels, those who are concerned about bone density, those who feel discomfort in their knee joints, those who are concerned about their skin condition, those who are concerned about their stomach condition, those who are concerned about their vision, those who feel fatigue, etc., or a similar display.

[0050] The dosage form of the preparation and composition of the present invention is not particularly limited, and includes oral preparations and parenteral preparations, etc., with oral preparations being preferred. The oral preparations include, for example, solid or powder preparations such as fine granules, granules, tablets, capsules, and pills, and liquid preparations such as suspensions, emulsions, syrups, and extracts. Parenteral preparations include, for example, injections, external preparations, and poultices.

[0051] The formulations and compositions of the present invention may consist of at least one selected from the group consisting of unicorn flower, unicorn bark, and extracts thereof, or may contain at least one selected from the group consisting of unicorn flower, unicorn bark, and extracts thereof.

[0052] When the formulation and composition of the present invention are oral preparations, they may contain other additives, such as excipients, binders, disintegrants, emulsifiers, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, flavorings, stabilizers, moisturizers, preservatives, antioxidants, or suspending agents. Examples of additives include, but are not limited to, gelatin, sodium alginate, starch, corn starch, white sugar, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate.

[0053] The formulations and compositions of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, horses, ponies, donkeys, cows, pigs, sheep, goats, rabbits, monkeys, hamsters, guinea pigs, mice, rats, ferrets, squirrels, and birds; livestock such as cows, chickens, and pigs; and animals kept on ranches, zoos, etc. (horses, sheep, goats, monkeys, elephants, lions, cheetahs, etc.).

[0054] The composition of the present invention may be a food or drink, a cosmetic, a medicine, a quasi-drug, a pet food, a feed, or the like.

[0055] The food and drink includes food and beverages. Examples of the food and beverage include beverages (soft drinks (coffee, cocoa, juice, mineral drinks, tea drinks (green tea, black tea, oolong tea), almond drinks, sports drinks, energy drinks, nutritional drinks, etc.), milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, alcoholic beverages, spreads (custard cream, butter cream, peanut cream, chocolate cream, cheese cream, etc.), pastes (fruit paste, vegetable paste, sesame paste, seaweed paste, etc.), Western confectionery (chocolate, donuts, pies, muffins, waffles, gum, Gummy candies, jellies, candies, cookies, crackers, biscuits, snacks, cakes, puddings, etc.), Japanese sweets (candy, rice crackers, karinto, arare, dumplings, ohagi, daifuku, bean mochi, mochi, bean paste, manju, castella, anmitsu, yokan, etc.), frozen desserts (ice cream, popsicles, sorbet, etc.), retort foods (curry, stew, beef bowl, Chinese rice bowl, porridge, porridge, miso soup, soup, meat sauce, demi-glace sauce, meatballs, hamburger steak, oden, red rice, yakitori, chawanmushi, etc.), instant foods (instant ramen, instant vermicelli soup, instant soup broth) Leaf noodles, instant udon noodles, instant soba noodles, instant yakisoba noodles, instant spaghetti, instant wonton noodles, instant shiruko, miso soup base, powdered soup base, powdered juice base, pancake mix, etc.), bottled and canned foods, jelly foods (jelly, agar, jelly drinks, etc.), seasonings (soy sauce, mirin, vinegar, miso, dressings, umami seasonings, compound seasonings, sauces, mayonnaise, ketchup, furikake, tempura sauce, noodle sauce, dashi stock base, Chinese soup base, bouillon, yakiniku sauce, hiyashabu sauce, curry roux, stew roux, etc.), dairy products (milk, cheese, yogurt, butter, fresh cream) etc.), processed fruits (jams, marmalades, fruits preserved in syrup, dried fruits, etc.), processed grain foods (noodles, pasta, bread, rice vermicelli, etc.), pickles (takuan, narazuke, kimchi pickles, fukujinzuke, scallion pickles, Chinese cabbage pickles, mustard pickles, eggplant pickles, light pickles, pickles, etc.), pickle bases (instant pickle bases, kimchi pickle bases, etc.), fish products (kamaboko, chikuwa, hanpen, etc.), livestock products (ham, sausage, salami, bacon, etc.), delicacies (skived dried squid, skived cod, salted sea urchin, salted squid, salted octopus, mirimboshi filefish, mirimboshi pufferfish, smoked squid, salted konowata, etc.),Examples of such foods include dried foods (seasoned seaweed, etc.), side dishes (mixed dishes, fried foods, stir-fried foods, grilled foods, simmered foods, vinegared foods, etc.), frozen foods (fried shrimp, croquettes, spring rolls, pork cutlets, shumai, dumplings, takoyaki, meat buns, red bean buns, etc.), baby foods (e.g., rice and noodle dishes, fish dishes, meat dishes, egg dishes, dairy dishes, vegetable dishes, soups, fruits, juices, hydrating drinks, tea, desserts, sweets, sauces, sprinkles, etc.); powdered milk (e.g., general infant formula, special infant formula, powdered milk for pregnant and lactating women, etc.); enteral nutritional foods (e.g., enteral nutritional agents, thickened liquid foods, nutritional supplements, nutritional supplements, enteral nutritional agents, flavors for thickened liquid foods, desserts, etc.); and other foods (e.g., nutritional supplements, blender foods, thickened foods, foods for examinations, cereal foods, foods for sports, emergency foods, etc.).

[0056] These foods or beverages may contain food additives and food ingredients, such as, but not limited to, antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meat, seafood, etc., either alone or in combination of two or more.

[0057] Tablets can be prepared by mixing a powdered active ingredient with a pharma- ceutically acceptable carrier component (such as an excipient) and compressing the mixture, and confectionery tablets such as candy can be prepared by pouring the mixture into a mold. Tablets can be sugar-coated to form sugar-coated tablets. Furthermore, tablets can be single-layer tablets or laminated tablets such as double-layer tablets.

[0058] Powdered granules such as granules may be prepared by various granulation methods (such as extrusion granulation, grinding granulation, dry compaction granulation, fluidized bed granulation, rolling granulation, and high-speed stirring granulation), and tablets can be prepared by an appropriate combination of the above-mentioned granulation methods and tableting methods (wet tableting, direct tableting), etc.

[0059] Capsules can be prepared by filling a capsule (soft or hard capsule) with a powdered agent (powder, granules, etc.) by a conventional method.

[0060] The liquid preparation can be prepared by dissolving or dispersing each component in an aqueous medium (purified water, purified water containing ethanol, etc.) which is a carrier component, filtering or sterilizing the mixture as necessary, filling the mixture into a predetermined container, and sterilizing the mixture. The preferred dosage form of the solid preparation is a capsule or tablet, and soft capsules (soft capsules) are more preferred.

[0061] Soft capsules have a smooth surface and are easy to swallow, and are therefore preferred by users. Common methods for producing soft capsules include the plate method, the rotary method, and the seamless method.

[0062] In the rotary method (punching method), a sheet-like capsule shell sandwiches the flowing filling contents and forms a capsule shape along the holes of a rotating cylindrical die. On the other hand, in the seamless method (dropping method), the capsule shell composition and contents are simultaneously discharged from multiple concentric nozzles to form a seamless capsule shape.

[0063] The base material for the soft capsule shell is not particularly limited, but starch, pullulan, cellulose, polyvinyl alcohol, gelatin, succinated gelatin, etc. can be used, with starch, gelatin, and succinated gelatin being preferred, and gelatin and succinated gelatin being more preferred. These may be used alone or in combination of two or more kinds.

[0064] The oral intake or administration amount of the flower of the bud, the bark of the bud, or the extract thereof per day for an adult can be appropriately determined according to the individual's condition, body weight, sex, age, activity of the material, route of intake or administration, schedule of intake or administration, formulation form, or other factors. The oral intake or administration amount of the flower of the bud, the bark of the bud, or the extract thereof per day for an adult is, for example, preferably 0.1 mg or more, more preferably 1 mg or more, even more preferably 10 mg or more, particularly preferably 20 mg or more, and most preferably 30 mg or more. It may also be 100 mg or more, 200 mg or more. The oral intake or administration amount of the flower of the bud, the bark of the bud, or the extract thereof per day for an adult is, for example, preferably 2000 mg or less, more preferably 1500 mg or less, even more preferably 1000 mg or less, and particularly preferably 500 mg or less. The daily oral intake or administration amount of the zygosperm flower, zygosperm bark, or an extract thereof for an adult is, for example, preferably 0.1 to 2000 mg, more preferably 1 to 1500 mg, even more preferably 10 to 1000 mg, even more preferably 20 to 500 mg, and even more preferably 30 to 500 mg.

[0065] The daily oral intake or dosage for an adult may be divided into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules depending on the dosage form.

[0066] The composition of the present invention can be ingested or administered once or several times a day, typically 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferred. EXAMPLES

[0067] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0068] [Preparation of test formulations] (Example 1. Ethanol extract 1) 3.0 g of dried powder of the rhododendron was added to 30 mL of 99.5% ethanol and stirred at room temperature for 30 minutes to perform extraction. After that, the residue was removed by the usual method, and the mixture was dried to obtain 0.1326 g of dried material.

[0069] (Example 2. Ethanol extract 2) 3.0 g of dried powder of the peanut bark was added to 30 mL of 99.5% ethanol and stirred at room temperature for 30 minutes to carry out extraction. After that, the residue was removed by the usual method, and the mixture was dried to obtain 0.1551 g of dried material.

[0070] (Example 3. Hot water extract 1) 27 mL of water was added to 3.0 g of dried powder of the rhododendron japonica, and the mixture was stirred at 90°C for 1 hour to carry out extraction. After that, the residue was removed by a conventional method, and the mixture was dried to obtain 0.7323 g of dried material.

[0071] (Example 4. Hot water extract 2) 27 mL of water was added to 3.0 g of dried powder of the peanut bark, and the mixture was stirred at 90°C for 1 hour to carry out extraction. After that, the residue was removed by a conventional method, and the mixture was dried to obtain 0.1851 g of dried material.

[0072] Example 5. Alkaline Extract Add 25mL of water to 3.0g of dried powder of the hyacinth flower, adjust the pH to 9.0 (±0.1) using 0.5mol / L sodium hydroxide solution, and add water to make a total of 33g. Stir at 70℃ for 1 hour to extract. After adjusting the pH to 7.0 using 0.5mol / L hydrochloric acid, remove the residue using the usual method, and then dry it to obtain 0.5802g of dried material.

[0073] [Test Example 1. Senescent cell removal ability confirmation test] (1) Preparation of senescent cells 10,000 human hepatocellular carcinoma-derived HepG2 cells were seeded on a 12-well plate and treated with 10 μM (containing 0.1% DMSO) etoposide (Sigma-Aldrich) for 2 days to induce cellular senescence. To confirm the induction of cellular senescence, the cells were treated with 1 μM SPiDER-βGal (Dojindo) and observed for fluorescent signals using an all-in-one fluorescence microscope BZ-9000 (Keyence) with a GFP filter (excitation wavelength: 470 / 40 nm, absorption wavelength: 525 / 50 nm). Conversion of SPiDER-βGal to a fluorescent substance caused by β-galactosidase activity in senescent cells was observed in etoposide-treated cells.

[0074] (2) Assessment of senescent cell elimination activity The ratio of the lethal concentration 50 (LC50) in normal (non-senescent) cells to the LC50 in senescent cells was determined. LC50 is the concentration required to kill half of the cells in a plate.

[0075] 8,000 HepG2 cells were seeded in a 96-well plate and treated with 10 μM (containing 0.1% DMSO) etoposide for two days to induce cell senescence. After that, the cells were treated with the above-prepared test preparations in RPMI medium (containing no HEPES) (ThermoFisher) containing 0.5% fetal bovine serum, with 0.179 to 40 μg / mL of the extracts of the flower and bark of the rhododendron amurense. After two days, the number of cells was measured using EnVision (PerkinElmer) based on the luminescence amount using CellTiter-Glo (Promega). Based on the measured values ​​of the treatment concentration, the LC50 was calculated using GraphPad Prism9 (GraphPad Software). The results of the ability to remove senescent cells were calculated based on the following formula and are shown in Figure 1.

[0076] How to determine senescent cell removal capacity Senescent cell elimination capacity = LC50 in normal (non-senescent) cells / LC50 in senescent cells

[0077] As shown in Figure 1, when the above formula was used to calculate the senescent cell removal ability, which is the ability to induce cell death specific to senescent cells, the values ​​obtained for the Hemerocallis flower extract were 9.8 (hot water extract), 5.0 (ethanol extract), and 4.7 (alkali extract), while the Hemerocallis bark extract gave values ​​of 4.2 (hot water extract) and 3.9 (ethanol extract). When the senescent cell removal ability of quercetin, which is known to have an effect of removing senescent cells, was examined under the same conditions, the result was 1.1, confirming that the Hemerocallis flower extract and Hemerocallis bark extract have a higher ability to remove senescent cells than the known quercetin.

Claims

1. An agent for removing senescent cells comprising an effective amount of at least one member selected from the group consisting of uniflora flowers, uniflora bark, and extracts thereof.

2. The senescent cell removing agent according to claim 1, which is labeled with at least one or more indications selected from the group consisting of "reducing senescent cells," "removing senescent cells," "reducing the proportion of senescent cells," and "promoting the reduction of senescent cells."

3. The senolytic agent according to claim 1 , wherein the senolytic agent selectively removes senescent cells relative to normal cells.

4. The senolytic agent according to claim 1, wherein the ratio (A / B) of (A) the 50% lethal concentration (LC50) in normal cells to (B) the 50% lethal concentration in senescent cells is greater than 1.1.

Images