Composition for removing senescent cells

Abstract

To provide a new composition for removing senescent cells.SOLUTION: A composition for removing senescent cells, comprising an aqueous solvent extract of turmeric.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for removing senescent cells. [Background technology]

[0002] Cellular senescence is a state in which cells irreversibly stop cell division in response to stress. Various stresses, such as ultraviolet light and oxidation, can induce cellular senescence. The DNA damage response (DDR) that occurs after exposure to stress is thought to be the trigger for cellular senescence. Cells undergoing cellular senescence (senescent cells) are known to secrete inflammatory cytokines, collagen-degrading enzymes, and other substances. This secretion is also known as the senescence-associated secretory phenotype (SASP).

[0003] Examples of approaches to inhibit cellular senescence include preventing cells from aging by reducing oxidative stress and repairing DNA damage (prevention of cellular senescence), suppressing inflammation and tissue destruction by inhibiting the SASP of senescent cells (SASP inhibition), and removing senescent cells themselves (removal of senescent cells (senolysis)). In recent years, it has been reported that removal of senescent cells is useful for preventing or treating age-related diseases (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] S. Chaib1 et al., Cellular senescence and senolytics: the path to the clinic, Nat Med., 28(8), 2022, 1556-1568. [Non-patent document 2] MJ Yousefzadeh et al., Fisetin is a senotherapeutic that extends health and lifespan, EBioMedicine, 36, 2018, 18-28 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present disclosure is to provide a new composition for removing senescent cells. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that an aqueous extract of turmeric can remove senescent cells. The present invention was completed through further research based on this finding, and the present disclosure encompasses, for example, the following representative inventions. Item 1. A composition for removing senescent cells, comprising an aqueous solvent extract of turmeric. Item 2. A composition for removing senescent cells according to Item 1, which is used for removing human senescent cells. Item 3. Use of an aqueous solvent extract of turmeric for the manufacture of a composition for removing senescent cells. Item 4. A method for removing senescent cells, comprising applying an effective amount of an aqueous solvent extract of turmeric to a subject in need of senescent cell removal. Item 5. An aqueous solvent extract of turmeric for use in removing senescent cells. [Effects of the Invention]

[0007] According to the present disclosure, a composition for removing senescent cells containing an aqueous solvent extract of turmeric can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] The results of the test examples are shown below. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments included in the present disclosure will be described in more detail. In the present disclosure, "comprise" also means "consist essentially of" or "consist of."

[0010] The present disclosure encompasses a composition for removing senolytic cells containing an aqueous solvent extract of turmeric. In the present disclosure, the composition for removing senolytic cells may be referred to as the "composition of the present disclosure."

[0011] Turmeric (Curcuma longa) is a plant belonging to the genus Curcuma in the family Zingiberaceae, and is a perennial plant distributed in tropical Asia and cultivated in India, Southeast Asia, southeastern China, Okinawa, etc. In the present disclosure, the part of turmeric used in the aqueous solvent extract is not limited as long as the effects of the present disclosure can be obtained, and may be any of the rhizome, stem, root, leaf, flower, fruit, etc., with the rhizome being preferred. One part may be used alone, or two or more parts may be used in combination.

[0012] The aqueous solvent extract of turmeric can be obtained by extracting the above-mentioned parts of turmeric directly or after cutting, crushing, drying, etc. as necessary using an aqueous solvent as an extraction solvent. The method for producing the aqueous solvent extract (extraction method) and extraction conditions, etc. are not particularly limited.

[0013] The aqueous solvent is not limited as long as the effects of the present disclosure are obtained, but examples thereof include water; lower (monohydric) alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, and isopropanol; lower (polyhydric) alcohols having 2 to 5 carbon atoms, such as propylene glycol, 1,3-butylene glycol, and glycerin (whether anhydrous or hydrous); lower aliphatic ketones having 2 to 5 carbon atoms, such as acetone and methyl ethyl ketone; and any mixtures of these solvents. Preferred examples of aqueous solvents include water; alcohols, such as methanol, ethanol, isopropanol, propylene glycol, and 1,3-butylene glycol; and any mixtures of water and these alcohols. More preferred examples of aqueous solvents include water, lower alcohols having 1 to 5 carbon atoms, and mixtures thereof. For example, when hydrous ethanol (a mixture of water and ethanol) is used as the aqueous solvent, the concentration of ethanol is not particularly limited, and the ethanol content in the hydrous ethanol is preferably 10% by volume or more and 80% by volume or less. The ethanol content in the aqueous ethanol is more preferably 20% by volume or more and 75% by volume or less, and 30% by volume or more and 70% by volume or less, for example. For example, when a solvent such as a lower alcohol is mixed with water instead of or together with ethanol to be used as an aqueous solvent, the total content of solvents other than water in the aqueous solvent (for example, the total content of alcohols and lower aliphatic ketones, etc.) is explained in the same manner. That is, the total content in the aqueous solvent is preferably 10% by volume or more and 80% by volume or less, for example. One type of aqueous solvent may be used alone, or two or more types may be used in combination.

[0014] The turmeric part and the aqueous solvent may be mixed in any amount. The extraction temperature is not limited as long as the effects of the present disclosure are obtained. Typical examples of the extraction temperature include temperatures from 40°C to the boiling point, and more preferably from 50°C to 80°C. While not limiting the present disclosure, when water is used as the aqueous solvent, preferred examples of the water temperature include temperatures from 40°C to 100°C, and more preferably from 50°C to 80°C. In the present disclosure, hot water is exemplified by temperatures from 70°C to 100°C. The extraction time is also not limited as long as the effects of the present disclosure are obtained. Examples of the extraction time include 1 hour to 72 hours, preferably 3 hours to 48 hours, and more preferably 6 hours to 24 hours. Shaking may or may not be performed during the extraction.

[0015] In the present disclosure, the extract obtained by solvent extraction may be used as an aqueous solvent extract as is. Alternatively, the extract may be subjected to processes such as concentration, drying, dilution, crude purification, or purification, resulting in a concentrate, dried product, diluted product, crudely purified product, or purified product, which may be used as the aqueous solvent extract. These processes may be performed using known procedures, such as vacuum concentration, filtration, freeze-drying, adsorption, or HPLC (High Performance Liquid Chromatography). Furthermore, the aqueous solvent extract of turmeric used in the present disclosure may be produced in accordance with the method for producing the herbal preparation "extract" described in the General Provisions for Preparations in the 18th Edition of the Japanese Pharmacopoeia. Specifically, this method involves measuring out a set amount of appropriately sized herbal medicine according to the prescription, adding 10 to 20 times the total amount of water, heating for a set period of time, separating the mixture into solid and liquid forms by centrifugation, or the like, and concentrating or drying the resulting infusion by an appropriate method. A soft extract with a starch syrup-like consistency is called a soft extract, while a crushable solid mass, granules, or powder is called a dry extract.

[0016] The aqueous solvent extract of turmeric incorporated in the composition of the present disclosure may be in any of liquid, semi-solid, and solid forms. Commercially available products may be used as the aqueous solvent extract. The aqueous solvent extract may be used alone or in combination of two or more.

[0017] The content of the aqueous solvent extract in the composition of the present disclosure is not limited and may be appropriately determined depending on the route of application (administration, ingestion), the form of the aqueous solvent extract, the formulation, etc., and is exemplified as 0.1% by mass or more and 100% by mass or less. The content of the extract in the composition is preferably 1% by mass or more and 99% by mass or less, more preferably 2% by mass or more and 80% by mass or less, even more preferably 5% by mass or more and 70% by mass or less, and particularly preferably 10% by mass or more and 60% by mass or less.

[0018] In addition to the aqueous solvent extract, the composition of the present disclosure may contain optional components such as pharmaceutically acceptable components, edible components, etc. Examples of optional components include solvents (water, alcohols (whether anhydrous or hydrous), such as lower (monohydric) alcohols (e.g., water, methanol, ethanol, isopropanol, etc.), and polyhydric alcohols (e.g., propylene glycol, 1,3-butylene glycol, etc.)), excipients, disintegrants, diluents, lubricants, flavorings, colorants, sweeteners, flavoring agents, suspending agents, humectants, emulsifiers, solubilizers, dispersants, buffers, binders, penetration enhancers, stabilizers, bulking agents, preservatives, thickeners, pH adjusters, surfactants, coating agents, absorption enhancers, fillers, antioxidants, cooling agents, gelling agents, amino acids, vitamins, various pharmacologically active components such as anti-inflammatory components, blood circulation promoting components, antioxidant components, and anti-fatigue components, as well as various nutritional components. The optional components may be used alone or in combination of two or more, and the content of the optional components may be determined appropriately.

[0019] The manner of use of the composition of the present disclosure is not limited and may be appropriately determined depending on the purpose, and can be used as a food composition (including beverages, health functional foods (including foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), supplements, foods for the sick, foods for nursing care), quasi-drug compositions, pharmaceutical compositions, feed compositions, etc. Furthermore, the composition of the present disclosure can be used as an additive to food compositions, quasi-drug compositions, pharmaceutical compositions, feed compositions, etc.

[0020] The form of the composition of the present disclosure is not limited and may be appropriately determined depending on the purpose, and may be any of solid, semi-solid, and liquid. Therefore, the composition may be any of powder, fine granules, granules, tablets, pills, capsules (including hard capsules and soft capsules), troches, chewable tablets, gels, pastes, creams, liquids, suspensions, emulsions, sprays, and lyophilized liquids. In addition, for example, the composition of the present disclosure may be used by mixing with water or the like.

[0021] The composition of the present disclosure may be applied (administered or ingested) by oral or parenteral administration such as transdermal administration, preferably oral or transdermal administration, as long as it provides a senescent cell removal effect. Preferred examples of the composition of the present disclosure include oral compositions and topical compositions. The composition of the present disclosure may also be an oral composition such as a toothpaste or liquid toothpaste.

[0022] In the present disclosure, senescent cells are cells in a state of irreversible cell division arrest, and it is said that senescent cells tend to accumulate in the body with age. As long as the effects of the present disclosure can be obtained, the senescent cells are not limited in the present disclosure. Preferable examples of senescent cells include cells of epithelial tissues, oral mucosa, etc. Therefore, preferred examples of the composition of the present invention include oral compositions, topical compositions, oral compositions, etc.

[0023] The composition of the present disclosure may be produced according to conventional procedures known in the art for the above-mentioned usage modes, various forms, etc., and may be produced by using the aqueous solvent extract and, if necessary, further mixing any of the above-mentioned optional components within a range that does not interfere with the effects of the present disclosure.

[0024] The subject (target animal) of the composition of the present disclosure is not limited, and examples thereof include humans, non-human mammals, etc. Examples of non-human mammals include mice, rats, guinea pigs, rabbits, dogs, cats, monkeys, pigs, cows, etc. The subject (target animal) is preferably a human.

[0025] It has been shown that aging and stress cause the accumulation of senescent cells in tissues, and that inflammation induced by these senescent cells affects the onset and progression of age-related diseases or symptoms. Because the composition of the present disclosure can remove senescent cells, it can be said that the composition of the present disclosure can be used for the purpose of preventing or suppressing various symptoms and diseases caused by the presence or accumulation of senescent cells, such as age-related diseases. Therefore, a preferred example of the composition of the present disclosure is applied to subjects from whom senescent cells should be removed, or subjects from whom senescent cells should be prevented or suppressed.

[0026] The amount of the composition of the present disclosure to be administered to a subject (subject animal) is not particularly limited and may be appropriately determined depending on the subject's (subject animal's) physique, age, symptoms, the form of the aqueous solvent extract, and the application method. For example, when administered orally, the amount of the aqueous solvent extract (in terms of dry matter) per day for an adult (body weight 60 kg) may be approximately 5 mg to 2000 mg, with preferred examples being approximately 8 mg to 1800 mg, 10 mg to 1500 mg, 10 mg to 1200 mg, 15 mg to 1000 mg, and 20 mg to 500 mg. Here, "dry matter" refers to a dried extract (dried aqueous solvent extract) conforming to the Pharmacopoeia. It may be administered (administered or ingested) once or multiple times per day. When administered via a route other than oral administration or to a non-human subject, the amount and number of applications may be appropriately determined based on the explanation for oral administration.

[0027] The composition of the present invention can remove (kill) senescent cells, and therefore the composition of the present invention can be used for the purpose of removing senescent cells, i.e., for the purpose of senolysis.

[0028] Therefore, the present disclosure encompasses a method for removing senescent cells, which comprises applying an effective amount of an aqueous solvent extract of turmeric to a subject. The conditions for the method, such as the composition of the present disclosure, the aqueous solvent extract of turmeric, the subject, and the amount applied, are as described above. [Example]

[0029] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0030] Test Example Test Procedure 1. Flask (culture surface area 75 cm) 2 Normal human dermal fibroblasts (NB1RGB) pre-cultured in 10% FBS-MEMα using 10% FBS were seeded onto a 96-well plate at 50,000 cells / well and pre-cultured overnight at 37°C and 5% CO2. 2. After pre-incubation, doxorubicin was added to the wells to a final concentration of 200 ng / mL, and the cells were cultured at 37°C and 5% CO2 for 7 days to induce cellular senescence, yielding NB1RGB senescent cells (hereafter simply referred to as "senescent cells"). Doxorubicin is known to induce cellular senescence by acting on DNA and inhibiting cell division. 3. Separately, cells that had not been subjected to senescence induction treatment (hereinafter sometimes simply referred to as "non-senescent cells") were prepared by culturing in the same manner except that doxorubicin was not added. 4. The senescent cells obtained in step 2 and the non-senescent cells obtained in step 3 were washed twice with PBS(-), and then turmeric extract was added to the medium (10% FBS-MEMα) at ​​20 mg / mL for each of the senescent cells and non-senescent cells, and the cells were cultured at 37°C and 5% CO2 for 4 days. 5. After culturing, the medium was aspirated, and 200 μL / well of 0.5 mg / mL MTT solution was added to the cells, followed by incubation at 37°C and 5% CO2 for 2 hours. 6. After the reaction, the plate was washed once with PBS(-), and then propanol was added to a volume of 100 μL / well. 7. The absorbance at 570 nm and 655 nm was measured using a microplate reader (Molecular Device), and the cell viability was calculated using the following formula: The control was a sample in which the cells were cultured, reacted, and added with propanol in the same manner as in 4 above, except that they were not contacted with the extract, and the absorbance was measured.

[0031]

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[0032] turmeric extract In this test example, turmeric rhizomes were mixed with a mixed solvent of water and 1,3-butylene glycol (aqueous solvent), and the extract obtained by extraction was used as the turmeric extract.

[0033] Curcumin Curcumin has been reported to have a senolytic effect on mouse embryonic fibroblasts (Non-Patent Document 2). Curcumin is known to be a type of polyphenol contained in turmeric. Therefore, the present inventors determined cell viability in the same manner as described above, except that curcumin was used instead of turmeric extract. In step 4 above, curcumin was added to both senescent and non-senescent cells at a concentration of 0.02 μg / mL. In this test example, curcumin commercially available as a reagent (Wako Special Grade, 038-04921, Fujifilm Wako Pure Chemical Industries, Ltd.) was used.

[0034] result The results are shown in Figure 1. In Figure 1, (a) shows the results when turmeric extract was used, and (b) shows the results when curcumin was used.

[0035] As shown on the right side of Figure 1(a), when senescent cells were treated with turmeric extract, the cell viability of the senescent cells was significantly reduced compared to the control (senescent cells not treated with turmeric extract). In contrast, as shown on the left side of Figure 1(a), no reduction in cell viability was observed when non-senescent cells were treated with turmeric extract. These results demonstrate that turmeric extract can induce cell death in senescent cells and selectively remove them. It was confirmed that turmeric extract has the ability to selectively remove senescent cells (senolysis effect).

[0036] In contrast, as shown on the right side of Figure 1(b), no decrease in cell viability was observed when senescent cells were treated with curcumin. Based on these results, it is believed that the senescent cell-eliminating effect of turmeric extract observed in this test example is due to components other than curcumin.

Claims

1. A composition for removing senescent cells, comprising an aqueous solvent extract of turmeric.

2. The composition for removing senescent cells according to claim 1, which is used for removing human senescent cells.

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