Composition for removing senescent cells and method for producing the same

Abstract

The object of this invention is to provide a novel anti-aging composition that can promote the function of removing senescent cells. [Solution] To solve the above problems, an anti-aging composition containing agrimol B is provided. According to the present invention, an anti-aging composition that can promote the function of removing senescent cells can be provided. In the anti-aging composition, agrimol B is preferably contained as a plant extract, and is particularly preferably contained as an extract of Agrimonia pilosa.

Description

TECHNICAL FIELD

[0001] The present invention relates to an anti-aging composition. More specifically, the present invention relates to an anti-aging composition for use in fields such as pharmaceuticals and health foods.

BACKGROUND ART

[0002] Aging caused by aging leads to functional decline and metabolic decline in various organs, and triggers the onset of many diseases. Recently, it has been clarified that senescent cells increase in organs with aging, and these senescent cells induce chronic inflammation and the like, thereby causing systemic metabolic insufficiency and metabolic disorders that occur with aging. Furthermore, it is known that the increase of senescent cells is not only caused by aging, but also by disturbances in lifestyle habits such as high-fat diet intake, excessive stress, ultraviolet rays and the like. It has been reported that removing these senescent cells can improve insulin resistance through suppression of chronic inflammation, suppress arteriosclerosis, and further suppress pathological conditions that develop with aging (Non-Patent Document 1). Attempts have also been made to suppress aging by ingesting antioxidant substances to eliminate active oxygen generated to a level that cannot be coped with by the biological defense mechanism due to oxidative stress. Representative antioxidant substances include tocopherols, carotenoids, and flavonoids contained in various plants.

[0003] However, although the intake of antioxidant substances contributes to the elimination of active oxygen generated in the living body, it does not contribute at all to the removal of already accumulated senescent cells. Therefore, the removal of senescent cells is essential for the prevention and treatment of various diseases involving senescent cells accumulated in the living body with aging.

[0004] On the other hand, the present inventors have continuously focused on and explored the mechanism of action of agrimol and the extract of Agrimonia pilosa containing the same, and have already proposed a composition for anti-Helicobacter pylori (Patent Document 1), a composition for nerve activation (Patent Document 2), an immune aging improving function (Patent Document 3, Japanese Patent Application No. 2020-108408), and the like.

[0005] [Patent Document 1] Japanese Patent Publication No. 2003-342190 [Patent Document 2] Japanese Patent Publication No. 2018-008888 [Non-Patent Document 1] Nat.Med., Vol.15, Number9, P1082-1087, 2009 [Overview of the project] [Problems that the invention aims to solve]

[0006] As described above, removing senescent cells can improve and treat various diseases and aging, so there is a need for novel compositions that can promote the function of removing senescent cells.

[0007] The object of this invention is to provide a novel anti-aging composition that can promote the removal of senescent cells. [Means for solving the problem]

[0008] As a result of diligent research into the above-mentioned problems, the inventors of this invention have found that agrimol B has a strong senescent cell removal effect, and have completed the present invention.

[0009] In other words, the present invention provides the following [1] to [5]. [1] An anti-aging composition containing agrimol B. The present invention can promote the body's ability to remove senescent cells. [2] The anti-aging composition according to [1], characterized in that it contains agrimol B as a plant extract. This characteristic makes it easy to obtain anti-aging compositions. [3] The anti-aging composition according to [2], characterized in that the plant extract is Agrimonia pilosa extract. [4] A composition for removing senescent cells containing agrimol B. [5] A life-extending composition containing agrimol B. This characteristic makes it possible to easily obtain novel anti-aging compositions, senescent cell removal compositions, and life-extending compositions that are highly effective. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide an anti-aging composition that can promote the function of removing senescent cells. [Brief explanation of the drawing]

[0011] [Figure 1] Images showing WI38 cells induced to senescence by doxorubicin, and the senescent cell removal effects of kinmizuhiki extract and agrumol B. [Figure 2] A graph showing the concentration-dependent decrease in SPiDER-βGal fluorescence values ​​in WI38 cells senescence-induced by doxorubicin, induced by the addition of Agrimonia pilosa extract and agrimol B. [Figure 3] A graph showing the decrease in SA β-gal-expressing CD4+ T cells and CD8+ T cells in peripheral blood mononuclear cells of aged mice administered with Agrimonia pilosa extract. [Figure 4] A graph showing the reduction of senescent cells in the kidneys of aged mice administered with Agrimonia pilosa extract. [Figure 5] A graph showing the life-extending effect of administering Agrimonia pilosa extract to aged mice. [Modes for carrying out the invention]

[0012] The following describes the anti-aging composition, the senescent cell removal composition, and the life-extending composition of the present invention. Hereinafter, these will also be referred to collectively as "compositions." In this specification, the notation "A~B" (where A and B are numerical values) refers to a numerical range that includes both ends of that range. The compositions described in the embodiments are merely illustrative examples for the purpose of explaining the present invention and are not limiting.

[0013] [Anti-aging composition] The anti-aging composition of the present invention is characterized by containing Agrimol B. According to the present invention, the senescent cell clearance function can be promoted.

[0014] [Senescent cell clearance composition] The senescent cell clearance function is a function typified by the autophagy system that degrades part of the cytoplasm such as cytoplasmic components including proteins and intracellular organelles such as mitochondria, and has the function of degrading proteins to supply amino acids under starvation conditions and the function of degrading intracellular waste products.

[0015] Diseases caused by the decline of the senescent cell clearance function include, for example, neurodegenerative diseases, metabolic disorders, immune disorders, infectious diseases, cancers, and other diseases. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, and dementia. Examples of metabolic disorders include type 2 diabetes, nephropathy, metabolic syndrome, and fatty liver. Examples of immune disorders include Crohn's disease and autoimmune diseases. Examples of infectious diseases include opportunistic infections. Examples of other diseases include arteriosclerosis, muscular atrophy, myopathy, and anemia.

[0016] In addition, aging caused by the decline of the senescent cell clearance function includes, for example, symptoms resulting from the accumulation of intracellular waste products, such as dysfunction of organs and tissues. That is, the senescent cell clearance composition of the present invention can be expected to have the effect of preventing and improving dysfunction of organs and tissues.

[0017] The anti-aging composition, senescent cell clearance composition and lifespan-extending composition of the present invention can be used in pharmaceuticals, foods, veterinary drugs, feeds and the like.

[0018] <Agrimol B> The agrimol B mentioned above is the compound shown in the following chemical formula (molecular weight: 682.75 g / mol). Agrimol B can be used by chemical synthesis or by extraction, isolation, and purification from natural products such as plants. It may also be purchased commercially. Furthermore, agrimol B may be included as a plant extract of a plant containing agrimol B.

[0019] [ka]

[0020] Agrimol B can be chemically synthesized using phloroglucinol or the like as starting materials. For example, it can be synthesized by the method described in Acta Pharmaceutica Sinica 1989, 24(6): pp. 431-437, or by the method disclosed in Japanese Patent Publication No. 11-335256.

[0021] In addition, while there are two types of agrimol B obtained by synthesis, (S)-(+)-agremol B and its optical isomer (R)-(-)-agremol B, the term "agremol B" as used herein may refer to a single compound consisting of one of these types, or a mixture consisting of both types.

[0022] The content of agrimol B in the anti-aging composition of the present invention is not particularly limited, but is, for example, 0.001 to 100% by mass. The lower limit is preferably 0.005% by mass or more, more preferably 0.02% by mass or more, and the upper limit is preferably 98% by mass or less. By setting the content within the above range, the function of removing senescent cells can be further promoted.

[0023] <Plant extracts> As described above, the anti-aging composition of the present invention may contain agrimol B as a plant extract. Such a plant extract is not particularly limited as long as it contains agrimol, but preferably it is Agrimonia pilosa extract. Agrimonia pilosa is a perennial herb belonging to the genus Agrimonia in the family Rosaceae, and grows wild in forest edges, fields, and roadsides in Honshu, Shikoku, and Kyushu. It is also known as Ryugasō. In the present invention, the whole plant, whether wild or cultivated, can be harvested and then naturally dried or heat-dried. Alternatively, dried Agrimonia pilosa, commercially available as a Chinese herbal medicine, folk remedy, or health food (herbal tea) ingredient, can also be used. Dried whole Agrimonia pilosa is commercially available under the herbal medicine name Senkakusō.

[0024] Examples of plant extracts in the present invention include extracts with water or various organic solvents, distillates obtained by steam distillation, dried powders, grated products, and juices extracted from grated products. Preferably, from the viewpoint of promoting the function of autophagy, the extract is an extract with water or various organic solvents, and more preferably, an extract is an extract with ethyl alcohol or aqueous ethyl alcohol containing water. Extraction with water or various organic solvents can be carried out using water, various organic solvents, and mixtures thereof as solvents, according to known extraction methods. Before performing the extraction, the raw materials, either whole or roughly chopped, may be washed with a hydrophobic or hydrophilic solvent alone or a mixture thereof to remove unwanted substances such as pigments. Examples of hydrophobic solvents include chloroform, ether, hexane, cyclohexane, toluene, dichloromethane, petroleum ether, and benzene, while examples of hydrophilic solvents include lower alcohols (e.g., methyl, ethyl, n-propyl, isobutyl, or butyl alcohol), ethyl acetate, acetone, isopropyl alcohol, and acetonitrile. Water may be added to the raw materials as needed to wet them.

[0025] To perform extraction with water, for example, 0.5 to 10 parts by weight of water can be added to 1 part by weight of the raw material, and extraction can be carried out at 40 to 120°C for 5 minutes to 2 hours, preferably 10 to 60 minutes, and more preferably 10 to 20 minutes. During this time, stirring can be used to increase the extraction efficiency. Extraction can also be carried out by heating under reflux. After extraction, filtration or pressing is performed using a filter cloth, filter, or mesh screen. In this case, pressurization may be used to increase work efficiency. Alternatively, the extract can also be obtained by centrifugal separation (1000 to 20000 rpm). To increase the recovery rate, this extraction and filtration process can be repeated 1 to 5 times.

[0026] To perform extraction with an organic solvent, for example, 0.5 to 10 parts by weight of solvent can be added to 1 part by weight of the raw material, and the extract can be obtained in the same manner as with water extraction. The solvents used can be the hydrophobic and hydrophilic solvents mentioned above, either alone or in combination of two or more, and aqueous solvents can also be used. In particular, when washing the raw material with a hydrophobic solvent before extraction to remove unwanted substances such as pigments, as described above, and when washing the crude extract after extraction with a hydrophobic solvent, as described below, water, lower alcohols, and 50-80% (v / v) aqueous lower alcohols are preferred as solvents for extraction. Examples of these lower alcohols are the same as those mentioned above. Solvent removal can be carried out by conventional methods such as filtration, centrifugation, and distillation.

[0027] A particularly preferred method for extracting plant extracts, especially Agrimonia pilosa extract, can be carried out as follows, for example. The whole plant, whether wild or cultivated, is harvested and then air-dried or heat-dried before use. This is then finely chopped and immersed in approximately 10 times its volume of water or 0-99.5% (v / v) ethyl alcohol, preferably 1-50% ethyl alcohol, and especially preferably 5-10% ethyl alcohol, for 3-5 days at room temperature for extraction, or extracted at 50-80°C for 5-24 hours using a reflux condenser, and the extract is filtered to collect the liquid. This extract is then subjected to a vacuum drying device such as a rotary evaporator, or a freeze-drying device to remove water and ethyl alcohol and obtain a plant extract (solid).

[0028] Next, the plant extract is concentrated. In this process, the plant extract is concentrated by washing it with 0-40% ethyl alcohol to remove 0-40% ethyl alcohol-soluble components. Specifically, the plant extract (solid) is washed with 0-40% ethanol, particularly preferably 10-30% ethanol, and the 0-40% ethanol, particularly preferably 10-30% ethanol-soluble components are removed by means of filtration or other means. The filtered residue is then removed using a vacuum drying device such as a rotary evaporator or a freeze-drying device to remove water and ethyl alcohol, thereby obtaining a more concentrated plant extract. The concentrated plant extract is then redissolved in 50-99.5% ethanol, particularly preferably 70-90% ethanol, and excipients used for formulation are added and freeze-dried to obtain a concentrated plant extract of the desired concentration.

[0029] As described above, the composition of the present invention exhibits therapeutic and ameliorative effects against diseases such as neurodegenerative diseases, metabolic disorders, immune disorders, infectious diseases, and cancer, as well as aging conditions. Therefore, formulations containing the composition of the present invention can be used as agents for the following purposes. (a) Therapeutic agents for neurodegenerative diseases: Agents that treat neurodegenerative diseases caused by a decline in the function of removing senescent cells. (b) Therapeutic agents for metabolic disorders: Agents that treat metabolic disorders caused by a decrease in the function of removing senescent cells. (c) Immune function improving agents: Agents that enhance the body's natural immune function by promoting the removal of senescent cells. Agents that treat and improve immune abnormalities and treat and prevent infectious diseases. (d) Cancer treatment and suppression agents: Agents that improve the body's natural immunity by promoting the function of removing senescent cells, thereby treating cancer or suppressing cancer metastasis and recurrence. (e) Anti-aging agents: Agents that suppress and improve aging caused by a decline in the function of removing senescent cells.

[0030] In addition to ordinary foods, the composition of the present invention can be added to nutritional supplements, functional foods, health foods, foods for specified health uses, etc., for the purpose of achieving the above-mentioned effects.

[0031] The form of the composition of the present invention is not particularly limited as long as it is acceptable for use in pharmaceuticals, foods, veterinary drugs, feed, etc. Examples include tablets such as sugar-coated tablets, buccal tablets, coated tablets, and chewable tablets; pills, powders, soft capsules, hard capsules, granules, suspensions, emulsions, syrups, liquid preparations such as elixirs, lozenges, processed foods, health foods (nutritional supplements, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.); supplements; foods for the sick (hospital food, sick person food, nursing care food, etc.); confectionery; oils and fats; dairy products; retort foods; microwaveable foods; frozen foods; seasonings; health supplements; beverages; and nutritional drinks. [Examples]

[0032] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention.

[0033] The senescent cell removal effect of an anti-aging composition containing agrimol B, and an anti-aging composition containing agrimol B as Agrimonia pilosa extract, was confirmed by the following procedure.

[0034] (1) Preparation of test samples (Agrimol B) The standard sample of Agrimol B was obtained from Biorbyt. Agrimol B was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, which was used in the following tests.

[0035] (Kinmizuhiki extract) The goldenrod extract was prepared using the following procedure. 100 g of dried aerial parts of Agrimonia pilosa (Fukuda Ryu) was mixed with 10 times the amount (1 kg) of 90% ethyl alcohol (EtOH), and reflux extraction was repeated twice. The resulting extract was subjected to solid-liquid separation by conventional methods, activated carbon treatment, and diatomaceous earth filtration. The filtrate was concentrated under reduced pressure and freeze-dried to obtain Agrimonia pilosa extract (solid content 9.28 g, yield 9.28% (relative to raw material)). 8.51 g of this Agrimonia pilosa extract was dissolved and suspended (solid content 10%) in a 20% EtOH solution heated to 60°C. The supernatant was obtained as the soluble component in 20% EtOH, and the precipitate as the insoluble component. The supernatant was removed by diatomaceous earth filtration, and the precipitate was recovered. The recovered precipitate was dissolved again in 90% EtOH, filtered by diatomaceous earth using a conventional method, and then concentrated and freeze-dried again to obtain concentrated Agrimonia pilosa extract (solid content 0.59 g, yield 6.93% (compared to Agrimonia pilosa extract)). This concentrated Agrimonia pilosa extract is obtained by removing 20% ​​EtOH-soluble components from the Agrimonia pilosa extract extracted with 90% EtOH, resulting in a higher concentration of hydrophobic components, including agrimol B, per unit solid. This concentrated Agrimonia pilosa extract was used in the following experiments.

[0036] (Measurement of agrimol B content in goldenrod extract) The agrimol B content of the test sample used in this embodiment was measured by a conventional method, and it was found to contain 0.447% by mass of agrimol B. The HPLC conditions used for the measurement are shown below. Column: Wakosil-II5C18 AR 4.6mm x 150mm (Manufactured by Wako Pure Chemical Industries, Ltd.) Mobile phase: Distilled water: Acetonitrile: Phosphate = 100:900:1 Flow rate: 1 mL / min Detection: UV 288 nm Injection volume: 10 μL

[0037] (2) Culture and treatment methods WI38 cells 5x10 4 The cells were turbidified with MEM medium (Sigma) containing 10% FBS, 1% penicillin-streptomycin (Sigma), and 1% non-essential amino acid solution (Sigma) to a concentration of cells / mL. 100 μL of the mixture was seeded into 96-well plates (Sumitomo Bakelite) or 96-well black clear bottom plates (Greiner) and cultured at 37°C under 5% CO2. After 24 hours of culture, doxorubicin was added to a final concentration of 62.5 nM. After another 24 hours of culture, the medium was changed and cultured for a further 2 days. Agrimonia extract or agrimol B was added to the respective concentrations and cultured for 3 days.

[0038] (3)SA-X-gal staining After removing the culture medium from the cultured cells in a 96-well plate (Sumitomo Bakelite) and washing with PBS, staining was performed using the Senescence β-Galactosidase Staining Kit (Cell signaling) according to the attached instructions. After adding the staining solution and reacting at 37°C for 24 hours, photographs were taken under a microscope. The results are shown in (Figure 1).

[0039] (4) SPiDER-βgal assay Cells cultured in a 96-well black clear bottom plate (Greiner) were measured using the Cell Count Normalization Kit (Dojindo) according to the instructions and a fluorescence plate reader (Ex: 350 nm / Em: 461 nm) to determine the cell count. After removing the reaction solution and washing with PBS, the Cellular Senescence Plate Assay Kit - SPiDER-βGal (Dojindo) was used to measure fluorescence levels using a fluorescence plate reader according to the instructions (Ex: 500 nm / Em: 550 nm). The obtained fluorescence values ​​were corrected using the fluorescence values ​​measured previously with the Cell Count Normalization Kit. The results are shown in Figure 2.

[0040] (result) As shown in Figure 1, the blue-stained senescent cells (spot-like substances other than the nucleus) induced by the addition of doxorubicin (Senescence β-Galactosidase) were reduced by the addition of Agrimonia pilosa extract and agrumol B. Furthermore, as shown in Figure 2, the addition of Agrimonia pilosa extract and Agrimol B resulted in a concentration-dependent decrease in SPiDER-βGal fluorescence values, i.e., a decrease in senescent cells. These results confirmed that Agrimonia pilosa extract and agrimol B have the effect of removing senescent cells.

[0041] <Verification of the senescent cell removal effect of Agrimonia pilosa extract in aged mice> (1) Preparation of test samples 50 kg of dried Agrimonia pilosa leaves (Tochimoto Tenkaido) were mixed with 90% EtOH, and reflux extraction was repeated twice. The amount of 90% EtOH added was 14 times the weight of the dried Agrimonia pilosa leaves (1st time) and 13 times the weight (2nd time). After extraction, solid-liquid separation was performed using a mesh, and the filtrate was concentrated under reduced pressure to obtain the extract. Subsequently, water was added to the obtained extract to make it 20% EtOH, and it was redissolved and dispersed to obtain the supernatant as the soluble component and the precipitate as the insoluble component. The supernatant was removed by diatomaceous earth filtration, and the precipitate was recovered as the residue. 90% EtOH was added to the recovered residue and stirred with activated carbon at 60°C for 1 hour. After that, the activated carbon was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure, freeze-dried, pulverized, and sieved to obtain Agrimonia pilosa extract. This Agrimonia extract has been refined by removing 20% ​​of the EtOH-soluble components, resulting in a concentration of hydrophobic components, including agrimol B. HPLC analysis of the agrimol B content in the Agrimonia extract used in this study revealed a concentration of 0.207% by mass.

[0042] (2) Animal experiments 82-week-old C57BL / 6J mice (Charles River, Japan) were received and subjected to a one-week acclimatization period. After acclimatization, the mice were switched to a control diet of AIN-93M, or a diet containing AIN-93M with 0.2% or 0.4% goldenrod extract, and administered as mixed diet for 16 weeks. On the final day, after isoflurane absorption anesthesia, cardiac blood was collected, and liver and kidney tissue was collected and frozen for measurement.

[0043] (3) Effects on senescent cells in immune cells - SA β-gal high CD4 + T, CD8 + Measurement of T cells Cardiac blood was collected using a syringe containing 6 μL of 10% EDTA (pH 7.5), and the plasma and blood cells were separated by centrifugation at 3,000 rpm for 15 minutes. 500 μL of PBS(-) was added to the blood cells to suspend them, and this was gently added to the upper layer of a tube containing Ficol (Cytiva), and centrifuged at 400 × g for 30 minutes. Peripheral blood mononuclear cells in the middle layer were collected into a separate tube. 10 mL of PBS(-) was added to this tube, and the cells were washed by centrifugation at 2,000 rpm for 5 minutes. Peripheral blood mononuclear cells were treated with anti-CD16 / CD32 antibody (Biolegend) diluted in FACS buffer (2% FBS, PBS(-) containing 2 mM EDTA), and the mixture was allowed to react on ice for 20 minutes. Furthermore, anti-TCRb-Alexafluor700 antibody (Biolegend), anti-CD4-BV786 antibody (Biolegend), and anti-CD8-PerCP / Cy5.5 antibody (Biolegend) were added, and the mixture was reacted on ice for 30 minutes to stain the cell surface antigens. Subsequently, the cells were washed by treating them with 500 μL of FACS buffer and centrifugation at 2,000 rpm for 5 minutes, and dead cells were stained using the LIVE / DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen). As described above, the stained cells were fixed by treating them with 4% paraformaldehyde at room temperature for 3 minutes. The cells were then washed by adding 1 mL of McIlvaine Buffer (pH 6.0) and centrifugation at 2,000 rpm for 5 minutes. Next, SPiDER-βGal (Dojin), diluted 500-fold with McIlvaine Buffer (pH 6.0), was treated and incubated at 37°C for 1 hour to stain the cells with SA β-gal. Afterward, the cells were washed by treating them with 1,000 μL of FACS buffer and centrifugation at 2,000 rpm for 5 minutes. The cells prepared in this manner were then subjected to FACS analysis. The obtained data were statistically analyzed using Dunnett's method, and significance testing was performed. A p-value of 0.05 or less was considered statistically significant.

[0044] As a result, as shown in Figure 3, compared to young mice, aged mice (9 weeks old) showed high expression of SA β-gal in peripheral blood mononuclear cells of CD4. + T cells, CD8 + Although the proportion of T cells was high, it was confirmed that this proportion decreased after administration of Agrimonia pilosa extract. These results confirm that Agrimonia pilosa extract has a senescent cell-removing effect in vivo.

[0045] (4) Effects on senescent cells in the kidney - SA β-gal staining of renal tissue sections SA-βGal staining of the kidney using X-Gal To evaluate senescent cells in the kidneys of the animals evaluated in the above examples, SA β-Gal staining, a senescent cell marker, was performed using the following procedure. (A) Preparation of sections Kidneys were collected from mice and immersed in 10% neutral buffered formalin at 4°C for 6 hours. They were then washed with PBS and immersed in 30% sucrose at 4°C for 24 hours. The kidneys were removed from the sucrose and embedded in OTC compound (Sakura Finetech Japan) within cryomold (Sakura Finetech Japan). After freezing with dry ice, they were stored at -80°C until sectioning. Furthermore, the kidneys were sectioned into 6 μm sections using a cryomicrotome and mounted on glass slides. The glass slides were washed three times with PBS and then air-dried. (B) SA-βgal staining While the glass slide (A) above was air-drying, a fresh SA-βgal staining solution was prepared with the following composition. For 20 mL staining solution: 4 mL Citric acid / Na phosphate buffer 1 mL 100 mM Potassium ferrocyanide 1 mL 100 mM Potassium ferricyanide 0.6 mL 5 M Sodium Chloride 0.04mL 1M Magnesium chloride 1 mL 20 mg / mL X-gal in N',N'-dimethylformamide During the preparation of the staining solution, X-gal was added last, and after adjusting the pH of the final solution to 6.0, it was made up with dH2O. Air-dried glass slides were placed in this SA-βgal staining solution and stained at 37°C for 16 hours. After staining, the glass slides were washed three times with PBS, and the kidneys were observed using an all-in-one fluorescence microscope BZ-X800 (Keyence).

[0046] As a result, older mice showed a greater number of cells stained a deep blue compared to younger mice. This is because X-Gal was degraded by SA β-gal, which is used as a marker for senescent cells, resulting in a blue color. This indicates that older mice have more SA β-gal, and therefore more senescent cells. On the other hand, in mice administered with Agrimonia pilosa extract, the blue color became lighter in a dose-dependent manner, confirming that Agrimonia pilosa extract reduces senescent cells in a dose-dependent manner (Figure 4). In other words, it was confirmed that senescent cells were removed by Agrimonia pilosa extract containing agrimol B. The senescent cell-removing effect of agrimol B was confirmed in Figures 1 and 2, and as shown in Figures 3 and 4, a similar effect was confirmed in mice.

[0047] <Life-extending effect of Agrimonia extract> (1) Preparation of test samples Approximately 400 g of dried Agrimonia pilosa (Tochimoto Tenkaido) was ground in a mill, 10 times its volume of 99.5% EtOH was added, and the mixture was thoroughly immersed. Extraction was carried out by stirring at room temperature for 4 to 7 days. Solid-liquid separation was performed by filtration, and the filtrate was concentrated under reduced pressure to obtain Agrimonia pilosa extract. The presence of agrimol B in the obtained Agrimonia pilosa extract was confirmed and used in the following tests. (2) Animal experiments 78-week-old male C57BL / 6J mice (Charles River, Japan) were acclimatized for one week on purified feed AIN-93M (Oriental Yeast Co., Ltd.), and then divided into two groups: a control group (n=15) and a group administered Agrimonia pilosa extract (n=14). The control group was given AIN-93M ad libitum for four months, and the Agrimonia pilosa extract group was given AIN-93M containing 0.5% Agrimonia pilosa extract. Survival rates until the end of the study were calculated.

[0048] As a result, compared to the control group, the group administered Agrimonia pilosa extract showed a suppressed decline in survival rate during the administration period (Figure 5). This confirmed that Agrimonia pilosa extract has a life-extending effect. In the life-extending effect test, Agrimonia pilosa extract that had not undergone treatment to concentrate the hydrophobic components, including agrimol B, was used. However, since a life-extending effect was confirmed even with this unconcentrated Agrimonia pilosa extract, it is naturally expected that a similar effect would be observed with Agrimonia pilosa extract that contains a higher concentration of hydrophobic components, including agrimol B. [Industrial applicability]

[0049] The present invention can provide a novel anti-aging composition that can promote the function of removing senescent cells. Furthermore, it can provide pharmaceuticals, quasi-drugs, oral cosmetics, oral beauty products, health foods (nutritional supplements, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.), supplements, animal feed, etc., containing the novel anti-aging composition.

Claims

1. It contains Agrimolophia pilosa extract containing Agrimolophia B, The aforementioned Agrimonia extract is a composition for removing senescent cells, in which the raw material is only the above-ground parts of Agrimonia.

2. It contains Agrimolophia pilosa extract containing Agrimolophia B, A composition for removing senescent cells, wherein the agrimol B content in the aforementioned goldenrod extract is 0.207% by mass or more.

3. An extraction process to obtain an extract from the above-ground parts of Agrimonia pilosa containing agrimol B using primary ethyl alcohol with a water content of 1-50%, A drying step to remove at least a portion of the first ethyl alcohol from the extract using a vacuum drying apparatus or a freeze-drying apparatus to obtain a solid, A concentration step is performed to wash the solid with a second ethyl alcohol with a water content of 0 to 40% to remove the second ethyl alcohol-soluble component from the solid, thereby obtaining a concentrated goldenrod extract. A method for producing a composition for removing senescent cells, which includes the composition.

Images