Anti-aging composition

Abstract

To provide an anti-aging composition capable of removing senescent cells without causing side effects.SOLUTION: The present invention relates to an anti-aging composition containing a fermented barley extract and an organic acid, an anti-aging composition containing a culture or a processed product thereof of lactic acid bacteria cultured in a medium containing a fermented barley extract, and an anti-aging composition containing pyrogallol or gallic acid, wherein theses anti-aging compositions enable a reduction in the number of senescent cells (senolysis) by a senescent-cell-eliminating action that induces apoptosis in senescent cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anti-aging composition, and more particularly to an anti-aging composition having a senolytic activity that selectively induces apoptosis (cell death) in senescent cells. [Background technology]

[0002] Aging is a phenomenon in which physical, physiological, and mental functions decline with age. Physical changes caused by aging include decreased motor function, decreased muscle strength and activity, deterioration of skin and hair, tooth loss, decreased eyesight and hearing, and decreased bone mass. Aging due to functional decline caused by aging is not a disease, but it is problematic because it increases the risk of geriatric diseases such as glucose and lipid metabolism disorders, arteriosclerosis, osteoporosis, and cataracts, which are known as age-related diseases.

[0003] In recent years, it has become clear that the accumulation of cellular senescence is involved in the onset and progression of age-related diseases, and research is being conducted on the development of new drugs and food ingredients that have the senolytic effect of removing senescent cells. Cellular senescence is a cellular program that forces damaged or old cells to undergo a stable growth arrest to avoid replication. Senescent cells, which are cells in which senescence is induced, lose their ability to proliferate and cease division, but maintain metabolic activity in a viable state and freeze the apoptosis (cell death) function. Senescent cells do not divide or die, and have distinctive appearances such as different cell morphology and size from normal cells and abnormal nuclear structure.

[0004] Senescent cells have been found to accumulate in tissues and organs during the aging process, often at sites adjacent to age-related lesions, such as age-related diseases and disorders. Furthermore, senescent cells exhibit chromatin remodeling, increased β-galactosidase activity (referred to as senescence-associated β-galactosidase, or SA-β-Gal), and secrete multiple factors, primarily proinflammatory, such as inflammatory cytokines, chemokines, and matrix metalloproteinases. These secreted substances are termed the senescence-associated secretory phenotype (SASP). These SASP factors are thought to be associated with the development of age-related diseases such as arteriosclerosis, fibrotic lung disease, and osteoporosis (Non-Patent Document 1), and they also serve as markers for senescent cells. Furthermore, even in neurons, which are known not to divide, SASP factors secreted by surrounding senescent cells have been shown to adversely affect neurons and contribute to the deposition of amyloid β, one of the causes of Alzheimer's disease.

[0005] Genetic analysis using a premature aging model mouse has reported that artificially removing senescent cells from an elderly individual significantly delays the onset of geriatric diseases such as arteriosclerosis and renal damage and extends the individual's lifespan (Non-Patent Document 2).Furthermore, it has been suggested that in non-premature aging mice, when p16-positive cells, a biomarker of senescent cells, accumulate in the body, the lifespan tends to be shortened, and conversely, removing p16-positive cells may extend the individual's healthy lifespan (Non-Patent Document 3).

[0006] Since these reports, the screening and development of pharmacological compounds that act as senolytic agents, which can selectively eliminate senescent cells in vivo by inducing cell death, has accelerated, and promising compounds have been identified. For example, it has been reported that administering the drug dasatinib in combination with quercetin, a yellow pigment found in onions, eliminates senescent cells (Non-Patent Document 4), and fisetin, a polyphenol abundant in strawberries, has also been shown to have senolytic properties. Other Bcl-2 family inhibitors have also been identified, but some senolytic compounds have severe side effects, necessitating the identification of safe substances with senolytic properties (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2021-112181 [Non-patent literature]

[0008] [Non-Patent Document 1] EMBO Molecular Medicine (2019) Vol.11, No.12, PMID:31746100 [Non-patent document 2] Nature (2011) Vol.479, p.232-236 [Non-patent document 3] Nature (2016) Vol.530, p.184-189 [Non-patent document 4] Aging Cell (2015) Vol.14, p.644-658 [Non-patent document 5] Biotechnology Journal (2010) Vol.5, No.10, p.1050-1059 [Non-patent document 6] Aging (Albany NY)(2021) Vol.13, No.5, p.6375-6405 [Non-Patent Document 7] Nature medicine (2019) Vol.25, No.8, p.1234-1242 [Non-patent document 8] J.Gerontol :Series A (2021) Vol.76, No.11, p.1895-1905 [Non-Patent Document 9] Antioxidants (2024) Vol.13, No.3, p.304 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a safe, side-effect-free anti-aging composition that acts as a senolytic agent that selectively induces cell death in senescent cells. Another problem that the present invention aims to solve is to produce food and beverage products that have a senolytic effect, which is the effect of removing senescent cells, and to provide supplements, food and beverage products, feed, cosmetics, or pharmaceuticals that have an anti-aging effect by reducing the number of senescent cells (senolysis). [Means for solving the problem]

[0010] As a result of intensive research conducted by the inventors to solve the above problems, they discovered that a composition containing fermented barley extract and an organic acid acts as an anti-aging composition with senolytic effects. Furthermore, the present inventors have discovered that cultures and processed products of lactic acid bacteria, such as Lactiplantiobacillus bacteria, cultivated in a medium containing fermented barley extract have a senolytic effect. Based on this discovery, the present inventors have developed the present invention, which relates to an anti-aging composition that can be used as a supplement, food or drink, pharmaceutical, etc.

[0011] The present invention relates to the following anti-aging compositions (1) to (15). (1) An anti-aging composition containing fermented barley extract and an organic acid. (2) The composition according to (1) above, wherein the organic acid is one or more selected from the group consisting of lactic acid, citric acid, malic acid, and acetic acid. (3) An anti-aging composition containing a culture of lactic acid bacteria cultured in a medium containing fermented barley extract, or a processed product thereof. (4) The composition according to (3) above, wherein the lactic acid bacteria are bacteria of the genus Lactiplantiobacillus, Leuconostoc, Lacticaseibacillus, or Limosilactobacillus. (5) The composition according to (4) above, wherein the lactic acid bacteria are bacteria of the species Lactiplantiobacillus plantarum, Lacticaseibacillus casei, or Limosilactobacillus fermentum. (6) The composition according to (5) above, wherein the lactic acid bacterium is Lactipranthiobacillus plantarum SNW0181 strain (NITE AP-04054). (7) The composition described in (3) above, wherein the lactic acid bacteria are lactic acid bacteria having gallic acid decarboxylation activity. (8) An anti-aging composition containing pyrogallol and / or gallic acid. (9) The composition according to (8) above, further comprising an organic acid.

[0012] (10) The anti-aging composition according to any one of (1) to (9) above, which has a senescent cell removing effect. (11) The anti-aging composition according to (10) above, wherein the senescent cell removal effect is based on activation of caspase-3 in senescent cells. (12) The anti-aging composition according to any one of (1) to (9) above, which is for oral or transdermal administration. (13) The anti-aging composition according to (12) above, which has an effect of removing senescent cells and / or an effect of suppressing muscle weakness associated with aging. (14) The anti-aging composition according to any one of (1) to (9) above, which is a preparation, a food or drink, a cosmetic, or a pharmaceutical. (15) The anti-aging composition according to (14) above, which has an effect of removing senescent cells and / or an effect of suppressing muscle weakness associated with aging. [Effects of the Invention]

[0013] According to the anti-aging composition of the present invention, the accumulation of senescent cells in tissues can be suppressed by removing the senescent cells, and diseases and symptoms associated with or caused by SASP factors secreted from senescent cells can be improved and prevented. Furthermore, it is possible to provide supplements, foods and drinks, pharmaceuticals, and feeds that have an anti-aging effect through senolysis and can be safely and easily ingested. [Brief explanation of the drawings]

[0014] [Figure 1] This shows the weight change over the test period in aging model mice that were administered lactic acid-containing fermented barley extract (FBE) via a sonograph for four weeks. The FBE+Lac group is the group administered lactic acid-containing FBE (N=7), and the vehicle group is the group administered distilled water (control) (N=7). [Figure 2] This shows the progress of muscle strength (grip strength) during the test period in the same test as in Figure 1. [Figure 3] This shows the progress of muscle strength (hanging time) during the test period in the same test as in Figure 1. [Figure 4] This shows the change in viable cell count observed under a microscope after adding a candidate substance to progeria model MEF (mouse embryonic fibroblast) cells (Wrn / Recq15 DKO-MEF cells). [Figure 5]This is a graph showing the change in SA-β-Gal activity as a function of fluorescence intensity following the addition of a candidate substance using progeria model MEF cells (Wrn / Recq15 DKO-MEF cells). [Figure 6] Cytotoxicity test results of SNW0181 using normal (WT) MEF cells (mouse embryonic fibroblasts) and C2C12 cells. [Figure 7] 1 is a graph showing the change in cell number resulting from the co-addition of SNW0181 and QVD-OPh, a caspase-3 inhibitor, to progeria model MEF cells. [Figure 8] This shows the cell elimination effect of SNW0181 culture medium on normal human fibroblasts (TIG-1-20) in which cellular senescence was induced by exposure to a DNA damaging agent. [Figure 9] This shows the cell elimination effect of SNW0181 culture medium on senescent cells (Wrn / Recq15 DKO-MEF cells). [Figure 10] This shows the cell removal effect of SNW0181 culture medium on normal cells (MEF cells). [Figure 11] This shows the cell removal effect of Lactiplantiobacillus plantarum NBRC3070 culture medium on senescent cells (Wrn / Recq15 DKO-MEF cells). [Figure 12] This shows the cell removal effect of Lactiplantiobacillus plantarum NBRC3070 culture medium on normal cells (MEF cells). [Figure 13] 1 shows the cell removal effect of various lactic acid bacteria culture solutions on progeria model MEF cells (Wrn / Recq15 DKO-MEF cells). [Figure 14] 1 shows the cell elimination effect of a combination of FBE and various organic acids on progeria model MEF cells (Wrn / Recq15 DKO-MEF cells). [Figure 15] HPLC analysis results showing the gallic acid decarboxylation activity of each lactic acid bacteria strain. [Figure 16]1 shows the cell removal effect of pyrogallol and / or lactic acid on progeria model MEF cells (Wrn / Recq15 DKO-MEF cells) (after 7 days). [Figure 17] 1 shows the cell removal effect of pyrogallol and / or lactic acid on normal cells (MEF cells) (after 7 days). [Figure 18] The results are shown below for omics data on the intestinal microbiota and metabolites in the liver and feces of aged mice that had taken pyrogallol. (a) Using three types of machine learning, we selected features of the intestinal microbiota and metabolites in the liver and feces, and (b) performed cluster classification using exploratory factor analysis. [Figure 19] Based on the factor analysis in Figure 18, (a) the results of structural equation modeling for the selected factor group and (b) the results of differential analysis of the constituent molecule group of the structural equation modeling are shown. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to an anti-aging composition, particularly a composition having the effect of removing senescent cells. Anti-aging refers to the prevention or amelioration of aging, and senolytic refers to the induction of cell death in senescent cells in vivo or in vitro, thereby selectively removing senescent cells from a cell population containing senescent cells.

[0016] As used herein, senescent cells refer to cells in which cellular senescence has been induced, that is, cells that have lost their ability to proliferate and ceased to divide, but which maintain metabolic activity in a viable state and whose apoptosis (cell death) mechanism is frozen. Senescent cells are characterized by their lack of cell division and cell death, their morphology and size differ from normal cells, abnormal nuclear structure, and DNA damage. Cell markers include the specific expression of p16, accumulation of senescence-associated β-galactosidase (SA-β-Gal), and secretion of SASP factors.

[0017] Approximately 70% of patients worldwide with Werner syndrome (WRN), a known hereditary progeria disorder, are Japanese. The present inventors have isolated mouse embryonic fibroblast (MEF) cells (Wrn / Recq15 DKO mice) derived from the Werner syndrome causative gene (wrn), a progeria model mouse. - We succeeded in generating senescent cells (MEF cells), which exhibit all the characteristics of senescent cells, including specific expression of p16, accumulation of SA-β-Gal, expression of SASP genes, division arrest, and DNA damage. Conventionally, test cells used to screen for substances with senolytic effects have been prepared by inducing senescence in normal fibroblasts by exposing them to drugs such as DNA damaging agents for 1 to 2 weeks. However, by using MEF cells derived from these progeria model mice, it has become possible to identify substances with senolytic effects more quickly and accurately, leading to the completion of the present invention.

[0018] The anti-aging composition of the present invention having the effect of removing senescent cells is a composition containing fermented barley extract and an organic acid, a composition containing a culture of lactic acid bacteria cultured in a medium containing fermented barley extract or a processed product thereof, or a composition containing pyrogallol and / or gallic acid, preferably a composition containing pyrogallol and / or gallic acid and an organic acid.

[0019] The fermented barley extract (hereinafter sometimes referred to as "FBE; Fermented Barley Extracts") of the present invention is not particularly limited as long as it is an extract derived from fermented barley. For example, it may be the bottoms after distillation of alcoholic beverages obtained when producing alcoholic beverages such as shochu by fermenting barley. Among fermented barley extracts, the bottoms after distillation of barley shochu are preferred, and commercially available products such as Barlex (Sanwa Shurui Co., Ltd.) and Barlex S (Sanwa Shurui Co., Ltd.) can be used.

[0020] The organic acid of the present invention is not particularly limited, but is preferably lactic acid, citric acid, malic acid, or acetic acid, and is particularly preferably lactic acid. The organic acid includes organic acids present in the human body. The organic acid may also be secreted by bacteria such as lactic acid bacteria, and is contained in a composition containing, for example, a culture of lactic acid bacteria cultured in a medium containing fermented barley extract, or a processed product thereof.

[0021] Examples of the lactic acid bacteria of the present invention include bacteria of the genus Lactiplantiobacillus, Leuconostoc, Lacticaseibacillus, or Limosilactobacillus. Of the lactic acid bacteria of these genera or other lactic acid bacteria, lactic acid bacteria having gallic acid decarboxylation activity are preferably used.

[0022] The genus Lactiplantiobacillus is a lactobacillus that was reclassified from the genus Lactobacillus in 2020. Previously, over 200 species of Lactobacillus were classified into the genus Lactobacillus. As a result of a reevaluation of the genus classification at the genome level, Lactiplantiobacillus is one of eight genera whose names were changed as a result of reclassification. Lactiplantiobacillus plantarum was reclassified from Lactobacillus plantarum. Lactiplantiobacillus plantarum is a lactobacillus isolated from saliva and present in plants and many fermented foods. It is available from the National Institute of Technology and Evaluation (NBRC) Biological Resource Center and the American Type Culture Collection (ATCC) in the United States.

[0023] The lactic acid bacteria of the present invention are preferably Lactiplantiobacillus plantarum, Lacticaseibacillus casei, or Limosilactobacillus fermentum, and specific examples include Lacticaseibacillus casei NBRC15883 and Limosilactobacillus fermentum NBRC3071.

[0024] In the examples of the present specification, the Lactiprantiobacillus plantarum strain SNW0181 is used as Lactiprantiobacillus plantarum. The Lactiprantiobacillus plantarum SNW0181 strain was received on December 21, 2023, at the National Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number NITE AP-04054 and deposited under the accession number NITE P-04054. The Lactipranthiobacillus plantarum SNW0181 strain includes not only the deposited strain itself but also substantially equivalent strains, such as descendants of the deposited strain, such as bred strains and subcultured strains, which have the same mycological properties as the deposited strain.

[0025] In the anti-aging composition of the present invention, a culture of lactic acid bacteria cultured in a medium containing the fermented barley extract or a processed product thereof is used as the medium for bacterial culture, which is a medium containing fermented barley extract and glucose added. When MRS (deMan, Rogosa, Sharpe) medium, which is commonly used for culturing lactic acid bacteria, is used, the senolytic effect is not observed. The lactic acid bacteria culture contained in the composition of the present invention may be, for example, the culture obtained by culturing, or a diluted or concentrated culture, or a culture supernatant or culture fraction. As a processed bacterial culture, a culture obtained by crushing, heating, or freeze-drying, or a fraction or dried product thereof may be used.

[0026] On the other hand, the senolytic effect was not observed when the SNW0181 strain was fermented in FBE medium alone, which lacked lactic acid bacteria, or in media other than FBE, such as MRS medium. Therefore, a search for substances produced by fermenting the SNW0181 strain in FBE medium revealed that pyrogallol has a senolytic effect. Furthermore, cell experiments confirmed that compositions containing pyrogallol and organic acids have a stronger senolytic effect.

[0027] Therefore, the anti-aging composition of the present invention includes a composition containing pyrogallol, preferably a composition containing pyrogallol and an organic acid. Pyrogallol is a compound known to have anti-inflammatory activity and was found to be present in the FBE medium fermentation product of the SNW0181 strain. Furthermore, it has been reported that gallic acid is metabolized to pyrogallol in the small intestine (Non-Patent Document 5), and compositions containing gallic acid are also expected to have a senolytic effect when orally ingested.

[0028] In the present invention, senescent cell removal refers to inducing cell death in senescent cells in vivo or in vitro and selectively removing senescent cells from a cell population containing senescent cells. This senolytic effect, which induces apoptosis in cells that have already become senescent, resulting in their death and removal, does not usually affect normal cells in which senescence has not been induced. Furthermore, by inducing cell death in senescent cells, the composition of the present invention can suppress the secretion of SASP factors by senescent cells, which can damage surrounding tissues.

[0029] The composition of the present invention is expected to improve or prevent aging-related diseases and disorders associated with or caused by SASP factors secreted from senescent cells due to its senescent cell-eliminating effect. Age-related diseases and disorders include, for example, progeria, atherosclerosis, arrhythmia, cardiomyopathy, congestive heart failure, myocardial infarction, coronary artery disease, cerebral aneurysm, stroke, coronary artery thrombosis, hypertension, hyperlipidemia, diabetes, renal glomerulosclerosis, osteoarthritis, arthritis, inflammatory bowel disease, Alzheimer's disease, Parkinson's disease, dementia, mild cognitive impairment, pulmonary fibrosis, interstitial pneumonia, chronic obstructive pulmonary disease, asthma, myelofibrosis, liver fibrosis, macular degeneration, glaucoma, cataracts, skin diseases, cancer, sarcoma, lymphoma, and the like.

[0030] The senescent cell removal effect of the present invention can be achieved, for example, by using MEF cells (Wrn / Recq15 DKO) derived from a progeria model mouse, which is a mouse lacking the Werner syndrome causative gene (wrn). - This can be confirmed by a decrease in the number of viable cells after addition to MEF cells. Alternatively, normal fibroblasts can be treated with a DNA-damaging agent for 1 to 2 weeks to produce cells in which senescence has been induced. Specific expression of p16, accumulation of SA-β-Gal, expression of SASP genes, mitotic arrest, DNA damage, etc. can be detected in these cells. The composition for removing senescent cells of the present invention can then be applied to these cells, and the reduction in the number of viable cells can be confirmed.

[0031] When the composition of the present invention is a composition to be taken orally, it is preferably in the form of a food or drink. The form of the food or drink is not particularly limited, and it may be formed into powder, granules, capsules, or tablets.Other forms include food ingredients, food additives, syrups, suspensions, energy drinks, liquid diets, soft drinks, milk drinks, lactic acid bacteria drinks, functional seasonings, gel foods, puddings, yogurt, confectionery, cakes, breads, noodles, pasta, chocolate, candy, chewing gum, etc. Furthermore, foods and beverages are not limited to those for humans, but also include food for mammals such as dogs and cats kept as pets or livestock. The concept of foods and beverages also encompasses not only ordinary foods and beverages, but also beverages, so-called supplements, health foods, enteral nutritional foods, foods for special dietary uses, foods with nutrient functions, foods for specified health uses, and the like.

[0032] One embodiment of the anti-aging composition of the present invention is preferably used for at least one of pharmaceuticals, beverages, food compositions (supplements), and food additives. When used as pharmaceuticals, examples of oral administration agents include, but are not limited to, powders, granules, capsules, pills, and tablets. The effective dosage of a pharmaceutical composition varies depending on the condition of the subject (including age, physical condition, etc.) and dosage form, but the oral dosage for a human (adult weighing 60 kg) can be set within the above-mentioned range of daily intake for a human. For example, the dosage of the composition of the present invention is 0.5 to 50 g / day for an adult for the invention of fermented barley extract and organic acid or the invention of lactic acid bacteria culture of fermented barley extract, and 0.5 to 5 g / day for the invention of pyrogallol and / or gallic acid.

[0033] When the composition of the present invention is a transdermal composition, it is preferably in the form of a cosmetic or quasi-drug. The form of the cosmetic or quasi-drug is not particularly limited, but may be various forms such as a cream (ointment), a milky lotion (emulsion), a lotion (liquid), a gel, a mousse, a shampoo, or a treatment. Furthermore, when producing these products, bactericidal or antibacterial ingredients, anti-inflammatory agents, and various other active medicinal ingredients may be appropriately blended.

[0034] Next, specific examples of the present invention will be described with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0035] [Test Example 1] Anti-aging effect of the combination of fermented barley extract (FBE) and lactic acid To investigate the anti-aging effect of lactic acid-containing FBE, an in vivo study was conducted using aging model mice. The lactic acid-containing FBE sample was prepared by mixing 15 g / L of lactic acid with FBE (Brix 8), adjusting the pH to 4.5 with 25% NaOH, and then sterilizing it in an autoclave. As test animals, C57BL / 6N mice (7 weeks old) were intraperitoneally transplanted with cultured MEF cells derived from progeria model mice, and used as aging model mice.

[0036] Aging model mice were divided into a distilled water-treated group (Vehicle group, N=7) and a lactic acid-containing FBE-treated group (FBE+Lac group, N=7). The dose was 200 μL per mouse, administered five times a week for four weeks via a sonograph. During the study period, weight, grip strength, and hanging tests were performed to examine the effect of treatment on improving muscle weakness. The results are shown in Figures 1 to 3. These results show that administration of lactic acid-containing FBE significantly increased the grip strength of aging model mice at 2 weeks, and also significantly increased the grip strength at 3 to 4 weeks in the hanging test.

[0037] This animal experiment used an aging model mouse in which senescent cells were transplanted into young mice. Therefore, it is presumed that muscle strength was affected by the increase in muscle mass due to growth up until the second week, and no significant differences were observed in the hanging test. However, from the third week onwards, the vehicle group showed a decrease in muscle strength in the hanging test, whereas the FBE+Lac group showed an increase. This confirmed the anti-aging effect of oral intake of lactic acid-containing FBE, i.e., its ability to suppress the muscle strength loss associated with aging.

[0038] [Preparation of Lactiprantiobacillus culture medium] Lactipranthiobacillus plantarum SNW0181 strain NITE AP-04054 was subcultured in a medium containing fermented barley extract (Brix 4), 1% glucose, and adjusted to pH 7.0. The preculture was inoculated at 1% (V / V) into a medium containing fermented barley extract (Brix 8), 1% glucose, and adjusted to pH 6.0, and cultured at 30°C for 24 hours. The Brix of the culture after culture was 8, the residual glucose concentration was 0.36 g / L, and the pH was 4.1. The culture containing live cells was sterilized by autoclave and then frozen. Hereinafter, the resulting sample will be referred to as "SNW0181 culture." As a negative control, a Brix 8 fermented barley extract with a glucose concentration of 0.36 g / L and a pH of 4.1 was frozen. Hereinafter, this negative control will be referred to as "FBE."

[0039] [Test Example 2] Senescent cell removal effect of SNW0181 culture medium (Part 1) A 24-well plate (BioLite) was filled with high-glucose DMEM medium (NACALAI TESQUE) and MEF cells derived from a progeria model mouse (Wrn / Recq15 DKO) prepared by the present inventors. - A suspension of 10 MEF cells was added. 5 Cells were seeded at 1000 cells / well. SNW0181 culture medium and negative control FBE were added to four wells at a 2% (v / v) solution volume. For comparison, DMSO (dimethyl sulfoxide), an organic solvent that inhibits cell proliferation, was added to four wells of the plate at 0.2% / well (DMSO) as a positive control. Additionally, ABT-263, a Bcl-2 inhibitor and known senolytic agent, was added at 1 μM / well (ABT-263), and Fisetin, a known senolytic agent, was added at 5 μM / well (Fisetin), respectively, to four wells of the plate. The plates were then incubated at 37°C, 5% CO2. For reference, a 24-well plate was similarly filled with a suspension of high-glucose DMEM medium (manufactured by NACALAI TESQUE) and normal (WT) MEF cells (mouse embryonic fibroblasts). 5The cells were seeded at 10 cells / well, and 0.2% DMSO (dimethyl sulfoxide) was added to three wells (WT-DMSO), followed by incubation at 37°C, 5% CO2. The number of cells in each well of the two plates a. 48 hours after the start of culture and b. 7 days after the start of culture was counted using a fluorescence microscope by the following method.

[0040] For fluorescent staining, the medium was removed and the cells were washed with Hanks Buffer (Sigma-Aldrich). Cellular Sensation Detection Kit-SPIDER-β-Gal (Dojindo) and DAPI (Dojindo) were mixed with Hanks Buffer to final concentrations of 0.4 μM and 220 μM, respectively. 300 μL of the staining solution was added per well and incubated at 37°C, 5% CO2, for 30 minutes. After removing the staining solution and washing with Hanks Buffer, 300 μL of 70% ethanol pre-cooled at -30°C was added per well to fix the cells. The cells were then observed and their fluorescence intensity was measured using a fluorescence microscope (BZ-9000, KEYENCE). For DAPI and SA-β-Gal, fluorescent filters were used: DAPI-V (OP-88359, KEYENCE) and TRITC (OP-87764, KEYENCE), respectively. The observed fluorescent images were saved, and the number of DAPI-stained cells was counted using analysis software.

[0041] The results are shown in Figure 4. The vertical axis shows the multiplication factor of the cell number in each sample, assuming that the number of cells in the DMSO well was 1. Forty-eight hours after the addition of a., the cell counts of two known senolytic agents, ABT-263 and Fisetin, were approximately 0.5 and 0.6, respectively, when DMSO was set to 1, while FBE was approximately 0.8 and SNW0181 culture medium was 0.1. Furthermore, 7 days after the addition of b., the cell counts of ABT-263 and Fisetin, when DMSO was set to 1, were approximately 0.9 and 0.7, respectively, while FBE was 0.9 and SNW0181 culture medium was approximately 0.3 (Student's t-test, p-value < 0.05). This indicates that the addition of SNW0181 culture medium significantly reduced senescent cells by decreasing cell counts. These results strongly suggest that the SNW0181 culture medium has a senolytic effect.

[0042] [Test Example 3] Senescent cell removal effect of SNW0181 culture medium (Part 2) In Test Example 2, the change in cell number of MEF cells derived from a progeria model mouse was used as an indicator of the senolytic effect, while in Test Example 3, the change in senescence-associated β-galactosidase (SA-β-Gal) activity was used as an indicator. In Test Example 3, the same samples as in Test Example 2 were used, and the wells containing each sample were cultured under the same conditions at 37°C, 5% CO2. Changes in SA-β-Gal activity in each well of the two plates were detected using a fluorescence microscope a. 48 hours and b. 7 days after the start of culture. After 48 hours and 7 days of culture, cells were stained with senescence-associated acid β-galactosidase (SA-β-Gal), which specifically stains senescent cells, using the Cellular Senescence Detection Kit-SPIDER-β-Gal (Dojindo) and DAPI. SA-β-Gal stained cells were observed under a fluorescence microscope (BZ-9000, KEYENCE), and the fluorescence intensity was measured.

[0043] The results are shown in Figure 5. The vertical axis shows the magnification of the fluorescence intensity of each sample when the fluorescence intensity in the DMSO well was set to 1. Forty-eight hours after the addition of a., the SA-β-Gal activity of two known senolytic agents, ABT-263 and Fisetin, was approximately 0.3 and 0.5, respectively, when DMSO was taken as 1, whereas that of FBE was approximately 0.7 and that of SNW0181 culture medium was approximately 0.3. Seven days after the addition of b., the SA-β-Gal activity of ABT-263 and Fisetin, when DMSO was taken as 1, was approximately 0.5 and 0.6, respectively, whereas that of FBE was approximately 1.1 and that of SNW0181 culture medium was 0.4 (Student's t-test, p-value < 0.05). This indicates that the addition of SNW0181 culture medium significantly reduced senescent cells through a decrease in SA-β-Gal activity. As described above, the results of cell count measurement in Test Example 2 and SA-β-Gal activity measurement in Test Example 3 demonstrated that the SNW0181 culture medium has the effect of removing senescent cells.

[0044] [Test Example 4] Evaluation of cytotoxicity (toxicity) against normal cells The cytotoxicity of SNW0181 culture medium was evaluated using normal (WT) MEF cells (mouse embryonic fibroblasts) and C2C12 cells (a mouse immortalized striated muscle cell line). Cell suspensions of normal MEF cells and C2C12 cells were prepared using high-glucose DMEM medium (NACALAI TESQUE), and 10 cells were plated in separate 24-well plates (BioLite). 5 The cells were seeded at 10 cells / well. After 24 hours, SNW0181 culture medium was added to each well at a concentration of 0% (no addition), 0.25%, 0.75%, 1%, 1.5%, or 2% (v / v) and cultured for 7 days. After 7 days of culture, 300 μL of crystal violet staining solution was added to each well and the cells were shaken for 30 minutes to stain only live cells. After gently rinsing with running water to remove the staining solution, the absorbance (OD ) of each sample at 590 nm was measured using an absorbance meter (Molecular Devices). 590 ) was measured and compared with the value when SNW0181 culture medium was not added to determine cytotoxicity.

[0045] The results are shown in Figure 6. The absorbance of normal MEF cells and C2C12 cells without SNW0181 culture medium and the absorbance of each cell type with varying concentrations of SNW0181 culture medium added decreased slightly depending on the amount of SNW0181 culture medium added, but there was no significant difference compared to the no-addition group, so cytotoxicity (toxicity) against the two types of normal cells was not observed. It was estimated that the addition of 2% SNW0181 culture medium reduced the number of viable cells by approximately 20% in normal MEF cells and by approximately 10% in C2C12 cells. When this was compared with the senescent cell-eliminating effect in Test Examples 2 and 3, which also added 2% SNW0181 culture medium, the senescent cell-eliminating effect on senescent cells in Test Examples 2 and 3 was several times greater than the reduction in normal cells, confirming the senescent cell-eliminating effect of SNW0181 culture medium.

[0046] Furthermore, a toxicity test of the SNW0181 strain was conducted using the following method to confirm its safety. 1.Acute oral toxicity test A single oral administration of 2000 mg / kg of the bacterial cells was administered to male and female mice, and they were observed for 14 days. No abnormalities or deaths were observed during the observation period. 2. Reverse mutation test Tests were conducted using bacteria (Salmonella tryphimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA) under conditions without and with metabolic activation. Test doses were set at 2500, 1250, 625, 313, and 156 μg / plate, with a maximum dose of 5000 μg / plate. As a result, for both the non-metabolically activated and metabolically activated strains, there was no test dose that showed a mean number of revertant colonies that was more than twice that of the negative control group.

[0047] [Test Example 5] Senolytic effect of SNW0181 culture medium with the addition of a caspase-3 inhibitor The final stage of apoptosis requires the action of caspase-3. Therefore, we added the caspase-3 inhibitor QVD-OPh together with the SNW0181 culture medium to MEF cells, a model of progeria, and examined the changes in cell number. Similarly, using the same sample as in Test Example 2, MEF cells derived from a progeria model mouse (Wrn / Recq15 DKO) were cultured in a 24-well plate (BioLite). - MEF cells) at 10 5 The cells were seeded at 10 cells / well. SNW0181 culture medium was added to 18 wells, and FBE was added to 9 wells, each at a solution volume of 2% (v / v). QVD-OPh was further added to 9 of the 18 wells to which SNW0181 culture medium was added at 10 μM / well, and the plate was cultured under conditions of 37°C, 5% CO2. After 24 hours of culture, the number of cells in each well was counted using a fluorescence microscope using the same method as in Test Example 2.

[0048] The results are shown in Figure 7. When FBE was taken as 1, the cell number was approximately 0.4 in the SNW0181 culture medium and approximately 0.7 in the SNW0181 culture medium and QVD-OPh co-addition culture. The effect of 2% (v / v) SNW0181 culture medium on the progeria model MEF cells was attenuated by the co-addition of a caspase-3 inhibitor. These results confirmed that adding SNW0181 culture medium to the culture medium of progeria model MEF cells activates caspase-3 and that SNW0181 culture medium promotes the induction of apoptosis in progeria model MEF cells.

[0049] [Test Example 6] Senolytic effect of SNW0181 culture medium on normal human fibroblasts (TIG-1-20) induced to undergo cellular senescence by exposure to DNA damaging agents Normal human fibroblasts (TIG-1-20) were treated with etoposide, a DNA damaging agent, for two weeks to induce cellular senescence. 10 mL of high-glucose DMEM medium (manufactured by NACALAI TESQUE) was dispensed into a 10 cm cell culture dish (manufactured by BioLite), and etoposide was added to a concentration of 100 μM. Normal human fibroblasts (provided by the JCRB Cell Bank) were then cultured at a concentration of 4.0 × 10 5 The cells were seeded and cultured for 2 weeks at 37°C in 5% CO2. After incubation, etoposide-treated human fibroblasts were plated in 24-well plates at 10 4 The cells were seeded at 100 cells / well and cultured for 24 hours at 37°C in 5% CO. SNW0181 culture medium or FBE was added to a solution volume of 2% (v / v), and the cells were cultured for 48 hours at 37°C in 5% CO. After 48 hours, the number of cells in each well was counted using a fluorescence microscope using the same method as in Test Example 2.

[0050] The results are shown in Figure 8. The cell number for SNW0181 was approximately 0.4, assuming that FBE was 1. Similar to the results of Test Example 2 on progeria model MEF cells, the SNW0181 culture medium also exerted a senolytic effect on drug (etoposide)-induced human senescent cells.

[0051] [Test Example 7] Senolytic effect of FBE lactic acid bacteria culture solution or combination of FBE and organic acids (1) Preparation of lactic acid bacteria culture samples Strain used Lactiplantiobacillus plantarum SNW0181 Lactiplantiobacillus plantarum NBRC3070 Lactococcus lactis ATCC11454 Leuconostoc mesenteroides subsp.dextranicum NBRC3349 Lacticaseibacillus casei NBRC15883 Limosilactobacillus fermentum NBRC3071

[0052] After refreshing the frozen stock with MRS medium, the strain was inoculated at 1% (v / v) into a test tube containing FBE medium (Brix 4, 1% glucose, pH 7.0, 5 mL / tube) and allowed to stand at 30°C for 16 hours for pre-culture. Then, 40 mL of FBE medium (Brix 4, 1% glucose, pH 6.0) was prepared in a 300 mL Erlenmeyer flask, and the pre-culture solution was inoculated at 1% (v / v) and cultured at 30°C for 24 hours at 100 rpm. After culture, the strain was autoclaved and used for testing. To test the cells or supernatant of the SNW0181 culture medium, 100 μL of the above culture medium was centrifuged (15,000 rpm, 5 minutes), and the supernatant was used, while the precipitate was suspended in 100 μL of PBS(-).

[0053] (2) Organic acid sample preparation To FBE-Brix 8, pH 6.0, 15 g / L (w / v) of organic acid (lactic acid, citric acid monohydrate, malic acid, acetic acid) was added, and the pH was adjusted to 4.1 with 25% NaOH aqueous solution.

[0054] (3) Senolysis test A 24-well plate (TPP) was filled with high-glucose DMEM medium (Fujifilm Wako) and MEF cells derived from a progeria model mouse (Wrn / Recq15 DKO - A suspension of 1 x 10 MEF cells 5 Cells were seeded at 100 cells / well and cultured at 37°C in 5% CO2. Samples were added to six wells at a 2% (v / v) solution volume. A control was used without sample. Similarly, a 24-well plate was filled with high-glucose DMEM medium (Fujifilm Wako) and a suspension of normal (WT) MEF cells (mouse embryonic fibroblasts) at 1 × 10 5 The cells were seeded at 10 cells / well and cultured at 37°C in 5% CO2. The number of viable cells in each well was counted using trypan blue 48 hours after the start of culture as follows: First, the culture supernatant was removed from each well and washed with 500 μL of PBS(-). Then, 200 μL of 0.1% trypsin (Gibco) was added, and the wells were incubated at 37°C for 5 minutes. The cells were then detached by pipetting. The detached solution was mixed with an equal volume of trypan blue solution, and the number of viable cells was counted using an automated cell counter (Countess, Invitrogen).

[0055] (4) Identification of the senolytic active fraction in the SNW0181 culture medium The results are shown in Figures 9 and 10. The vertical axis shows the ratio of the cell number of each sample to the control cell number, which is set to 1. Figure 9 shows senescent cells (Wrn / Recq15 DKO - Figure 10 shows the results of investigating whether the killing effect of SNW0181 culture medium on normal cells (MEF cells) is due to the supernatant or the bacterial cells. The results showed that the number of viable cells significantly decreased when culture medium supernatant was added compared to the control, but there was no significant difference in the bacterial cells. On the other hand, no killing effect was observed in normal cells with either fraction. These results indicate that the senolytic effect of SNW0181 culture medium is due to the culture medium supernatant.

[0056] (5) Senolytic effect of the culture medium of the same microorganism (Lactiplantiobacillus plantarum NBRC3070) We investigated whether a senolytic effect could be observed in the FBE culture medium of Lactiplantiobacillus plantarum NBRC3070, the same species as SNW0181. The results are shown in Figures 11 and 12. The vertical axis shows the multiplication of the cell number of each sample when the control cell number is set to 1. Figure 11 shows the results of examining the killing effect of NBRC3070 culture medium on senescent cells, and Figure 12 shows the results of examining the killing effect of NBRC3070 culture medium on normal cells. As a result, NBRC3070 culture medium had no killing effect on either cell. These results suggest that the activity differs depending on the bacterial species, as the culture medium of NBRC3070, which is the same species as SNW0181, did not have a senolytic effect.

[0057] (6) Senolytic effect of various lactic acid bacteria cultures We investigated whether the senolytic effect was observed in the FBE culture medium of various lactic acid bacteria. The results are shown in Figure 13. The vertical axis shows the multiplication of the cell number of each sample when the cell number of the control was set to 1. The results showed that the number of viable cells significantly decreased when culture media from Lacticaseibacillus casei NBRC15883 and Limosilactobacillus fermentum NBRC3071 were added compared to the control group, indicating that the culture media from these two strains have a senolytic effect. Lacticaseibacillus casei NBRC15883 and Limosilactobacillus fermentum NBRC3071 are lactic acid bacteria that have gallic acid decarboxylation activity.

[0058] (7) Senolytic effect of the combination of FBE and organic acids We investigated whether the senolytic effect could be observed in FBE medium alone, adjusted to pH 4.5 with organic acids. The results are shown in Figure 14. The vertical axis shows the multiplication of the cell count for each sample, with the control cell count set at 1. As a result, when a combination of FBE and lactic acid, citric acid, malic acid, or acetic acid was added, the number of viable cells was significantly reduced compared to the control group.

[0059] (8) Discussion The senolytic effect was observed in the supernatant of the SNW0181 culture medium (Figures 9 and 10), suggesting that the secondary metabolic products of SNW0181 or substances produced by extracellular enzymes may be the relevant components. We investigated whether the senolytic effect was also present in the culture medium of the same species of lactic acid bacteria (NBRC3070) or lactic acid bacteria of a different genera, and confirmed whether the activity varied depending on the bacterial species (Figures 11 to 13), so we conducted the experiment in Test Example 8. On the other hand, the senolytic effect was observed even when the FBE medium was adjusted to pH 4.5 with an organic acid such as lactic acid (Figure 14). In preliminary tests, the senolytic effect was not observed when the pH of the FBE medium was adjusted with hydrochloric acid. Therefore, in the composition of the present invention containing FBE and an organic acid, it is thought that the senolytic effect is exerted by the combination of some component in the FBE and an organic acid such as lactic acid.

[0060] [Test Example 8] Gallic acid decarboxylation activity of each lactic acid bacteria strain A comprehensive analysis of the SNW0181 culture medium was performed to examine the increase in phenolic compounds before and after incubation. The results showed that pyrogallol increased approximately 8.5-fold (0.36 μmol / L) compared to before incubation, suggesting that it may be one of the components involved. Pyrogallol is produced by decarboxylation of gallic acid, and some lactic acid bacteria are known to possess gallic acid decarboxylase. Furthermore, barley contains bound gallic acid, and gallic acid liberated by koji tannase is thought to be present in shochu lees. In this study, the gallic acid decarboxylation activity of the six strains used in the senolysis test in Test Example 7 was examined to investigate the relationship between pyrogallol production and senolysis.

[0061] The lactic acid bacteria shown in Test Example 7(1) above were cultured in an FBE medium containing gallic acid, and the gallic acid decarboxylation activity was confirmed based on the presence or absence of a pyrogallol peak after the culture. First, the bacteria were inoculated into a pre-culture medium (FBE medium (Brix 4, 1% glucose, pH 7.0)) and cultured statically at 30°C for 16 hours. Next, the pre-culture was inoculated into a 1% main culture medium (FBE medium (Brix 8, 1% glucose, 1 mg / L gallic acid monohydrate, pH 6.0)) and cultured at 30°C for 24 hours at 100 rpm, followed by sterilization at 121°C for 15 minutes. Gallic acid and pyrogallol in the culture medium were then measured by HPLC. The HPLC conditions were as follows: HPLC conditions Eluent A: 0.1% TFA in H2O, Eluent B: 0.1% TFA in AcCN Column: Unison UK-C18, 4.6 x 250 mm (Imtakt) Elution method: Isocratic B concentration 5% 10 min Column temperature: 40℃ Detection: 210nm

[0062] The results of the HPLC analysis are shown in FIG. Of the six lactic acid bacteria tested for gallic acid decarboxylation activity, the culture media of L. plantarum SNW0181, L. casei NBRC15883, and L. fermentum NBRC3071 showed a decrease in gallic acid before incubation and a pyrogallol peak was detected, suggesting that these strains possess gallic acid decarboxylation activity. All of these strains exhibited a senolytic effect, suggesting that the combination of lactic acid and pyrogallol may exhibit the senolytic effect.

[0063] [Test Example 9] Senolytic effect of the combination of pyrogallol and organic acids A 24-well plate (TPP) was filled with high-glucose DMEM medium (Fujifilm Wako) and MEF cells derived from a progeria model mouse (Wrn / Recq15 DKO - A suspension of 1 x 10 MEF cells 5 Cells were seeded at 1000 cells / well and cultured at 37°C in 5% CO2. Samples were added to five wells at a 2% (v / v) solution volume. A control was used without sample. Similarly, a 24-well plate was filled with high-glucose DMEM medium (Fujifilm Wako) and a suspension of normal (WT) MEF cells (mouse embryonic fibroblasts) at 1 × 10 5 The cells were seeded at 10 cells / well and cultured at 37°C in 5% CO2. Seven days after the start of culture, the number of viable cells in each well was counted using trypan blue as follows. First, the culture supernatant was removed from each well and washed with 500 μL of PBS(-). Then, 200 μL of 0.1% trypsin (Gibco) was added, and the wells were incubated at 37°C for 5 minutes. The cells were then detached by pipetting. The detached solution was mixed with an equal volume of trypan blue solution, and the number of viable cells was counted using an automated cell counter (Countess, Invitrogen). The entire data was analyzed.

[0064] The results for senescent cells are shown in Figure 16, and the results for normal cells are shown in Figure 17. The vertical axis shows the multiplication factor of the number of viable cells in each sample when the number of viable cells in the control is set to 1. When pyrogallol was added alone, the viable cell count was 0.83, while when pyrogallol and lactic acid were added, the viable cell count was 0.77. This suggests that the senolytic effect was observed with pyrogallol alone, but that the addition of pyrogallol and lactic acid enhanced the senolytic effect. However, the senolytic effect was not observed with lactic acid alone.

[0065] [Test Example 10] Effect of pyrogallol intake on aged mice Test Example 7 revealed that a sample containing fermented barley extract (FBE) and organic acids has a senolytic effect in an in vitro test using senescent cells, and Test Example 9 suggested that pyrogallol is an important candidate molecule derived from FBE as a functional molecule for aging control. Test Example 10 evaluated the functionality of pyrogallol in an in vivo system using aged mice.

[0066] C57BL / 6 aged mice (23-29 months old) and young mice (16 weeks old) were used as test animals. The aged mice were divided into a water-treated group (10 males and 10 females) and a pyrogallol-treated group (9 females and 10 males), while the young mice (8 males and 8 females) were assigned to the water-treated group. The water-treated group received 10 mL / g BW and the pyrogallol group received 40 mg / g BW, administered five times weekly for 4 weeks. After the feeding experiment, the mice were dissected, and their livers and cecal feces were collected. Metabolic analysis of the liver and cecal feces was performed using triple quadrupole GC-MS. Additionally, the cecal feces were comprehensively analyzed for bacterial flora using meta-16S analysis, and these omics data were used for analysis.

[0067] Using the omics data, we first selected features using three types of machine learning (association analysis, random forest, and XGBoost), and then clustered the data using exploratory factor analysis (Figure 18(a) and (b)). As a result, we were able to select the intestinal microbiota and metabolites in the liver and feces that were strongly associated with the pyrogallol intake group. Structural equation modeling was performed on these selected factor groups, and computationally demonstrated that the increase in Akkermansia species, fecal ascorbic acid, and liver orotic acid in aged mice showed optimal values ​​as a causal structure group that could be controlled by pyrogallol administration (Figure 19(a) and (b)).

[0068] The Akkermansia genus has been reported to have various anti-aging effects. For example, oral administration of Akkermansia muciniphila to mice has been shown to increase polyamines, which have been reported to have anti-aging effects, in the intestines and liver (Non-Patent Document 6), and fecal transplantation of Akkermansia muciniphila into progeria mice has been reported to extend lifespan (Non-Patent Document 7). It has also been reported that administration of dasatinib and quercetin, which have senolytic effects, reduces intestinal senescent cells and increases Akkermansia in aged mice (Non-Patent Document 8), suggesting that pyrogallol administration exerts a senolytic effect, resulting in an increase in Akkermansia.

[0069] Furthermore, it has been reported that the relative abundance of Akkermansia muciniphila in the intestine and the amount of reactive oxygen species show a significant negative correlation (Non-Patent Document 9). Therefore, it is expected that an increase in ascorbic acid in feces will increase the antioxidant capacity in the intestine, potentially bringing about a positive change in the growth environment of the Akkermansia genus. Furthermore, orotic acid is biosynthesized in mitochondria, and its biosynthesis decreases with age-related decline in mitochondrial function. Therefore, an increase in orotic acid in the liver is thought to be data suggesting an improvement in mitochondrial function with age. These results indicate that pyrogallol can affect the intestinal microbiota, liver and fecal metabolites, suggesting a senolytic effect of pyrogallol in vivo.

Claims

1. An anti-aging composition containing fermented barley extract and an organic acid.

2. The composition according to claim 1, wherein the organic acid is one or more selected from the group consisting of lactic acid, citric acid, malic acid, and acetic acid.

3. An anti-aging composition comprising a culture of lactic acid bacteria cultured in a medium containing fermented barley extract, or a processed product thereof.

4. 4. The composition of claim 3, wherein the lactic acid bacteria are bacteria of the genus Lactiplantiobacillus, Leuconostoc, Lacticaseibacillus, or Limosilactobacillus.

5. 5. The composition according to claim 4, wherein the lactic acid bacteria are bacteria of the species Lactiplantiobacillus plantarum, Lacticaseibacillus casei, or Limosilactobacillus fermentum.

6. The composition according to claim 5, wherein the lactic acid bacterium is Lactipranthobacillus plantarum SNW0181 strain (NITE AP-04054).

7. The composition according to claim 3 , wherein the lactic acid bacteria are lactic acid bacteria having gallic acid decarboxylation activity.

8. An anti-aging composition containing pyrogallol and / or gallic acid.

9. The composition of claim 8 further comprising an organic acid.

10. 10. The anti-aging composition according to claim 1, which has an effect of removing senescent cells.

11. The anti-aging composition according to claim 10, wherein the senescent cell-eliminating effect is based on activation of caspase-3 in senescent cells.

12. 10. The anti-aging composition according to claim 1, which is for oral or transdermal administration.

13. The anti-aging composition according to claim 12, which has an effect of removing senescent cells and / or an effect of suppressing muscle weakness associated with aging.

14. The anti-aging composition according to any one of claims 1 to 9, which is a preparation, a food or drink, a cosmetic, or a pharmaceutical.

15. The anti-aging composition according to claim 14, which has an effect of removing senescent cells and / or an effect of suppressing muscle weakness associated with aging.

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