eNAMPT increasing agent, sirtuin activator or expression enhancer, NAD+ increasing agent, and senescent cell inhibitor.

Abstract

Provided is a novel use of a composition that contains S-1-propenyl cysteine and / or a salt thereof.　The present invention can be an eNAMPT increasing agent in which S-1-propenyl cysteine or a salt thereof is an active ingredient. The present invention can be an eNAMPT increasing agent that contains S-1-propenyl cysteine or a salt thereof and that is administered to an animal. Particularly, it is preferable that the present invention be used for increasing eNAMPT in blood.

Description

[Technical Field]

[0001] This invention relates to increasing eNAMPT, activating or enhancing the expression of sirtuins, and NAD + This relates to agents used to increase and suppress senescent cells. [Background technology]

[0002] Sirtuins are known molecules in which high expression extends lifespan in nematodes, flies, and mammals, and lifespan is shortened when they are deficient. Sirtuins are NAD + Sirtuins are sirtuin-dependent deacetylases, and their genes are widely conserved from bacteria to eukaryotes. Sirtuins are attracting attention as candidate longevity genes in animals because deleting Sir2, a sirtuin homolog in yeast and nematodes, shortens lifespan, while overexpression extends it. Mammals have seven sirtuins (SIRT1-7), and among them, SIRT1, which is most similar in structure and function to yeast Sir2, has been shown to be involved in the regulation of a wide range of cellular functions, including gene expression associated with aging, intracellular metabolism, energy consumption, inflammation, and stress response pathways (Non-Patent Literature 1). Furthermore, in recent years, the potential relationship between sirtuins and certain diseases has also been revealed. For example, Non-Patent Document 2 describes how SIRT1 knockout mice exhibited impaired cognitive functions, including immediate memory, classical conditioning, and spatial learning. Conversely, SIRT1 overexpression was found to result in regular synaptic plasticity and memory. This indicates that SIRT1 plays a role in normal learning, memory, and synaptic plasticity. In addition, Non-Patent Document 3 describes a relationship between SIRT1 and hepatic steatohepatism, and Non-Patent Document 4 describes a relationship with cardiac function.

[0003] Nicotinamide mononucleotide (NAD + ) functions in vivo as a coenzyme for dehydrogenases and a substrate for sirtuins. Also, NAD +An increase in the amount is known to increase sirtuin activity. NAD synthesis is regulated by nicotinamide phosphoribosyltransferase (NAMPT). Since NAMPT function declines with age, NAD + It is known that the amount decreases and the activity of sirtuins declines. Non-patent document 5 mentions NAD + It has been disclosed that administration of nicotinamide riboside, a precursor of NAD, suppresses cellular senescence in the brain. + Regarding this, Non-Patent Document 6 describes the relationship with non-alcoholic fatty liver disease, and Non-Patent Document 7 describes the relationship with renal impairment.

[0004] Cellular senescence is a phenomenon in which excessive DNA damage occurs through the accumulation of DNA replication errors during cell division, oxidative stress, radiation, and activation of oncogenes, leading to the activation of the p16 / RB pathway and p53 / p21 pathway, and the induction of cyclin inhibitor kinase inhibitors, resulting in irreversible cell cycle arrest. Cells that have undergone senescence (senescent cells) accumulate in tissues due to the acceleration of cellular senescence and decreased removal ability caused by the decline in mitochondrial and immune function associated with aging. Senescent cells accumulated in tissues secrete inflammatory cytokines and proteases called senescence-related secretory factors, which damage surrounding tissues and accelerate the aging of tissues and the body. On the other hand, it has been suggested that the removal of senescent cells prevents or delays tissue dysfunction and suppresses aging (Non-Patent Literature 8). Furthermore, it has been revealed that an increase in senescent cells is involved in kidney damage (Non-Patent Literature 9) and fatty liver (Non-Patent Literature 10).

[0005] On the other hand, S-1-propenylcysteine ​​is a cysteine ​​derivative represented by the following formula (1), and is one of the sulfur-containing components that can be found in Allium plants such as garlic. [ka]

[0006] S-1-propenyl cysteine has been reported to have pharmacological effects such as immunomodulatory effects (Patent Document 1), blood pressure lowering effects (Patent Document 2), antioxidant effects (Patent Document 3), blood flow improving effects (Patent Document 4), autophagy activating effects (Patent Document 5), and periodontal disease prevention, treatment or improving effects (Patent Document 6). However, regarding the increase in eNAMPT, activation of sirtuin, suppression of senescent cells, and + increase in NAD, nothing is known at all.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-patent Document 6

Non-patent Document 7

Non-patent Document 8

Non-patent Document 9

Non-patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention relates to a novel use of S-1-propenylcysteine, a salt thereof, or a composition containing these.

Means for Solving the Problems

[0010] The present invention relates to the following 1) to 11). 1) An eNAMPT increasing agent containing S-1-propenylcysteine or a salt thereof as an active ingredient. 2) An eNAMPT increasing agent containing S-1-propenylcysteine or a salt thereof and administered to an animal. 3) The agent according to 2) used for increasing eNAMPT in blood. 4) An agent containing S-1-propenylcysteine or a salt thereof, administered to an animal, and used for activating or enhancing the expression of sirtuin or suppressing senescent cells in one or more organs. 5) An agent containing S-1-propenylcysteine or a salt thereof, administered to an animal, and used for increasing NAD + in one or more organs. 6) The agent according to 4) or 5), wherein the organ is one or more selected from the group consisting of the brain, kidneys, skeletal muscles, liver, and heart. 7) The agent according to 6), wherein the organ is one or more selected from the group consisting of skeletal muscle, liver, and heart. 8) The agent according to 7), further comprising one or more organs selected from the group consisting of the brain and the kidneys. 9) An agent described in any one of items 4) to 8) for acting on two or more organs. 10) The agent described in 9) for acting on three or more organs. 11) An agent described in any one of items 1) to 10), which is in the form of a food additive or food. [Effects of the Invention]

[0011] According to the present invention, novel uses for S-1-propenylcysteine, its salts, or compositions containing these can be provided. [Brief explanation of the drawing]

[0012] [Figure 1] S-1-propenylcysteine ​​administration exhibits sirtuin activation in the cerebral cortex. [Figure 2] This shows the change in SIRT1 protein levels in the hippocampus after administration of S-1-propenylcysteine. [Figure 3] This shows the change in NAD+ levels in the hippocampus after administration of S-1-propenylcysteine. [Figure 4] This shows the changes in the p53 protein, a senescent cell marker, in the hippocampus following administration of S-1-propenylcysteine. [Figure 5] This shows the change in renal SIRT1 expression levels after administration of S-1-propenylcysteine. [Figure 6] This shows the changes in the expression levels of the senescent cell markers p16 and p21 genes in the kidney following administration of S-1-propenylcysteine. [Figure 7]This shows the changes in KIM-1 and NGAL gene protein levels, which are markers of renal impairment in the kidneys, following the administration of S-1-propenylcysteine. [Figure 8] This study demonstrates the maintenance of cognitive function and memory through the administration of S-1-propenylcysteine. [Figure 9] This shows the changes in NAD+ levels in the kidneys, skeletal muscle, and liver following the administration of S-1-propenylcysteine. [Figure 10] This shows the changes in SIRT1 protein levels in the cerebral cortex, hippocampus, heart, lungs, liver, kidneys, and skeletal muscle following administration of S-1-propenylcysteine. [Figure 11] This shows the change in SIRT1 protein levels in the hypothalamus following administration of S-1-propenylcysteine. [Figure 12] This shows the change in blood eNAMPT levels after administration of S-1-propenylcysteine. [Figure 13] This shows the changes in SIRT1 protein levels in the liver and heart following administration of S-1-propenylcysteine. [Modes for carrying out the invention]

[0013] <agent> S-1-propenylcysteine ​​exists in cis or trans stereoconfigurations, as shown by the wavy line in formula (1) below. In the present invention, it is preferable that S-1-propenylcysteine ​​has a high proportion of the trans isomer, and it is more preferable that the proportion of the trans isomer is 50-100%, 75-100%, 80-100%, and even more preferable that it is 90-100%, when the sum of the trans and cis isomers is 100%. Furthermore, because an asymmetric carbon is present in the cysteine ​​structure, optical isomers exist, which may be the D-form, L-form, or racemic mixture. [ka]

[0014] Salts of S-1-propenylcysteine ​​are physiologically acceptable salts and may be either acid addition salts or base addition salts. Examples of acid addition salts include (a) salts with mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; (b) salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, fumaric acid, gluconic acid, malic acid, succinic acid, tartaric acid, trichloroacetic acid, and trifluoroacetic acid; and (c) salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Examples of base addition salts include (a) salts with alkali metals such as sodium and potassium. Examples include salts of (b) calcium, magnesium, and other alkaline earth metals, (c) ammonium salts, and (d) salts of nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-efenamine, and N,N′-dibenzylethylenediamine.

[0015] Furthermore, S-1-propenylcysteine ​​or its salts can exist not only in an unsolvated form, but also as hydrates and solvates, and such hydrates or solvates can exist in any crystalline form depending on the manufacturing conditions. Therefore, S-1-propenylcysteine ​​or its salts in the present invention encompass all stereoisomers, hydrates, solvates, and all polymorphic or amorphous crystalline forms.

[0016] S-1-propenylcysteine ​​or its salts can be obtained by organic synthesis ([1] Tetrahedron Letter, 1975, 37, 3201-3202; [2] Synthesis, 2006, 20, 3367-3369; [3] Bull. Korean Chem. Soc. 2011, 32(1), 319-320). Furthermore, S-1-propenylcysteine ​​or its salts can be obtained by processing Allium plants in various ways. For example, there are methods for obtaining S-1-propenylcysteine ​​by treating heat-treated Allium plants with an enzyme containing protease or lactase derived from Bacillus subtilis (Japanese Patent Publication No. 6105871), methods for obtaining S-1-propenylcysteine ​​by co-administering gamma-glutamyl transpeptidase or glutaminase activity to the juice or extract of Allium plants (Japanese Patent Publication No. 2015-144571), methods for obtaining S-1-propenylcysteine ​​by inoculating garlic with yeast and fermenting it (Korean Patent No. 2010-072874-5), and methods for obtaining S-1-propenylcysteine ​​by aging Allium plants in an ethanol aqueous solution for more than one month (International Publication No. 2016 / 088892, Molecules. 2017;22:570.).

[0017] Examples of Allium species include garlic (Allium sativum L.), onion (Allium cepa L.), elephant garlic (Allium ampeloprasum L.), chives (Allium tuberosum), and leeks (Allium fistulosum L.). These plants may be used individually or in combination. The above Allium species can be used raw, or, if necessary, with the outer skin removed and cut or shredded, or as a powder, or extracted using a solvent suitable for pharmaceutical or food production. Examples of solvents include water, alcohol, or solvents to which acids or basic substances are added.

[0018] The S-1-propenylcysteine ​​or salt thereof of the present invention can be used not only as an isolated and purified product, but also as a crude product, the above-mentioned plant or a processed product thereof, or a fraction in which the content of S-1-propenylcysteine ​​or salt thereof has been increased by extraction from the above-mentioned plant.

[0019] In the present invention, S-1-propenylcysteine ​​or a salt thereof may be included in an amount of 1 ppm or more, preferably 10 ppm or more, 100 ppm or more, 500 ppm or more, or 1000 ppm or more on a dry basis. The upper limit of the content is not particularly limited, but may be 100,000 ppm or less, 50,000 ppm or less, or 10,000 ppm or less.

[0020] For example, as shown below, it can be obtained by 1) extracting Allium plants in a 10-50% ethanol aqueous solution at 0-80°C for more than one month (Step 1), and 2) separating the obtained extract into solid and liquid components and recovering the ethanol-eluted fraction (Step 2).

[0021] The ethanol aqueous solution used in step 1) can be a 10-50% ethanol aqueous solution, but is preferably prepared to an ethanol concentration of 20-40%. The processing temperature can be set in the range of 0-80°C, but is preferably 10-60°C, and more preferably 20-40°C. The processing period can be at least one month for extraction under the above conditions, but is preferably 1-20 months, and more preferably 1-10 months. Furthermore, considering hygiene and the volatilization of ethanol, this step can be carried out in an airtight, sealed, or airtight container, but the use of an airtight container is preferred.

[0022] In step 2), the extract obtained in step 1) is subjected to solid-liquid separation, and the ethanol-eluted fraction is recovered. By appropriately concentrating the recovered material, an extract fraction containing S-1-propenylcysteine ​​or its salt can be obtained. This extract fraction can be used as is, but it can also be dried by spray drying or other means as appropriate before use.

[0023] Furthermore, the isolation of S-1-propenylcysteine ​​or its salt from the extract fraction containing the above-mentioned S-1-propenylcysteine ​​or its salt can be performed by dialysis using a dialysis membrane with a molecular exclusion size of 3000 to 4000 as needed, followed by appropriate combination of separation and purification methods such as adsorption and separation using a cation exchange resin, normal-phase chromatography, or reverse-phase chromatography. Here, adsorption and separation using cation exchange resins can be performed by adsorbing the material onto a cation exchange resin (for example, Amberlite (Dow Chemical), DOWEX (Dow Chemical), DIAION (Mitsubishi Chemical), etc.) and then eluting it with 0.1-3N aqueous ammonia. Normal-phase chromatography can be performed, for example, by using a silica gel column and eluting with a chloroform / methanol / water mixture. Reverse-phase chromatography can be performed, for example, by using an octadecylsilyl column and eluting with a 0.01-3% formic acid aqueous solution.

[0024] Preferably, the ethanol extract fraction is dialyzed (dialysis membrane: molecular exclusion size 3000-4000), then adsorbed onto a cation exchange resin, eluted with 0.5-2N aqueous ammonia, the eluted material is subjected to silica gel column chromatography (solvent: chloroform / methanol / water mixture) to recover the fraction containing the target substance, and then subjected to preparative reverse-phase column chromatography (solvent: 0.1-0.5% aqueous formic acid solution) to recover the target substance. The S-1-propenylcysteine ​​obtained by steps 1) and 2) above has a trans isomer proportion of approximately 70-90% when the total of the trans and cis isomers is taken as 100%.

[0025] In this invention, S-1-propenylcysteine ​​or its salt is, for example, the LD50 of a dilute ethanol extract of garlic (extract content 14.5%, number of alcohols 1.18), which is one of the raw materials. 50The toxicity is generally low, as the value is 50 ml / kg or higher regardless of the route of administration (oral, intraperitoneal, or subcutaneous) (The Journal of Toxicological Sciences. 1984;9:57.), and because Allium plants such as garlic and onions are commonly used as food.

[0026] As shown in the test examples below, S-1-propenylcysteine ​​or its salts increased blood eNAMPT levels. Therefore, S-1-propenylcysteine, its salts, or compositions containing them can be eNAMPT-increasing agents. eNAMPT, an extracellular nicotinamide phosphoribosyltransferase secreted from adipose tissue, circulates in bodily fluids such as blood and is delivered to the cells of one or more organs throughout the body, where it contributes to NAD production. + It promotes synthesis. In fact, NAD is found in the brain, kidneys, skeletal muscle, and liver of animals. + (It is known to have a function that improves the function of sirtuins.) This increased it. Furthermore, SIRT1 activity was increased in the cerebral cortex of aging-accelerated mice, and SIRT1 expression was increased in the hippocampus, kidney, hypothalamus, heart, lung, liver, and skeletal muscle. Furthermore, S-1-propenylcysteine ​​or its salt suppressed cells expressing p16 and p21, which are markers of senescent cells, in the kidney, and cells expressing p53 in the hippocampus.

[0027] Dynamics and effects of eNAMPT in the blood, and the relationship between increased eNAMPT levels and NAD levels in various organs. + Considering the phenomena of increased sirtuin activity, enhanced sirtuin expression, and suppression of senescent cells, S-1-propenylcysteine, its salts, or compositions containing these, when administered to animals, results in increased sirtuin activity or expression, enhanced NAD activity, and increased NAD activity in one or more organs (preferably two, three, four, five, six, or seven independently for each use). + It can be used to increase and suppress senescent cells.

[0028] The organs are not particularly limited and include, for example, one or more of the brain, kidneys, skeletal muscles, liver, and heart. In particular, the organs include one or more of the skeletal muscles, liver, and heart, and may also include one or more of the brain and kidneys.

[0029] However, the present invention does not have to include embodiments that act solely on the brain. Furthermore, the present invention does not have to include embodiments that act solely on the kidney with respect to the activation or enhancement of sirtuin expression and the suppression of senescent cells. Furthermore, the present invention does not have to include embodiments that act solely on the brain and kidney with respect to the activation or enhancement of sirtuin expression.

[0030] The sirtuin activator or expression enhancer and senescent cell inhibitor of the present invention may be used to prevent, treat, or improve symptoms caused by low sirtuin activity or low expression, or symptoms caused by an increase in senescent cells. As previously mentioned, impaired SIRT1 function and accelerated cellular senescence are thought to be involved in symptoms and diseases such as renal impairment, cognitive / memory impairment (including short-term and long-term), hepatic steatosis, cardiac dysfunction, and fatty liver (Non-patent literature 2-4, 9-10). Renal impairments include diseases with high expression of KIM-1 and NGAL, acute kidney injury, diabetic nephropathy, and nephrotic syndrome. Cognitive / memory impairments include neuropathy of the cerebral cortex or hippocampus, learning disabilities, and memory impairment (including short-term and long-term).

[0031] In this invention, "sirtuin" refers to sirtuin proteins and their homologs. Human sirtuins 1 to 7 (SIRT1 to 7) are known, but in this invention, SIRT1 is preferred.

[0032] "Sirtuin expression enhancement" refers to an increase in the transcript and / or sirtuin protein of sirtuin genes, and includes, for example, activation of gene transcription and / or translation, improvement of the stability of transcripts and / or proteins, and inhibition of transcript and / or protein degradation.

[0033] In this invention, "cellular senescence" refers to the phenomenon in which irreversible cell cycle arrest occurs in cells. Cellular senescence has been observed, for example, in brain cells (e.g., hippocampal cells, cerebral cortical cells), kidney cells, liver, and skeletal muscle. "Senosis of senescent cells" refers to either or both of the removal of senescent cells, or the prevention or delay of the cellular senescence phenomenon.

[0034] While the p16, p21, and p53 genes are known as molecular markers for DNA damage that causes cellular senescence, the senescence cell inhibitor of the present invention is more preferably one that suppresses mitochondrial dysfunction and DNA damage that promote cellular senescence. Furthermore, senescence cells can be suppressed more effectively by enhancing immune function.

[0035] NAD of the present invention + The growth agent is NAD + It may be used for the prevention, treatment, or improvement of symptoms caused by the low presence of [the substance]. NAD + As previously mentioned, the low presence of these substances is thought to be involved in symptoms and diseases such as non-alcoholic fatty liver disease and renal impairment (Non-patent literature 2-5, 10-12). Examples of renal impairment include diseases with high expression of KIM-1 and NGAL, acute kidney injury, diabetic nephropathy, and nephrotic syndrome.

[0036] The present invention's eNAMPT and / or NAD + The enhancers, sirtuin activators / expression enhancers, and senescent cell inhibitors may be in the form of pharmaceuticals or foods, or as materials or formulations added to them.

[0037] Furthermore, the food contains eNAMPT and / or NAD + This category includes foods, functional foods, foods with functional claims, foods for the sick, foods for specified health uses, and nutritional supplements, all based on the concepts of increasing, sirtuin activation / expression enhancement, or suppressing senescent cells, and which, as necessary, display explanations and indications of their effects.

[0038] The dosage form of the pharmaceutical product is preferably one suitable for oral administration. Specific dosage forms of oral preparations include, for example, tablets, capsules, granules, pills, and granules as solid preparations, and emulsions, solutions, suspensions, and syrups as liquid preparations. Such pharmaceutical preparations can be prepared by conventional methods, by appropriately blending S-1-propenylcysteine ​​or a salt thereof with excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters, etc., as needed. However, the dosage form of the pharmaceutical product is not particularly limited and may be a dosage form suitable for parenteral administration, such as an intravenous (injection, catheter), transmucosal (liquid or ointment), or intracranial (catheter) administration.

[0039] The form of food is not particularly limited and can take various forms, such as solid foods, semi-liquid foods, gel-like foods, tablets, caplets, capsules, etc. More specifically, it can take various forms of food such as confectionery, beverages, seasonings, processed seafood products, processed meat products, bread, and health foods. Such foods can be manufactured by conventional methods, using food ingredients typically used in the production of these foods, and appropriately blending them with S-1-propenylcysteine ​​or its salt.

[0040] The above pharmaceuticals or foods contain eNAMPT and / or NAD + It may also contain other substances involved in increasing, activating, enhancing the expression of sirtuins, and suppressing senescent cells, such as resveratrol and nicotinamide mononucleotide. Furthermore, it may contain vitamins, lipids, and minerals that soothe inflammation, such as vitamin C, vitamin E, vitamin B2, vitamin B6, niacin, hesperidin, alpha-lipoic acid, glutathione, coenzyme Q10, zinc, magnesium, and omega-3 fatty acids.

[0041] The preferred daily intake of the above-mentioned pharmaceutical or food product varies depending on factors such as the target of intake, the form of intake, the type of materials or additives taken simultaneously, and the interval between intakes. However, it is preferable to take 0.001 to 10 mg / kg of S-1-propenylcysteine ​​or its salt per day, and more preferably 0.1 to 1 mg / kg. Furthermore, if desired, this daily amount can be divided into 2 to 4 doses.

[0042] Targets for administration or intake include those with decreased sirtuin activity or expression, increased senescent cells, or NAD + Examples include organisms in which the amount of eNAMPT is reduced, but these may also be healthy organisms without specific diseases (e.g., diabetes, kidney disease). The organisms are not limited to the animals described above, but also include nematodes and cells (which may be derived from the aforementioned animals or plants), but animals are preferred. Examples of animals include vertebrates (preferably rodents and primates), fish, birds, insects and reptiles, with rodents (especially rats and mice) and primates (especially humans and monkeys) being particularly preferred.

[0043] When processed products of Allium plants or sulfur-containing components contained in Allium plants are administered to animals, it increases eNAMPT, activates or enhances sirtuin expression, and promotes NAD production in the animals. + To the best of our knowledge, there are no facts demonstrating an increase in this substance, nor any fact demonstrating the suppression of aging in non-cancer cells within animals. These were unexpected discoveries made as a result of our diligent research.

[0044] Examples include humans suffering from renal impairment, cognitive / memory impairment (including short-term and long-term), hepatic steatohepatosis, cardiac failure, fatty liver, non-alcoholic fatty liver disease, etc., or animals (e.g., humans) for whom prevention of such diseases is desired, but healthy organisms are also preferred.

[0045] <Another form> In another embodiment, the present invention may also relate to S-1-propenylcysteine ​​or a salt thereof, or a composition containing S-1-propenylcysteine ​​or a salt thereof, for use in the various applications described above. In another embodiment, the present invention may also relate to a method of administering S-1-propenylcysteine ​​or a salt thereof, or a composition containing S-1-propenylcysteine ​​or a salt thereof, for use in the various applications described above. In another embodiment, the present invention may also relate to the use of S-1-propenylcysteine ​​or a salt thereof, or a composition containing S-1-propenylcysteine ​​or a salt thereof, for the various uses described above. In another embodiment, the present invention may also relate to the use of S-1-propenylcysteine ​​or a salt thereof, or a composition containing S-1-propenylcysteine ​​or a salt thereof, in the manufacture of agents used for the various applications described above. Details of these embodiments are the same as those of the agent described above. [Examples]

[0046] Manufacturing Example 1: Ethanol Extraction Fraction of Garlic Approximately 1 kg of garlic bulbs with the outer skin removed and approximately 1000 mL of 30% ethanol were placed in a sealed container. This container was left at room temperature for 1 to 10 months, with occasional stirring. The solid and liquid were separated from this mixture, and the liquid was dried by spray drying to obtain a yellowish-brown powder.

[0047] Production Example 2: Isolation of S-1-propenylcysteine ​​from ethanol extract fraction of garlic (1) The ethanol extract fraction of garlic obtained in Production Example 1 was placed in a dialysis tube with a pore size of 3500 and dialyzed against purified water. The dialysis solution was passed through a cation exchange resin Dowex 50Wx8 (H+) and the resin was thoroughly washed with purified water. The amino acids adsorbed on the resin were eluted with 2N ammonia and concentrated under reduced pressure. The concentrate was attached to a silica gel column and subjected to column chromatography using a chloroform / methanol / water mixture as the solvent. The fraction containing the target product (S-1-propenylcysteine) was recovered and concentrated. The concentrate was dissolved in water and chromatographed using a preparative reverse-phase column (octadecylsilyl column) with 0.1% formic acid as the solvent to recover the target product, and the solvent was removed by freeze-drying. The obtained freeze-dried product was examined using NMR (solvent: heavy water) and mass spectrometry, and its structure was compared with spectra obtained from the standard substances shown below, confirming that it was a mixture of trans-S-1-propenylcysteine ​​and cis-S-1-propenylcysteine ​​(trans isomer:cis isomer = 8:2).

[0048] trans-S-1-propenylcysteine 1 H-NMR (500 MHz, in D2O-NaOD, δ): 1.76 (d, 3H, J = 7.0 Hz), 2.98 (dd, 1H, J = 7.5, 14.5 Hz), 3.14 (dd, 1H, J = 4.5, 14.5 Hz) 3.69 (dd, 1H, J = 4.5, 7.5Hz), 5.10-5.14 (m, 1H), 6.02(d, 1H, J = 15.5 Hz); 13 C-NMR (125 MHz, in D2O-NaOD, δ): 17.61, 33.53, 53.70, 119.92, 132.12, 172.73, HRMS: observed [M+H] + = 162.0583, calculated [M+H] + = 162.0581

[0049] cis-S-1-propenylcysteine 1H-NMR (500 MHz, in D2O, δ): 1.74 (d, 3H, J = 7.0 Hz), 3.21 (dd, 1H, J = 7.5, 15.0 Hz), 3.31 (dd, 1H, J = 4.5, 15.0 Hz), 3.95 (dd, 1H, J = 4.5, 7.5 Hz), 5.82-5.86 (m, 1H), 6.01(d, 1H, J = 9.5 Hz); 13 C-NMR (125 MHz, in D2O-NaOD, δ): 13.89, 33.88, 54.16, 122.58, 127.78, 172.63. HRMS: observed [M+H] + = 162.0580, calculated [M+H] + = 162.0581

[0050] (2) Measurement of S-1-propenylcysteine ​​in the ethanol extract fraction of garlic 500 mg to 1 g of the ethanol extract fraction of garlic obtained in Production Example 1(1) was taken into a container, and a 20 mM hydrochloric acid solution of Sn-3-butenylcysteine ​​was added as an internal standard. The mixture was then diluted to 20 mL with 20 mM hydrochloric acid. After thorough mixing, a portion was taken and centrifuged at 1750 G for approximately 10 minutes. A portion of the supernatant was taken and centrifuged using a centrifugal filtration unit (Amicon Ultra, cutoff: 3000) (15000 rpm, 10 minutes). 20 μL of the resulting filtrate was taken and derivatized using the AccQ·Tag Derivatization Kit (Waters). Separately, a standard compound was dissolved in 20 mM hydrochloric acid, and the same procedure as for the sample was performed to prepare a calibration standard solution. The sample solution and standard solution were chromatographed using the Acquity UPLC system (Waters) to determine their content. As a result, the S-1-propenylcysteine ​​content was 3.7 ± 0.3 mg / g dry weight.

[0051] Test example Sirtuin activation (1) Sample preparation The test solutions for evaluating biological activity were prepared as follows. All test solutions were prepared immediately before use for the biological activity evaluation. (a) Approximately 5 mg of S-1-propenylcysteine ​​(cis / trans mixture) prepared in Manufacturing Example 2 was accurately weighed out and dissolved in 10 mL of purified water to prepare the administration solution. (b) Approximately 6 mg of S-1-propenylcysteine ​​(cis / trans mixture) prepared in Production Example 2 was accurately weighed out and dissolved in 1 mL of culture medium. This solution was used as the stock solution and was diluted as appropriate for in vitro testing.

[0052] (2) Animals used for evaluation tests Animals for evaluating biological activity were bred as follows: Test aging-accelerated mice SAMP8 (male) were purchased from SLC Japan. After purchase, they underwent a one-week acclimatization period before being used as evaluation test animals.

[0053] (3) Preparation of human neuroblasts for evaluation testing Human neuroblastocyte cells, specifically the SH-SY5Y cell line, were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, the antibiotic penicillin, and streptomycin solution to obtain cells for evaluation.

[0054] (4) Sirtuin activation test I purchased the SIRT1_GLO kit from Promega to measure sirtuin activity. I placed lysate of cerebral cortex in a 96-well plate, added various reaction reagents, and then measured the luciferase luminescence intensity using a plate reader.

[0055] (a) SIRT1 activation effect (in vivo): Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally as a single dose, and the cerebral cortex was excised at 15, 30, 60, and 180 minutes. The cerebral cortex was lysed using an automated tissue lysator, and then solubilized by adding cell lysate. Centrifuge was performed, and the activity of sirtuins in the supernatant was measured. The results are shown in Figure 1. Sirtuin activity increased 180 minutes after administration of S-1-propenylcysteine ​​(Figure 1).

[0056] (b) Increased SIRT1 protein (in vivo): Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally for two weeks, after which the hippocampus was excised. The hippocampus was lysed using an automated tissue lysator, and the cells were lysed with Millipore RIPA cell lysis buffer, diluted 10-fold with purified water containing a protease / phosphatase mixed inhibitor from Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were anti-SIRT1 antibody (Biolegend) and anti-β-actin antibody (Fujifilm Wako Pure Chemical Industries). The results are shown in Figure 2. S-1-propenylcysteine ​​increased the amount of SIRT1 protein.

[0057] (C)NAD + In vivo dose-increasing effect: Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally as a single dose, and the cerebral cortex was removed 180 minutes later. Alternatively, using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was mixed into their feed, and after feeding for one month, the cerebral cortex was removed. NAD in the cerebral cortex + The quantity is NAD manufactured by Dojin Chemical Laboratories. + Measurements were taken using the / NADH Assay Kit. The results are shown in Figure 3. S-1-propenylcysteine ​​is NAD + The quantity was increased.

[0058] (5) Inhibitory effect on the expression of the cellular senescence marker p53 protein in the hippocampus (in vivo): Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally for 6 weeks, after which the hippocampus was excised. The hippocampus was lysed using an automated tissue lysator, and the cells were lysed with Millipore RIPA cell lysis buffer diluted 10-fold with purified water containing a Roche protease inhibitor and a phosphatase inhibitor. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were anti-p53 antibody (ProteinTec) and anti-β-actin antibody (Medical & Biological Laboratories). The results are shown in Figure 4. S-1-propenylcysteine ​​reduced the amount of p53 protein.

[0059] (6) Enhancement of SIRT1 gene expression in the kidney (in vivo): The animals used for the evaluation test described in (2) above were repeatedly administered the sample prepared in (1)(a) above for two weeks, after which the kidneys were removed. The kidneys were lysed using an automated tissue lithograph, and RNA was extracted using TRIzol solution from Thermo Fisher Scientific. cDNA was synthesized using the PRIMEScript RT reagent Kit with gEraser from TAKARA. The synthesized cDNA was subjected to Real-Time PCR using the KAPA SYBR Fast qPCR kit from Nippon Genetics. The results are shown in Figure 5. S-1-propenylcysteine ​​increased SIRT1 expression in mouse kidneys.

[0060] (7) Suppression of senescent cell markers p16 and p21 gene expression in the kidney (in vivo): The animals used for evaluation tests described in (2) above were repeatedly administered the sample prepared in (1)(a) above for two weeks, after which the kidneys were removed. The kidneys were lysed using an automated tissue lithograph, and RNA was extracted using TRIzol solution from Thermo Fisher Scientific. cDNA was synthesized using the PRIMEScript RT reagent Kit with gEraser from TAKARA. The synthesized cDNA was subjected to Real-Time PCR using the KAPA SYBR Fast qPCR Kit from Nippon Genetics. The results are shown in Figure 6. S-1-propenylcysteine ​​reduced the number of cells expressing the p16 and p21 genes in the kidneys of senescence-accelerated mice.

[0061] (8) Inhibitory effect on renal impairment (in vivo): Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally for two weeks, after which the kidneys were removed. The kidneys were lysed using an automated tissue lysator, and the cells were lysed with Millipore's RIPA cell lysis buffer, which was diluted 10-fold with purified water containing Roche's protease inhibitor and phosphatase inhibitor. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were the kidney injury molecule (KIM-1) antibody (R&D Systems), lipocalin-2 (NGAL) antibody (Proteintech), and anti-GAPDH antibody (Fujifilm Wako Pure Chemical Industries), all of which are markers of kidney damage. The results are shown in Figure 7. S-1-propenylcysteine ​​reduced KIM-1 and NGAL levels.

[0062] (9) Cognition / memory maintenance effect (in vivo): Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was mixed into their feed, and after feeding for four months, a passive avoidance test was conducted. The passive avoidance test utilizes the mouse's preference for dark places. On the first day, mice placed in a bright room were given an electric shock (50V for 1 second) when they moved to a dark room. The time it took to move to the dark room was defined as the response latency of the acquisition trial. After two months, the mice were again placed in a bright room, and the time it took to move to the dark room (response latency of the retention trial) was used as an indicator of memory. The results are shown in Figure 8. S-1-propenylcysteine ​​improved age-related decline in cognitive and memory function.

[0063] (10) NAD in the kidneys, skeletal muscle and liver + In vivo dose-increasing effect: Using the evaluation test animals described in (2) above, the sample prepared in (1)(a) above was administered orally as a single dose, and 60 minutes later the cerebral cortex, liver, kidneys, and skeletal muscle were excised. NAD in the cerebral cortex, liver, kidneys, and skeletal muscle + The quantity is NAD manufactured by Dojin Chemical Laboratories. + Measurements were taken using the / NADH Assay Kit. The results are shown in Figure 9. S-1-propenylcysteine ​​is NAD + The quantity was increased.

[0064] (11) Increased SIRT1 protein activity in the cerebral cortex, hippocampus, heart, lungs, liver, kidneys, and skeletal muscle (in vivo): Approximately 10 mg of S-1-propenylcysteine ​​(cis / trans mixture) prepared in Manufacturing Example 2 was accurately weighed out and dissolved in 10 mL of purified water to prepare the administration solution. The prepared sample was repeatedly administered orally for two weeks to the evaluation test animals described in (2) above, after which the cerebral cortex, hippocampus, heart, lungs, liver, kidneys, and skeletal muscle were excised. The cerebral cortex, hippocampus, heart, lungs, liver, kidneys, and skeletal muscle were lysed using an automated tissue lysator, and then the cells were lysed with Millipore RIPA cell lysis buffer, diluted 10-fold with purified water containing a protease / phosphatase mixed inhibitor from Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were anti-SIRT1 antibody (Biolegend) and anti-β-actin antibody (Fujifilm Wako Pure Chemical Industries). The results are shown in Figure 10. S-1-propenylcysteine ​​increased the amount of SIRT1 protein.

[0065] (12) Increased SIRT1 protein in the hypothalamus (in vivo): The S-1-propenylcysteine ​​(cis / trans mixture) prepared in Production Example 2 was dissolved in purified water to a concentration of 0.001% to prepare the administration solution. Animals for evaluating biological activity were bred as follows: SAMR1 (male) and SAMP8 (male) aging-accelerated test mice were purchased from SLC Japan. After purchase, they underwent a one-week acclimatization period before being used as test animals. Using these test animals, the hypothalamus was excised after feeding them either a normal diet or SAMP8 mixed with the prepared sample for 10 months. The hypothalamus was then lysed using an automated tissue lysator, and the cells were lysed with Millipore RIPA cell lysis buffer diluted 10-fold with purified water containing a protease / phosphatase mixed inhibitor from Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were anti-SIRT1 antibody (Biolegend) and anti-β-actin antibody (Fujifilm Wako Pure Chemical Industries). The results are shown in Figure 11. S-1-propenylcysteine ​​increased the amount of SIRT1 protein.

[0066] (13) Effect of increasing eNAMPT in the blood (in vivo): In animals from which the hypothalamus was removed in (12), blood eNAMPT levels were measured according to the method of YOSHIDA M et al. Cell Metab. 2019;30(2):329-342.e5. The results are shown in Figure 12. S-1-propenylcysteine ​​increased blood eNAMPT levels.

[0067] (14) Increased SIRT1 protein levels in the liver and heart (in vivo): Using evaluation animals prepared in the same manner as in (12), the sample prepared in (1)(a) above was administered orally for two weeks, after which the liver and heart were excised. The liver and heart were lysed using an automated tissue lysator, and the cells were lysed with Millipore RIPA cell lysis buffer diluted 10-fold with purified water containing a protease / phosphatase mixed inhibitor from Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 min, 4°C), and the supernatant was used as the cell extract. This cell extract was analyzed by Western blotting according to standard procedures. Antibodies used were anti-SIRT1 antibody (Biolegend) and anti-β-actin antibody (Fujifilm Wako Pure Chemical Industries). The results are shown in Figure 13. S-1-propenylcysteine ​​increased the amount of SIRT1 protein.

Claims

1. An eNAMPT increasing agent comprising isolated S-1-propenylcysteine ​​or a salt thereof as an active ingredient.

2. An eNAMPT increasing agent comprising isolated S-1-propenylcysteine ​​or a salt thereof, which is administered to an animal.

3. The agent according to claim 2, used for increasing eNAMPT in the blood.

4. An agent containing isolated S-1-propenylcysteine ​​or a salt thereof, which is administered to an animal and used to activate or enhance the expression of sirtuins or to suppress senescent cells in one or more organs.

5. A solution containing isolated S-1-propenylcysteine ​​or a salt thereof, administered to an animal, which increases NAD levels in one or more organs. ＋ An agent used to increase something.

6. The agent according to claim 4 or 5, wherein the organ is one or more selected from the group consisting of the brain, kidneys, skeletal muscles, liver, and heart.

7. The agent according to claim 6, wherein the organ is one or more selected from the group consisting of skeletal muscle, liver, and heart.

8. The agent according to claim 7, wherein the aforementioned organ further comprises one or more selected from the group consisting of the brain and the kidneys.

9. The agent according to claim 4 or 5 for acting on two or more organs.

10. The agent according to claim 9 for acting on three or more organs.

11. The agent according to claim 1, 4, or 5, in the form of a food additive or food product.

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