ENAMPT-increasing agent, histone deacetylase type 3 activation or expression enhancer, NAD +-increasing agent, and senescence cell inhibitor

Abstract

The present invention provides a novel use of S-1-propenylcysteine, a salt thereof, or a composition containing the S-1-propenylcysteine and the salt thereof. The present invention may be an eNAMPT increasing agent containing S-1-propenylcysteine or a salt thereof as an active ingredient. The present invention may be an eNAMPT-increasing agent that contains S-1-propenylcysteine or a salt thereof and is administered to an animal. The compounds are particularly preferably used for increasing eNAMPT in blood.

Description

Technical Field

[0001] The present invention relates to a method for enhancing eNAMPT, enhancing the activation or expression of histone deacetylase type 3, and NAD + Preparations used to enhance and inhibit senescent cells. Background Art

[0002] Sirtuin is a molecule that prolongs lifespan when highly expressed in nematodes, flies, mammals, etc., and shortens lifespan when missing. Histone deacetylase 3 is a molecule that deacetylates NAD. + It is a histone deacetylase, whose genes are preserved in a wide range from bacteria to eukaryotes. If Sir2, a homolog of histone deacetylase type 3 in yeast or nematodes, is missing, the lifespan will be shortened, and if it is overexpressed, the lifespan will be extended. Therefore, histone deacetylase type 3 has attracted attention as a candidate for longevity gene in animals. There are 7 histone deacetylases type 3 (SIRT1~7) in mammals. Among them, SIRT1, which is most similar to yeast Sir2 in structure and function, is likely to be related to the control of a wide range of cellular functions such as the expression of genes associated with aging, intracellular metabolism, energy consumption, inflammation and stress response pathways (Non-patent Document 1).

[0003] In addition, in recent years, it has also been known that histone deacetylase type 3 may be associated with specific diseases. For example, in non-patent document 2, SIRT1 gene knockout mice showed decreased cognitive functions including short-term memory, classical conditioning, and spatial learning. On the contrary, it was observed that overexpression of SIRT1 exhibited regular synaptic plasticity and memory. It can be seen that SIRT1 plays a role in normal learning, memory, and synaptic plasticity. In addition, the correlation between hepatic steatosis and SIRT1 is recorded in non-patent document 3, and the correlation between cardiac function and SIRT1 is recorded in non-patent document 4.

[0004] Nicotinamide mononucleotide (NAD + ) functions as a coenzyme of dehydrogenase or a substrate of histone deacetylase type 3 in vivo. + Increased amounts of NAD increase the activity of histone deacetylase type 3. The synthesis of NAD can be controlled by nicotinamide phosphoribosyltransferase (NAMPT). It is known that the role of NAMPT decreases with age, so NAD + The amount of NAD decreases, and the activity of histone deacetylase type 3 decreases. Non-patent document 5 discloses + Administration of nicotinamide riboside, a precursor of NAD, inhibits senescent cells in the brain.+ , and its association with non-alcoholic fatty liver disease is recorded in Non-Patent Document 6, and its association with renal damage is recorded in Non-Patent Document 7.

[0005] Cell aging refers to the following phenomenon, that is, excessive DNA damage is mediated by the accumulation of DNA replication errors accompanying cell division, oxidative stress, radiation, activation of oncogenes, etc., thereby activating the p16 / RB path or the p53 / p21 path, and inducing inhibitors of cell cycle protein inhibitory kinases, thereby causing irreversible cell cycle arrest. Cells (senescent cells) that cause cell aging will accumulate in tissues due to the promotion of cell aging or the decline in the ability to remove it, and the above-mentioned cell aging is caused by the decline in mitochondria and immune function with age. Senescent cells accumulated in tissues will secrete inflammatory cytokines or proteases, etc., which are called cell aging-related secretory factors, thereby damaging the surrounding tissues and promoting the aging of tissues or organisms. On the other hand, there is a revelation that the removal of senescent cells will prevent or delay the dysfunction of tissues and inhibit aging (non-patent literature 8). In addition, it is known that the increase of senescent cells is related to renal injury (non-patent literature 9) and fatty liver (non-patent literature 10).

[0006] On the other hand, S-1-propenylcysteine ​​is a cysteine ​​derivative represented by the following formula (1), and is a sulfur-containing component that may be contained in Allium plants such as garlic.

[0007] [Chemical formula 1]

[0008]

[0009] According to reports, S-1-allylcysteine ​​has pharmacological effects such as immunomodulatory effects (Patent Document 1), hypotensive effects (Patent Document 2), antioxidant effects (Patent Document 3), blood flow improvement effects (Patent Document 4), autophagy activation effects (Patent Document 5), and periodontal disease prevention, treatment, or improvement effects (Patent Document 6). However, S-1-allylcysteine ​​increases eNAMPT, histone deacetylase type 3, activates or inhibits senescent cells, and NAD + The increase is completely unknown.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: International Publication No. 2016 / 088892

[0013] Patent Document 2: International Publication No. 2016 / 199885

[0014] Patent Document 3: Japanese Patent Application Publication No. 2020-029529

[0015] Patent Document 4: International Publication No. 2019 / 031442

[0016] Patent Document 5: International Publication No. 2019 / 235597

[0017] Patent Document 6: International Publication No. 2020 / 230813

[0018] Non-patent literature

[0019] Non-patent literature 1: Trends Cell Biol. 2014; 24(8): 464-71.

[0020] Non-patent literature 2: J Neurosci 2010; 30(29): 9695-9707.

[0021] Non-patent literature 3: Molecular Medicine Reports 2018; 18: 1609-1615

[0022] Non-patent literature 4: Aging 2021; 13(10): 14482-14498

[0023] Non-patent literature 5: Cell Metab. 2018 Mar 6; 27(3): 513-528.

[0024] Non-patent literature 6: British Journal of Pharmacology 2016; 173: 2352-2368

[0025] Non-patent literature 7: Nature 2016; 531(7595): 528-532

[0026] Non-patent literature 8: Nature. 2011 Nov 2; 479(7372): 232-6.

[0027] Non-patent literature 9: Front Pharmacol. 2019; 10: 770.

[0028] Non-patent literature 10: Nature Communications 2017; 8:15691 Summary of the invention

[0029] The present invention relates to a novel use of S-1-propenylcysteine, its salt or a composition containing them.

[0030] The present invention relates to the following 1) to 11).

[0031] 1) An eNAMPT increasing agent comprising S-1-allylcysteine ​​or a salt thereof as an active ingredient.

[0032] 2) An eNAMPT increasing agent, which contains S-1-allylcysteine ​​or a salt thereof and is administered to an animal.

[0033] 3) The preparation according to 2), which is used for increasing eNAMPT in blood.

[0034] 4) A preparation containing S-1-allylcysteine ​​or a salt thereof and being administered to an animal, the preparation being used for activation or enhancement of expression of histone deacetylase type 3 in one or more organs, or inhibition of senescent cells.

[0035] 5) A preparation containing S-1-allylcysteine ​​or a salt thereof and administered to an animal, the preparation being used for NAD in more than one organ + Increase.

[0036] 6) The preparation according to 4) or 5), wherein the organ comprises one or more selected from the group consisting of brain, kidney, skeletal muscle, liver and heart.

[0037] 7) The preparation according to 6), wherein the organ comprises one or more selected from skeletal muscle, liver and heart.

[0038] 8) The preparation according to 7), wherein the above-mentioned organ further includes one or more selected from the group consisting of brain and kidney.

[0039] 9) The preparation according to any one of 4) to 8), which is used to act on two or more organs.

[0040] 10) The preparation according to 9), which is used to act on three or more organs.

[0041] 11) The preparation according to any one of 1) to 10), which is in the form of a food additive or a food.

[0042] According to the present invention, novel uses of S-1-propenylcysteine, a salt thereof, or a composition containing them can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results showed that administration of S-1-allylcysteine ​​resulted in activation of histone deacetylase type 3 in the cerebral cortex.

[0044] Figure 2 The figure shows the change in the amount of SIRT1 protein in the hippocampus caused by the administration of S-1-allylcysteine.

[0045] Figure 3Showing that administration of S-1-allylcysteine ​​leads to NAD in the hippocampus + Changes in quantity.

[0046] Figure 4 The results show that administration of S-1-allylcysteine ​​leads to changes in p53 protein, a marker of senescent cells, in the hippocampus.

[0047] Figure 5 The figure shows the change in SIRT1 expression level in the kidney caused by the administration of S-1-allylcysteine.

[0048] Figure 6 The figure shows the changes in the expression levels of p16 and p21 genes, which are senescent cell markers in the kidney, caused by the administration of S-1-allylcysteine.

[0049] Figure 7 The figure shows changes in the amount of KIM-1 and NGAL gene proteins, which are kidney injury markers, in the kidneys caused by the administration of S-1-allylcysteine.

[0050] Figure 8 Administration of S-1-allylcysteine ​​resulted in maintenance of cognition / memory.

[0051] Fig. 9 The results show that the administration of S-1-allylcysteine ​​resulted in the increase of NAD in kidney, skeletal muscle and liver + Changes in quantity.

[0052] Fig.10 The results show changes in the amount of SIRT1 protein in the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle caused by the administration of S-1-allylcysteine.

[0053] Fig.11 The figure shows the change in the amount of SIRT1 protein in the hypothalamus caused by the administration of S-1-allylcysteine.

[0054] Fig.12 The figure shows the change in the amount of eNAMPT in the blood caused by the administration of S-1-allylcysteine.

[0055] Fig.13 The figure shows the changes in the amount of SIRT1 protein in the liver and heart caused by the administration of S-1-allylcysteine. DETAILED DESCRIPTION

[0056] <Preparation>

[0057] "S-1-propenylcysteine" has a cis or trans spatial configuration as indicated by the wavy line in the following formula (1). In the present invention, S-1-propenylcysteine ​​preferably has a higher ratio of the trans isomer, and when the total of the trans isomer and the cis isomer is set to 100%, the ratio of the trans isomer is more preferably 50 to 100%, more preferably 75 to 100%, more preferably 80 to 100%, and even more preferably 90 to 100%.

[0058] In addition, since there is a chiral carbon in the structure of cysteine, there are optical isomers, which may be any of the D-isomer, the L-isomer or the racemic isomer.

[0059] [Chemical formula 2]

[0060]

[0061] The salt of S-1-propenylcysteine ​​is a physiologically acceptable salt, and may be either an acid addition salt or a base addition salt. Examples of the acid addition salt include: (a) salts with inorganic 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; (c) salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid; and examples of the base addition salt include: (a) salts with sodium, (a) salts with alkali metals such as potassium, (b) salts with alkaline earth metals such as calcium and magnesium, (c) ammonium salts, (d) salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine (1-Ephenamine), and N,N'-dibenzylethylenediamine.

[0062] Furthermore, S-1-propenylcysteine ​​or its salt may exist not only in an unsolvated form but also in the form of a hydrate or a solvate, and the hydrate or solvate may exist in any crystalline form depending on the production conditions. Therefore, S-1-propenylcysteine ​​or its salt in the present invention includes all stereoisomers, hydrates, solvates, and all polymorphic forms or amorphous forms.

[0063] S-1-Propylene cysteine ​​or a salt thereof can be obtained by an organic synthesis method ([1] Tetrahedron Letter, 1975, 37, 3201-3202; [2] Synthesis, 2006, 20, 3367-3369; [3] Bull. Korean Chem. Soc. 2011, 32(1), 319-320).

[0064] In addition, S-1-propenylcysteine ​​or a salt thereof can be obtained by processing Allium plants by various methods. For example, the following methods can be cited: a method for obtaining S-1-propenylcysteine ​​by allowing an enzyme containing protease or lactase from Bacillus subtilis to act on a heat-treated Allium plant (Japanese Patent No. 6105871); a method for obtaining S-1-propenylcysteine ​​by allowing an enzyme having γ-glutamyl transpeptidase or glutaminase activity to coexist with a juice or an extract of an Allium plant (Japanese Patent Laid-Open No. 2015-144571); a method for obtaining S-1-propenylcysteine ​​by inoculating yeast into garlic and fermenting it (Korean Patent No. 2010-072874-5); a method for obtaining S-1-propenylcysteine ​​by aging an Allium plant in an ethanol aqueous solution for more than one month (International Publication No. 2016 / 088892, Molecules. 2017; 22: 570.), etc.

[0065] Here, as the Allium plant, garlic (Allium sativum L.), onion (Allium cepa L.), southern European garlic (Allium ampeloprasum L.), leek (Allium tuberosum) and shallot (Allium fistulosum L.) can be mentioned. These plants can be used alone or in combination. In addition, the above-mentioned Allium plants can be used fresh directly, or the skin can be removed and cut or chopped as needed; or the powdered material can be used, and the solvent that can be used to make medicines or foods can be used to extract. As the solvent, water or alcohol, or a solvent obtained by adding an acid or alkaline substance to the solvent can be mentioned.

[0066] The S-1-propenylcysteine ​​or its salt of the present invention can be used not only in the form of an isolated and purified substance but also in the form of a crude product, the above-mentioned plant or its processed product, or a fraction having an increased content of S-1-propenylcysteine ​​or its salt by extraction from the above-mentioned plant.

[0067] In the present invention, S-1-propenylcysteine ​​or its salt is contained 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, in terms of dry matter. The upper limit of the content is not particularly limited, and may be 100,000 ppm or less, 50,000 ppm or less, or 10,000 ppm or less.

[0068] As shown below, for example, it can be obtained by: 1) extracting Allium plants in a 10-50% ethanol aqueous solution at 0-80°C for more than 1 month (step 1), 2) subjecting the obtained extract to solid-liquid separation, and recovering the ethanol elution component (step 2).

[0069] The ethanol aqueous solution used in step 1) can use 10-50% ethanol aqueous solution, preferably an ethanol aqueous solution prepared with an ethanol concentration of 20-40%. In addition, the treatment temperature can be set in the range of 0-80°C, preferably 10-60°C, and more preferably 20-40°C. The treatment period can be extracted under the above conditions for at least 1 month, preferably 1-20 months, and more preferably 1-10 months. In addition, considering the hygiene aspect or the volatilization of ethanol, this step can be carried out in an airtight, sealed or closed container, preferably using a closed container.

[0070] In step 2), the extract obtained in step 1) is subjected to solid-liquid separation, and the ethanol elution fraction is recovered. By appropriately concentrating the recovered product, an extraction fraction containing S-1-propenylcysteine ​​or its salt or its salt can be obtained. The extraction fraction can be used directly or can be appropriately dried by spray drying or the like.

[0071] Furthermore, when separating S-1-propenylcysteine ​​or its salt from the extraction fraction containing the above-mentioned S-1-propenylcysteine ​​or its salt, it can be carried out as follows: dialysis is performed using a dialysis membrane with a molecular exclusion size of 3000 to 4000 as needed, and then, adsorption and separation using a cation exchange resin are appropriately combined with separation and purification means using normal phase chromatography or reverse phase chromatography.

[0072] Here, as adsorption and separation using a cation exchange resin, the following method can be cited: the above-mentioned extraction component is adsorbed on a cation exchange resin (for example, Amberlite (Dow Chemical Company), DOWEX (manufactured by Dow Chemical Company), DIAION (manufactured by Mitsubishi Chemical)), etc.), and eluted with 0.1 to 3N ammonia water.

[0073] Examples of normal phase chromatography include a method using a silica gel column and eluting with a chloroform / methanol / water mixture or the like.

[0074] Examples of the reverse phase chromatography include a method using an octadecylsilane column and eluting with a 0.01 to 3% formic acid aqueous solution or the like.

[0075] The following method can be preferably cited: the above-mentioned ethanol extraction component is dialyzed (dialysis membrane: molecular exclusion size 3000-4000), and then adsorbed on a cation exchange resin, and then eluted with 0.5-2N ammonia water, and the eluate is used for silica gel column chromatography (solvent: chloroform / methanol / water mixture) to recover the component containing the target substance, and then the target substance is recovered using preparative reverse phase column chromatography (solvent: 0.1-0.5% formic acid aqueous solution).

[0076] Regarding S-1-propenylcysteine ​​obtained by the above steps 1) and 2), when the total of the trans isomer and the cis isomer is 100%, the ratio of the trans isomer is approximately 70 to 90%.

[0077] S-1-propenylcysteine ​​or its salt in the present invention is generally less toxic due to the following reasons: the LD50 of a dilute ethanol extract of garlic (extract volume 14.5%, alcohol number 1.18) is 1.34. 50 The value is 50 ml / kg or more in any of the oral, intraperitoneal and subcutaneous administration routes (The Journal of Toxicological Sciences. 1984; 9: 57.); and Allium plants such as garlic and onion are often used as food.

[0078] As shown in the test examples described below, S-1-allylcysteine ​​or its salt increases eNAMPT in the blood. Therefore, S-1-allylcysteine, its salt or a composition containing them can be an eNAMPT increasing agent.

[0079] eNAMPT and extracellular nicotinamide phosphorylation transferase secreted from adipose tissue circulate in body fluids such as blood and are delivered to cells in one or more organs throughout the body, promoting the production of NAD + In fact, NAD is used in the brain, kidney, skeletal muscle and liver of animals. + Increase (It is known that it has the function of increasing the function of histone deacetylase type 3.).

[0080] Thus, SIRT1 activity was increased in the cerebral cortex and SIRT1 expression was increased in the hippocampus, kidney, hypothalamus, heart, lung, liver, and skeletal muscle of senescence-promoting mice.

[0081] In addition, S-1-allylcysteine ​​or a salt thereof inhibited the expression of cells in the kidney that express p16 and p21, which are markers of aging cells, and cells in the hippocampus that express p53.

[0082] If we consider the dynamics and effects of eNAMPT in the blood, the increase of eNAMPT in the blood and the NAD +In the present invention, if the phenomenon of increasing the activation or expression of histone deacetylase type 3 and inhibiting senescent cells is detected, S-1-allylcysteine, a salt thereof or a composition containing them can be administered to animals to increase the activation or expression of histone deacetylase type 3, NAD in one or more organs (preferably two or more, three or more, four or more, five or more, six or more or seven or more organs for each purpose independently), and inhibit the activation or expression of histone deacetylase type 3 and NAD + Increase and inhibition of senescent cells.

[0083] The organ is not particularly limited, and examples thereof include one or more of the brain, kidney, skeletal muscle, liver, and heart. Among them, the organ includes one or more of the skeletal muscle, liver, and heart, and in addition, may include one or more of the brain and kidney.

[0084] However, the present invention may not include a method that acts only on the brain. In addition, in the present invention, regarding the activation or expression enhancement of histone deacetylase type 3 and the inhibition of senescent cells, the method may not include a method that acts only on the kidney. In addition, in the present invention, regarding the activation or expression enhancement of histone deacetylase type 3, the method may not include a method that acts only on the brain and kidney.

[0085] The histone deacetylase type 3 activation or expression enhancer and senescent cell inhibitor of the present invention can be used to prevent, treat or improve symptoms caused by low activity or low expression of histone deacetylase type 3, or symptoms caused by an increase in senescent cells.

[0086] As described above, the reduced function of SIRT1 and the hypersensitivity of cell aging are said to be related to symptoms and diseases such as kidney damage, cognitive / memory impairment (including short-term and long-term), hepatic steatosis, heart failure, fatty liver, etc. (Non-patent literature 2-4, 9-10). Examples of kidney damage include: diseases with high expression of KIM-1 and NGAL, acute kidney injury, diabetic nephropathy, nephrotic syndrome, etc. Examples of cognitive / memory impairment include: neurological disorders of the cerebral cortex or hippocampus, learning disorders, memory impairment (including short-term and long-term), etc.

[0087] In the present invention, "histone deacetylase type 3" refers to histone deacetylase type 3 protein and its homologs. Among human histone deacetylase type 3, 1 to 7 (SIRT1 to 7) are known, and in the present invention, SIRT1 is preferred.

[0088] "Enhanced histone deacetylase type 3 expression" refers to an increase in the transcription product of the histone deacetylase type 3 gene and / or histone deacetylase type 3 protein, including, for example: activation of gene transcription and / or translation, improvement of the stability of the transcription product and / or protein, inhibition of the degradation of the transcription product and / or protein, etc.

[0089] In the present invention, "cell aging" refers to the phenomenon of irreversible cell cycle arrest in cells. Cell aging is observed, for example, in brain cells (e.g., hippocampal cells, cerebral cortex cells), kidney cells, liver, and skeletal muscle. "Senescent cell inhibition" refers to one or both of the removal of senescent cells, the prevention or delay of cell aging.

[0090] The p16, p21 and p53 genes are known as molecular markers of DNA damage that causes cell aging. The senescent cell inhibitor of the present invention preferably inhibits mitochondrial dysfunction or DNA damage that promotes cell aging. In addition, by improving immune function, senescent cells can be more effectively inhibited.

[0091] NAD of the present invention + Increasers can be used to prevent, treat or improve NAD + Symptoms of low presence.

[0092] NAD + As mentioned above, low levels of KIM-1 are said to be associated with symptoms and diseases such as non-alcoholic fatty liver disease and renal damage (Non-patent Documents 2-5, 10-12). Examples of renal damage include diseases with high expression of KIM-1 and NGAL, acute renal damage, diabetic nephropathy, and nephrotic syndrome.

[0093] eNAMPT and / or NAD of the present invention + The enhancer, histone deacetylase type 3 activation / expression enhancer, and senescent cell inhibitor may be in the form of a drug or food, or may be in the form of a material or preparation added thereto.

[0094] Additionally, the food includes: eNAMPT and / or NAD + Foods, functional foods, foods with functional claims, foods for patients, foods for specified health purposes, and nutritional supplements that are labeled as concepts of increase, enhancement of histone deacetylase type 3 activation / expression, or inhibition of senescent cells, and that explain the effects based on these functions as necessary.

[0095] The dosage form of the drug is preferably a dosage form suitable for oral administration. As specific dosage forms of oral administration preparations, for example, tablets, capsules, fine granules, pills, granules as solid preparations; emulsions, solutions, suspensions, syrups and the like as liquid preparations. The drug preparation can be prepared by appropriately mixing excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters and the like with the S-1-propenylcysteine ​​or its salt of the present invention as needed, and prepared according to conventional methods.

[0096] However, the dosage form of the drug is not particularly limited, and may be a dosage form suitable for parenteral administration, such as a dosage form suitable for intravenous administration (injection, catheter), transmucosal administration (liquid or ointment), or intracranial administration (catheter).

[0097] The form of the food is not particularly limited, and may be, for example, solid food, semi-fluid food, gel food, tablets, capsule-type tablets, capsules, and more specifically, may be snacks, beverages, seasonings, processed seafood, processed meat, bread, health food, and other forms of food.

[0098] The food can be produced by appropriately mixing food materials commonly used in producing such food with the S-1-propenylcysteine ​​or a salt thereof of the present invention and following a conventional method.

[0099] The above medicine or food may contain eNAMPT and / or NAD + Other substances related to the increase of leukemia, activation of histone deacetylase type 3, enhanced expression or inhibition of senescent cells, such as resveratrol or nicotinamide mononucleotide. In addition, it can contain vitamins, lipids, and minerals that relieve inflammation, such as vitamin C, vitamin E, vitamin B2, vitamin B6, niacin, hesperidin, α-lipoic acid, glutathione, coenzyme Q10, zinc, magnesium, ω-3 fatty acids, etc.

[0100] The preferred daily intake of the above-mentioned medicine or food varies depending on the object of intake, the form of intake, the type of materials or additives taken at the same time, the intake interval, etc., and as S-1-propenylcysteine ​​or its salt, it is preferably taken 0.001 to 10 mg / kg per day, and more preferably 0.1 to 1 mg / kg per day. In addition, the daily amount can be taken in 2 to 4 times as needed.

[0101] Examples of the subjects for administration or ingestion include decreased activity or expression of histone deacetylase type 3, increased senescent cells, or NAD + and / or an organism with a reduced amount of eNAMPT, but may also be a healthy organism without these or specific diseases (e.g., diabetes, renal damage). The organism is not limited to the animals described above, but also includes: nematodes, cells (which may be derived from the above animals or plants.), but preferably animals. Examples of the animal include: vertebrates (preferably rodents and primates), fish, birds, insects and reptiles, particularly preferably rodents (especially rats and mice) and primates (especially humans and monkeys).

[0102] To the best of the knowledge of the present inventors, there is no evidence that the administration of processed products of Allium plants or sulfur-containing components contained in Allium plants to animals will cause an increase in eNAMPT, activation or enhanced expression of histone deacetylase type 3, or NAD + These are the results of intensive research conducted by the inventors and others, and are unexpected findings.

[0103] Examples include humans suffering from renal damage, cognitive / memory impairment (including short-term and long-term), hepatic steatosis, heart failure, fatty liver, non-alcoholic fatty liver disease, and animals (e.g., humans) for whom prevention of such diseases is desired, but healthy organisms are also preferred.

[0104] <Other methods>

[0105] In another embodiment, the present invention may also relate to S-1-propenylcysteine ​​or a salt thereof used for the above-mentioned various purposes, or a composition containing S-1-propenylcysteine ​​or a salt thereof.

[0106] In another embodiment, the present invention may also relate to a method for using S-1-propenylcysteine ​​or a salt thereof, or a composition containing S-1-propenylcysteine ​​or a salt thereof, for the above-mentioned various purposes.

[0107] 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 above-mentioned various purposes.

[0108] 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 production of a preparation used in the above-mentioned various applications.

[0109] The details of these means are the same as those of the above-mentioned agents.

[0110] Example

[0111] Preparation Example 1 Ethanol Extract of Garlic

[0112] About 1 kg of garlic bulbs with the skin removed and about 1000 mL of 30% ethanol are placed in a sealed container. The container is placed at room temperature for 1 to 10 months with appropriate stirring. Solids and liquids are separated from the mixture, and the liquid is dried by spray drying to obtain a yellow-brown powder.

[0113] Preparation Example 2 Isolation of S-1-propenylcysteine ​​from the ethanol extract of garlic

[0114] (1) The ethanol extract fraction of garlic obtained in Preparation Example 1 was placed in a dialysis tube with a pore size of 3500 and dialyzed against purified water. The dialyzed external solution was passed through a cation exchange resin Dowex50Wx8 (H+), and the resin was well 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 a solvent. The fraction containing the target product (S-1-propenylcysteine) was recovered and concentrated. The concentrate was dissolved in water, chromatographed using a preparative reverse phase column (octadecylsilane column) with 0.1% formic acid as a solvent, the target product was recovered, and the solvent was removed by freeze drying. The structure of the obtained freeze-dried product was compared with the spectrum obtained from the standard substance shown below by NMR (solvent: heavy water) and mass spectrometry, and it was confirmed that it was a mixture of trans-S-1-propenylcysteine ​​and cis-S-1-propenylcysteine ​​(trans isomer: cis isomer = 8:2).

[0115] trans-S-1-allylcysteine

[0116] 1 H-NMR(500MHz,in D2O-NaOD, δ): 1.76 (d, 3H, J = 7.0Hz), 2.98 (dd, 1H, J = 7.5, 14.5Hz), 3.14 (dd, 1H, J = 4 .5,14.5Hz)3.69(dd,1H,J=4.5,7.5Hz),5.10-5.14(m,1H),6.02(d,1H,J=15.5Hz);

[0117] 13 C-NMR(125MHz,in D2O-NaOD,δ):17.61,33.53,53.70,119.92,132.12,172.73,

[0118] HRMS: observed[M+H] + =162.0583, calculated [M+H] + =162.0581

[0119] cis-S-1-Propylene Cysteine

[0120] 1H-NMR(500MHz,in D2O, δ): 1.74 (d, 3H, J = 7.0Hz), 3.21 (dd, 1H, J = 7.5, 15.0Hz), 3.31 (dd, 1H, J = 4.5 ,15.0Hz),3.95(dd,1H,J=4.5,7.5Hz),5.82-5.86(m,1H),6.01(d,1H,J=9.5Hz);

[0121] 13 C-NMR (125MHz, in D2O-NaOD, δ): 13.89, 33.88, 54.16, 122.58, 127.78, 172.63.

[0122] HRMS: observed[M+H] + =162.0580, calculated [M+H] + =162.0581

[0123] (2) Determination of S-1-allylcysteine ​​in ethanol extract of garlic

[0124] 500 mg to 1 g of the ethanol extract of garlic obtained in Production Example 1 (1) was placed in a container, and a 20 mM hydrochloric acid solution of Sn-3-butenylcysteine ​​was added as an internal standard, and the solution was made up to 20 mL with 20 mM hydrochloric acid. After sufficient stirring, a portion was taken and centrifuged at 1750 G for about 10 minutes. A portion of the obtained supernatant was taken and centrifuged (15000 rpm, 10 minutes) using a centrifugal filter unit (Amicon Ultra, cutoff: 3000). 20 μL of the obtained filtrate was taken and derivatized using the AccQ·Tag Derivatization Kit (Waters). In addition, the standard compound was dissolved in 20 mM hydrochloric acid, and the same operation as the sample was performed to prepare a standard solution for the calibration curve. The sample solution and the standard solution were chromatographed using an Acquity UPLC system (Waters) to determine the content. As a result, the content of S-1-butenylcysteine ​​was 3.7±0.3 mg / g dry matter.

[0125] Test example

[0126] Histone deacetylase type 3 activation

[0127] (1) Sample preparation

[0128] Preparation of the test solution for evaluating biological activity was performed as follows: When evaluating biological activity, the test solution was prepared before use.

[0129] (a) About 5 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 10 mL of purified water to prepare a dosing solution.

[0130] (b) About 6 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 1 mL of culture medium. This solution was used as a stock solution and appropriately diluted for use in an in vitro test.

[0131] (2) Evaluation of experimental animals

[0132] Animals used for evaluation of biological activity were bred as follows: Experimental accelerated aging mice SAMP8 (male) were purchased from Japan SLC and acclimated for one week after purchase to be used as evaluation test animals.

[0133] (3) Preparation of human neuroblasts for evaluation experiments

[0134] Human neuroblastoma cell line SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotic penicillin and streptomycin solution, and the obtained cells were used as evaluation test cells.

[0135] (4) Histone deacetylase type 3 activation test

[0136] In order to measure the activity of histone deacetylase type 3, a SIRT1_GLO kit manufactured by Promega was purchased. The cerebral cortex lysate was placed in a 96-well plate, and after adding various reaction reagents, the luminescence intensity of luciferase was measured using a plate reader.

[0137] (a) SIRT1 activation (in vivo):

[0138] The test animals of the evaluation method of (2) were used to orally administer a single dose of the sample prepared in (1) (a) above, and then the cerebral cortex was removed after 15, 30, 60, and 180 minutes. The cerebral cortex was crushed using an automatic tissue crusher and then dissolved by adding a cell lysate. The cells were centrifuged and the activity of histone deacetylase type 3 in the supernatant was measured. The results are shown in Figure 1 .

[0139] 180 minutes after administration of S-1-allylcysteine, histone deacetylase type 3 activity increased. Figure 1 ).

[0140] (b) SIRT1 protein increase effect (in vivo):

[0141] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 2 weeks, and then the hippocampus was removed. The hippocampus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by ThermoFisher Scientific, and then centrifuged (10000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. Using this cell extract, analysis was performed by protein blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 2 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0142] (C)NAD + Volume-increasing effect (in vivo):

[0143] Using the test animals described in (2), the sample prepared in (1) (a) was orally administered once, and then the cerebral cortex was removed 180 minutes later. Also, using the test animals described in (2), the sample prepared in (1) (a) was mixed with the feed, and the feed was fed for 1 month, and then the cerebral cortex was removed. NAD in the cerebral cortex + NAD produced by Tongren Chemical Research Institute + / NADH detection kit (Assay Kit) was used for determination. The results are shown in Figure 3 . S-1-Propylenecysteine ​​increases NAD + quantity.

[0144] (5) Inhibition of p53 protein expression, a marker of cellular aging, in the hippocampus (in vivo):

[0145] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 6 weeks, and then the hippocampus was removed. The hippocampus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with protease inhibitors and phosphatase inhibitors manufactured by Roche. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-p53 antibody (manufactured by Proteintech) and anti-β-actin antibody (manufactured by the Institute of Medical Biology). The results are shown in Figure 4 S-1-allylcysteine ​​reduced the amount of p53 protein.

[0146] (6) Enhancement of SIRT1 gene expression in kidney (in vivo):

[0147] Using the evaluation test animals described in (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and RNA was extracted using a TRIzol solution manufactured by Thermo Fisher Scientific. cDNA was synthesized using the PRIMEScript RT reagent Kit with gEraser manufactured by TAKARA. Real-time polymerase chain reaction (Real-Time PCR) was performed on the synthesized cDNA using the KAPA SYBR Fast qPCR kit manufactured by NIPPON Genetics. The results are shown in Figure 5 S-1-allylcysteine ​​increased SIRT1 gene expression in mouse kidneys.

[0148] (7) Inhibition of p16 and p21 gene expression, markers of senescent cells in the kidney (in vivo):

[0149] The test animals of the evaluation method of (2) were repeatedly orally administered with the sample prepared in (1) (a) for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and RNA was extracted using TRIzol solution manufactured by Thermo Fisher Scientific.

[0150] cDNA was synthesized using PRIMEScript RT reagent Kit with gEraser manufactured by TAKARA. Real-time PCR was performed on the synthesized cDNA using KAPA SYBR Fast qPCR Kit manufactured by NIPPON Genetics. The results are shown in Figure 6 S-1-allylcysteine ​​reduces p16 and p21 gene-expressing cells in the kidneys of senescent mice.

[0151] (8) Kidney damage inhibition effect (in vivo)

[0152] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was repeatedly orally administered for 2 weeks, and then the kidneys were removed. The kidneys were crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with protease inhibitors and phosphatase inhibitors manufactured by Roche. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. Using this cell extract, analysis was performed by Western blotting according to a conventional method. Antibodies used were kidney injury molecule (KIM-1) antibody (manufactured by R&D system) and Lipocalin-2 (NGAL) antibody (manufactured by Proteintech), which are kidney injury markers, and anti-GAPDH antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 7 S-1-Propylenecysteine ​​reduced KIM-1 and NGAL.

[0153] (9) Cognitive / memory maintenance effect (in vivo):

[0154] Using the evaluation test animals of (2) above, the sample prepared in (1) (a) above was mixed into the bait, and after feeding for 4 months, a passive avoidance test was conducted. The passive avoidance test is a test that utilizes the habit of mice to prefer darker places. If the mice placed in the bright room move to the dark room, they will be subjected to electrical stimulation (50V·1 second). The time before the mice move to the dark room on the first day is used as the reaction latency of the collection test. After 2 months, the mice are placed in the bright room again, and the time to move to the dark room (the reaction latency of the retention test) is used as the evaluation method of the memory pointer. The results are shown in Figure 8 S-1-Propylenecysteine ​​improves cognitive / memory function decline associated with age.

[0155] (10) NAD in kidney, skeletal muscle and liver + Volume-increasing effect (in vivo):

[0156] The test animals of the evaluation method of (2) were orally administered once with the sample prepared in (1) (a) above, and then 60 minutes later, the cerebral cortex, liver, kidney, and skeletal muscle were removed. NAD in cerebral cortex, liver, kidney, and skeletal muscle + NAD produced by Tongren Chemical Research Institute + / NADH Assay Kit was used for determination. The results are shown in Fig. 9 . S-1-Propylenecysteine ​​increases NAD + quantity.

[0157] (11) Increased SIRT1 protein in the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle (in vivo):

[0158] About 10 mg of S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was accurately weighed and dissolved in 10 mL of purified water to prepare a dosing solution.

[0159] Using the evaluation test animals of (2) above, the prepared sample was repeatedly orally administered for 2 weeks, and then the cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle were removed. The cerebral cortex, hippocampus, heart, lung, liver, kidney, and skeletal muscle were crushed using an automatic tissue crusher, and then lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to conventional methods. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako PureChemical Industries, Ltd.). The results are shown in Fig.10 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0160] (12) Increased effect of SIRT1 protein in the hypothalamus (in vivo):

[0161] S-1-propenylcysteine ​​(cis / trans mixture) produced in Production Example 2 was dissolved in pure water at a concentration of 0.001% to prepare a dosing solution.

[0162] Animals used for evaluation of biological activity were bred as follows: Experimental aging-promoting mice SAMR1 (male) and SAMP8 (male) were purchased from Japan SLC. After purchase, they were acclimated for one week and used as evaluation test animals.

[0163] The evaluation test animals were fed with normal food or, for SAMP8, the sample prepared above was mixed with the feed, and the hypothalamus was removed after feeding for 10 months. The hypothalamus was crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific, and then centrifuged (10000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to conventional methods. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Fig.11 S-1-allylcysteine ​​increased SIRT1 protein amount.

[0164] (13) Increased effect of eNAMPT in blood (in vivo):

[0165] The amount of eNAMPT in the blood of the animals whose hypothalamus had been removed in (12) was measured according to the method of Yoshidaam et al. Cell Metab. 2019; 30(2): 329-342.e5. The results are shown in Fig.12 S-1-allylcysteine ​​increases the amount of eNAMPT in the blood.

[0166] (14) Increased amount of SIRT1 protein in liver and heart (in vivo):

[0167] The sample prepared in (1) (a) was repeatedly orally administered to the evaluation test animals prepared in the same manner as (12) for 2 weeks, and then the liver and heart were removed. The liver and heart were crushed using an automatic tissue crusher, and the cells were lysed using RIPA cell lysis buffer manufactured by Millipore, which was diluted to 10 times with purified water supplemented with a protease / phosphatase mixed inhibitor manufactured by Thermo Fisher Scientific. The cells were then centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was used as a cell extract. The cell extract was analyzed by Western blotting according to a conventional method. Antibodies used were anti-SIRT1 antibody (manufactured by Biolegend) and anti-β-actin antibody (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The results are shown in Fig.13 S-1-allylcysteine ​​increased SIRT1 protein amount.

Claims

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

2. An eNAMPT increasing agent, which contains S-1-allylcysteine ​​or a salt thereof and is administered to an animal. The preparation according to claim 2, which is used for increasing eNAMPT in blood.

4. A preparation containing S-1-allylcysteine ​​or a salt thereof and being administered to an animal, wherein the preparation is used for activation or expression enhancement of histone deacetylase type 3 in one or more organs, or inhibition of senescent cells.

5. A preparation containing S-1-allylcysteine ​​or a salt thereof and administered to an animal, the preparation being used for NAD in more than one organ + Increase.

6. The preparation according to claim 4 or 5, wherein The organ comprises at least one selected from the group consisting of brain, kidney, skeletal muscle, liver and heart.

7. The preparation according to claim 6, wherein The organ comprises at least one selected from skeletal muscle, liver and heart.

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

9. The preparation according to claim 4 or 5, which is used to act on two or more organs.

10. The preparation according to claim 9, which is used to act on three or more organs.

11. The preparation according to any one of claims 1, 4 or 5, which is in the form of a food additive or a food.

Images