Cell senescence inhibitor

Abstract

To provide a cell senescence inhibitor that exhibits an inhibitory effect on cell senescence in mammals.SOLUTION: A cell senescence inhibitor comprises pyrroloquinoline quinone or a salt thereof, and arginine or leucine or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cellular aging inhibitor. [Background technology]

[0002] Aging is considered a risk factor for various diseases. Age-related predisposition to various diseases is known, including cancer, arteriosclerosis, pulmonary fibrosis, osteoporosis, osteoarthritis, and neurodegenerative diseases. Senescent cells are strongly associated with age-related pathologies that play an important role in the onset and progression of age-related diseases and disorders, and accumulate in tissues and organs during the aging process. The chronic accumulation of senescent cells in tissues and organs during the aging process is thought to be a major driving force behind the onset and progression of age-related diseases and disorders. Therefore, inhibiting the accumulation of senescent cells is believed to have the effect of suppressing aging overall. Therefore, methods for inhibiting senescent cell accumulation are needed. Senescent cells accumulate senescence-associated acid β-galactosidase (SA-β-gal), and senescence can be assessed by measuring the amount of accumulation. Pyrroloquinoline quinone has been known to inhibit aging (inducing longevity) (Patent Document 1). This anti-aging effect is strongly related to the quinone structure that undergoes oxidation and reduction, and is achieved by increasing the activity of oxidoreductases (Non-Patent Document 1).

[0003] However, imidazopyrroloquinoline compounds having a substituent at the 5-position, which are synthesized from pyrroloquinoline quinone and an amino acid, are typical derivatives of pyrroloquinoline quinone and are tricarboxylic acid compounds characterized by having an amino acid residue (Non-Patent Document 2), but the anti-aging function of such imidazopyrroloquinoline compounds is not known.

[0004] Until now, it has been thought that imidazopyrroloquinoline does not have the redox activity required for the anti-aging mechanism of pyrroloquinoline quinone, and therefore cannot act on redox enzymes, which are an important mechanism for anti-aging. In fact, it has been reported that imidazopyrroloquinoline (7-oxo-7,10-dihydroimidazo[4,5,1-ij]pyrrolo[2,3-f]quinoline-1,3,9-tricarboxylic acid) does not have the lifespan extension effect in nematodes (Non-Patent Document 1).

[0005] However, it is known that unsubstituted imidazopyrroloquinoline esters are effective in scavenging active oxygen and treating diabetes (Patent Documents 2 and 3).

[0006] On the other hand, pyrroloquinoline quinone is known to increase mitochondria. Mitochondria are intracellular organelles that play an important role in metabolism. The amount of mitochondria in cells is important for cellular activation. Improvement of mitochondrial function can improve metabolic disorders, muscle disorders, and neurodegenerative diseases that occur with aging, so it is desirable for it to have this improving function. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 047637 [Patent Document 2] Japanese Patent Application Publication No. 10-045594 [Non-patent literature]

[0008] [Non-Patent Document 1] Journal of Cell Science, 2017, p2631-2643 [Non-patent document 2] BioFnctors vol.-5 no.2 pp.75-81. 199.5 / 1996 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a cell aging inhibitor and a food composition that have the effect of inhibiting cell aging in mammals. [Means for solving the problem]

[0010] In order to achieve the objects of the present invention, the present inventors have conducted extensive research and have unexpectedly found that imidazopyrroloquinoline compounds represented by the following formulae (1) and (2) have excellent anti-aging effects. Furthermore, they have confirmed that when a mammal orally ingests pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof, these imidazopyrroloquinoline compounds are produced during digestion in the digestive tract, thereby completing the present invention.

[0011] That is, the present invention is as follows. [1] A cell aging inhibitor comprising pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof. [2] The cell aging inhibitor described in [1] above, further comprising an edible protein. [3] The cell aging inhibitor according to the above [1], wherein the content of arginine or leucine or a salt thereof is 1 to 10 parts by weight relative to the content of pyrroloquinoline quinone or a salt thereof. [4] The cell aging inhibitor according to [1] above, which increases the amount of mitochondria in cells. [5] The agent for inhibiting cellular senescence according to [1] above, which inhibits cellular senescence accompanied by increased activity of senescence-associated acid β-galactosidase (SA-β-gal). [6] A food composition for inhibiting cellular aging, comprising pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof. [7] A cellular aging inhibitor comprising, as an active ingredient, a compound represented by the following formula (1) or a salt thereof:

[0012] [ka]

[0013] [8] The cell aging inhibitor according to [7] above, which increases the amount of mitochondria in cells. [9] A cellular aging inhibitor comprising, as an active ingredient, a compound represented by the following formula (2) or a salt thereof:

[0014] [ka]

[0015]

[10] The cell aging inhibitor according to [9] above, which increases the amount of mitochondria in cells. [Effects of the Invention]

[0016] The cellular aging inhibitor and food composition of the present invention have an inhibitory effect on cellular aging in mammals. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a cellular aging inhibitor or a food composition comprising, as an active ingredient, a compound represented by the following formula (1) or (2) or a salt thereof:

[0018] [ka]

[0019] The inventors have confirmed that when a mammal orally ingests pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof, an imidazopyrroloquinoline compound represented by formula (1) or formula (2) is generated during digestion in the digestive tract. Therefore, the present invention provides a cellular aging inhibitor or food composition containing pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof.

[0020] The cellular senescence inhibitor and food composition of the present invention have the effect of inhibiting cellular senescence in mammals. The cellular senescence inhibitor and food composition of the present invention have the effect of inhibiting cellular senescence, particularly cellular senescence accompanied by increased activity of senescence-associated acid β-galactosidase (SA-β-gal). Examples of mammals include humans, pigs, cows, horses, goats, sheep, wild boars, rabbits, mice, rats, hamsters, guinea pigs, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, dogs, and cats.

[0021] The cellular aging inhibitor or food composition of the present invention preferably has the function of increasing the amount of mitochondria in mammalian cells.

[0022] From the viewpoint of achieving high efficacy while keeping costs low, the content of arginine or leucine, or a salt thereof, in the cellular aging inhibitor or food composition of the present invention is, by weight, preferably 1 to 100, more preferably 1 to 50, and even more preferably 1 to 10, relative to the content of pyrroloquinoline quinone or a salt thereof of 1. The cellular aging inhibitor of the present invention is preferably a solid.

[0023] When the cellular aging inhibitor or food composition of the present invention contains a salt of pyrroloquinoline quinone, the salt of pyrroloquinoline quinone is preferably an alkali metal salt, more preferably a disodium salt. The salt of pyrroloquinoline quinone may be a hydrate crystal, or other solvents may be incorporated into the crystal structure. Pyrroloquinoline quinone or its salt can be produced by known methods such as fermentation or organic synthesis, and purified by recrystallization or column chromatography can be used. When the cellular aging inhibitor of the present invention contains a salt of arginine, the salt of arginine is preferably arginine hydrochloride.

[0024] The cell aging inhibitor or food composition of the present invention preferably further contains an edible protein. The inclusion of an edible protein can further promote the production of imidazopyrroloquinoline compounds in vivo. Usable edible proteins are not particularly limited, but examples include whey protein, casein protein, soy protein, and pea protein, with soy protein and whey protein being particularly preferred. Foods containing edible protein that are provided as food can be used as is, and foods with a protein content of preferably 20% by mass or more can be used as edible proteins.

[0025] The cellular senescence inhibitor or food composition of the present invention may further contain other known components that inhibit cellular senescence, such as sodium ascorbate, ascorbic acid, and D-α-tocopherol.

[0026] The cellular aging inhibitor of the present invention may be a product in the pharmaceutical or food field, or may be a raw material used in the production of a product in the pharmaceutical or food field.

[0027] The product may be, for example, an oral preparation. Such oral preparations may be in the form of, but are not limited to, capsules, tablets, granules, powders, pills, suspensions, emulsions, infusions, decoctions, syrups, liquids, etc. Oral preparations may also be in the form of jelly, gummies, or crushed jelly. Such oral preparations may contain, but are not limited to, typical formulation additives such as excipients, binders, disintegrants, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, bacterial inhibitors, flavors, sweeteners, and coloring agents.

[0028] Examples of excipients include sugars such as glucose, lactose, sucrose, mannitol, and sorbitol; starches such as corn starch, potato starch, dextrin, α-starch, and carboxymethyl starch; celluloses such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; silicates such as gum arabic, dextran, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphates such as calcium phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate.

[0029] Examples of binders include gelatin, polyvinylpyrrolidone, and macrogol.

[0030] Examples of disintegrants include chemically modified starches or celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.

[0031] Examples of lubricants include talc, stearic acid, glycerin monostearate, metal stearates such as calcium stearate and magnesium stearate, colloidal silica, waxes such as pea gum and gaelt, carboxylic acids such as boric acid, glycol, fumaric acid and adipic acid, sodium carboxylates such as sodium benzoate, sulfates such as sodium sulfate, lauryl sulfates such as leucine, sodium lauryl sulfate and magnesium lauryl sulfate, and silicic acids such as silicic anhydride and silicic acid hydrate.

[0032] Examples of dispersing agents include propylene glycol alginate, ethanol, carmellose sodium, citric acid hydrate, sodium citrate hydrate, glycerin, glycerin fatty acid esters, magnesium silicate, light anhydrous silicic acid, crystalline cellulose, crystalline cellulose-carmellose sodium, titanium oxide, sucrose fatty acid esters, stearic acid, magnesium stearate, oleic acid, sorbitan fatty acid esters, low-substituted hydroxypropyl cellulose, dextrin, sodium starch glycolate, corn starch, concentrated glycerin, hydroxypropyl starch, hydroxypropyl cellulose, hypromellose, propylene glycol, povidone, polysorbate 80, macrogol (such as macrogol 300 and macrogol 4000), anhydrous sodium citrate, magnesium aluminometasilicate, glycerin monooleate, sorbitan monooleate, aluminum monostearate, glycerin monostearate, sodium lauryl sulfate, lauromacrogol, and calcium hydrogen phosphate hydrate.

[0033] Examples of suspending agents include polysorbate 80, polyoxyethylene hydrogenated castor oil (N), sucrose fatty acid esters, soybean lecithin, macrogol (such as macrogol 4000 and macrogol 6000), sodium hydroxide, hydroxypropyl cellulose, glycerin, D-sorbitol, magnesium aluminometasilicate, carboxyvinyl polymer, dried aluminum hydroxide gel, xanthan gum, butylhydroxyanisole, propylene glycol, crystalline cellulose, gum arabic, powdered gum arabic, hypromellose, powdered agar, povidone, methylcellulose, kaolin, carrageenan, carmellose sodium, glycerin fatty acid esters, and magnesium aluminum silicate.

[0034] Examples of emulsifiers include glycerin monostearate, sodium lauryl sulfate, polyoxyethylene hydrogenated castor oil (N), polysorbate 80, medium-chain fatty acid triglyceride, soybean lecithin, and lauromacrogol.

[0035] Examples of diluents include dibasic calcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Examples of buffers include anhydrous citric acid, sodium citrate hydrate, sodium bicarbonate, lactic acid, citric acid hydrate, sodium benzoate, tartaric acid, DL-malic acid, sodium chloride, acetic acid, sodium acetate hydrate, anhydrous sodium monohydrogen phosphate, L-glutamic acid, and dilute hydrochloric acid.

[0036] Examples of antioxidants include sodium ascorbate, ascorbic acid, calcium ascorbate, D-α-tocopherol, ethoxyquin, dibutylhydroxytoluene, ascorbyl palmitate, butylhydroxyanisole, etc. Examples of bacteria inhibitors include butylhydroxyanisole, dibutylhydroxytoluene, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, etc.

[0037] Examples of flavoring agents include menthol, peppermint oil, etc. Examples of sweeteners include D-sorbitol, xylitol, refined white sugar, lactose hydrate, white sugar, glucose, D-mannitol, aspartame, reduced maltose syrup, licorice extract, saccharin, saccharin sodium hydrate, sucralose, purified stevia extract, maltitol, acesulfame potassium, etc.

[0038] Examples of coloring agents include yellow ferric oxide, indigo carmine, titanium oxide, carmine, black iron oxide, ferric oxide, synthetic food colors (food blue No. 1, food blue No. 2, food yellow No. 4, food yellow No. 5, food red No. 2, food red No. 3, food red No. 102, etc.), natural food colors, etc.

[0039] Furthermore, the product may be, for example, a health food, functional food, nutritional supplement, health supplement (supplement), health food, food for specified health uses, nutritionally functional food, food with functional claims, etc. Such foods may contain additives commonly used in foods, such as sweeteners, coloring agents, preservatives, thickening and stabilizing agents, antioxidants (antioxidants), color formers, bleaching agents, antibacterial and antifungal agents, gum bases, bittering agents, enzymes, glazing agents, acidulants, emulsifiers, strengthening agents, manufacturing agents, flavorings, and spice extracts.

[0040] Examples of preservatives include sodium dehydroacetate, potassium sorbate, etc. Examples of thickening stabilizers include guar gum, locust bean gum, carrageenan, gum arabic, alginic acids, pectin, xanthan gum, pullulan, tamarind seed gum, psyllium seed gum, crystalline cellulose, carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, agar, glucomannan, gelatin, dextrin, starch, dextran, etc.

[0041] Examples of color formers and bleaching agents include sodium nitrite. Examples of antibacterial and antifungal agents include acetic acid and citric acid. Examples of gum bases include methyl acetylricinoleate, urushi wax, ester gum, elemi resin, cucumber wax, ozokerite, and opopanax resin. Examples of bittering agents include isoalpha-bitter acid, caffeine, Coriolus versicolor extract, Cinchona extract, Phellodendron amurense extract, and Gentian extract. Examples of enzymes include nattokinase. Examples of brightening agents include silicon dioxide and titanium dioxide. Examples of acidulants include acetic acid, lactic acid, and citric acid. Examples of strengthening agents include vitamins such as retinol and D-α-tocopherol. Examples of manufacturing agents include calcium silicate and cyclodextrin. Examples of spice extracts include extracts from mustard, pepper, sesame, cinnamon, onion, garlic, basil, paprika, rosemary, and wasabi.

[0042] The product may also be, for example, a food additive. Such food additives can be added to, for example, dairy products, fermented foods, beverages, spreads, pastes, Western confectioneries, frozen desserts, seasonings, cooked foods, etc. More specifically, they can be added to miso, soy sauce, miso soup, ramen, yakisoba, curry, corn soup, mapo tofu, mapo eggplant, pasta sauce, pudding, cake, bread, yogurt, cheese, coffee, juice, cocoa, tea drinks, sports drinks, soft drinks, dairy drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, sake, Western liquor, fruit liquor, custard cream, fruit paste, chocolate, donuts, pies, cream puffs, gum, gummy candies, jelly, candy, cookies, cakes, puddings, biscuits, ice cream, popsicles, sherbet, beef bowls, rice porridge, pickles, jam, ham, sausages, bacon, dressings, furikake (seasoning toppings), umami seasonings, soup bases, ketchup, oyster sauce, etc.

[0043] The food composition of the present invention is in the form of a food additive, or in the form of a food such as a health food, functional food, nutritional supplement, health supplement (supplement), health food, food for specified health uses, functional nutritional food, food with functional claims, dairy product, fermented food, beverage, spread, paste, Western confectionery, frozen dessert, seasoning, or prepared food.

[0044] The effective dose for inhibiting cellular senescence of the cellular senescence inhibitor or food composition of the present invention is preferably 5 mg to 150 mg per day (for adults) based on the amount of pyrroloquinoline quinone, or the compound represented by formula (1) or (2), or a salt thereof. In the case of a food product, the intake amount based on the amount of pyrroloquinoline quinone, or the compound represented by formula (1) or (2), or a salt thereof is preferably 1.66 to 50 mg per meal, and in the case of an oral preparation, the dosage per dose is preferably 3 to 40 mg based on the same standard. In particular, capsules or tablets are preferably used, with one tablet containing 3 to 40 mg per dose.

[0045] The effect of the cellular senescence inhibitor of the present invention can be evaluated by detecting the amount of senescent cells. Mammalian cellular senescence can be detected using a microscope or plate reader by staining with senescence-associated acid β-galactosidase (SA-β-Gal), a marker for detecting senescent cells, and assay kits for such tests are commercially available. Furthermore, an increase in mitochondrial mass can be evaluated using a mitochondrial staining reagent. Measurements can be performed using a microscope or plate reader.

[0046] The generation of imidazopyrroloquinoline compounds in the digestive tract when pyrroloquinoline quinone or a salt thereof and arginine or leucine or a salt thereof are orally ingested can be evaluated using HPLC (high-performance liquid chromatography), NMR (nuclear magnetic resonance spectroscopy), UV (ultraviolet absorption spectroscopy), etc. By directly measuring a sample with HPLC, the presence of imidazopyrroloquinoline compounds can be confirmed from the peaks in the chromatogram. By analyzing the generation under conditions similar to the in vivo environment, the generation of imidazopyrroloquinoline compounds in the in vivo environment can be evaluated. Specifically, this can be clarified by analysis in the gastrointestinal environment at around 37°C. [Example]

[0047] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. In the following description, unless a temperature is specified, the temperature condition is room temperature (23±3°C), and unless a pressure is specified, the pressure condition is atmospheric pressure (1 atm). Unless otherwise specified, pyrroloquinoline quinone disodium manufactured by Mitsubishi Gas Chemical Company (product name BioPQQ (registered trademark)) was used. Unless otherwise specified, reagents used were manufactured by Fujifilm Wako Co., Ltd. For ultraviolet absorption spectra, Shimadzu Corporation's product name "UV1800" was used.

[0048] Example 1: Synthesis of disodium 5-(3-guanidinopropyl)-7-oxo-7,10-dihydroimidazo[4,5,1-ij]pyrrolo[2,3-f]quinoline-1,3,9-tricarboxylate (disodium salt of the compound represented by formula (1)) (hereinafter, the compound represented by formula (1) will be abbreviated as "ArgIPQ" and the disodium salt thereof will be abbreviated as "ArgIPQ salt") 1 g of pyrroloquinoline quinone disodium and 1 g of arginine were mixed with 20 mL of water and stirred at room temperature for at least 30 minutes to obtain a mixture. This mixture was heated to 70°C and reacted for 1 day. After the reaction, a yellow solid precipitated in the liquid. The mixture was then cooled to room temperature and filtered to obtain a yellow solid. The yellow solid was washed with 10 mL of ethanol and dried under reduced pressure at room temperature for 12 hours to obtain 1.3 g of yellow crystals.

[0049] HPLC condition 1: Shimadzu LC-2010 Column: YMC-Pack ODS-A Detection wavelength: 259 nm Mobile phase: 30 mM acetic acid - 70 mM ammonium acetate Column temperature: 40°C The amount of ArgIPQ was calculated under these conditions.

[0050] Example 2: Synthesis of 5-isobutyl-7-oxo-7,10-dihydroimidazo[4,5,1-ij]pyrrolo[2,3-f]quinoline-1,3,9-tricarboxylic acid (compound represented by formula (2)) (hereinafter abbreviated as "LeuIPQ") 3.5 g of leucine was dissolved in 200 mL of water. The pH was 6 (test paper). Air was blown in at 1 L / min, and 1.0 g of pyrroloquinoline quinone was added and stirred. After 2 hours, the pH was 7 (test paper). After overnight reaction, 4 mL of concentrated hydrochloric acid was added and the pH was adjusted to 1. 12 g of NaCl was added and the mixture was chilled in the refrigerator. After overnight centrifugation, the precipitated cake was recovered. 200 mL of ethyl acetate was added to the cake and thoroughly dissolved. The ethyl acetate layer was decanted and filtered using a No. 2 filter paper funnel. This process was repeated four times. 800 mL of ethyl acetate layer was obtained. The ethyl acetate was removed using an evaporator, yielding 0.75 g of solid (80% yield, 98.0% HPLC purity). 0.75 g of the resulting solid was dissolved in 50 mL of methanol and filtered. This methanol solution was concentrated to 15 mL and chilled in the refrigerator for 3 hours. The precipitated solid was centrifuged, washed with 1 mL of ether, and dried under reduced pressure at 50° C. 159 mg of a solid was obtained (HPLC purity 99.4%). 1H-NMR(DMSO-d6, DMSO2.5ppm) δ 0.87d,(Me), 2.07m(CH), 3.00d(CH2), 7.17(aromatic), 7.86(aromatic)

[0051] HPLC condition 2: Shimadzu LC-2010 Column: Inert Sustain Detection wavelength: 259 nm Mobile phase: 35% methanol-phosphoric acid Column temperature: 40°C The amount of LeuIPQ was calculated under these conditions.

[0052] Test Example 1: Cell-based assay The cells used were IEC-6 intestinal cells. The medium used was 500 mL of DMEM low glucose (1 g / L) (GIBCO 11885-084) + 5 mL of 100% penicillin-streptomycin (WAKO 168-23191) + 200 μL of insulin (10 mg / mL) (SIGMA). The cells were cultured in 5% CO2 at 37°C.

[0053] Senescent cell inhibition experiment Senescent cells can be induced by treating cells with D-galactose. IEC6 cells were seeded at 10,000 cells / well in a 96-well microplate. The following day, D-galactose (20 g / L) in serum-free medium and the test compounds (ArgIPQ salt in Example 1 and LeuIPQ in Example 2) (20 μM) were added. The cells were incubated for 48 hours at 37°C and 5% CO2. A control experiment was performed without adding galactose.

[0054] Senescent cell detection test (Cellular Senescence Plate Assay Kit - SPiDER-βGal): Cellstain Hoechst 33342 solution was diluted 500 times with HBSS(+) buffer (nucleic acid staining solution). The β-gal solution was diluted 10 times with Assay buffer. (β-gal assay solution) 100uL of nucleic acid staining solution was added to each well and incubated at 37℃ for 30 minutes. The liquid was removed from the plate and washed once with HBSS(+) buffer. 100uL HBSS(+) buffer was added and the fluorescence intensity was measured using a microplate reader. Nucleic acid: 355 / 460 nm filter The liquid was removed from the plate, 50uL of lysis buffer was added, and the plate was incubated at room temperature for 10 minutes. 50 μL of β-gal assay solution was added and incubated at 37°C for 2 hours. 100µL Stop Solution was added, and the fluorescence intensity was measured using a microplate reader with a GFP filter (n=8).

[0055] The number of senescent cells without the addition of galactose was set to 100, and the number of senescent cells without the addition of the substance to be evaluated was 129. This increase in senescent cells was set to 100. As an example, the anti-aging effect of 20 μM of the additives (ArgIPQ salt in Example 1 and LeuIPQ in Example 2) was calculated. The results are summarized below.

[0056] [Table 1]

[0057] The compound of the present invention has the effect of inhibiting cell aging. It was an unexpected result that a substance with this skeleton can inhibit aging.

[0058] Test Example 2: Mitochondrial Increase Assay IEC6 cells were seeded at 6,000 cells / well in a 96-well microplate. The next day, the substances to be evaluated (ArgIPQ salt of Example 1, LeuIPQ of Example 2, and pyrrolopyrroline quinone) (20 μM) were added. The mixture was incubated at 37°C and 5% CO for 48 hours. Mitochondrial Assay (MitoBright LT Green): Cellstain Hoechst 33342 solution was diluted 500 times with HBSS(+) buffer (nucleic acid staining solution). MitoBright LT Green solution was diluted 1000 times with HBSS(+) buffer (Mito staining solution). 100uL of nucleic acid staining solution was added to each well and incubated at 37℃ for 30 minutes. The liquid was removed from the plate and washed once with HBSS(+) buffer. 100uL of Mitochondrial Staining Solution was added to each well and incubated at 37℃ for 30 minutes. The liquid was removed from the plate and washed once with HBSS(+) buffer. 100uL HBSS(+) buffer was added and the fluorescence intensity was measured using a microplate reader. Nucleic acids: 355 / 460nm filter Mitochondria: 485 / 535nm filter (n=8)

[0059] The number of mitochondria without addition was calculated as 100.

[0060] [Table 2]

[0061] The compound of the present invention effectively inhibited senescent cells, and also significantly increased mitochondrial function, more so than pyrroloquinoline quinone, which has been known to be highly effective.

[0062] Test Example 3: Generation test of ArgIPQ of formula (1) Pyrroloquinoline quinone disodium, arginine hydrochloride, and other additives were mixed. To replicate the in vivo environment, the amount of compound of formula (1) produced was measured under neutral conditions (intestine) at 37°C. Phosphate buffer (GIBCO PBS pH 7.2) was used. 2 mg of pyrroloquinoline quinone disodium was placed in a 2 mL test tube, and 1 mL of phosphate buffer was added. The mixture was allowed to react at 37°C for 1 hour.

[0063] The amount of ArgIPQ generated by formula (1) was measured using a UV spectrometer or HPLC, and the results are shown in the table below.

[0064] [Table 3]

[0065] Under the conditions of the example, the senescent cell inhibitory effect was maintained and the compound was efficiently converted into a component with a higher mitochondria-increasing effect than pyrroloquinoline quinone.

[0066] Test Example 4: Generation test of LeuIPQ of formula (2) Pyrroloquinoline quinone disodium, leucine, and other additives were mixed together. To reproduce the in vivo environment, the amount of compound of formula (2) produced was examined under neutral conditions (in the intestine) at 37°C. Phosphate buffer (GIBCO PBS pH 7.2) was used. 2 mg of pyrroloquinoline quinone disodium was placed in a 2 mL test tube, and 1 mL of phosphate buffer was added, followed by reaction at 37°C for 1 hour.

[0067] The amount of LeuIPQ generated by formula (2) was measured by UV spectrometer or HPLC, and the results are shown in the table below.

[0068] [Table 4]

[0069] Under the conditions of the examples, the compound was efficiently converted into a component that had an anti-aging cell effect and a higher mitochondria-increasing effect than pyrroloquinoline quinone. The present invention makes it possible to effectively generate compounds that have anti-aging cell and mitochondria-increasing effects in vivo. [Industrial Applicability]

[0070] The cellular aging inhibitor of the present invention is useful in various fields such as pharmaceuticals, cosmetics, foods (particularly functional foods), and feeds.

Claims

1. A cell aging inhibitor comprising pyrroloquinoline quinone or a salt thereof, and arginine or leucine or a salt thereof.

2. The cellular aging inhibitor according to claim 1 , further comprising an edible protein.

3. 2. The cell aging inhibitor according to claim 1, wherein the content of arginine or leucine or a salt thereof is 1 to 10 parts by weight relative to the content of pyrroloquinoline quinone or a salt thereof.

4. The cell aging inhibitor according to claim 1, which increases the amount of mitochondria in cells.

5. The agent for inhibiting cellular senescence according to claim 1, which inhibits cellular senescence accompanied by increased activity of senescence-associated acid β-galactosidase (SA-β-gal).

6. A food composition for inhibiting cellular aging, comprising pyrroloquinoline quinone or a salt thereof, and arginine or leucine or a salt thereof.

7. A cellular aging inhibitor comprising, as an active ingredient, a compound represented by the following formula (1) or a salt thereof: 【Chemical 1】

8. The cell aging inhibitor according to claim 7, which increases the amount of mitochondria in cells.

9. A cellular aging inhibitor comprising, as an active ingredient, a compound represented by the following formula (2) or a salt thereof: 【Chemistry 2】

10. The cell aging inhibitor according to claim 9, which increases the amount of mitochondria in cells.

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