Telomere shortening inhibitor and method for inhibiting telomere shortening

Abstract

The present invention provides a telomere shortening inhibitor and a method for inhibiting telomere shortening that can suppress telomere shortening associated with cellular senescence. [Solution] A telomere shortening inhibitor comprising at least one component (A) selected from the group consisting of α-glucosylrutin and ferulic acid.

Description

Technical Field

[0001] The present invention relates to a telomere shortening inhibitor and a telomere shortening inhibition method.

Background Art

[0002] A telomere is a complex of DNA having a tandem repeat sequence of TTAGGG (also referred to as a telomere sequence) and a telomere-binding protein, and is present at the end of a chromosome. The telomere connects the end of the chromosome and the nuclear membrane, and plays a role in protecting the chromosome from degradation and loss of genetic information.

[0003] Since the telomere sequence is not completely replicated in terms of its structure during chromosome replication, it shortens every time cell division is repeated. As a result, cells gradually show signs of aging and cell aging progresses. When the telomere sequence is shortened to a certain length, cell division stops (reaching the Hayflick limit), and it is known to induce cell death (apoptosis).

[0004] And because the accumulation of aging cells causes the tissues and organs in the body to decline, leading to individual aging, it is considered that the length of telomeres is related to lifespan. In addition, it is known that lack of sleep, obesity, smoking, stress, overeating, etc. accelerate telomere shortening (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). Therefore, the length of telomeres can be said to be not only an indicator of cell aging but also an indicator of healthy lifespan. Suppressing telomere shortening is considered to have an important meaning in terms of improving healthy lifespan.

[0005] As a method related to the length of telomeres, a method of increasing the length of telomeres by an aqueous extract of a species of the genus Smilax has been reported (Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] [Non-Patent Document 1] Jeong-Hwa Jin et al., Association between sleep parameters and longitudinal shortening of telomere length, Aging (Albany NY). 2022 Apr 2;14(7):2930-2944 [Non-Patent Document 2] AM Valdes et al., Obesity, cigarette smoking, and telomere length in women, The Lancet, Volume 366, Issue 9486, 20-26 August 2005: 662-664 [Non-Patent Document 3] Elissa S Epel et al., Accelerated telomere shortening in response to life stress, Proc Natl Acad Sci US A. 2004 Dec 7;101(49):17312-5 [Non-Patent Document 4] Lucia Alonso-Pedrero et al., Ultra-processed food consumption and the risk of short telomeres in an elderly population of the Seguimiento Universidad de Navarra (SUN) Projec, Am J Clin Nutr., 2020 Jun 1;111(6):1259-1266 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a telomere shortening inhibitor and a method for inhibiting telomere shortening that can suppress telomere shortening associated with cellular senescence. [Means for solving the problem]

[0009] The inventors diligently studied to solve the aforementioned problems. As a result, they found that the aforementioned problems can be solved by having the following configuration, and thus completed the present invention. The present invention relates, for example, to the following [1] to [5]. [1] A telomere shortening inhibitor comprising at least one component (A) selected from the group consisting of α-glucosylrutin and ferulic acid. [2] The telomere shortening inhibitor according to [1], wherein the α-glucosylrutin comprises α-monoglucosylrutin. [3] The telomere shortening inhibitor according to [2], wherein the α-monoglucosylrutin content in the α-glucosylrutin is 65% by mass or more. [4] The telomere shortening inhibitor according to any one of [1] to [3], wherein the ferulic acid is contained as a composition comprising 65% by mass or more of ferulic acid and 15% by mass or more of α-monoglucosyl hesperidin. [5] A method for inhibiting telomere shortening, comprising the step of administering a telomere shortening inhibitor described in any of [1] to [4]. [Effects of the Invention]

[0010] According to the present invention, it is possible to suppress telomere shortening associated with cellular senescence. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a graph showing the results of the telomere maintenance test. [Modes for carrying out the invention]

[0012] Next, the present invention will be described in detail. The description of "A~B" regarding the numerical range means A or more and B or less, unless otherwise specified. Also, % means mass %. The telomere shortening inhibitor of the present invention contains at least one component (A) selected from the group consisting of α-glucosylrutin and ferulic acid. The component (A) may be α-glucosylrutin and ferulic acid, or either α-glucosylrutin or ferulic acid. However, from the viewpoint of the telomere shortening inhibitory effect, ferulic acid is preferred.

[0013] [α-glucosylrutin] α-glucosylrutin (also referred to as α-glucoside rutin) is a general term for compounds in which one or more molecules of glucose are added to the glucose residue in the rutinose residue of rutin by an α1→4 bond. The α-glucosylrutin in the present invention may consist of one kind alone of such compounds having such a structure, or may be a mixture of two or more kinds.

[0014] α-glucosylrutin can be represented by the following formula (1). In formula (1), n is 0 or an integer of 1 or more, for example, an integer of 1~19.

[0015]

Chemical formula

[0016] α-glucosylrutin is a compound contained as a main component in a product known as "enzymatically treated rutin" (sometimes called "glycosyltransferase rutin"). Among α-glucosylrutins, those with only one glucose bonded are called "α-monoglucosylrutin", and those with two or more glucoses bonded are called "α-polyglycosylrutin". That is, in formula (1), α-monoglucosylrutin is a compound with n = 0, and α-polyglycosylrutin is generally a compound with n = 1~19.

[0017] Enzyme-treated rutin is an aggregate of compounds produced by enzymatic treatment of the sugars in rutin, and typically includes a mixture of compounds with different numbers of glucose molecules attached to rutin, such as a mixture of α-monoglucosylrutin and α-polyglucosylrutin. Furthermore, because enzyme-treated rutin is generally produced by enzymatic treatment, it may also contain unreacted rutin or other derivatives, such as isoquercitrin. Isoquercitrin (sometimes called "isoquercitrin") is a compound in which β-D-glucose is attached to the hydroxyl group at position 3 of the quercetin skeleton; in other words, it is a compound in which the rhamnose residue in the rutinose residue of rutin is cleaved.

[0018] Enzyme-treated rutin is a product obtained, for example, by treating rutin with a glycosyltransferase (such as cyclodextrin glucanotransferase (CGTase, EC2.4.1.19), an enzyme that has the function of adding glucose to rutin) in the presence of α-glucosyl sugar compounds (such as cyclodextrin or partially hydrolyzed starch) (referred to as "first enzyme-treated rutin" in this specification).

[0019] The first enzyme-treated rutin is a composition containing aggregates of various α-glucosylrutins with different numbers of bound glucose molecules, namely α-monoglucosylrutin and α-polyglucosylrutin, and unreacted rutin. If necessary, the first enzyme-treated rutin can be purified using, for example, a porous synthetic adsorbent and an appropriate eluate to remove sugar donors and other impurities, further reduce the rutin content, and increase the purity of α-glucosylrutin to obtain the first enzyme-treated rutin (purified α-glucosylrutin).

[0020] Furthermore, by treating the first enzyme-treated rutin with an enzyme having glucoamylase activity that cleaves α-1,4-glucosidic bonds at the glucose level, such as glucoamylase (EC3.2.1.3), in α-glucosylrutin to which multiple glucose molecules are attached, all glucose residues except for one glucose residue directly attached to the glucose residue (in the rutinose residue) of the rutin itself are cleaved, thereby obtaining enzyme-treated rutin containing a large amount of α-monoglucosylrutin (referred to herein as "second enzyme-treated rutin"). This enzyme treatment does not cause the glucose residue in the rutinose residue directly bound to the quercetin skeleton to be cleaved from the quercetin skeleton.

[0021] In the telomere formation inhibitor, considering the effects of the present invention, it is preferable to use enzyme-treated rutin, which is a composition containing α-glucosylrutin, and either a composition containing first enzyme-treated rutin or second enzyme-treated rutin may be used.

[0022] Considering the effects of the present invention, the enzyme-treated rutin is preferably a mixture containing at least α-glucosylrutin and further containing isoquercitrin. Such a mixture can be produced by the following procedure: (i) preparing the first enzyme-treated rutin described above; (ii) treating the first enzyme-treated rutin with an enzyme having glucoamylase activity to convert almost all of the α-glucosylrutin to α-monoglucosylrutin; and (iii) simultaneously treating it with an enzyme having rhamnosidase activity to convert almost all of the unreacted rutin to isoquercitrin.

[0023] The enzyme-treated rutin is preferably a composition containing α-monoglucosylrutin and isoquercitrin, and more preferably a composition containing 20 to 100% by mass of α-monoglucosylrutin and 1 to 20% by mass of isoquercitrin.

[0024] Commercially available enzyme-treated rutin products include, for example, "αG Rutin PS-C," "αG Rutin PS," "αG Rutin P," and "αG Rutin H" from Toyo Sugar Refining Co., Ltd. "αG Rutin PS-C" and "αG Rutin PS" are compositions containing 65% by mass of α-monoglucosylrutin and 15% by mass of isoquercitrin. "αG Rutin P" is a composition containing 60% by mass of α-glucosylrutin, 10% by mass of rutin, and 1% of isoquercitrin.

[0025] α-monoglucosylrutin is preferred as the α-glucosylrutin. This is because the molecular weight of α-monoglucosylrutin is smaller than that of α-polyglucosylrutin, resulting in a higher number of molecules per unit mass for α-monoglucosylrutin, which is considered advantageous in terms of its effects.

[0026] The presence of various α-glucosylrutins and other components in enzyme-treated rutin can be confirmed by HPLC chromatogram, and the content of each component, or the purity of a specific desired component, can be calculated from the peak area of ​​the chromatogram.

[0027] The method for producing α-glucosylrutin is not particularly limited, and known methods can be used. As mentioned above, it is preferable to produce it by enzymatic treatment of rutin because it yields a good yield and is easy to manufacture. The method for obtaining and preparing rutin is not particularly limited, and compounds that are generally manufactured and sold as reagents or purified products may be used, or compounds prepared by extraction from raw materials such as the peels of citrus fruits (mandarins, oranges, etc.) or buckwheat seeds may be used.

[0028] The amount of α-glucosylrutin contained in the telomere shortening inhibitor is not particularly limited. For example, the lower limit of the α-glucosylrutin content in the telomere shortening inhibitor may be 30% by mass, 40% by mass, 45% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass. The upper limit of the α-glucosylrutin content in the telomere shortening inhibitor may be 100% by mass, 99% by mass, 98% by mass, 95% by mass, 90% by mass, or 85% by mass. The range of the α-glucosylrutin content in the telomere shortening inhibitor can be arbitrarily set by arbitrarily combining the lower and upper limits, for example, ranges such as 30-100% by mass, 60-100% by mass, or 60-90% by mass can be set. From the viewpoint of inhibiting telomere shortening, it is preferable that the α-glucosylrutin content in the telomere shortening inhibitor is 65% by mass or more.

[0029] Since α-glucosylrutin, the active ingredient of the telomere shortening inhibitor, has good water solubility, it can be uniformly dissolved or dispersed even when added to water or formulations with a high water content.

[0030] [Ferulic acid] Ferulic acid is a compound also known as 3-(4-hydroxy-3-methoxyphenyl)2-propenoic acid. Ferulic acid may be in free form or as a salt. Examples of ferulic acid salts include salts with inorganic bases such as sodium, potassium, magnesium, and calcium, and salts with organic bases such as ammonium and triethylamine. Of these, salts with inorganic bases are preferred, and salts with sodium are more preferred.

[0031] The method for producing ferulic acid is not particularly limited; it may be extracted from plants or chemically synthesized, but it is preferably extracted from plants.

[0032] Examples of plants from which ferulic acid can be extracted include coffee, onion, radish, lemon, apple, orange, pineapple, peanut, artichoke, angelica tree, pine, Coptis japonica, sedge, sweet potato, corn, barley, wheat, rye, and rice, with rice being preferred and rice bran more preferred. Rice refers to the raw or dried seeds of the grass species Oryza sativa LINNE.

[0033] Methods for extracting ferulic acid from plants include known plant extraction methods. For example, one method involves adding an extraction solvent commonly used by those skilled in the art, such as ethanol, water, aqueous ethanol, or hexane, to a plant or its fragments, and then extracting by stirring or heating as needed. The extract may be concentrated as needed.

[0034] One method for extracting ferulic acid from rice bran is to partition rice bran oil obtained from rice bran with aqueous ethanol and hexane at room temperature and in a weakly alkaline environment, and then hydrolyze the ferulic acid ester obtained in the aqueous ethanol fraction with sulfuric acid under pressure and heating, and purify it.

[0035] The method for chemically synthesizing ferulic acid is not particularly limited, and known methods can be used, but one example is the condensation reaction of vanillin and malonic acid. Ferulic acid is commercially available in a highly purified powder form (for example, ferulic acid purity of 98% or higher) extracted from rice bran, and this can be used.

[0036] Ferulic acid has poor water solubility, and its use at high concentrations may be limited when added to water or water-rich formulations. Therefore, it is preferable to use a composition in which ferulic acid has been solubilized with a solubilizing agent. Examples of the solubilizing agent include surfactants, cyclodextrins, alkaline agents, and α-glucosyl hesperidin, but α-glucosyl hesperidin is preferred. When the solubilizing agent is α-glucosyl hesperidin, the composition preferably contains 10 to 95% by mass of ferulic acid and 5 to 90% by mass of α-glucosyl hesperidin, more preferably 30 to 90% by mass of ferulic acid and 10 to 70% by mass of α-glucosyl hesperidin, based on 100% by mass of the composition. When using the above composition, it is possible to uniformly dissolve or disperse it even when added to water or formulations with a high water content, and ferulic acid can be used at high concentrations. The method for producing the above composition is not particularly limited and can be produced by known methods.

[0037] As for ferulic acid, for example, one that conforms to the 10th edition of the Japanese Food Additives Standards for "ferulic acid" can be used. Examples of commercially available ferulic acid include the product "Ferulic Acid" from Oryza Oil & Fat Chemical Co., Ltd. "Ferulic Acid SL," currently under development by Toyo Sugar Refining Co., Ltd., is a composition in which ferulic acid is solubilized by α-glucosyl hesperidin, and contains 65% by mass of ferulic acid and 15% by mass of α-glucosyl hesperidin.

[0038] The amount of ferulic acid contained in the telomere shortening inhibitor is not particularly limited. For example, the lower limit of the ferulic acid content in the telomere shortening inhibitor can be 5% by mass, 10% by mass, 20% by mass, 30% by mass, 40% by mass, 50% by mass, or 60% by mass. The upper limit of the ferulic acid content in the telomere shortening inhibitor can be 65% by mass, 70% by mass, 75% by mass, 80% by mass, 90% by mass, or 100% by mass. The range of the ferulic acid content in the telomere shortening inhibitor can be arbitrarily set by arbitrarily combining the lower and upper limits, for example, ranges such as 5-100% by mass, 10-90% by mass, or 20-80% by mass can be set. From the viewpoint of telomere shortening inhibitory effect, it is preferable that the ferulic acid content in the telomere shortening inhibitor is 60% by mass or more.

[0039] [Telomere shortening inhibitors] In this specification, a telomere shortening inhibitor means an agent containing a component that has the effect of suppressing telomere shortening, and includes at least one component (A) selected from the group consisting of α-glucosylrutin and ferulic acid.

[0040] The telomere shortening inhibitory effect can be evaluated by whether or not it inhibits telomere shortening in a test that detects telomere shortening associated with cellular senescence in vitro (telomere maintenance test), as shown in the examples described later. For example, if the telomere length of cells that have been passaged with the test substance added is longer than the telomere length of cells that have been passaged under the same conditions without the test substance added, then the test substance can be evaluated as having a telomere shortening inhibitory effect.

[0041] The cells used in the telomere maintenance test are not particularly limited and any known cells can be used, but are preferably human fibroblasts, more preferably TIG cells, and even more preferably TIG1-20 cells. The degree of cellular senescence of the cells used in the telomere maintenance test is not limited, but typically the population doubling level (PDL) before the addition of the test substance is 50 or less, preferably 40 or less, more preferably 30 or less, and even more preferably 15 to 25. The aforementioned passage should be performed a number of times sufficient to detect telomere shortening, for example, 5 to 15 times, preferably 7 to 12 times, and more preferably 8 to 10 times.

[0042] The method for measuring telomere length is not particularly limited, and known methods can be used, such as the method described in the literature "Nathan J O' Callaghan & Michael Fenech, A quantitative PCR method for measuring absolute telomere length, Biological Procedure Online, 2011".

[0043] The telomere shortening inhibition rate is not particularly limited, but can be calculated using, for example, the following formula (1), and in the telomere maintenance test, a rate of 10% or more is preferred, and a rate of 30% or more is more preferred. Telomere shortening inhibition rate (%) = 100 - (L young -L old-sample ) / (L young -L old-control )×100...Equation (1) Telomere length of cells before passage: L young Telomere length of cells after passage without the addition of the test substance: L old-control Telomere length of cells after passage with the test substance added: L old-sample

[0044] The telomere shortening inhibitor may contain any component (A), consist solely of component (A), or may further contain any known components such as excipients, stabilizers, wetting agents, or emulsifiers, as long as they do not interfere with the telomere shortening inhibitory effect of component (A).

[0045] <Uses of telomere shortening inhibitors> Since the telomere shortening inhibitor has the effect of suppressing telomere shortening, it can be administered to the body once or multiple times for the purpose of suppressing telomere shortening in the body.

[0046] The dosage of the telomere shortening inhibitor can be appropriately selected depending on the age, sex, race, etc. of the recipient. For example, when the telomere shortening inhibitor is administered orally, 10 mg to 1000 mg of component (A) per day is preferred, and 100 mg to 300 mg is more preferred. The number of administrations of the telomere shortening inhibitor can be one or multiple times, and the daily administration frequency can be, for example, divided into 1 to 3 times per day, or divided into 2 or 3 times. The duration of administration of the telomere shortening inhibitor can be appropriately selected depending on the age, sex, race, etc. of the recipient. From the viewpoint of telomere shortening inhibitory effect, it is preferably 1 to 90 days, more preferably 14 to 60 days.

[0047] The telomere shortening inhibitor preferably suppresses telomere shortening associated with cellular senescence, and more preferably suppresses telomere shortening associated with cellular senescence in vitro. The telomere shortening inhibitor preferably suppresses telomere shortening accelerated by factors such as sleep deprivation, obesity, smoking, stress, and overeating.

[0048] The telomere shortening inhibitor can suppress telomere shortening associated with cellular senescence, thereby contributing to the delay of the progression of cellular senescence. Furthermore, the telomere shortening inhibitor contributes to improving healthy life expectancy under conditions that accelerate telomere shortening, such as lack of sleep, obesity, smoking, stress, and excessive eating and drinking.

[0049] [formulation] The telomere shortening inhibitor may be administered directly to the body, or it may be administered as a formulation in which the telomere shortening inhibitor is combined with a pharmaceutically acceptable carrier. Examples of formulations include food and beverages, pharmaceuticals, and quasi-drugs. The method of administration is not particularly limited and can be oral or parenteral. Oral administration is preferred because the formulation is easy to administer.

[0050] In the case of oral administration, the preparation may be a food or beverage (including health functional foods such as Foods for Specified Health Uses, Foods with Nutrient Function Claims, Foods with Function Claims, and other so-called health foods and supplements), a pharmaceutical product, a quasi-drug, etc.

[0051] Oral preparations can be solid or liquid (including paste). Dosage forms are not limited; specific examples of solid preparations include powders, granules, tablets, capsules, and lozenges. Examples of liquid preparations include oral solutions, suspensions, emulsions, syrups, and drinks. These and other dosage forms are selected as appropriate depending on the purpose.

[0052] In the case of parenteral administration, the preparation may be a topical skin preparation, an injection, a suppository, etc.

[0053] The aforementioned formulations can be manufactured by adding the telomere shortening inhibitor in accordance with methods commonly used for these formulations. The telomere shortening inhibitor may be added at the beginning of the manufacturing process of the formulation, or at the middle or end of the manufacturing process. The method of addition may be selected appropriately from mixing, kneading, dissolving, immersion, spraying, misting, coating, etc., depending on the form of the formulation. [Examples]

[0054] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto.

[0055] <Example 1: Telomere maintenance test> The telomere maintenance test was conducted based on the following literature. Nathan J O'Callaghan & Michael Fenech, A quantitative PCR method for measuring absolute telomere length, Biological Procedure Online, 2011

[0056] (reagent) • Test substance: αG-Resveratrol (manufactured by Toyo Sugar Refining Co., Ltd.): A composition containing resveratrol, comprising 5% or more by mass of α-glucosylresveratrol. αG-Rutin PS (manufactured by Toyo Sugar Refining Co., Ltd.): A composition containing 65% by mass of α-monoglucosylrutin and 15% by mass of isoquercitrin. αG-Hesperidin PA-T (manufactured by Toyo Sugar Refining Co., Ltd.): A composition containing hesperidin, comprising 75% by mass or more of α-monoglucosylhesperidin. αG-naringin PS (manufactured by Toyo Sugar Refining Co., Ltd.): A composition containing 75% by mass of α-monoglucosylnaringin and 10% by mass of 7-glucosylnaringenin. Ferulic acid SL (manufactured by Toyo Sugar Refining Co., Ltd.): A composition containing 65% by mass of ferulic acid and 15% by mass of α-monoglucosyl hesperidin.

[0057] • TIG1-20 cells (human fetal lung-derived fibroblasts, PDL = approximately 20) ·THUNDERBIRD Next SYBR qPCR Mix:TOYOBO ·LightCycler 480 Multiwell Plate 96, white:Roche ·qPCR Primer / Telomere&36B4 standard oligo:SIGMA-ALDRICH ·Ambion Nuclease-Free Water (Not DEPC Treated):Invitrogen(Thermo Fisher) Cell culture medium DMEM (1X) GlutaMAX: Gibco (Thermo Fisher) • Fetal bovine serum (FBS): SIGMA-ALDRICH ·Penicillin-Streptomycin Mixed Solution: nacalai tesque ·2.5g / L-Trypsin 1mM EDTA Solution: nacalai tesque ·BioLite 6Well Multidish:Thermo Scientific ·DNeasy Blood&Tissue Kit:QIAGEN

[0058] Measurement equipment: Light Cycler 480 System II (Roche) Test Flow

[0059] (method) 1. Long-term cell culture and gDNA extraction Cells were passaged 10 times under conditions with and without each test substance to induce cellular senescence, and telomere shortening associated with cellular senescence was detected. As a sample before telomere shortening, gDNA was extracted from a portion of cells at the start of the experiment, and this was designated as the Young sample. Each test substance was added to a culture medium (DMEM + 10% FBS + penicillin streptomycin 100 units / mL) at a concentration of 25 ng / μL, and cell culture was performed under the respective conditions. Cells without the test substance were also cultured in the same manner and used as the control (Old) sample.

[0060] Cell culture was performed in a 6-well multi-well plate, with a total of 10 passages, and 1.5 × 10⁶ cells per passage. 5 The concentration was adjusted to be equivalent to the number of cells per well. After 10 passages, gDNA was extracted from the cells. The extraction method followed the kit protocol. The gDNA was stored at 4°C.

[0061] 2. Measurement of telomere length by qPCR (i) Create a dilution series of the oligonucleotides to be used as standard substances. For both 36B4 standard oligo and telomere standard oligo, a dilution series (2x dilution each) was prepared so that the final concentration ranged from 100 pg / well to 1.56 pg / well.

[0062] [Creating a calibration curve] (i) First, primer mixes were prepared for both telomere primer and 36B4 primer as follows: Primer mix:Forward Primer 2μM+Reverse Primer 2μM

[0063] (ii) Master Mix solutions were prepared for both the Telomere standard and the 36B4 standard as follows: Total 16μL (qPCR mix 10μL, Primer mix 2μL, Nuclease Free water 4μL)

[0064] (iii) 4 μL of the standard substance and 16 μL of the Master Mix were loaded into each well. (iv) The telomere length was measured by qPCR. qPCR was performed for 45 cycles with cycles of 95°C for 15 seconds and 60°C for 1 minute after 95°C for 10 minutes. (v) Using the measurement results (Ct values), a calibration curve was created by the following calculation method.

[0065] <In the case of Telomere standard> Let the standard substance concentration be c (pg / well). a. The number of base pairs of the telomere sequence present in the well at c (pg / well) was calculated by the following formula (I). logTL (kb / reaction) = log(c × 10^-12 / 4.43 × 10^-20 × 1000 × 84) ··· Formula (I) Note that "4.43 × 10^-20: telomere standard" is the weight per molecule, and "84" is the number of bases per molecule of telomere standard. b. A calibration curve was created using the Ct value obtained from the sample with concentration c and the calculated logTL.

[0066] <In the case of 36B4 standard> Let the standard substance concentration be x (pg / well). a. The number of 36B4 sequences present in the well at x (pg / well) was calculated by the following formula (II). log copies ( / rection) = log(x × 10^-12 / 3.9 × 10^-20) ··· Formula (II) Note that "3.9 × 10^-20" is the weight per molecule of 36B4 standard. b. A calibration curve was created using the Ct value obtained from the sample with concentration x and the calculated log copies.

[0067] [Measurement of samples] (i) Each gDNA was diluted to 5 ng / μL to prepare a sample with a final concentration of 20 ng / well. (ii) Primer mixes were prepared for both telomere primer and β-globin primer (HK primer) as follows. Primer mix:Forward Primer 2μM+Reverse Primer 2μM

[0068] (iii) Master Mix solutions were prepared using each primer as follows: Total 16μL (qPCR mix 10μL, Primer mix 2μL, NF water 4μL) (iv) The sample was loaded into the wells (4 μL of sample and 16 μL of Master Mix per well). For each sample, a total of 4 wells were prepared: 2 wells to which Master Mix containing Telomere primer was added, and 2 wells to which Master Mix containing HK primer was added.

[0069] (v) Telomere length was measured by qPCR. The qPCR was performed for 45 cycles of 95°C for 10 min followed by 95°C for 15 seconds and 60°C for 1 min. (vi) Telomere length was calculated by comparison with the calibration curve. (vii) The test was administered three times, and the average value was calculated.

[0070] <Specific calculation method> • Ct values ​​from samples using Telomere primer Using a calibration curve obtained from the telomere standard, the log TL value corresponding to the Ct value was calculated. The logarithm was then removed to calculate the "telomere length per well."

[0071] • Ct values ​​from samples using HK primer Using a calibration curve obtained from the 36B4 standard, the number of log copies corresponding to the Ct value was calculated. The "number of DNA molecules per well" was then calculated by removing the logarithm. By dividing these values, we calculated the telomere length (Kb / human diploid) contained in the DNA.

[0072] (result) The results of the telomere maintenance test are shown in Table 1 and Figure 1.

[0073] [Table 1]

[0074] Table 1 and Figure 1 show that αG-resveratrol, αG-hesperidin PA-T, and αG-naringin PS hardly inhibit telomere shortening, while αG-rutin PS and ferulic acid SL inhibit telomere shortening. Ferulic acid SL (a composition containing 65% by mass of ferulic acid and 15% by mass of α-monoglucosylhesperidin) showed a telomere shortening inhibitory effect, while αG-hesperidin PA-T containing 75% by mass or more of α-monoglucosylhesperidin did not show a telomere shortening inhibitory effect. Therefore, it can be concluded that the telomere shortening inhibitory effect of ferulic acid SL is due to ferulic acid.

Claims

1. A telomere shortening inhibitor comprising at least one component (A) selected from the group consisting of α-glucosylrutin and ferulic acid.

2. The telomere shortening inhibitor according to claim 1, wherein the α-glucosylrutin comprises α-monoglucosylrutin.

3. The telomere shortening inhibitor according to claim 2, wherein the α-monoglucosylrutin content in the α-glucosylrutin is 65% by mass or more.

4. The telomere shortening inhibitor according to claim 1, wherein the ferulic acid is contained in a composition comprising 65% by mass or more of ferulic acid and 15% by mass or more of α-monoglucosyl hesperidin.

5. A method for inhibiting telomere shortening, comprising the step of administering a telomere shortening inhibitor according to any one of claims 1 to 4.

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