Clock gene expression regulator

Abstract

To provide a novel technology that regulates the expression of the clock gene.SOLUTION: A clock gene expression regulator contains α-monoglucosylrutin as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a clock gene expression regulator. [Background technology]

[0002] Humans and many other mammals have an internal clock (circadian clock) with a cycle of approximately 24 hours. It is known that disruptions to the internal clock due to recent environmental changes can cause sleep disorders and metabolic disorders, and that the function of the internal clock declines with age. Bright light therapy is known as a treatment for disruptions to the internal clock.

[0003] Furthermore, a technology relating to improving sleep is described in Patent Document 1 (JP 2019-202958 A). This document describes a composition containing hesperidin and tea polyphenols for improving sensitivity to cold, alleviating stiff shoulders, recovering from fatigue, or improving sleep (claim 1), and states that administration of this composition improves the absorbability of hesperidin, thereby concentrating blood in the periphery and increasing skin temperature, thereby improving sleep (paragraph 0035).

[0004] Furthermore, clock genes are involved in the mechanism of the biological clock. Patent Document 2 (JP 2008-266319 A) describes a technology relating to the regulation of the expression rhythm of clock genes. This document describes (paragraph 0014) a circadian rhythm regulating composition containing a specific substance as an active ingredient (claim 1) as a technology aimed at preventing, improving, and controlling abnormalities in physiological phenomena (sleep, wakefulness, hormone secretion, body temperature, digestion, blood pressure, metabolism, etc.) caused by disruption of circadian rhythms. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2019-202958 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-266319 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a new technique for regulating the expression of clock genes. [Means for solving the problem]

[0007] According to the present invention, the following clock gene expression regulators are provided: [1] A clock gene expression regulator containing α-glucosylrutin as an active ingredient. [2] The clock gene expression regulator described in [1], which has the effect of regulating circadian rhythm. [3] The clock gene expression regulator according to [1] or [2], wherein the clock gene is at least one selected from the group consisting of Bmal gene, Per gene, Cry gene and Clock gene. [4] A clock gene expression regulator described in any one of [1] to [3], which is taken orally. [5] A clock gene expression regulator described in any one of [1] to [4], which is an agent for improving at least one symptom selected from the group consisting of fatigue, drowsiness, and lethargy at any time from the time of waking up to daytime. [Effects of the Invention]

[0008] According to the present invention, a new technique for regulating the expression of clock genes can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of evaluation of the expression level of the Per1 gene in an example. [Figure 2] FIG. 1 shows the results of evaluation of the expression level of the Per2 gene in an example. [Figure 3] FIG. 1 shows the results of evaluation of the expression level of the Bmal1 gene in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an embodiment of the present invention will be described. In this embodiment, the composition may contain each component either alone or in combination of two or more. In this specification, the symbol "to" indicating a numerical range means "greater than or equal to" or "less than or equal to," and both of the numerical values ​​at both ends are included.

[0011] (regulator of clock gene expression) In this embodiment, the clock gene expression regulator (hereinafter also simply referred to as "regulator") contains α-glucosylrutin as an active ingredient. Specifically, the regulator is a composition that has the effect of regulating clock gene expression and contains α-glucosylrutin as an active ingredient.

[0012] In this embodiment, the regulator contains α-glucosylrutin as an active ingredient, and therefore can improve quality of life.

[0013] The regulator preferably regulates circadian rhythms by regulating the expression of clock genes, and more preferably resets the body clock. This can further improve the quality of life during active hours. Furthermore, resetting the body clock can also improve sleep disorders and suppress the decline in vitality and energy that accompanies aging, including the improvement of lifestyle balance.

[0014] The regulator is preferably at least one agent selected from the group consisting of fatigue, sleepiness, and lethargy that occurs any time from the time of waking up to daytime, thereby improving the quality of life during active hours.

[0015] Specifically, the clock gene is a gene that controls the biological clock, and is preferably at least one selected from the group consisting of Bmal (Brain and Muscle Arnt-like) genes such as Bmal1 and Bmal2; Per (Period) genes such as Per1 and Per2; Cry (Cryptochrome) genes such as Cry1 and Cry2; and Clock genes, and more preferably at least one of the Per gene and the Bmal gene. This allows for more stable improvement of the circadian rhythm regulation effect.

[0016] The expression of clock genes specifically refers to the expression of clock genes or proteins encoded by clock genes. The regulator preferably has the effect of transiently enhancing the expression of clock genes, thereby enabling the biological clock to be reset more stably. Similarly, the regulator has the effect of enhancing the expression of the Per gene, and more preferably has the effect of enhancing the expression of the Per gene and suppressing the expression of the Bmal gene.

[0017] Specific indices that characterize the expression rhythm of clock genes include amplitude, period, and phase. Specifically, amplitude refers to the width from the peak to the trough in the clock gene expression rhythm, and peak height refers to the height from the baseline (time axis) of the clock gene expression rhythm to the peak. Specifically, the period is the time taken for one cycle (for example, from one peak to the next peak) in the clock gene expression rhythm. Specifically, the phase refers to a specific position on the time axis of the clock gene expression rhythm.

[0018] The regulator may not change the amplitude of the clock gene expression rhythm, or may cause a change such as an increase in amplitude or a suppression of attenuation. Furthermore, in terms of reducing the impact on normal daily schedules, the regulating agent preferably does not have at least one of the effects of lengthening and shortening the period length of the clock gene expression rhythm, and more preferably does not have either of these effects. In a similar respect, the regulating agent does not have at least one of the effects of advancing and delaying the phase of the clock gene expression rhythm, and more preferably has neither of these effects.

[0019] Next, the constituent components of the regulator will be described.

[0020] (α-Glucosylrutin) α-Glucosylrutin is a quercetin glycoside, specifically a compound in which one or more (approximately 2 to 20) glucose residues are bound to the glucose residue in the rutinose residue of rutin via an α1→4 bond. The structure of α-glucosylrutin is shown in the following general formula (I).

[0021] [ka]

[0022] In the above general formula (I), n is an integer of 1 or greater, preferably 1 or an integer of 2 to 20, more preferably 1. This makes it possible to more stably obtain the effect of regulating clock gene expression.

[0023] α-Glucosylrutin can be obtained as a component contained in enzyme-treated rutin produced by, for example, a known method, more specifically, the method described in JP 2019-43952 A. Alternatively, α-glucosylrutin may be obtained by appropriately purifying the enzyme-treated rutin. Specifically, enzymatically modified rutin can be obtained by treating rutin with a glycosyltransferase in the presence of an α-glucosyl sugar compound (a sugar donor). Specific examples of α-glucosyl sugar compounds include cyclodextrin and partial starch hydrolysates. A specific example of a glycosyltransferase is cyclodextrin glucanotransferase (CGTase, EC2.4.1.19).

[0024] The product obtained here (hereinafter also referred to as "crude product") is a mixture containing α-glucosylrutins (α-monoglucosylrutin and other α-polyglucosylrutins) with different numbers of glucose bound to rutin, unreacted rutin, and α-glucosyl sugar compounds. Therefore, the crude product may be purified to obtain a purified product with improved α-glucosylrutin purity.

[0025] For example, the purity of α-glucosylrutin can be improved by using a porous synthetic adsorbent and an appropriate eluent to remove α-glucosyl sugar compounds and other impurities in the crude product and further reducing the content of rutin. Alternatively, the crude product may be treated with an enzyme having glucoamylase activity to cleave the remaining glucose residues, leaving only the glucose residues directly attached to the glucose residues constituting the rutinose residues in α-glucosylrutin to which two or more glucose residues have been attached, thereby increasing the concentration of α-monoglucosylrutin. Examples of enzymes having glucoamylase activity include enzymes that cleave α-1,4-glucosidic bonds in glucose units, such as glucoamylase (EC 3.2.1.3). In this case, the glucose residues constituting the rutinose residues directly attached to the quercetin skeleton are not cleaved from the quercetin skeleton.

[0026] The amount of rutin in the enzyme-treated rutin is preferably 10% by mass or more, more preferably 40% by mass or more, even more preferably 70% by mass or more, and preferably 85% by mass or less, thereby enabling more efficient regulation of clock gene expression. The enzyme-treated rutin obtained by the above-mentioned method may contain, in addition to α-glucosylrutin, a small amount of unreacted rutin or a rutin degradation product such as quercetin, or may also contain isoquercitrin, which has a structure in which a rhamnose residue has been removed from rutin.

[0027] The amount of rutin in enzyme-treated rutin can be calculated, for example, in accordance with the method for quantifying enzyme-treated rutin, described on page 202 of "Voluntary Standards for Food Additives Other than Chemically Synthesized Products, Second Edition" (published October 1, 1993, edited by the Voluntary Standards Committee of the Japan Food Additives Association, published by the Japan Food Additives Association). A rutin standard solution with a known concentration can be prepared using a commercially available rutin reagent (e.g., manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0028] Specific examples of commercially available products containing α-glucosylrutin include commercially available enzyme-treated rutin such as "αG Rutin PS" (rutin equivalent: 80-86% by mass), "αG Rutin P" (rutin equivalent: 40-46% by mass), and "αG Rutin H" (rutin equivalent: 22-26% by mass) (all manufactured by Toyo Sugar Refining Co., Ltd.). These commercially available products may be used as is, or may be purified.

[0029] The amount of α-glucosylrutin contained in a single dose of the regulator is, for example, 10 mg or more, preferably 50 mg or more, more preferably 100 mg or more, and even more preferably 200 mg or more, which can more reliably obtain the effect of regulating clock gene expression. The content of α-glucosylrutin in a single dose of the preparation is, for example, 500 mg or less, preferably 400 mg or less, and more preferably 300 mg or less.

[0030] The amount of α-glucosylrutin per daily dose is, for example, 10 mg or more, preferably 50 mg or more, more preferably 100 mg or more, and even more preferably 200 mg or more, which can more reliably achieve the effect of regulating clock gene expression. The amount of α-glucosylrutin per daily application is, for example, 500 mg or less, preferably 400 mg or less, and more preferably 300 mg or less.

[0031] The amount of the regulator to be applied can be determined, for example, depending on the form of the regulator. The frequency of application of the conditioner per day may be, for example, 1 to 10 times, preferably 1 to 5 times, and more preferably 1 to 3 times.

[0032] (Other ingredients) The adjuster may contain ingredients other than those described above. The other ingredients can be selected, for example, from ingredients used in foods. Furthermore, the other ingredients can be selected, for example, depending on the dosage form of the adjuster.

[0033] When the conditioning agent is a food composition, the other ingredients may be, for example, one or more selected from the group consisting of excipients, binders, disintegrants, lubricants, coating agents, suspending agents, preservatives, antioxidants (excluding polyphenols), and flavoring agents.

[0034] Examples of excipients include one or more components selected from the group consisting of corn starch, crystalline cellulose, calcium carbonate, kanbai powder, sodium dehydroacetate, hypromellose, medicinal charcoal, shellac, potato starch, light anhydrous silicic acid, hydrous silicon dioxide, silicon dioxide, precipitated calcium carbonate, anhydrous calcium hydrogen phosphate, magnesium oxide, calcium lactate, calcium silicate, magnesium aluminometasilicate, synthetic hydrotalcite, synthetic aluminum silicate, lactose, sucrose, maltose, D-mannitol, erythritol, glucose, and fructose.

[0035] Examples of binders include one or more components selected from the group consisting of powdered acacia, dextrin, carboxymethyl cellulose, hydroxypropyl cellulose, and polyvinyl alcohol.

[0036] Examples of disintegrants include one or more components selected from the group consisting of carmellose, carmellose calcium, croscarmellose sodium, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, crospovidone, alginic acid, sodium starch glycolate, partially pregelatinized starch, and bentonite.

[0037] Examples of lubricants include one or more components selected from the group consisting of magnesium stearate, calcium stearate, talc, sucrose fatty acid esters, glycerin fatty acid esters, polyethylene glycol, and hardened oils.

[0038] Examples of coating agents include one or more components selected from the group consisting of talc, hypromellose, aminoalkyl methacrylate copolymer, gum arabic, ethyl cellulose, carnauba wax, carboxyvinyl polymer, magnesium stearate, shellac, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, pullulan, povidone, polyvinyl alcohol, and macrogol.

[0039] The suspending agent may include, for example, at least one component selected from the group consisting of sodium alginate and polyvinylpyrrolidone.

[0040] The preservative may include, for example, at least one component selected from the group consisting of ethyl parahydroxybenzoate and butyl parahydroxybenzoate.

[0041] Antioxidants (excluding polyphenols) include, for example, tocopherol.

[0042] Examples of flavoring agents include one or more components selected from the group consisting of sucrose, honey, aspartame, stevia, dipotassium glycyrrhizinate, and activated carbon.

[0043] From the viewpoint of improving the ease of continuous intake, the regulator is preferably a food composition, such as a food for specified health uses or a food with functional claims. In addition, from the viewpoint of ease of application to the subject, the regulator is taken orally.

[0044] The regulator is preferably a solid preparation from the viewpoint of ease of application to the subject. The dosage form of the solid preparation is, for example, a tablet, gummies, jellies, powders, or granules, preferably a tablet. These solid preparations may have a coating layer such as a sugar coating layer or a film coating layer.

[0045] (Manufacturing method) The method for producing the regulator includes, for example, a step of blending α-glucosylrutin and other ingredients as appropriate. The regulator can be produced, for example, according to its dosage form, and then formulated.

[0046] (packaging) The package is a container in which the regulator of this embodiment is housed. By using the package, for example, it is possible to improve the convenience of using the regulator. Specific examples of packaging forms include bottle packaging, jar packaging, PTP packaging (Press-Through Package), pouch packaging, stick packaging, and SP packaging (Strip Package). The regulator may be temporarily packaged in these packages and then airtightly stored. That is, the regulator may be contained in an airtight package. Furthermore, these may be pillow-packaged, or may be stored in a box or the like. Furthermore, for example, from the viewpoint of suppressing moisture absorption, a desiccant or the like may be contained in the packaging container together with the regulator.

[0047] Examples of materials for the packaging container include one or more materials selected from the group consisting of resin materials, metal materials, and inorganic materials such as glass. Furthermore, if necessary, environmentally friendly materials such as recycled plastics, biomass plastics, and biodegradable plastics may be used for part or all of the packaging material, and environmentally friendly containers and packaging may be used.

[0048] Specific examples of film materials that can be used to form packaging containers include resin films such as polypropylene film, polyethylene terephthalate film, and polyethylene film, as well as these resin films to which metal foil such as aluminum foil is attached by vapor deposition, etc. Either a single-layer film or a multi-layer film (for example, a laminate film) may be used.

[0049] The packaging container can be selected depending on the shape of the regulator, for example. When the regulator is a tablet, the packaging container is, for example, a pouch bag, preferably an aluminum pouch bag. The pouch bag may have a fastener function.

[0050] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations can also be adopted. [Example]

[0051] The present embodiment will be specifically described below with reference to examples, but the present embodiment is not limited to these examples.

[0052] (Test Example 1) In this example, human dermal fibroblasts (ATCC Detroit 551) were used as evaluation cells to evaluate the effect of α-glucosylrutin on gene expression rhythms. In the following, enzyme-treated rutin containing α-glucosylrutin as the main component ("αG Rutin P" manufactured by Toyo Sugar Refining Co., Ltd.) was used. The concentration of the test substance added in the following explanation is the concentration of the enzyme-treated rutin, and the concentration of α-glucosylrutin in the enzyme-treated rutin used was 67%.

[0053] 2.5 × 10 cells were plated in a 6-well plate. 4Cells were seeded per well and cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS. After culturing for 1 day, the cells were treated with a medium containing 100 μM dexamethasone for 2 hours, and then the above-mentioned enzyme-modified rutin containing α-glucosylrutin was added as a test substance. When adding the test substance, the medium in the well was removed and replaced with a medium containing the test substance at 23 μg / mL (BMAL1) or 77 μg / mL (PER1, PER2) (solvent: 0.3% dimethyl sulfoxide (DMSO)) in Example 1, or with a medium containing only solvent (0.3% DMSO) in Control Example 1. The time immediately after the replacement was designated as 0 hours, and cells were sampled 30, 60, and 120 minutes later. RNA was extracted from the collected cells using an RNeasy Mini Kit (QIAGEN), and cDNA was synthesized from 1 μg of RNA using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed using this cDNA with a TaqMan Gene Expression Assay (Thermo Fisher Scientific) to measure the mRNA expression levels of Per1 (Assay ID: Hs00242988_m1), Per2 (Assay ID: Hs01007553_m1), and Bmal1 (Assay ID: Hs00154147_m1). The values ​​obtained were compared with the control (no test substance added) at each time point using a t-test (two-sided significance level 5%). The measurement results for Per1, Per2, and Bmal1 are shown in Figures 1 to 3, respectively. The vertical axes in Figures 1 to 3 are relative values ​​to Control Example 1 (control). An "*" in the figures indicates "significant difference from Control Example 1 (p<0.05)."

[0054] 1 and 2, it was confirmed that the addition of α-glucosylrutin transiently enhanced the expression of Per1 and Per2 30 to 120 minutes after the addition. As shown in FIG. 3, the addition of α-glucosylrutin was confirmed to transiently suppress the expression of Bmal1 30 to 120 minutes after the addition.

Claims

1. A clock gene expression regulator containing α-glucosylrutin as an active ingredient.

2. The clock gene expression regulator according to claim 1, which has the effect of regulating circadian rhythm.

3. 3. The clock gene expression regulator according to claim 1, wherein the clock gene is at least one selected from the group consisting of a Bmal gene, a Per gene, a Cry gene, and a Clock gene.

4. The clock gene expression regulator according to claim 1 or 2, which is taken orally.

5. The clock gene expression regulator according to claim 1 or 2, which is an agent for improving at least one symptom selected from the group consisting of fatigue, sleepiness, and lethargy that occurs any time from the time of waking up to daytime.

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