Food composition for regulating clock gene expression, cosmetic composition for regulating clock gene expression, clock gene expression regulator, food composition for regulating biological clock, cosmetic composition for regulating biological clock, biological clock regulator, food composition for regulating circadian rhythm, cosmetic composition for regulating circadian rhythm, and circadian rhythm regulator

Abstract

Provided are a food composition for modulating CLOCK gene expression, a cosmetic composition for modulating CLOCK gene expression, a CLOCK gene expression modulator, a food composition for in-vivo CLOCK modulation, a cosmetic composition for in-vivo CLOCK modulation, an in-vivo CLOCK modulator, a food composition for modulating circadian rhythm, a cosmetic composition for modulating circadian rhythm, and a circadian rhythm modulator.　The problem addressed by the present invention is solved by a food composition for modulating CLOCK gene expression, a cosmetic composition for modulating CLOCK gene expression, a CLOCK gene expression modulator, a food composition for in-vivo CLOCK modulation, a cosmetic composition for in-vivo CLOCK modulation, an in-vivo CLOCK modulator, a food composition for modulating circadian rhythm, a cosmetic composition for modulating circadian rhythm, and a circadian rhythm modulator that are characterized by containing, as an active ingredient, at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena.

Description

[Technical Field]

[0001] The present invention relates to a food composition for regulating clock gene expression, a cosmetic composition for regulating clock gene expression, a clock gene expression regulator, a food composition for regulating the biological clock, a cosmetic composition for regulating the biological clock, a biological clock regulator, a food composition for regulating circadian rhythm, a cosmetic composition for regulating circadian rhythm, and a circadian rhythm regulator. [Background technology]

[0002] Most living organisms on Earth, including humans, maintain homeostasis by controlling various physiological functions in a roughly 24-hour cycle in accordance with the external environment (such as the light-dark cycle). This biological diurnal variation is called the circadian rhythm.

[0003] In recent years, various physical and mental disorders caused by abnormalities in circadian rhythms have become a social issue. In particular, many problems are caused by disruptions to circadian rhythms due to changes in living and working environments. Circadian rhythms are controlled by the expression of a group of genes called clock genes. Researchers are searching for substances that can regulate circadian rhythms by adjusting the expression of clock genes.

[0004] Patent Document 1 describes that a specific component contained in Kenyan purple tea has the effect of promoting Bmal1 gene expression and regulating circadian rhythm. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6227851 Summary of the Invention [Problem to be solved by the invention]

[0006] Meanwhile, Euglena (genus name: Euglena, Japanese name: Midorimushi) has attracted attention as a promising biological resource for use as food, feed, fuel, etc. Euglena is a primary producer in the food chain, and it has been considered difficult to mass-cultivate it for reasons such as the fact that it is preyed upon by predators and that the culture conditions, such as light, temperature, and agitation speed, are more difficult than those for other microorganisms. However, in recent years, the inventors have conducted extensive research to establish mass-cultivation techniques, opening the way for the mass supply of Euglena and Euglena-derived substances, such as paramylon extracted from Euglena.

[0007] While studying the physiological activities of Euglena-derived substances, the present inventors focused on circadian rhythms and investigated the effects of Euglena-derived substances on clock genes. As a result of extensive research, they discovered that Euglena-derived substances affect the expression levels of clock genes, leading to the completion of the present invention.

[0008] An object of the present invention is to provide a new use for Euglena-derived substances and to provide a food composition for regulating clock gene expression, a cosmetic composition for regulating clock gene expression, a clock gene expression regulator, a food composition for regulating the biological clock, a cosmetic composition for regulating the biological clock, a biological clock regulator, a food composition for regulating circadian rhythm, a cosmetic composition for regulating circadian rhythm, and a circadian rhythm regulator, which are capable of regulating the expression of clock genes and regulating the biological clock and circadian rhythm. [Means for solving the problem]

[0009] The above-mentioned problems are solved by a food composition for regulating clock gene expression, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the expression of clock genes. The above-mentioned problem is also solved by a cosmetic composition for regulating clock gene expression, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the expression of clock genes. The above-mentioned problem can also be solved by a clock gene expression regulator, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the expression of clock genes. The above-mentioned problems are also solved by a food composition for regulating the biological clock, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient, and is used to regulate the biological clock. The above-mentioned problem is also solved by a cosmetic composition for regulating the biological clock, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient, and is used to regulate the biological clock. The above-mentioned problem is also solved by a biological clock regulating agent, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the biological clock. The above-mentioned problems can also be solved by a food composition for regulating circadian rhythm, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient, and is used to regulate circadian rhythm. The above-mentioned problems are also solved by a cosmetic composition for circadian rhythm regulation, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate circadian rhythms. The above-mentioned problems can also be solved by a circadian rhythm regulating agent, which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate circadian rhythms. [Effects of the Invention]

[0010] The present invention provides a food composition for regulating clock gene expression, a cosmetic composition for regulating clock gene expression, and a clock gene expression regulator that can effectively regulate clock gene expression.The present invention also provides a food composition for regulating the biological clock, a cosmetic composition for regulating the biological clock, and a biological clock regulator that can regulate the biological clock.The present invention also provides a food composition for regulating circadian rhythm, a cosmetic composition for regulating circadian rhythm, and a circadian rhythm regulator that can regulate circadian rhythm. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the schedule for a test of administering a Euglena-derived substance to mice. [Figure 2] 1 is a graph showing the expression level of Per1 gene in the liver of mice in each group. [Figure 3] 1 is a graph showing the expression level of Per2 gene in the liver of mice in each group. [Figure 4] 1 is a graph showing the expression level of the Rev-erbα gene in the liver of mice in each group. [Figure 5] 1 is a graph showing the expression level of the Bmal1 gene in the liver of mice in each group. [Figure 6] 1 is a graph showing the expression level of the Per1 gene in the jejunum of mice in each group. [Figure 7] 1 is a graph showing the expression level of the Per2 gene in the jejunum of mice in each group. [Figure 8] FIG. 1 is a schematic diagram showing the protocol for a 3-day administration test of Euglena and paramylon to mice. [Figure 9] 1 is a graph showing the rhythm and phase of PER2 protein expression in the kidney. [Figure 10] 1 is a graph showing the rhythm and phase of expression of PER2 protein in the liver. [Figure 11] 1 is a graph showing the rhythm and phase of expression of PER2 protein in the submandibular gland. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described with reference to Figures 1 to 11. The present embodiments relate to a food composition for regulating clock gene expression, a cosmetic composition for regulating clock gene expression, a clock gene expression regulator, a food composition for regulating the biological clock, a cosmetic composition for regulating the biological clock, a biological clock regulator, a food composition for regulating circadian rhythm, a cosmetic composition for regulating circadian rhythm, and a circadian rhythm regulator, each of which contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient.

[0013] <Circadian rhythm> Circadian rhythms are physiological phenomena that fluctuate on a roughly 24-hour cycle and are present in most living organisms, including mammals, animals, plants, fungi, and algae. In humans, circadian rhythms are controlled primarily by the suprachiasmatic nucleus (SCN), which is regulated by a group of genes known as clock genes.

[0014] Specifically, the transcriptional activators BMAL1 and CLOCK activate the expression of the Period and Cryptochrome genes, which then transcribing and translating the Period (PER1, PER2) and Cryptochrome (CRY1, CRY2) proteins, which then interact with BMAL1 and CLOCK, repressing their own transcription, forming a negative feedback loop. Subsequently, PER and CRY proteins undergo phosphorylation and ubiquitination, followed by proteasome degradation, thereby releasing transcriptional repression. The next cycle is then initiated by BMAL1 and CLOCK, which takes approximately 24 hours. This is known as the molecular mechanism underlying circadian rhythm formation.

[0015] <Regulator of clock gene expression> The clock gene expression regulator of this embodiment is characterized by containing at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and being used to regulate the expression of clock genes.

[0016] "Clock gene expression" includes not only expression at the transcription level (expression as mRNA) but also expression at the translation level (expression as protein). Furthermore, "adjustment (change) of clock gene expression" includes not only an increase or decrease in the expression level of clock genes, but also a change in the expression rhythm of clock genes (at least one of amplitude, phase, and period length).

[0017] (clock genes) "Clock genes" are genes that function to control circadian rhythms (body clocks). Examples of clock genes include, but are not limited to, Period genes (Per1, Per2, Per3, etc.), Bmal genes (Arntl, Mop3, Tic, Jap3, Pasd3, genes also known as bHLHe5, Bmal1, Bmal2, etc.), Cryptochrome genes (Cry1, Cry2, etc.), Clock genes, Ror genes, Rev-erb genes, E4BP genes, GSK3β gene, CK1 gene, Dec gene, and Rev-erbα gene.

[0018] In this embodiment, the clock gene to be regulated is preferably one or more selected from the group including the Per1 gene, the Per2 gene, the Rev-erbα gene, and the Bmal1 gene.

[0019] <Body clock regulator> The biological clock regulating agent of this embodiment is a biological clock regulating agent characterized by containing at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and being used to regulate the biological clock.

[0020] The term "biological clock adjusting effect" refers to at least one of the effects of restoring the biological clock, which has a natural 24-hour cycle, from an abnormal state to a normal state and maintaining the normal state.

[0021] The biological clock regulating agent according to this embodiment is used to regulate (control) the biological clock by regulating the expression of clock genes. When an abnormality occurs in the biological clock, the expression rhythm (at least one of amplitude, phase, and period length) of the clock genes deviates from normal. However, by administering the biological clock regulating agent at an appropriate time, the biological clock can be brought closer to normal. In other words, the biological clock can be regulated by adjusting the expression level of clock genes at the appropriate time.

[0022] <Circadian rhythm regulator> The circadian rhythm regulator of this embodiment is characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate circadian rhythms.

[0023] "Circadian rhythm adjusting effect" refers to at least one of the effects of restoring the body's inherent circadian rhythm, which has a cycle of approximately 24 hours, from an abnormal rhythm to a normal rhythm and maintaining the normal rhythm.

[0024] The circadian rhythm regulating agent according to this embodiment is used to regulate (control) the circadian rhythm by regulating the expression of clock genes. When an abnormality occurs in the circadian rhythm, the expression rhythm (at least one of amplitude, phase, and period length) of the clock genes deviates from normal. However, by administering the circadian rhythm regulating agent at an appropriate time, the circadian rhythm can be brought closer to normal. In other words, by adjusting the expression level of the clock genes at the appropriate timing of the circadian rhythm, the phase, period, and amplitude of the circadian rhythm can be adjusted.

[0025] <Euglena> In this embodiment, "Euglena" includes microorganisms taxonomically classified in the genus Euglena, their varieties, mutants, and closely related species in the family Euglenaceae. Here, the genus Euglena refers to a group of organisms belonging to the Excavata, phylum Euglenozoa, class Euglenophyceae, order Euglenales, and family Euglenaceae among eukaryotes.

[0026] Specific examples of species belonging to the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis, etc. As Euglena, Euglena gracilis, particularly Euglena gracilis Z strain, can be used. However, other Euglena species such as Euglena gracilis Z strain mutant SM-ZK strain (chloroplast-deficient strain) and E. gracilis var. bacillaris, genetic mutants such as chloroplast mutants of these species, and Astasia longa can also be used.

[0027] The genus Euglena is widely distributed in freshwater such as ponds and marshes, and may be isolated from these and used. Alternatively, any Euglena species that has already been isolated may be used. The genus Euglena encompasses all mutant strains thereof. These mutant strains also include those obtained by genetic methods such as recombination, transduction, and transformation.

[0028] (Euglena algae) In this embodiment, Euglena algae bodies can be used as Euglena. Euglena live cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are. Euglena live cells can be used as they are after harvesting from the culture tank, but it is preferable to wash them with water or physiological saline. Euglena algae bodies may also be used in the form of a dispersion in which they are dispersed in a liquid such as water. In this embodiment, dried Euglena algae bodies (Euglena powder) obtained by freeze-drying or spray-drying live Euglena cells are preferably used as Euglena algae.

[0029] Furthermore, mechanically treated Euglena cells obtained by subjecting living Euglena cells to mechanical treatment such as ultrasonic irradiation or homogenization may be used as Euglena cells. Furthermore, the mechanically treated product may be dried and used as Euglena cells.

[0030] (Euglena extract) In this embodiment, a Euglena extract (Euglena extract) can be used as Euglena, and it is particularly preferable to use a Euglena aqueous solvent extract. In this embodiment, the term "Euglena aqueous solvent extract" refers to an extract extracted from Euglena using an aqueous solvent. In particular, it is preferable to use a water extract, hot water extract, alcohol extract, or glycol extract of Euglena extracted using water, alcohols, or glycols as the aqueous solvent at 5°C to 600°C for several seconds to several tens of hours. The water used for extraction does not necessarily have to be distilled water, pure water, or ultrapure water; for example, tap water or water containing impurities may be used, but water that does not contain components that interfere with the extraction of active ingredients is preferred.

[0031] In this embodiment, the term "water extract" refers to an extract using water at 0 to 50°C (excluding 0°C). Here, "water" refers to water at 0 to 50°C (excluding 0°C). The temperature of the water is not particularly limited as long as it is within a range that allows the active ingredient to be sufficiently extracted without affecting the active ingredient, but is preferably 1 to 40°C, more preferably 5 to 35°C, and particularly preferably 10 to 30°C.

[0032] In this embodiment, "hot water extract" refers to an extract using water at a temperature higher than 50°C, and can also be called "warm water extract." Here, "hot water" refers to water at a temperature higher than 50°C, and is a concept that includes "hot water," including water in a boiling state. It is not limited to hot water in a liquid state, but also includes hot water in a gaseous state and a supercritical state. The temperature of the hot water is not particularly limited as long as it is within a range that can sufficiently extract the active ingredient without affecting the active ingredient, but is preferably higher than 50°C and lower than 120°C, more preferably higher than 50°C and lower than 100°C.

[0033] The pH of the water used for extraction is not particularly limited as long as it is within a range that allows the active ingredient to be sufficiently extracted without affecting the active ingredient, but a pH of 4 to 10 is preferred, a pH of 5 to 9 is more preferred, and a pH of 6 to 8 is particularly preferred.

[0034] In this embodiment, the aqueous solvent may be one or more solvents that can sufficiently extract the active ingredient without affecting the active ingredient and that can be normally used for extraction. Examples of the aqueous solvent include, but are not limited to, water, alcohols, and glycols. Examples of alcohols include ethanol, methanol, n-propanol, and isopropanol. Examples of glycols include butylene glycol (BG) and propylene glycol. Other aqueous solvents include acetone. These solvents may be used alone or as an aqueous solution, or as a mixed solvent of any two or more of them.

[0035] The temperature of the aqueous solvent used for extraction is, for example, 0°C or higher, and is not particularly limited as long as it does not affect the active ingredient. Aqueous solvents in a boiling or supercritical state can also be used, but aqueous solvents at 5°C to 600°C are preferred, and aqueous solvents at 10°C to 200°C are more preferred. Therefore, aqueous solvents for extraction also include aqueous solvents in a boiling or supercritical state. The amount of aqueous solvent used for extraction is preferably an amount that can sufficiently dissolve the water-soluble active ingredient contained in Euglena.

[0036] The extraction method is not particularly limited, and can be, for example, the method described below. However, without being limited thereto, any conventional extraction method can be freely selected and used. Examples include a method in which dry powder of Euglena algae is soaked in an aqueous solvent for a predetermined time, followed by centrifugation or filtration, and a method in which dry powder of Euglena algae is added to an aqueous solvent, shaken to uniformly disperse the powder, and then centrifuged or filtrated. Furthermore, to promote extraction, the aqueous solvent after addition of Euglena can be heated.

[0037] Water extraction of Euglena can be performed by a conventional method such as, but not limited to, the following. For example, Euglena tissues and water are placed in a container and allowed to stand for a predetermined time while being stirred or shaken as appropriate. The resulting extract can be used as the water extract as is. Alternatively, for example, the supernatant obtained by centrifuging such an extract can be used as the water extract. Alternatively, the extract or supernatant can be concentrated and dried to remove water, and the resulting extract can be used as the water extract. Water extraction may be performed by adding a small amount of alcohol, for example, 10% by mass or less, preferably ethanol, to water to increase extraction efficiency and shorten the extraction time. The extraction time when water extraction is performed is not particularly limited as long as it is a time that allows the active ingredient to be extracted, and can be set appropriately depending on the extraction temperature, from a few seconds to several tens of hours.

[0038] Extraction with hot water can be performed by a commonly used method such as, but not limited to, the following. Euglena is introduced into a commonly used extractor together with water and then heated to perform extraction. When extracting with boiling water or water in a supercritical state, an extractor that can withstand the vapor pressure of water must be used. The pressure during extraction can be set to 1 to 5,000 atmospheres, preferably 60 to 400 atmospheres.

[0039] When extraction is performed under high temperature and pressure, if the extraction time is too long, the active ingredient may decompose or undergo a chemical reaction. Therefore, when extraction is performed under high temperature and pressure, the extraction time is preferably short, for example, within 3 minutes, more preferably within 1 minute, and particularly preferably within 30 seconds.

[0040] The extracted Euglena extract can be used as it is as an active ingredient in the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator according to this embodiment, but the extract can also be further fractionated by an appropriate separation method (e.g., partition extraction, gel filtration, silica gel chromatography, reverse-phase or normal-phase high-performance liquid chromatography, etc.) to obtain highly active fractions for use. Alternatively, the Euglena extract or a fraction thereof can be concentrated and dried to remove the aqueous solvent, and the resulting fraction can be used as an aqueous solvent extract.

[0041] Alternatively, the Euglena extract may be a hydrolyzed Euglena extract obtained by enzymatic hydrolysis of Euglena powder (Euglena algae). Extracts extracted using BG may also be used. The Euglena extract may be combined with other cosmetic ingredients, such as by encapsulating permeable collagen, permeable elastin, permeable hyaluronic acid, or hexapeptide-33 in a multilayered capsule.

[0042] <Paramylon> "Paramylon" is a porous storage polysaccharide (β-1,3-glucan) formed by the polymerization of approximately 700 glucose units via β-1,3-bonds, found in the genus Euglena. Paramylon particles are flat, spheroidal particles formed by helically entangled β-1,3-glucan chains.

[0043] Paramylon exists as granules within the cells of all species and varieties of Euglena, and the number, shape, and uniformity of the particles vary by species. Paramylon consists solely of glucose, and the average degree of polymerization of paramylon obtained from the wild-type strain of E. gracilis Z and the chloroplast-deficient strain SM-ZK is approximately 700 glucose units. Paramylon is insoluble in water and hot water, but is soluble in dilute alkali, concentrated acid, dimethyl sulfoxide, formaldehyde, and formic acid. The average density of paramylon is 1.53 in E. gracilis Z and 1.63 in E. gracilis var. bacillaris SM-L1.

[0044] According to X-ray analysis using powder diffraction analysis, paramylon has a loosely spiral structure in which three linear β-1,3-glucans are twisted together like a right-handed rope. Several of these glucan molecules gather together to form paramylon granules. Paramylon granules have a very high crystalline structure, accounting for approximately 90%, making it the compound with the highest crystalline structure rate among polysaccharides (Euglena Physiology and Biochemistry, edited by Masasaburo Kitaoka, Academic Press Center). The particle size distribution of paramylon (manufactured by Euglena Co., Ltd.) measured using a laser diffraction / scattering particle size distribution analyzer yields a median diameter of 1.5 to 2.5 μm.

[0045] Paramylon particles are isolated from cultured Euglena cells by any suitable method, purified into fine particles, and typically provided as a powder. For example, paramylon particles can be obtained by (1) culturing Euglena cells in any suitable medium, (2) separating Euglena cells from the medium, (3) isolating paramylon from the separated Euglena cells, (4) purifying the isolated paramylon, and, if necessary, (5) cooling and subsequent freeze-drying. Paramylon isolation is performed, for example, using a nonionic or anionic surfactant of a type that is largely biodegradable. Paramylon purification is performed substantially simultaneously with isolation.

[0046] (Processed paramylon products) Processed paramylon products include water-soluble paramylon obtained by chemically or physically treating paramylon using various known methods, sulfated paramylon, and paramylon derivatives.

[0047] Examples of processed paramylon products include amorphous paramylon and emulsion paramylon. Amorphous paramylon is a substance obtained by amorphizing crystalline paramylon derived from Euglena. Amorphous paramylon has a relative crystallinity of 1 to 20% compared to crystalline paramylon produced from Euglena by known methods. This relative crystallinity was determined by the method described in JP 2011-184592 A.

[0048] Specifically, amorphous paramylon and paramylon were each pulverized in a pulverizer (Retsh's Ball Mill MM400) at a frequency of 20 rpm for 5 minutes, and then scanned using an X-ray diffractometer (Spectris's H'PertPRO) at a tube voltage of 45 kV and a tube current of 40 mA over a 2θ range of 5° to 30° to obtain the diffraction peaks Pc,Pa of paramylon and amorphous paramylon near 2θ = 20°. Using these Pc,Pa values, the relative crystallinity of amorphous paramylon was calculated using the formula: Relative crystallinity of amorphous paramylon = Pa / Pc × 100(%).

[0049] Amorphous paramylon is prepared by treating crystalline paramylon powder with alkali, neutralizing it with acid, washing, removing moisture, and drying, according to the method described in JP 2011-184592 A. Paramylon processed products also include water-soluble paramylon, sulfated paramylon, and paramylon derivatives obtained by chemically or physically treating paramylon using various known methods.

[0050] "Emulsion paramylon" is a substance also known as emulsion paramylon because its processing method and physical properties are similar to those of an emulsion. It is obtained by adding water to paramylon and then performing a collision process in which the resulting fluid is ejected from a fine-pore nozzle at ultra-high pressure and collided with an object, according to the method described in JP 2016-199650 A, resulting in processed paramylon that has swollen by combining with more than four times its amount of water.

[0051] Emulsion paramylon can be obtained by performing a collision process at least once using a known property modification device (for example, the device described in JP 2011-88108 A and JP 6-47264 A) in which a slurry of a solid such as powder to which a water-soluble solvent has been added is sprayed at ultra-high pressure from a fine-pore nozzle and caused to collide with an object to be impacted, at a nozzle pressure of 245 MPa during spraying.

[0052] When the particle size of emulsion paramylon was measured using a laser diffraction / scattering particle size distribution analyzer, the median diameter was 7 μm or more, more than five times that of paramylon, and using an optical electron microscope, it was observed that the particles were attached to adjacent particles, and they swelled by binding to more than four times as much water as paramylon.

[0053] While a slurry made by mixing raw paramylon with water is a thin fluid, emulsion paramylon has increased viscosity as the paramylon disperses in the water molecules, giving it a sticky, elastic texture that sticks to your hand when you touch it, and a glue-like feel. Based on its processing method and physical properties, the resulting processed paramylon is referred to as emulsion paramylon in this specification, but it is unclear whether it is emulsified or not; the paramylon is in a swollen state due to binding with water.

[0054] <Application> The clock gene expression regulator, biological clock regulator, and circadian rhythm regulator according to the present embodiment are formulated as food compositions such as health foods, cosmetic compositions, or pharmaceutical compositions, and are taken or administered prophylactically to maintain normal circadian rhythms. Euglena-derived substances such as paramylon and Euglena can be taken as foods and have no side effects, allowing for continuous intake or administration.

[0055] (Food composition) In the food field, the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator of this embodiment can be used to provide a food composition having the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator functions by incorporating an effective amount of a Euglena-derived substance as a food ingredient into various foods. That is, in the food field, the present invention can provide a food composition labeled for circadian rhythm regulation (biological clock regulation), etc. Examples of such food compositions include general foods, foods for specified health uses, foods with nutrient functions, foods with functional claims, foods for hospital patients, and supplements. They can also be used as food additives.

[0056] Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria drinks, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered drinks (powdered juice, powdered soup, etc.), concentrated drinks, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candy, chocolate, etc.), bread, cereal, etc. Furthermore, in the case of foods for specified health uses, foods with nutrient functions, foods with functional claims, etc., they may be in the form of capsules, lozenges, syrup, granules, powder, etc.

[0057] Here, a specified health food is a food containing functional ingredients that affect physiological functions, etc., and can be labeled as suitable for a specific health use with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, it is a food that is sold with a label indicating a specific health use related to regulating the circadian rhythm (body clock).

[0058] A food with nutrient functions is a food used to supplement nutrients (vitamins, minerals), and displays the function of the nutrients. To be sold as a food with nutrient functions, the amount of nutrients contained in the recommended daily intake must be within the specified upper and lower limits, and in addition to displaying the nutritional function, warning labels must also be displayed.

[0059] Foods with functional claims are foods that display scientifically based functionality at the responsibility of the business operator, and information on the basis of safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale.

[0060] In addition to the Euglena-derived substance, the food composition according to this embodiment can contain one or more selected ingredients that can be used in conventional food compositions, such as various seasonings, preservatives, emulsifiers, stabilizers, flavorings, colorants, antiseptics, and pH adjusters.

[0061] (Cosmetic composition) The clock gene expression regulator, biological clock regulator, and circadian rhythm regulator according to this embodiment can be suitably used in cosmetic compositions, taking advantage of their clock gene expression regulator, biological clock regulator, and circadian rhythm regulator effects. The cosmetic composition can be applied to any type of cosmetic, for example, skin care cosmetics such as lotions, milky lotions, creams, and serums, makeup cosmetics such as foundations, concealers, makeup bases, lipsticks, blushers, eye shadows, and eyeliners, and sunscreen cosmetics.

[0062] In addition to the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator of this embodiment, the cosmetic composition of this embodiment can also contain one or more freely selected ingredients that can be used in regular cosmetic compositions. For example, all additives that can be commonly used in the field of cosmetics, such as base materials, preservatives, emulsifiers, colorants, antiseptics, surfactants, ultraviolet absorbers, antioxidants, moisturizers, ultraviolet absorbers, fragrances, antiseptic and antifungal agents, extender pigments, color pigments, alcohol, and water, can be contained.

[0063] In the cosmetic composition according to this embodiment, the contents of the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator are not particularly limited and can be freely set according to the purpose.

[0064] (Pharmaceutical composition) In the pharmaceutical field, the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator of this embodiment can be provided as a pharmaceutical composition having the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator by combining an amount of a Euglena-derived substance that effectively exerts the clock gene expression regulator, biological clock regulator, and circadian rhythm regulator effects with pharmaceutically acceptable carriers and additives. The pharmaceutical composition may be a drug or a quasi-drug. The pharmaceutical composition may be applied internally or externally, and may be used in the form of an oral preparation, an injection such as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, and / or an intraperitoneal injection, a transmucosal application preparation, a transdermal application preparation, or the like. The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, and examples thereof include solid preparations such as tablets, granules, capsules, powders, and dustings, liquid preparations such as solutions and suspensions, ointments, and semi-solid preparations such as gels.

[0065] The pharmaceutical composition according to this embodiment can contain one or more pharmaceutically acceptable additives selected freely. For example, when the pharmaceutical composition according to this embodiment is applied to an oral preparation, it may contain all additives that are commonly used in the field of pharmaceutical preparations, such as excipients, binders, disintegrants, surfactants, preservatives, colorants, flavoring agents, fragrances, stabilizers, antiseptics, antioxidants, etc. Furthermore, it may also be made into a sustained-release preparation by utilizing a drug delivery system (DDS). [Example]

[0066] The present invention will be described in detail below based on specific examples, but the present invention is not limited to these.

[0067] <Substances derived from Euglena> (Euglena) Euglena gracilis powder (Euglena algae, manufactured by Euglena Co., Ltd.) was used as Euglena.

[0068] (paramylon) Paramylon, a Euglena-derived substance, was prepared by the following procedure. Euglena gracilis powder (Euglena Co., Ltd.) was added to distilled water and stirred at room temperature for two days. The mixture was sonicated to disrupt the cell membranes, and crude paramylon particles were collected by centrifugation. The collected paramylon particles were dispersed in a 1% aqueous solution of sodium dodecyl sulfate and treated at 95°C for two hours. The collected paramylon particles were then dispersed in a 0.1% aqueous solution of sodium dodecyl sulfate and treated at 50°C for 30 minutes. This procedure removed lipids and proteins, and the particles were then washed with acetone and ether and dried at 50°C to obtain purified paramylon particles (paramylon powder).

[0069] <Test 1: Administration test to mice> As shown in Figure 1, 6-week-old ICR male mice were fasted for 24 hours and then divided into a fasting group (n = 7), a normal diet group (n = 7 (liver), n = 6 (jejunum)), a Euglena group (n = 8), and a paramylon group (n = 8). At ZT 5.5, the normal diet group, Euglena group, and paramylon group received 1 g of normal diet (AIN-93M), 5% Euglena diet, and 5% paramylon diet, respectively. In the Euglena and paramylon groups, the β-cornstarch in the normal diet was replaced with 5% Euglena or paramylon. At 1.5 hours after ingestion (ZT 7), the mice were dissected, and the liver and jejunum were removed. ZT (Zeitgeber time) 0 is defined as the start of the light period.

[0070] Next, total RNA was extracted from the excised liver and jejunum, reverse transcribed into cDNA, and the expression levels of the clock genes Per1, Per2, Rev-erbα, and Bmal1 were analyzed by real-time PCR. The results are shown in Figures 2 to 7.

[0071] <Result 1> As shown in Figure 2, intake of a normal diet during fasting reduced Per1 expression in the liver, and expression was further reduced in the Euglena group. As shown in Figure 3, intake of a normal diet during fasting did not change Per2 expression in the liver, but a tendency toward increased expression was observed in the Euglena and paramylon groups. As shown in Figure 4, the intake of a normal diet during fasting did not significantly change Rev-erbα expression in the liver, but expression was significantly reduced in the Euglena and paramylon groups. As shown in Figure 5, no inter-group difference was observed in Bmal1 expression in the liver after feeding during fasting. As shown in Figure 6, intake of a normal diet during fasting significantly reduced Per1 expression in the jejunum, and expression was further reduced in the Euglena and paramylon groups. As shown in Figure 7, no intergroup differences were observed in Per2 expression in the jejunum after feeding during fasting.

[0072] <Test 2: 3-day administration test on mice> As shown in Figure 8, 14-week-old ICR male mice were divided into a control group (n = 4), a Euglena group (n = 4), and a paramylon group (n = 4). After acclimatization with a normal diet (AIN-93M) for 7 days, they were orally gavaged with 0.4 mL of saline, 0.4 mL of Euglena (50 mg / water), or 0.4 mL of paramylon (50 mg / water) for 3 days at ZT5. The expression rhythm of PER2 protein was measured using an in vivo imaging system (IVIS Kinetic, Caliper) starting 4 hours after the gavage on the third day.

[0073] <Result 2> As shown in Figure 9, the phase of the PER2 protein expression rhythm in the kidney was advanced in the Euglena and paramylon groups compared to the control group. As shown in Figure 10, the phase of the PER2 protein expression rhythm in the liver was advanced in the Euglena and paramylon groups compared to the control group. As shown in Figure 11, the phase of the PER2 protein expression rhythm in the submandibular gland was advanced in the Euglena and paramylon groups compared to the control group. Additionally, there were significant differences in phase changes in both paramylon groups.

[0074] <Summary> These results suggest that the intake of paramylon and / or Euglena affects the expression of clock genes in the liver and jejunum.

[0075] In other words, it was shown that by using at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient, it is possible to regulate the expression of clock genes and circadian rhythms.

Claims

1. A food composition for regulating clock gene expression, characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the expression of clock genes.

2. A clock gene expression regulator characterized by containing as an active ingredient at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena, and being used to regulate the expression of clock genes.

3. A food composition for regulating clock gene expression as described in claim 1 or a clock gene expression regulator as described in claim 2, containing paramylon as an active ingredient.

4. A food composition for regulating the biological clock, characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient and is used to regulate the biological clock.

5. A biological clock regulating agent characterized by containing, as an active ingredient, at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena, and being used to regulate the biological clock.

6. A food composition for regulating the body clock described in claim 4 or a body clock regulating agent described in claim 5, containing paramylon as an active ingredient.

7. A food composition for regulating circadian rhythm, characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena as an active ingredient, and is used to regulate circadian rhythm.

8. A circadian rhythm regulating agent characterized by containing, as an active ingredient, at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena, and being used to regulate circadian rhythms.

9. A food composition for regulating circadian rhythm as described in claim 7 or a circadian rhythm regulating agent as described in claim 8, containing paramylon as an active ingredient.

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