Sirtuin 3 activator

Abstract

To provide a novel sirtuin 3 activator.SOLUTION: A sirtuin 3 activator comprising effusol and / or dehydroeffusol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a sirtuin 3 activator and a skin-beautifying food composition containing the sirtuin 3 activator. [Background technology]

[0002] In recent years, sirtuins, which are NAD+-dependent deacetylases, have been attracting attention. Seven sirtuin genes, sirtuin 1 to sirtuin 7, have been identified in mammals, including humans. Sirtuin 3 is localized in mitochondria and has been reported to be involved in energy metabolism and antioxidants, and may be involved in cancer suppression (Non-Patent Documents 1 to 5, etc.). Due to these effects of sirtuins, sirtuin genes are known as "longevity genes." To date, various polyphenols, Euglena, carnosine and / or anserine, black turmeric, piceatannol, eggshell membrane components, resveratrol, and the like have been reported as activators of various sirtuins, including sirtuin 3 (Patent Documents 1 to 6, etc.). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2020-127398 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-97508 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-231487 [Patent Document 4] Japanese Patent Application Publication No. 2018-199680 [Patent Document 5] Japanese Patent Application Publication No. 2018-48152 [Patent Document 6] JP 2016-41680 A [Non-patent literature]

[0004] [Non-Patent Document 1] J Biol Chem. 2012; 287(51): 42419-42427 [Non-patent document 2] Biochimie Vol.179 Page.1-13 (2020) [Non-patent document 3] Front. Cell Dev. Biol. 2020, doi.org / 10.3389 / fcell.2020.00822 [Non-patent document 4] Cell. 2010 Nov 24;143(5):802-12. doi: 10.1016 / j.cell.2010.10.002. [Non-patent document 5] Oxid Med Cell Longev. 2020 Oct 23;2020:7308386. doi: 10.1155 / 2020 / 7308386. [Non-patent document 6] PLoS ONE 14(5): e0217394, May 28, 2019, doi.org / 10.1371 / journal.pone.0217394 [Non-Patent Document 7] Wako Pure Chemical News Vol.87 No.2 (June 2019 issue), p.9-11 [Non-patent document 8] Yoshinori Katakura, Searching for Anti-Brain-Aging Foods and Elucidating the Molecular Basis of Their Functionality, Asahi Beer Foundation, April 2010 [Non-Patent Document 9] Oxid Med Cell Longev. Volume 2016, Article ID 2927131, doi.org / 10.1155 / 2016 / 2927131 [Non-Patent Document 10] Journal of Functional Foods, 23, 444-452(2016), doi.org / 10.1016 / j.jff.2016.01.016. Summary of the Invention [Problem to be solved by the invention]

[0005] The main object of the present inventors was to provide a novel sirtuin 3 activator. [Means for solving the problem]

[0006] The present inventors discovered that efsole and / or dehydroefsole may have the effect of activating sirtuin 3, and made further improvements, leading to the completion of the present disclosure.

[0007] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. An agent for activating sirtuin 3 in skin cells via intestinal cells, comprising at least one component selected from the group consisting of efsol and dehydroefsol as an active ingredient. Section 2. Item 1. A sirtuin 3 activator according to Item 1, which activates sirtuin 3 in skin cells when taken orally. Section 3. Item 3. A skin-beautifying food composition containing the sirtuin 3 activator according to Item 1 or 2. Section 4. A skin-beautifying food composition containing at least one component selected from the group consisting of efsol and dehydroefsol as a skin-beautifying component that has the function of activating sirtuin 3 when taken orally. Section 5. Item 4. The skin-beautifying food composition according to Item 3, which contributes to beautiful skin by suppressing active oxygen in the skin. [Effects of the Invention]

[0008] According to the present disclosure, novel sirtuin 3 activators are provided. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows an HPLC chart of the rush core extract obtained in Test Example 1. [Figure 2]The results of Test Example 4-4-3 are shown below. The top of the bar graph indicates the average value for each group, and the error bars indicate the standard deviation. * indicates p<0.05, and ** indicates p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment of the present disclosure is described in more detail below. The present disclosure preferably includes, but is not limited to, a sirtuin 3 activator, a skin-beautifying food composition containing a sirtuin 3 activator, and the like. The present disclosure includes all of the disclosures herein that are recognizable to a person skilled in the art.

[0011] Sirtuin 3 activators encompassed by the present disclosure contain at least one selected from efsole and dehydroefsole. Hereinafter, such sirtuin 3 activators encompassed by the present disclosure may be referred to as "sirtuin 3 activators of the present disclosure."

[0012] Efsol is a compound represented by the following structural formula: [ka]

[0013] In the present disclosure, efsole may be chemically synthesized, or may be extracted and / or purified from a naturally occurring substance. If chemically synthesized, the synthesis method is not particularly limited as long as efsole can be synthesized, and efsole can be obtained by synthesis using known methods or methods that can be easily derived from known methods.

[0014] Efsol is known to be present in plants such as rush (wick grass), and can be obtained, for example, by extracting it from rush and purifying it as necessary. The extraction and / or purification method is not particularly limited as long as the desired effect is obtained, and for example, it may be extracted using an extraction solvent such as water, methanol, ethanol, or a mixture of at least two of these, and then purified as necessary.

[0015] The extraction solvent is not particularly limited, but methanol or 40 to 60 v / v % ethanol-water is preferred. If necessary, the resulting extract may be further fractionated and / or purified. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partition can be performed using, for example, water, n-hexane, ethyl acetate, etc. Purification can be performed, for example, by silica gel column chromatography and / or high-performance liquid chromatography (HPLC).

[0016] More specifically, for example, an ethanol-water extract of rush grass is concentrated under reduced pressure, followed by liquid-liquid partition extraction with methanol and n-hexane, the resulting methanol layer is concentrated under reduced pressure, and then water and ethyl acetate are added to perform further liquid-liquid partition extraction, and the ethyl acetate layer is purified by chromatography (silica gel column chromatography and high performance liquid chromatography). There are also no particular restrictions on the part of the plant used for extraction; for example, when the plant is rush grass, either the whole plant or the core of the rush grass may be used for extraction. Furthermore, commercially available ef-sol can also be purchased and used.

[0017] Dehydroefsol is a compound represented by the following structural formula: [ka]

[0018] In the present disclosure, dehydroefusole may be chemically synthesized, or may be extracted and / or purified from a naturally occurring substance. When chemically synthesized, the synthesis method is not particularly limited as long as dehydroefusole can be synthesized, and it can be obtained by synthesis using a known method or a method that can be easily derived from a known method.

[0019] Dehydroefsol is known to be present in plants such as rush, and can be obtained, for example, by extracting it from rush and purifying it as necessary. The extraction and / or purification method is not particularly limited as long as the desired effect is obtained, and the extract may be extracted using water, ethanol, or a mixture thereof as an extraction solvent, and purified as necessary.

[0020] The extraction solvent is not particularly limited, but 40 to 60 v / v % ethanol-water is preferred, and 50 v / v % ethanol-water is particularly preferred. If necessary, the resulting extract may be further fractionated and / or purified. Fractionation can be performed, for example, by liquid-liquid partition extraction. The partition can be performed using, for example, water, n-hexane, ethyl acetate, etc. Purification can be performed, for example, by silica gel column chromatography and / or high-performance liquid chromatography (HPLC).

[0021] More specifically, for example, an ethanol-water extract of rush grass is concentrated under reduced pressure, followed by liquid-liquid partition extraction with water and ethyl acetate, and the resulting ethyl acetate layer is purified by chromatography (silica gel column chromatography and high performance liquid chromatography). The part of the plant used for extraction is not particularly limited; for example, when the plant is rush grass, the whole plant or the rush core may be used for extraction. Since rush grass core contains a large amount of dehydroefsol, it is particularly preferable to use rush grass core. Alternatively, commercially available dehydroefsol can be purchased and used.

[0022] The present disclosure also encompasses a skin-beautifying food composition (hereinafter, sometimes referred to as the "composition of the present disclosure") containing the sirtuin 3 activator of the present disclosure. As described above, the sirtuin 3 activator of the present disclosure contains at least one selected from efsol and dehydroefsol, and efsol and dehydroefsol are contained in plants such as rush. Therefore, a plant (preferably rush) extract containing efsol and / or dehydroefsol may be used as is as the sirtuin 3 activator of the present disclosure or the composition of the present disclosure. In the present disclosure, the term "plant extract" preferably encompasses any of an extract obtained by an extraction procedure using a plant, a diluted solution of the extract, a concentrated solution of the extract, and a dried product obtained by drying the extract. Furthermore, the extract may be appropriately fractionated and / or purified, so long as it contains efsol and / or dehydroefsol. That is, the term "plant extract" in the present disclosure encompasses fractions, crude products, purified products, etc. of the extract. Furthermore, the plant extract may be a dried product obtained by drying using a conventional method (e.g., spray drying).

[0023] The sirtuin 3 activators and compositions of the present disclosure are used to activate sirtuin 3. In particular, they are preferably used to activate sirtuin 3 in skin cells via intestinal cells. In the present disclosure, the term "sirtuin 3 activation" preferably encompasses all of the following: promotion of promoter activity of the sirtuin 3 gene, an increase in the transcription level of the sirtuin 3 gene, and an increase in the amount of the sirtuin 3 translation product. Sirtuin 3 activation can be confirmed by conventionally known methods or by methods that can be easily derived by a person skilled in the art from conventionally known methods.

[0024] Enhancement of promoter activity of the sirtuin 3 gene can be determined, for example, by introducing into cells a plasmid in which the coding region of a reporter gene such as a fluorescent protein is linked downstream of the promoter region of the sirtuin 3 gene, and detecting and analyzing the expression of the reporter gene. More specifically, if the expression level of the reporter gene when the test substance is administered is greater than the expression level of the reporter gene when the test substance is not administered, it can be determined that the promoter activity of the sirtuin 3 gene is enhanced by the test substance.

[0025] Increased transcription of the sirtuin 3 gene can be determined by, for example, reverse transcription quantitative PCR (RT-qPCR) or RNA-seq. Increased translation of the sirtuin 3 gene can be determined by Western blotting or immunohistochemical staining.

[0026] In the present disclosure, "activation of sirtuin 3 in skin cells via intestinal cells" refers to the activation of sirtuin 3 in skin cells as a result of administering a certain component to intestinal cells. More specifically, the term "activation of sirtuin 3 in skin cells via intestinal cells" preferably encompasses the following: a component is absorbed into intestinal cells, resulting in the same component reaching skin cells, thereby activating sirtuin 3 in skin cells; a component is absorbed into intestinal cells and then reaches skin cells in a decomposed and / or modified state, thereby activating sirtuin 3 in skin cells; a component different from the component is released extracellularly due to absorption of the component by intestinal cells, and the different component acts on skin cells, thereby activating sirtuin 3 in skin cells; and a series of signal transduction caused by absorption of the component by intestinal cells, resulting in activation of sirtuin 3 in skin cells.

[0027] An example of "activation of sirtuin 3 in skin cells via intestinal cells" is the activation of sirtuin 3 in skin cells as a result of oral administration of a certain component to a subject. More specifically, when the subject is a human, oral administration of the component or a composition containing the component can activate sirtuin 3 in skin cells.

[0028] In the present disclosure, whether or not a component has the effect of activating sirtuin 3 in skin cells via intestinal cells can be confirmed by a conventionally known method or a method that a person skilled in the art can easily derive from a conventionally known method. More specifically, if oral administration of the component to a subject activates sirtuin 3 in skin cells compared to administration without the component, it can be determined that the component has the effect of activating sirtuin 3 in skin cells via intestinal cells. Furthermore, if intestinal-derived cells are cultured with a component and the culture supernatant is added to a skin cell culture medium containing a plasmid containing a reporter gene coding region linked downstream of the promoter region of the sirtuin 3 gene, and the expression of the reporter gene in the skin cells is increased compared to when culture supernatant of intestinal-derived cells cultured without the component is added, it can also be determined that the component has the effect of activating sirtuin 3 in skin cells via intestinal cells. Specific examples of intestinal-derived cells include human colon cancer-derived Caco-2 cells (American Tissue Culture Collection: ATCC), which are an intestinal epithelial cell model. Specific examples of skin cells include human epidermal keratinocyte cell line HaCaT cells (Riken Bioresource Center).

[0029] Sirtuin 3 has been reported to be involved in antioxidation (Non-Patent Document 1). Therefore, it is expected that the sirtuin 3 activator or composition of the present disclosure will exert a sirtuin 3 activation effect, thereby achieving an antioxidant effect, i.e., an effect of inhibiting reactive oxygen species. In the present disclosure, the term "inhibition of reactive oxygen species" preferably encompasses both preventing the generation of reactive oxygen species and quickly eliminating generated reactive oxygen species.

[0030] Reactive oxygen species are known to cause generally undesirable phenomena in the skin, such as age spots, wrinkles, and sagging. As described above, the sirtuin 3 activator and composition disclosed herein can exert an active oxygen inhibitory effect on the skin, and are therefore suitable for use in the prevention, improvement, and treatment of age spots, wrinkles, sagging, and the like (hereinafter, sometimes collectively referred to as "age spots, etc."). In this disclosure, the term "prevention of age spots, etc." encompasses application to a subject before age spots, etc. are visible, thereby preventing the occurrence of age spots, etc. In this disclosure, the terms "improvement of age spots, etc." and "treatment of age spots, etc." encompass application to a subject where age spots, etc. are visible, thereby inhibiting the progression of age spots, etc., stopping the progression of age spots, etc., inhibiting the increase of age spots, etc., and reducing age spots, etc. In addition, in this disclosure, use for the prevention, improvement, and treatment of age spots, etc. may be referred to as "for skin beautification."

[0031] The subjects who take the sirtuin 3 activator and composition of the present disclosure are not particularly limited. For example, preferred subjects include those who wish to activate longevity genes, those who wish to activate sirtuin 3, those who wish to suppress reactive oxygen species, those who wish to prevent, improve, or treat age spots, wrinkles, sagging skin, and those who wish to achieve skin-beautifying effects. The subject may also fall into two or more of these categories. Furthermore, the subject may be not only humans, but also non-human mammals. Mammals kept as pets or livestock are particularly preferred. Specific examples include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, and the like.

[0032] The sirtuin 3 activators and compositions of the present disclosure can be preferably used as pharmaceutical compositions or food compositions. When used as a pharmaceutical composition, the composition (sometimes referred to as the "pharmaceutical composition of the present disclosure") may contain efsol and / or dehydroefsol (or a plant extract containing efsol and / or dehydroefsol) and, if necessary, pharmaceutically acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavoring agents, odorants, surfactants, humectants, preservatives, pH adjusters, thickeners, etc.). Such bases, carriers, additives, etc. are specifically described, for example, in the Pharmaceutical Additives Dictionary 2021 (Yakuji Nipposha), and those described therein can be used. The dosage form is also not particularly limited, and the active ingredient and other ingredients can be mixed by conventional methods to prepare preparations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewable tablets, and soft tablets. For example, tablets can be prepared by tableting. Either direct tableting, in which the mixed raw materials are directly compressed, or granule tableting, in which the mixed raw materials are granulated and then compressed, can be used. For example, capsules can be either soft capsules or hard capsules.

[0033] The content of efsole and / or dehydroefsole in the pharmaceutical composition of the present disclosure is not particularly limited as long as it can exert a sirtuin 3 activating effect, and can be appropriately determined depending on the subject. For example, it may be 0.0005 to 100% by mass, preferably 0.005 to 90% by mass, more preferably 0.05 to 80% by mass, and even more preferably 0.1 to 70% by mass. The upper or lower limit of the range may be 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% by mass.

[0034] The timing and duration of administration of the pharmaceutical composition according to the present disclosure are not particularly limited, and can be appropriately selected taking into consideration, for example, the dosage form, the age of the subject, the severity of the symptoms of the subject, etc. The administration form is not particularly limited, but oral administration is preferred.

[0035] The dosage of the pharmaceutical composition according to the present disclosure can be selected appropriately depending on the age and condition of the subject, other conditions, etc. In particular, it can be set appropriately based on the amount of efsol and / or dehydroefsol contained therein, as long as the effect is not impaired. Although not particularly limited, for example, the amount of efsol and / or dehydroefsol administered per day to an adult may be about 0.1 to 100 mg, preferably about 0.5 to 80 mg, more preferably about 1 to 50 mg, and even more preferably about 5 to 30 mg, about 6 to 24 mg, about 10 to 24 mg, or about 12 to 24 mg. The upper or lower limit of the range may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mg. The dosage may be administered once a day or multiple times (preferably 2 to 3 times a day). For non-human mammals, the dosage can be appropriately determined with reference to that for humans.

[0036] When the composition of the present disclosure is used as a food composition (e.g., a food or beverage or a food additive), the composition (hereinafter sometimes referred to as the "food composition of the present disclosure") may be blended with efsol and / or dehydroefsol (or a plant extract containing efsol and / or dehydroefsol), as well as a base, carrier, additive, or other ingredient or material that is acceptable from a food hygiene perspective and that can be used in foods or beverages. Examples of food compositions that contain efsol and / or dehydroefsol include processed foods, beverages, health foods, functional foods, dietary supplements, supplements, foods with health claims, foods for specified health uses, foods with nutritional functionality, foods with functional claims, and foods for patients (hospital foods, sick foods, nursing care foods, etc.). Without being particularly limited, when the efsol and / or dehydroefsol contained in the food composition is a plant extract (preferably rush extract), the food composition may be, for example, a processed food, health food, foods with nutritional claims, foods for specified health uses, supplements, foods for patients, etc., that contain the extract. Furthermore, efsol and / or dehydroefsol may be made into a powder form, for example, and added to various foods and beverages such as beverages (juices, etc.), sweets, breads, soups (including powdered soups, etc.), processed foods, etc. Hospital food is food served when a patient is admitted to a hospital, sick food is food for sick people, and care food is food for people receiving care.

[0037] When preparing the food composition according to the present disclosure as a health food (nutritional functional food, food for specified health uses, etc.) or supplement, it is preferable to prepare it in the form of, for example, granules, capsules, tablets (including chewable tablets), beverages (drinkable preparations), etc. to facilitate continuous intake, and capsules, tablets, and pills are particularly preferred from the standpoint of ease of intake. However, the present disclosure is not particularly limited to these. The food composition according to the present disclosure in the form of granules, capsules, tablets, etc. can be appropriately prepared according to conventional methods using pharmaceutically and / or food hygienically acceptable carriers, etc. Furthermore, even when preparing it in other forms, conventional methods can be followed.

[0038] The content of efsole and / or dehydroefsole in the food compositions of the present disclosure is not particularly limited as long as the sirtuin 3 activation effect can be exerted, and can be appropriately set depending on the subject. For example, it may be 0.0005 to 100% by mass, preferably 0.005 to 90% by mass, more preferably 0.05 to 80% by mass, and even more preferably 0.1 to 70% by mass. The upper or lower limit of the range may be 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% by mass.

[0039] The intake amount, intake method, intake target, etc. of the food composition according to the present disclosure may be the same as, for example, the dosage, administration method, administration target, etc. described above for the pharmaceutical composition according to the present disclosure.

[0040] The food composition according to the present disclosure is not particularly limited, but is preferably a skin-beautifying food composition.

[0041] The present disclosure also encompasses methods for producing a sirtuin 3 activator and / or a skin-beautifying food composition, which include a step of blending efsol and / or dehydroefsol, as well as methods for activating sirtuin 3, suppressing reactive oxygen species, and contributing to skin beauty by administering efsol and / or dehydroefsol. With regard to these methods, the above descriptions regarding the sirtuin 3 activator and composition of the present disclosure are incorporated by reference.

[0042] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0043] Furthermore, the various characteristics (properties, values, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0044] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0045] Test Example 1. Preparation of rush core extract Hot water extraction was performed by adding 20 parts by weight of hot water (95°C or higher) to 1 part by weight of wick (dried pith from the stems of rushes of the Juncaceae family) and leaving it to stand for at least 8 minutes. The extract was filtered to remove the filtrate, and then 20 parts by weight of 50 v / v% ethanol-water was added to the residue. Reflux extraction was performed twice while heating to 80°C for 1 hour, followed by filtration to obtain a filtrate. The filtrate was concentrated under reduced pressure and spray-dried to obtain 0.01 parts by weight of rush core extract. Quantitative analysis of efsol and dehydroefsol in the rush core extract by HPLC revealed 0.73 w / w% efsol and 3.47 w / w% dehydroefsol. The results are shown in Figure 1.

[0046] HPLC conditions: Column: COSMOSIL 2.5 Cholester ID 3.0 × 100 mm (Nacalai Tesque), Mobile phase: 0.1% trifluoroacetic acid aqueous solution: acetonitrile = 60:40, Flow rate: 0.75 mL / min, Column temperature: 40°C, Detection: UV (280 nm)

[0047] Test Example 2: Isolation of Efsol 20 kg of commercially available whole rush (dried aboveground parts of rush of the Juncaceae family) was subjected to reflux extraction in 20 times its weight of 50 v / v% ethanol-water at 80°C for 2 hours. After reflux extraction, the filtrate was filtered and concentrated under reduced pressure to remove the solvent, yielding 292 g of a paste-like extract. 1.4 L of 90 v / v% methanol-water was added to the extract to form a suspension, and then 0.7 L of n-hexane was added and liquid-liquid partition extraction was carried out using a separatory funnel. This liquid-liquid partition extraction procedure was repeated three times, and the extract was separated into a methanol layer and a hexane layer.

[0048] The methanol layer was concentrated under reduced pressure, 1.4 L of distilled water was added to form a suspension, and 1.3 L of ethyl acetate was added and subjected to liquid-liquid partition extraction in a separatory funnel. The liquid-liquid partition extraction procedure was repeated three times to separate the aqueous layer and the ethyl acetate layer.

[0049] The ethyl acetate layer was evaporated under reduced pressure and dried to obtain 15.4 g of ethyl acetate extract. The ethyl acetate extract was fractionated into 13 fractions, A to M, using silica gel column chromatography (Purif-Pack®-EX SI-50 SIZE: 400 (φ46 x 220 mm, 200 g), Shoko Science Co., Ltd.) (eluent: chloroform containing 3 v / v% methanol to chloroform containing 20 v / v% methanol). Fraction E was purified using preparative liquid chromatography (column: COSMOSIL Cholester, Nacalai Tesque, elution solvent: 42-47 v / v% acetonitrile-water) to obtain compound 1 (0.123 g). The NMR spectrum of compound 1 was measured and it was identified as efsol.

[0050] Test Example 3: Isolation of dehydroefsol Five kg of commercially available wick (dried pith from the stems of rushes of the Juncaceae family) was subjected to reflux extraction twice for 2 hours at 80°C in 20 times its weight of 50 v / v% ethanol-water. After the reflux extraction, the mixture was filtered to obtain a filtrate, which was then concentrated under reduced pressure. 1.3 L of distilled water was added to the concentrated solution to form a suspension, and then 1.3 L of ethyl acetate was added, and liquid-liquid partition extraction was carried out in a separatory funnel. The liquid-liquid partition extraction procedure was repeated three times, and the solution was separated into an aqueous layer and an ethyl acetate layer.

[0051] The ethyl acetate layer was distilled under reduced pressure and dried to obtain 26.2 g of ethyl acetate extract. The ethyl acetate extract was fractionated into 13 fractions, A to M, by silica gel column chromatography (eluent: chloroform containing 2 v / v% methanol to chloroform containing 40 v / v% methanol). Fractions F and E were purified by preparative liquid chromatography (column: COSMOSIL Cholester, Nacalai Tesque, elution solvent: 40 v / v% acetonitrile-water) to obtain compound 2 (1.725 g). The NMR spectrum of compound 2 was measured and it was identified as dehydroefsole.

[0052] Test Example 4: Examination of Sirtuin 3 Activation 4-1. Cultivation of Caco-2 cells, a human colon cancer cell line Fetal bovine serum (FBS, Life Technologies) was heated in a 56°C incubator for 30 minutes to inactivate complement (referred to as inactivated FBS). 4.75 g of Dulbecco's modified Eagle's medium "Nissui" (Nissui Pharmaceutical) was dissolved in 470 mL of Milli-Q® water and sterilized by autoclaving. Then, 1 mL of 100 U / mL penicillin (Meiji Seika Pharma), 1 mL of 0.1 mg / mL streptomycin (Meiji Seika Pharma), and 6 mL of 10% NaHCO3 (Fujifilm Wako Pure Chemical Industries) were added. Furthermore, L-glutamine, sterilized by filtration through a 0.22 μm filter (Toyo Roshi), was added to a final concentration of 4 mM (referred to as DMEM medium).

[0053] The DMEM medium was supplemented with 10% (v / v) of the heat-inactivated FBS. Caco-2 cells (American Tissue Culture Collection: ATCC), a model of intestinal epithelial cells, derived from human colon cancer, were subcultured in a Petri dish at 37°C and 5% CO2 using the medium.

[0054] 4-2. Cultivation of human epidermal keratinocyte cell line HaCaT cells Human epidermal keratinocyte cell line HaCaT cells (Riken Bioresource Center) were subcultured in the same medium and under the same conditions as in Test Example 4-1.

[0055] 4-3. Preparation of HaCat(pSIRT3p-EGFP) cells A plasmid (referred to as pSIRT3p-EGFP) expressing Enhanced Green Fluorescent Protein (EGFP) under the control of the human sirtuin 3 (SIRT3) promoter was prepared according to the method described in Non-Patent Document 9. The pSIRT3p-EGFP plasmid was stably transfected into the human skin keratinocyte cell line HaCaT cells obtained in Test Example 4-2 according to the method described in Non-Patent Document 10 (referred to as HaCat(pSIRT3p-EGFP) cells). The HaCat(pSIRT3p-EGFP) cells were subcultured in the same manner as in Test Examples 4-1 and 4-2.

[0056] 4-4. Promotion of Sirtuin 3 expression in skin cells via intestinal cells We investigated the expression of Sirtuin 3 in skin cells via intestinal cells by adding Caco-2 cell culture supernatant to HaCaT (pSIRT3p-EGFP) cells. The specific method is described below. Unless otherwise specified, cell culture was performed at 37°C and 5% CO2.

[0057] 4-4-1. The Caco-2 cells obtained in Test Example 4-1 were cultured at a final concentration of 1.0 × 10 5 The cells were seeded onto a 24-well plate (FALCON) at a concentration of 100 cells / mL. The medium used was the DMEM medium supplemented with 10 v / v% heat-inactivated FBS, as described in Test Example 4-1. 24 hours after seeding, efsol obtained in Test Example 2 and dehydroefsol obtained in Test Example 3 were added to separate wells at a final concentration of 10 μM. Each test substance was dissolved in dimethyl sulfoxide (DMSO) before being added to the wells. DMSO was also added as a negative control. The final concentration of DMSO was the same for each well. After addition, the cells were cultured for an additional 24 hours.

[0058] 4-4-2. The HaCaT (pSIRT3p-EGFP) cells obtained in Test Example 4-3 were cultured at a final concentration of 6.0 × 10 5 The cells were seeded onto a 96-well black plate at a concentration of 100 μL / mL. The medium used was the DMEM medium supplemented with 10 v / v% inactivated FBS as described in Test Example 4-1. 24 hours after seeding, the medium was removed, and 100 μL / well of the culture supernatant of Caco-2 cells cultured in Test Example 4-4-1 was added. After addition, the cells were cultured for an additional 48 hours.

[0059] 4-4-3.SIRT3 expression analysis In the following tests, 1x PBS supplemented with 8 w / v% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) and 5 μL / mL of 2N NaOH was used as the cell fixative. The prepared cell fixative was stored at 4°C until use in the test. Hoechst 33342 diluted with 1x PBS to 2 μg / mL was used as the nuclear staining solution. The nuclear staining solution was prepared immediately before use and used in a light-protected environment.

[0060] After fixing the cells with the cell fixative, nuclear staining was performed using the nuclear staining solution. Cell count and SIRT3 promoter activity were analyzed using an IN Cell Analyzer 2200 (Cytiva). Specifically, 100 μL of the cell fixative was added to the culture medium of HaCaT (pSIRT3p-EGFP) cells after the procedure described in Test Example 4-4-2, and the cells were incubated at room temperature for 15 minutes. After incubation, the cell culture medium and cell fixative were removed from the wells, the cells were washed twice with 1x PBS, and the nuclear staining solution was added at 100 μL / well. After allowing the cells to stand at room temperature for 20 minutes, the nuclear staining solution was removed and the cells were washed twice with 1x PBS. After adding 100 μL / well of 1x PBS, EGFP fluorescence was analyzed using an IN Cell Analyzer 2200. Note that EGFP fluorescence intensity reflects SIRT3 promoter activity.

[0061] Images of HaCaT (pSIRT3p-EGFP) cells, which were subjected to the above procedure and detected for fluorescence using an IN Cell Analyzer 2200, were analyzed using IN Cell Investigator High-content image analysis software (GE Healthcare). Specifically, for each test substance, the fluorescence intensity when DMSO alone was added (control) was set at 1.00, and the relative fluorescence intensity was calculated. Note that the test was performed with n = 4, and fluorescence intensity analysis was performed for each well. Statistical analysis was performed using Student's t-test for each control group. The results are shown in Figure 2. In Figure 2, the top of the bar graph indicates the mean value for each group, and the error bars indicate the standard deviation. * indicates p < 0.05, and ** indicates p < 0.01.

[0062] As shown in Figure 2, when efsol and dehydroefsol were added, enhancement of SITR3 promoter activity was observed. These results suggest that efsol and dehydroefsol have the effect of activating sirtuin 3.

[0063] The culture supernatant of Caco-2 cells in the test substance group added to HaCaT(pSIRT3p-EGFP) cells in Test Example 4-4-2 contains efsol or dehydroefsol added in Test Example 4-4-1, but it is believed that the efsol or dehydroefsol is sufficiently diluted during the process of adding only a portion (approximately 1 / 5) of the culture supernatant to the culture medium of HaCaT(pSIRT3p-EGFP) cells. Methods for evaluating the effects of test substances via intestinal cells using Caco-2 cells are conventionally known. For example, Non-Patent Document 8 uses human colon cancer-derived Caco-2 cells as an intestinal epithelial cell model and human neuroblastoma SH-SY5Y cells as a neuronal cell model to investigate the possibility of brain function regulation via intestinal activation by foods and food-derived components.

[0064] Therefore, the enhancement of SITR3 promoter activity observed in Test Example 4-4-3 is thought to reflect the action of efsol or dehydroefsol via the intestinal epithelial cell model Caco-2 cells.

Claims

1. An agent for activating sirtuin 3 in skin cells via intestinal cells, comprising at least one component selected from the group consisting of efsol and dehydroefsol as an active ingredient.

2. The sirtuin 3 activator according to claim 1, which activates sirtuin 3 in skin cells when taken orally.

3. A skin-beautifying food composition containing the sirtuin 3 activator according to claim 1 or 2.

4. A skin-beautifying food composition containing at least one component selected from the group consisting of efsol and dehydroefsol as a skin-beautifying component that has the function of activating sirtuin 3 when taken orally.

5. The skin-beautifying food composition according to claim 3, which contributes to beautiful skin by suppressing active oxygen in the skin.

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