JP2020127398A - Sirtuin expression level enhancer - Google Patents

Abstract

To provide a sirtuin expression level enhancer.SOLUTION: A sirtuin expression level enhancer contains at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β1,3-glucan.SELECTED DRAWING: None

Description

The present invention relates to a sirtuin expression level enhancer and the like.

In recent years, with the increasing aging rate, technological developments regarding anti-aging have been actively conducted. The sirtuin gene, which is a histone deacetylase gene, is also called a longevity gene, and its activation causes various effects such as lifespan extension, aging suppression, wrinkle suppression, and spot suppression. It is said. Therefore, if the expression level of sirtuin can be increased, various effects represented by the anti-aging effect can be exhibited. Patent Document 1 describes that a specific polyphenol activates a sirtuin gene.

Euglena is a microalga belonging to Euglena (= Euglena) and is used as a food material. It is also practiced to apply the Euglena extract to the skin. In addition, paramylon is a β1,3-glucan produced by Euglena and is reported to be useful for wound treatment and allergy suppression. However, the relationship between Euglena or β1,3-glucan and the sirtuin gene has not yet been known.

International Publication No. 2014/042261

An object of the present invention is to provide a sirtuin expression level enhancer.

As a result of earnest research in view of the above problems, the present inventor has found that Euglena, paramylon, processed paramylon, and β1,3-glucan have a sirtuin expression level enhancing action. As a result of further research based on this finding, the present invention has been completed. That is, the present invention includes the following aspects:

Item 1. A sirtuin expression level enhancer containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan.

Item 2. Item 2. The sirtuin expression level enhancer according to Item 1, containing at least one selected from the group consisting of Euglena, paramylon, and processed paramylon.

Item 3. Item 3. The sirtuin expression level enhancer according to Item 1 or 2, which contains the Euglena and the Euglena is Euglena gracilis.

Item 4. Item 3. The sirtuin expression level enhancer according to Item 1 or 2, which contains the Euglena and is the Euglena gracilis EOD-1 strain (Accession No. FERM BP-11530).

Item 5. Item 3. The sirtuin expression level enhancer according to Item 1 or 2, which contains the paramylon and the paramylon is derived from Euglena gracilis.

Item 6. Item 3. The sirtuin expression level increasing agent according to Item 1 or 2, which contains the paramylon and is a paramylon derived from the Euglena gracilis EOD-1 strain (accession number FERM BP-11530).

Item 7. Item 7. The sirtuin expression level increasing agent according to any one of Items 1 to 6, which is a food composition, a food additive, a cosmetic, a cosmetic additive, or a medicine.

Item 8. Item 8. The sirtuin expression level enhancer according to any one of Items 1 to 7, which is an oral composition or an external composition.

Item 9. Life extension, anti-aging, wrinkle suppression, spot suppression, dementia prevention and/or improvement, sleep improvement, gray hair suppression, antioxidant, fatigue improvement, muscle enhancement, mitochondrial enhancement, energy metabolism improvement, anti-arteriosclerosis, anti-diabetes, anti Item 9. The sirtuin expression level enhancer according to any one of Items 1 to 8, for use in at least one selected from the group consisting of inflammation and antiobesity.

Item 10. Containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan,
Life extension, anti-aging, wrinkle suppression, spot suppression, dementia prevention and/or improvement, sleep improvement, gray hair suppression, antioxidant, fatigue improvement, muscle enhancement, mitochondrial enhancement, energy metabolism improvement, anti-arteriosclerosis, anti-diabetes, anti A composition for use in at least one selected from the group consisting of inflammation and antiobesity.

According to the present invention, a sirtuin expression level enhancer can be provided.

The result of Test Example 1 (Sirtuin expression level measurement test 1 (Caco-2)) is shown. The vertical axis represents the value obtained by dividing the hSIRT1 expression level measurement value by the β-actin expression level measurement value. In the horizontal axis, the control shows the case where the test substance is not added, and the others show the test substance added and the concentration thereof. ** indicates that the p-value (t-test) for the control is less than 0.01, and *** indicates that the p-value is less than 0.001. The result of using Euglena in Test Example 2 (Sirtuin expression level measurement test 2 (HaCaT)) is shown. The vertical axis represents the value obtained by dividing the hSIRT1 expression level measurement value by the β-actin expression level measurement value. In the horizontal axis, the control shows the case where the test substance is not added, and the others show the test substance added and the concentration thereof. *** indicates that the p-value (t-test) for the control is less than 0.001. The result when Paramylon is used in Test Example 2 (Sirtuin expression level measurement test 2 (HaCaT)) is shown. The vertical axis represents the value obtained by dividing the hSIRT1 expression level measurement value by the β-actin expression level measurement value. In the horizontal axis, the control shows the case where the test substance is not added, and the others show the test substance added and the concentration thereof. *** indicates that the p-value (t-test) for the control is less than 0.001. The measurement result of SIRT3 expression level in Test Example 3 (Sirtuin expression level measurement test 3 (Caco-2, HaCaT co-culture) is shown. The vertical axis represents the value obtained by dividing the hSIRT3 expression level measurement value by the β-actin expression level measurement value. In the horizontal axis, the control shows the case where the test substance is not added, and the others show the test substance added and the concentration thereof. ** indicates that the p-value (t-test) for the control is less than 0.01, and *** indicates that the p-value is less than 0.001. The measurement result of SIRT6 expression level in Test Example 3 (Sirtuin expression level measurement test 3 (Caco-2, HaCaT co-culture)) is shown. The vertical axis represents the value obtained by dividing the hSIRT6 expression level measurement value by the β-actin expression level measurement value. In the horizontal axis, the control shows the case where the test substance is not added, and the others show the test substance added and the concentration thereof. * Indicates that the p value (t test) for the control is less than 0.05, and *** indicates that the p value is less than 0.001.

In this specification, the expressions "containing" and "including" include the concepts of "containing", "including", "consisting essentially of" and "consisting solely of".

In the present specification, the expression "and/or" includes the meaning when either "and" or "or" is selected. That is, the expression “A and/or B” includes both the meanings of “A or B” and “A and B”.

The present invention, in one aspect thereof, comprises a sirtuin expression-enhancing agent containing at least one member selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan (herein, " Sometimes referred to as "inventive agent"). This will be described below.

1. Euglena Euglena is a microalgae belonging to Euglena (genus Euglena) and is not particularly limited as long as it is. As Euglena, specifically, for example, Euglena gracilis (Euglena gracilis), Euglena longa, Euglena caudata, Euglena oxyuris, Euglena tripteris, Euglena proxima, Euglena viridis, Euglena sociabilis, Euglena ehrenbergii, Euglena deses, Euglena pisciformis, Euglena spirogyra, Examples include Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicola . Among these, Euglena gracilis is preferable, and Euglena gracilis EOD-1 strain is more preferable from the viewpoint that the effect of the present invention can be exhibited more reliably. Under the provisions of the Budapest Treaty, deposit number FERM BP-11530 at the Japan Patent Organism Depositary {NITE-IPOD (Zip Code 292-0818 Kazusa Kamasa 2-5-8 120, Kisarazu City, Chiba Prefecture, Japan)} International Deposited].

The form of Euglena is not particularly limited, as long as it contains most of the Euglena cell body or its components. Examples of the form of Euglena include a dry powder form of Euglena, a suspension of Euglena, a Euglena extract, and the like, and among them, a dry powder form of Euglena is preferable.

The content of paramylon in the dry state of euglena is, for example, 50% or more, preferably 60% or more, more preferably 70% or more.

Euglena may be a single species or a combination of two or more species.

2. β1,3-glucan and paramylon β1,3-glucan are not particularly limited as long as they have one sugar chain (or sugar chain structure) as a main chain in which glucose is linked only by β1,3 bond. The β1,3-glucan is not limited to a linear one, but includes one having a branched chain.

The weight average molecular weight of the β1,3-glucan derivative is not particularly limited, but is 1×10 ⁴ from the viewpoint that the activity of improving metabolism of sugar and/or lipid can be more reliably exhibited (for example, at a lower concentration). To 2×10 ⁶ , preferably 5×10 ⁴ to 1×10 ⁶ , and more preferably 1×10 ⁵ to 1×10 ⁶ . The weight average molecular weight can be measured by the GPC method.

Although β1,3-glucan may be obtained by chemical synthesis, natural β1,3-glucan produced by various organisms is preferable from the viewpoint of availability and the like. Examples of natural β1,3-glucan include paramylon, curdlan, laminaran, callose, lentinan, schizophyllan and the like. Of these, paramylon is preferable. The paramylon will be described below.

Paramylon is a β-1,3-glucan derived from euglena and is not particularly limited as long as it is.

The euglena from which paramylon is derived is the same as described in “1. Euglena” above.

The mass average molecular weight of paramylon is not particularly limited, for example, 1 × 10 ^{4 to} 5 × 10 ⁶ , preferably 2 × 10 ⁴ to 1 × 10 ⁶ , more preferably 5 × 10 ⁴ to 1 × 10 ⁶ , more preferably Is 1×10 ^{5 to} 5×10 ⁵ .

The mass average molecular weight can be measured by SEC-MALS analysis under the following conditions:
SEC device: LC-10AVP system (Shimadzu Co., Japan),
Column used: KD-806M (shodex., Japan),
MALS detector: DAWN HELEOSII (wyatt Technologies., USA),
Eluent: 1% LiCl/DMI,
Flow rate: 0.5 mL/min.

In Euglena cells, paramylon usually exists as paramylon particles in which a triple helix structure formed by β-1,3-glucan chains is highly accumulated in a regular group.

The shape of the paramylon particles is not particularly limited, but is usually a flat spheroid.

The particle size distribution of the paramylon particles is not particularly limited, but is, for example, 0.5 to 15 μm, preferably 1 to 6 μm. The average particle size of the paramylon particles is also not particularly limited, but is, for example, 1 to 10, preferably 2 to 4 μm.

The form of paramylon is not particularly limited as long as it contains paramylon. Examples of the form of Euglena include a dry powder form of paramylon, a suspension of paramylon, and the like. Among them, a dry powder form of paramylon is preferable.

Paramylon may be a single type or a combination of two or more types.

3. Euglena and Paramylon Production Method Euglena can be prepared in a large amount by a method including a step of culturing Euglena contained in a liquid (culture step). The culturing step can be performed, for example, according to a known method (for example, the method described in Japanese Patent No. 5883532). In the culturing step, typically, a euglena genus microalga is cultivated under aerobic conditions while stirring a liquid (culture solution) containing water, euglena, and nutrients that can be used by euglena.

As nutrients, sugars (glucose (glucose), fructose (fructose) and other monosaccharides, or sucrose (sucrose), maltose (maltose) and other disaccharides), minerals (for example, sodium, potassium, magnesium, calcium, Iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron), vitamin Bs (for example, vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or Pyridoxamine), vitamin B12 (cyanocobalamin), folic acid, biotin, etc.) and the like. The concentration of nutrients in the culture solution is not particularly limited as long as it is a concentration at which Euglena can survive and grow.

The light conditions in the culturing step are not particularly limited, and the culturing step may be performed under either bright or dark conditions. When culturing in heterotrophic culture, it is cultivated under dark conditions. As the light condition, normal light intensity for growing algae can be adopted. The dark conditions include, for example, less than 10 μmol/m ² /s, and preferably complete dark conditions where no light is applied.

The culture temperature in the culture step is not particularly limited as long as it is a temperature at which Euglena can grow. As the culture temperature (temperature of the culture solution), for example, 20°C to 35°C is adopted.

The pH of the liquid in the culture step is not particularly limited as long as it is a pH at which Euglena can grow. The pH at which Euglena can grow is, for example, 3.0 to 5.5.

After the culturing step, it is preferable to concentrate the Euglena by centrifugal separation or gravity separation of the liquid. The Euglena obtained can be subjected to additional treatments (eg suspension in liquid, dispersion in water or oil, extract extraction, dry powderization, etc.) depending on the desired form.

Paramylon particles can be produced by separating, isolating, or purifying from Euglena, according to a known method (for example, the method described in Japanese Patent No. 5883532) or in accordance therewith. Paramylon particles can be easily obtained by, for example, recovering the cell content component obtained by disrupting the cell membrane of Euglena. Moreover, you may refine a paramylon particle as needed. Various types of purification of paramylon particles are known (for example, Japanese Patent No. 5883532) and can be performed according to these methods. Examples of the purification step include a surfactant treatment step and a washing step. The resulting Euglena can be subjected to additional treatments (eg suspension in liquid, dispersion in water or oil, dry powderization, etc.) depending on the desired morphology.

Four. Paramylon Processed Product The paramylon processed product is obtained by processing the paramylon, for example, physical treatment, chemical treatment, etc., and is not particularly limited as long as it is. Examples of the paramylon compound include fibrous paramylon and amorphous paramylon. Amorphalparamylon can be obtained according to a known method or in a similar manner, for example, by chemically treating it using the method described in JP-A-2011-184592.

As the paramylon compound, fibrous paramylon is preferable. The fibrous paramylon will be described below.

The fibrous paramylon is a β-1,3-glucan derived from Euglena and is not particularly limited as long as it is in a fibrous form. Until now, amorphous paramylon obtained by chemically treating paramylon particles (alkali treatment, etc.) has been reported, but this is not recognized as fibrosis when observed with an electron microscope, and its shape and size are Since it is an amorphous mass, it is not included in fiberized paramylon.

The weight average molecular weight of the fiberized paramylon is not particularly limited, but is, for example, 1×10 ⁴ to 2×10 ⁷ , and preferably 1×10 ^{5 to} 5×10 ⁵ .

The weight average molecular weight can be measured by the following method by SEC-MALS analysis: SEC device: LC-10ADvp system (Shimadzu Co., Japan), column used: KD-806M (shodex., Japan). ,
MALS detector: DAWN HELEOSII (wyatt Technologies., USA), eluent: 1% LiCl/DMI,
Flow rate: 0.5 mL/min.

The fiber diameter of the fiberized paramylon is not particularly limited, but is, for example, 10 to 500 nm, preferably 20 to 300 nm, more preferably 50 to 200 nm. The fiber diameter of the fiberized paramylon can be usually measured based on an electron microscope image of the fiberized paramylon.

The sedimentation volume of fibrous paramylon in water is not particularly limited, but is, for example, 30 to 300 mL/g, preferably 50 to 250 mL/g, and more preferably 70 to 200 mL/g.

Submerged volume can be measured according to or according to the following method:
Measurement is carried out according to the method described in "Dietary Fiber Society of Japan, Editorial Committee of the Dietary Fiber Society of Japan (2008) Dietary Fiber-Basics and Applications-3rd Edition, p.111 Daiichi Shuppan, Tokyo". .. Specifically, it is as follows. Weigh 125 mg of the test sample in the form of slurry into a 25 mL volume plastic tube in terms of dry mass, shake the plastic tube by hand and stir the contents. Then, transfer the contents to a graduated cylinder with a volume of 25 mL, and add pure water to 25 mL. After stirring the liquid in the graduated cylinder, leave it at 37°C for 24 hours. This causes the sample to settle, resulting in two layers, a layer containing predominantly the precipitated sample (lower layer) and a layer mainly containing water (upper layer), separated through the interface. The volume of the lower layer is obtained from the scale of the graduated cylinder, and the obtained volume is divided by the sample mass (dry mass) to calculate the volume of water settled (mL/g). Perform the test 3 or 4 times and calculate the average value and standard deviation.

Fibrotic paramylon is relatively resistant to enzymatic degradation. For example, the amount of the monomer (glucose) produced by the decomposition of β-glucanase is, for example, 0.1 to 50 mg, preferably 1 to 10 mg, per 1 g of fibrous paramylon.

This amount can be measured according to or according to the following method:
Reaction solution [Test substance 30 mg (dry weight), buffer solution (Tokyo Chemical Industry Co., Ltd. B0156, potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.0)) 5 mL, enzyme solution (Nippon Biocon Co. endo- 1,3-β-Glucanase (Lot 91102c) (enzyme content: 50 units/mL)) 0.1 mL, pure water, reaction solution volume 10 mL] is prepared and shaken horizontally at 40 rpm for 24 hours at 45 rpm. .. Immediately after shaking, freeze-preserve and freeze-dry for concentration. After freeze-drying, add 0.5 mL of pure water to each sample and stir (20-fold concentration). Centrifuge (10000 G, 5 minutes, 4°C) and collect the supernatant, which is repeated twice. The glucose concentration in the recovered supernatant is measured using a measurement kit (Wako Pure Chemical Industries, Ltd., glucose CII-Test Wako). Based on the measured value, the glucose production amount (mg) per 1 g of the test substance is calculated.

Fiberized paramylon has a relatively low solubility in an alkaline solution. For example, fibrous paramylon does not dissolve in 0.1-0.3 M aqueous sodium hydroxide solution. Here, "does not dissolve" means, for example, that the absorbance (660 nm) of the solution after suspending the fibrous paramylon in the aqueous solution (e.g., immediately after the passage of 1 hour) is, for example, 0.1 or more, preferably 1.0. It means that it is above.

Solubility can be measured according to or according to the following method:
Suspend 250 mg (dry weight) of the test substance in 10 mL of the test solution (pure water, 0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution, 1 M NaOH aqueous solution) in the vial. After shaking the vial vigorously by hand for 20 seconds and shaking on a shaker at 80 rpm for 1 hour, the absorbance at 660 nm of the solution in the vial is measured. The absorbance is measured with a spectrophotometer V-730 manufactured by JASCO Corporation.

The relative value of the crystallinity of the fiberized paramylon to the granular paramylon (the crystallinity of the fiberized paramylon/the crystallinity of the granular paramylon) is, for example, 0.60 to 0.90, preferably 0.65 to 0.80.

Crystallinity can be measured according to or according to the following method:
XRD measurement is performed on the test substance. The conditions are as follows. Equipment: PANalytical X'Pert3 Powder, tube voltage: 45kV, tube current: 40mA, measurement range: 5.005 to 50.018°, measurement interval: 0.013°, analysis software: HighScore. The crystallinity is analyzed by the ratio of the strength of the amorphous part and the strength of the crystal part at 2θ=5 to 80°. The analysis was performed after removing the background from each measurement table (background setting Auto, venting factor 0, granularity 100), and the amorphous part was determined by the tangent line passing through 2θ=14, 29°. To do. The conditions for the pending factor and granularity that determine each amorphous part are 0/20.

The fibrous paramylon may be in a form of being dispersed in a solvent such as water, or may be in a dry form. Fiberized paramylon can be redispersed in water, even in dry form.

In the present specification, “dry form” means that the water content is 15 mass% or less, preferably 10 mass% or less, more preferably 5 mass% or less.

As the fiberized paramylon, a defibrated product of paramylon particles, which is obtained by physically defibrating the paramylon particles, can be preferably used. Further, the defibrated material of Euglena obtained by applying this defibration treatment to Euglena can also be used as fiberized paramylon.

The defibration treatment hardly breaks the hydrogen bonds of β-1,3 glucan present in the paramylon particles (for example, 10% or less, 5% or less, 2% or less of the hydrogen bonds of β-1,3 glucan). , A process capable of defibration (cutting less than 1% or less), or a part or all of the β-1,3-glucan chain present in the paramylon particle or the triple helix structure formed by this There is no particular limitation as long as it is a process that can Preferably, it is preferable that the β-1,3 glucan present in the paramylon particles is subjected to a fibrillation treatment with almost no breaking of hydrogen bonds to form a fibrous form. A known treatment that can grind (shear) or grind (preferably grind (shear)) fine particles such as paramylon particles can be adopted as the defibration treatment.

The defibration treatment can be performed using a known milling machine (shearing machine), crushing machine or the like. Examples of the apparatus used for the defibration treatment include a stone mill grinder, a jet mill, a twin-screw kneader, a high-pressure homogenizer, a high-pressure emulsifier, a twin-screw extruder, and a bead mill. Among these, a stone mill type grinder and a bead mill are preferable.

The defibration treatment can be performed by a wet method or a dry method. It is preferable to perform the defibration treatment by a wet method because the fiberized paramylon can be more efficiently dispersed in the solution. The solvent used in the wet method is not particularly limited as long as it can disperse the fiberized paramylon, and water can be preferably used.

The defibration treatment may be a single type or a combination of two or more types. Further, it may be a partially defibrated paramylon, and is intended by the present invention as long as it includes the defibrated paramylon.

Five. Use Euglena, paramylon, paramylon processed product, and β1,3-glucan have an effect of enhancing sirtuin expression, and thus at least one selected from the group consisting of euglena, paramylon, paramylon processed product, and β1,3-glucan. Can be used as an active ingredient of a sirtuin expression level enhancer.

In addition, at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan is used in various applications based on the sirtuin expression-enhancing action, such as life extension, anti-aging, wrinkle suppression, and spot stain. Use as an active ingredient for suppression, prevention and/or improvement of dementia, sleep improvement, gray hair suppression, antioxidant, fatigue improvement, muscle enhancement, mitochondrial enhancement, energy metabolism improvement, anti-arteriosclerosis, anti-diabetes, anti-inflammatory, anti-obesity, etc. It is also possible to do so. Preferably, it includes a plurality of applications (two or more, more preferably three or more, even more preferably four or more, even more preferably five or more, even more preferably six or more) of these applications. Can be used for various purposes.

In addition, the uses, purposes and targets listed below:
It can also be used for (a) anti-aging age care (b) fatigue reduction (c) vitality support (d) beautiful skin.

The sirtuin targeted by the agent of the present invention is not particularly limited, and examples thereof include SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7 and the like. Of these, SIRT1, SIRT3, SIRT6 and the like are preferable, and SIRT1 is more preferable. These may be used alone or in combination of two or more.

Various uses of sirtuins are also known. Some examples are shown below. Applications involving SIRT1 include, for example, energy metabolism improvement, anti-inflammatory and the like. Applications involving SIRT3 include, for example, antioxidants. Applications involving SIRT6 include, for example, life extension.

The target cells of the agent of the present invention are not particularly limited. Examples of target cells include epidermal cells, dermal cells, muscle cells, and nerve cells. One kind may be used alone, or a combination of two or more kinds may be used.

The agent of the present invention is used in various fields such as food compositions (including health foods, health promoters, dietary supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, medicines, reagents, feeds, etc. Can be used as The agent of the present invention is preferably an oral composition or an external composition.

The form of the agent of the present invention is not particularly limited and may be a form usually used in each application depending on the application.

As the form of the agent of the present invention, when the application is a food composition, liquid, gel-like or solid food, for example, juice, soft drink, tea, soup, soy milk and other beverages, salad oil, dressing, yogurt, jelly , Pudding, sprinkles, baby milk powder, cake mix, dairy products (eg, powder, liquid, gel, solid, etc.), bread, confectionery (eg, cookies) and the like.

As the form of the agent of the present invention, when the application is cosmetics, for example, emulsion, cosmetic liquid, face cream, hand cream, lotion, body soap, shampoo, rinse, cosmetic gel, pack, foundation, lip balm, face wash Agents and the like.

As the form of the agent of the present invention, when the use is a medicine, for example, an ointment, an external liquid agent (a liniment agent, a lotion agent, etc.), a spray agent (an external aerosol agent, a pump spray agent, etc.), a cream agent, a gel agent , Patches (plasters, tapes such as plasters (reservoir type, matrix type, etc.), poultices, patches, microneedles, etc.), eye drops, eye ointments, nasal drops, suppositories, semi-rectal Formulations suitable for parenteral ingestion such as solid preparations and enema preparations (particularly, external preparation preparations); tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops), Formulations suitable for oral ingestion such as pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups) and jellies ( Oral formulation form).

Examples of the form of the agent of the present invention include tablets (intraorally disintegrating tablets, chewable tablets, effervescent tablets, troches, jellies) when the use is an additive, a health promoting agent, a dietary supplement (such as a supplement), etc. Pills, pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), jellies and the like.

The agent of the present invention may further contain other components, if necessary. As other components, components that can be incorporated into food compositions (including health foods, health promoters, dietary supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, medicines, reagents, feeds, etc. It is not particularly limited as long as it is a base, carrier, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrating agent, lubricant, thickener, coloring. Fragrances, fragrances, chelating agents and the like.

The content of the active ingredient in the agent of the present invention depends on the use, the mode of use, the state of the application, etc., and is not limited, but for example 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. can do.

The application amount (eg, administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exerts a sirtuin expression level-enhancing effect. It is 0.1-10000 mg/kg body weight. The above-mentioned application amount is preferably applied once or more times a day (for example, 1 to 3 times) in a divided manner, and may be appropriately increased or decreased depending on age, disease state and symptoms.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Reference example 1
As Euglena, Euglena gracilis EOD-1 strain (Incorporated administrative agency Product Evaluation Technology Foundation, Patent Biological Deposit Center (NITE-IPOD) dry powder (Shinko Environmental Solutions Co., Ltd., Paramylon content 70% or more) was prepared.

Reference example 2
Paramylon particles were prepared as follows.

The prepared Euglena gracilis EOD-1 strain (after culture and before drying) was collected in 5 flasks of liquid, and the collected liquid was centrifuged (500 xg, 4 minutes, room temperature) in a centrifuge tube. .. The supernatant in the centrifuge tube was once removed and collected. The recovered supernatant was placed in a centrifuge tube to disperse the precipitate in the centrifuge tube, and the whole was transferred to a 100-mL graduated cylinder. Furthermore, the collected supernatant was added to a graduated cylinder to make up to 90 mL.

[Enzyme treatment process]
The liquid whose volume was increased to 90 mL was transferred to a 200 mL beaker, and the pH of the liquid was adjusted to 3 by adding an aqueous hydrochloric acid solution while stirring. A proteolytic enzyme (acid protease, product name "Protease YP-SS", optimum pH 2.5-3.0 manufactured by Yakult Chemical Industry Co., Ltd.) was added to the liquid to a concentration of 5 g/L. The liquid was subjected to enzyme treatment at 50° C. for 2 hours while stirring.

[Surfactant treatment process]
An aqueous solution of sodium dodecyl sulfate was added to the liquid that had been subjected to the enzyme treatment step so that the concentration of sodium dodecyl sulfate was 3.0 mass/volume (w/v)%. The pH of the liquid was adjusted to 3 by adding aqueous hydrochloric acid while stirring the liquid containing sodium dodecyl sulfate. Furthermore, the liquid was stirred with a propeller stirrer (rotation speed 200 rpm) at 60° C. for 30 minutes.

[Separation process]
Paramylon was precipitated by centrifugation (1000 xg, 2 minutes, room temperature), and paramylon was separated from the liquid that had been subjected to the surfactant treatment step. A surfactant treatment step was performed in the same manner except that the concentration of sodium dodecyl sulfate was changed to 1.0 mass/volume% and the pH was not adjusted. Then, the separation process was performed in the same manner as above. In this way, the surfactant treatment step and the separation step were performed three times each.

[Washing process]
Paramylon precipitated by centrifugation in the separation step was suspended in pure water and allowed to stand at 40°C for 10 minutes. Next, paramylon was precipitated by centrifugation (1000 xg, 2 minutes, room temperature). This operation was performed three times in total.

[Drying process]
The paramylon precipitated by centrifugation in the washing step was dried at 50° C. to obtain paramylon particles. The obtained paramylon particles were used as paramylon in the following test examples.

Test example 1. Sirtuin expression level measurement test 1 (Caco-2)
Test Example 1-1. Cells and cell culture
Caco-2 cells (human colon cancer-derived cell line) are Dulbecco's modified Eagle medium (DMEM; Dalbecco's Modified Eagle Medium; Nissui, Tokyo, Japan) containing 10% Fetal bovine serum (FBS; Life Technologies, CA, USA). ), 4 mM L-glutamine, 100 U/mL penicillin (Meiji. Tokyo, Japan) and 0.1 mg/mL streptomycin (Meiji. Tokyo, Japan) were added, and cultured at 37°C under 5% CO ₂ conditions. ..

Test Example 1-2. Measurement of endogenous SIRT1 expression level by real-time qPCR
(1) Sample processing and total RNA extraction
Caco-2 cells were seeded on a 5 mL dish at 3×10 ⁵ cells, and Euglena dissolved in DMSO in advance was added thereto at 1 μg/mL. In addition, DMSO was added in the same amount as a negative control, and Resveratrol was added in an amount of 1 μM as a positive control, and the mixture was cultured for 24 hours. This operation was repeated twice, and after 24 hours, the lysate was collected and total RNA was extracted. High Pure RNA Isolation Kit (Roche, Indianapolis, IN, USA) was used for total RNA extraction. The reagents and instruments used from the preparation of total RNA to the end of the reverse transcription reaction were RNase Free. Completely remove the medium, add 200 μL of PBS and 400 μL of cell lysate contained in the High Pure RNA Isolation Kit, and thoroughly pass the cell lysate over the dish until the cell lysate is no longer viscous. Collected into a 1.5 mL sample tube. The collected sample was well suspended. The filter tube and the collection tube included in the High Pure RNA Isolation Kit were assembled, the sample was pipetted into the buffer receiver on the upper part of the filter tube, and centrifuged at 8,000 xg for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and this collection tube were assembled again. 90 μL of DNase incubation buffer per sample was pipetted into the sterilized reaction tube, and 10 μL of Dnase I was added to this tube and mixed. This mixed solution was pipetted onto the buffer receiver on the upper part of the filter tube, added to the glass fiber fleece in the filter tube, and incubated at room temperature for 15 minutes. The washing buffer I (500 μL) contained in the High Pure RNA Isolation Kit was added to the filter tube upper buffer receiver, and the mixture was centrifuged at 8,000×g for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and this collection tube were assembled again. 500 μL of Washing Buffer II was added to the buffer receiver in the upper part of the filter tube and centrifuged at 8,000×g for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and this collection tube were assembled again. 200 μL of washing buffer II was added to the buffer receiver in the upper part of the filter tube and centrifuged at 13,000×g for 2 minutes to remove the washing buffer remaining in the filter tube. The collection tube was discarded, the filter tube was inserted into a sterilized reaction tube, 50 μL of elution buffer was added to the filter tube, and the mixture was centrifuged at 8,000×g for 1 minute. The eluate obtained by this operation was used as an RNA solution. The RNA concentration in the solution was calculated using a NanoDrop 2000/2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA) based on the absorbance value at 260 nm, and used for the subsequent experiments.

(2) 5 pmol of oligo(dT) ₂₀ primer was added to 1.0 μg of total RNA extracted from cells synthesizing cDNA, and sterilized water was added so that the total volume was 13 μL. A thermal cycler (Peltier Thermal Cycler PTC-200, MJ Research, Watertown, MA, USA) was subjected to a heat treatment reaction at 65°C for 5 minutes, immediately transferred to ice and rapidly cooled. During that time, the reverse transcriptase reaction program was advanced to the stage of 42°C and suspended. Reverse transcriptase reaction buffer 4 μL, 10 mM dNTPs (Amershan Pharmacia Biotech., Buckinghamshire, UK) 2 μL, reverse transcriptase ReverTra Ace (TOYOBO, Osaka, Japan) A solution prepared by mixing 0.5 μL was added and gently mixed. After that, cDNA was synthesized by reaction at 42° C. for 20 minutes and at 99° C. for 5 minutes and used as a template for PCR to be used later.

(3) Quantitative RT-PCR
The cDNA was diluted 1/10, and both Forward and Reverse primers were diluted to 10 μM. Add 51.5 μL of RNase Free water, 3.5 μL each of Forward/Reverse primers, template cDNA 7.0 μL, and KAPA SYBR FAST qPCR Kit (NIPPON Genetics, Tokyo, Japan) 22 μL to 0.2 mL PCR tube. , Well suspended. Then, 25 μL of each was added to a 96-well plate, and quantitative PCR was performed using the Thermal Cycle Dicer Real Time System (TaKaRa). As PCR reaction conditions, one cycle of 95° C. for 30 seconds, 40 cycles of 95° C. for 5 seconds and 60° C. for 30 seconds were performed. A β-actin primer was used as a primer for the calibration curve. The relative gene expression level was determined by dividing the measured value by the β-actin value. The primers used are shown below. The primers used were those synthesized by Takara (Shiga, Japan). The sequences of each primer are shown below.

Homo sapiens actin, beta (ACTB), mRNA
HA067803-F: 5'-TGGCACCCAGCACAATGAA-3' (SEQ ID NO: 1)
HA067803-R :5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (SEQ ID NO: 2).

Homo sapiens sirtuin (silent mating type information regulation 2 homolog) 1 (hSIRT1), mRNA
HA044263-F: 5'-GCCTCACATGCAAGCTCTAGTGAC-3' (SEQ ID NO: 3)
HA044263-R: 5'-TTCGAGGATCTGTGCCAATCATAA-3' (SEQ ID NO: 4).

Test Example 1-3. Results Results are shown in FIG. It was found that the sirtuin expression level was increased by adding Euglena to the medium. From this, it was found that Euglena has a sirtuin expression level enhancing action.

Test example 2. Sirtuin expression level measurement test 2 (HaCaT)
Test Example 2-1. Cells and Cell Culture HaCaT cells (human epidermal keratinocyte-derived cells) were used in this test example. HaCaT cells are Dalbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 1.0 x 10 ⁵ U/L penicillin (Meiji Seika), 100 mg/L streptomycin (Meiji Seika), 2.0 g/L NaHCO ₃ (Nissui Pharmaceutical) The cells were cultured at 37°C in the presence of 5% CO ₂ .

Test Example 2-2. Measurement of endogenous SIRT1 expression level by real-time qPCR
(1) Sample processing and total RNA extraction
HaCaT cells were seeded in a φ50 mm dish (FALCON, 353002) at 1.0 x 10 ⁵ cells, and Euglena or paramylon previously dissolved in DMSO was added at 10 µg/mL and cultured for 24 hours. In addition, DMSO was added as a negative control in the same amount, and Resveratrol was added as a positive control in an amount of 5 μM, followed by culturing. This operation was repeated twice, and after 24 hours, the lysate was collected and RNA was extracted. A High Pure RNA Isolation Kit from Roche (Roche, Indianapolis IN) was used for total RNA extraction. The reagents and instruments used from the extraction of total RNA to the end of the reverse transcription reaction were RNase Free. Completely remove the medium, add 200 μL of PBS and 400 μL of cell lysate contained in the High Pure RNA Isolation Kit, and thoroughly pass the cell lysate over the dish until the cell lysate is no longer viscous. Collected into a 1.5 mL tube. The collected sample was vortexed for 30 seconds and well suspended. The filter tube and collection tube included in the High Pure RNA Isolation Kit were assembled, the sample was pipetted onto the buffer receiver on the upper part of the filter tube, and the sample was centrifuged at 8,000 xg for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and this collection tube were assembled again. 90 μL of DNase Incubation Buffer per sample was pipetted into a sterilized reaction tube, and Dnase I was added thereto at 10 μL per sample and mixed. This mixture was added to the filter tube and incubated at room temperature for 15 minutes. The washing buffer I (500 μL) contained in the High Pure RNA Isolation Kit was added to the filter, and the mixture was centrifuged at 8,000×g for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and the collection tube were assembled again. 500 μL of wash buffer II was added to the filter tube and centrifuged at 8,000×g for 15 seconds. The liquid discharged to the collection tube was discarded, and the filter tube and the collection tube were assembled again. 200 μL of washing buffer II was added to the filter tube and centrifuged at 13,000×g for 2 minutes to remove the washing buffer remaining in the filter tube. The collection tube was discarded, the filter tube was inserted into a new sterile reaction tube, 50 μL of elution buffer was applied to the membrane of the filter tube, and incubated at room temperature for 3 minutes. It was then centrifuged at 8,000 xg for 1 minute. The eluate obtained by this operation was used as an RNA solution. The RNA concentration in the solution was calculated based on the absorbance value at 260 nm using a NanoDrop 2000/2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA) and used for the subsequent experiments.

(2) 25 pmol of random primer (Toyobo) was added to 1.0 μg of total RNA extracted from the cDNA-synthesized cells, and sterilized water was added so that the total volume was 13.5 μL. A thermal cycler (Peltier Thermal Cycler PTC-200, MJ Research, Watertown, MA, USA) was subjected to a heat treatment reaction at 65°C for 5 minutes, immediately transferred to ice and rapidly cooled. During that time, the reverse transcriptase reaction program was advanced to the stage of 42°C and suspended. Reverse transcriptase reaction buffer 4 μL, 10 mM dNTPs (GE Healthcare, Amersham Place, UK) 2 μL, reverse transcriptase ReverTra Ace (Toyobo) 0.5 μL were mixed per sample for 5 minutes in ice. The solution was added and mixed gently. Then, cDNA was synthesized by reaction at 42°C for 20 minutes and at 99°C for 5 minutes, and used as a template for PCR to be used later.

(3) Real-time qPCR
The prepared cDNA was used as a template. In a 0.2 mL PCR tube, 51.5 μL of RNase Free water and 3.5 μL each of primer Forward/Reverse diluted to 10 μmol/L, diluted template cDNA (1/5 for SIRT1 primer, internal standard such as β-actin) The product was diluted 1/50) 7.0 μL and KAPA SYBER FAST qPCR Kit (Nippon Genetics) 22 μL were added and mixed well. Then, 25 μL of each was added to 96 well plate in 3 wells, and qPCR was performed using Thermal Cycle Dicer Real Time System (Takara Bio). The PCR reaction conditions were as follows: 95° C. for 30 seconds for 1 cycle, 95° C. for 5 seconds, 60° C. for 60 seconds for 40 cycles, and detection by FAM. Β-actin was used as a primer for the calibration curve. The relative gene expression level was determined by dividing the measured value by the β-actin value. The primers used are the same as in Test Example 1.

Test Example 2-3. Results The results with Euglena are shown in Figure 2 and the results with Paramylon are shown in Figure 3. It was found that the expression level of sirtuin was increased by adding Euglena or paramylon to the medium. From this, it was found that each of Euglena and Paramylon has a sirtuin expression level enhancing action.

Test example 3. Sirtuin expression level measurement test 3 (Caco-2, HaCaT co-culture)
Test Example 3-1. Culture of Caco-2 cells Human colon cancer-derived Caco-2 cells were used as model cells of human intestinal epithelium. Caco-2 cells were 10% inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating for 40 minutes in a thermostat at 56°C), 100,000 U/L penicillin (Meiji, Tokyo, Japan), and Dalbecco's Modified Eagle Medium (DMEM) medium (Nissui Pharmaceutical, Tokyo, Japan) containing 100 mg/L streptomycin (Meiji) and 2.0 g/L NaHCO ₃ at 37° C. in the presence of 5% CO _2. Subcultured in.

Test Example 3-2. Culture of HaCaT cells HaCaT cells were used as model cells of human skin. HaCaT cells are 10% inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating for 40 minutes in a thermostat at 56°C), 100,000 U/L penicillin (Meiji, Tokyo, Japan). , 100 mg/L streptomycin (Meiji), 2.0 g/L NaHCO3 in Dalbecco's Modified Eagle Medium (DMEM) medium (Nissui Pharmaceutical, Tokyo, Japan) at 37°C in the presence of 5% CO ₂ did.

Test Example 3-3. Paramylon sample preparation method Paramylon was prepared at 100 mg/mL using PBS and stored at -20°C, and thawed appropriately before use.

Test Example 3-4. Co-culture of Caco-2 cells and HaCaT cells (Co-Culture System)
2×10 Caco-2 cells on the insert (Apical (AP) side) (Cell Culture Insert, Transparent PET Membrane 24 Well 0.4 μm pore size, 353095) to be placed on the 24-well plate for cell culture insert (Corning, 353504). ^The cells were seeded at ⁵ cells/ml at 250 μL/insert. The cells used were DMEM medium containing inactivated 10% FBS. Induction culture was performed for 14 days, and the DMEM medium was replaced every 3 days.

The bottom of the insert (Basolateral (BL) compartment) contained 750 μL of DMEM medium (containing inactivated 10% FBS). As a blank, the same amount of DMEM medium was added to both AP and BL.

The formation of tight junctions was confirmed by differentiating Caco-2 cells by the above method and measuring the transepithelial electric resistance value (TER) using an electric resistance value measuring system (Millicell ERS-2 System).

The day before the sample addition, HaCaT cells were seeded in a 24-well plate at 1×10 ⁵ cells/well. After confirming the differentiation of Caco-2 cells, on day 15, the paramylon sample was mixed with DMEM medium to a final concentration of 500 μg/mL, added to AP, and mixed with HaCaT cells in a 24-well plate on the BL side and Co-culture was carried out for 48 hours in the presence of 5% CO _{2 at} ℃. PBS was added in the same amount as a negative control sample. Resveratrol (final concentration 10 μM) was used as a positive control sample.

Test Example 3-5. Measurement of endogenous SIRT3 and SIRT6 expression levels
Using a High Pure RNA Isolation Kit (Roche, Diagnostics Gmbh, Mannheim, Germany), HaCaT cell lysate was collected and RNA was extracted. The concentration of total RNA was measured using Nano Drop 2000c (Thermo Fisher Scientific, Waltham, MA).

RNase-free sterilized water was added to 0.5 μL of RNA 1 μg, oligo dT (20) primer (10 μM) in a 0.2 mL tube (SEIKO, Fukuoka, Japan) to adjust the total amount to 13.5 μL. Annealing was performed at 65° C. for 5 minutes using a PTC-200 Peltier Thermal Cycler (BIO-RAD, Laboratories, Hercules, CA, USA). Immediately thereafter, the plate was left on ice for 5 minutes. 5×RT Buffer (TOYOBO, Osaka, Japan) 4 μL, 10 mM dNTP set (GE Healthcare, Amersham Place, UK) 2 μL, ReverTra Ace (TOYOBO) 0.5 μL, and 5% at 42°C for 20 minutes at 99°C. After reacting for minutes, cDNA synthesis was performed.

Primer synthesis was outsourced to Sigma. The primer sequence of β-actin, which is an internal control, is the same as in Test Example 1. The primer sequences for detecting the SIRT3 and SIRT6 genes are shown below.

hSIRT3
Forward 5'-GGACACACAACGGAGGGGAA-3' (SEQ ID NO: 5)
Reverse 5'-TGCCATAGGAAGAAGTGGGCT-3' (SEQ ID NO: 6).

hSIRT6
Forward 5'-AACCGGCCTTTCCTGCTTCT-3' (SEQ ID NO: 7)
Reverse 5'-ACGCAGCAGTTCTTCTCCGT-3' (SEQ ID NO: 8).

A 0.2 mL tube was prepared (number of samples) x (number of dilution ratios) + (calibration curve sample). The cDNAs of the calibration curve sample and the measurement sample were diluted with sterilized water. Six samples for the calibration curve were prepared at 10, 50, 250, 1250, 6250, and 31250-fold dilution, and the measurement samples were each diluted 10-fold. A new 0.2 mL tube was prepared for the required number of calibration curve samples, measurement samples, and negative controls. For the calibration and measurement samples, 49 μL of sterilized water, 3.5 μL of each primer (10 μM), and 7 μL of diluted cDNA were mixed to prepare a mixed solution. For the negative control, a mixed solution of 17 μL of sterilized water and 0.5 μL of each primer (10 μM) was prepared. After removing the liquid on the tube wall with a flash, 25.5 μL of THUNDERBIRD (R) qPCR Mix was added to each of the calibration curve and measurement samples, and pipetting was performed 20 times or more. To the negative control, 7 μL of enzyme was added and then pipetting was performed in the same manner. 25 μL of each sample was dispensed in 3 wells on a 96 well plate. As a negative control, 25 μL was dispensed into 1 well. A seal was attached to the plate, and real-time PCR was performed using Thermal Cycler Dice Real Time System TP800 (Takara, Shiga, Japan). The reaction conditions were 95°C for 30 seconds as one cycle, 95°C for 5 seconds → 55°C for 10 seconds → 72°C for 20 seconds for 45 cycles, and 95°C for 15 seconds → 60°C for 30 seconds → 95°C for 15 seconds as one cycle. The average value of 3 wells of each sample was taken, and the relative value of each sample was calculated based on DMSO.

Test Example 3-6. result
The measurement result of SIRT3 expression level is shown in FIG. 4, and the measurement result of SIRT6 expression level is shown in FIG. In the test system of this test example, orally administered paramylon acts on intestinal cells (corresponding to Caco-2 cells), and secretions from intestinal cells (corresponding to the above culture supernatant) pass through body fluids (blood, etc.). To act on skin cells (corresponding to HaCaT cells) to increase the expression level of sirtuin. From FIGS. 4 and 5, it was revealed that the expression level of sirtuin in HaCaT cells was increased by the culture supernatant obtained after adding paramylon to the medium. From this, it was found that paramylon has a sirtuin expression level enhancing action. In particular, it was suggested that orally administered paramylon acts on various cells such as skin cells via body fluid (blood etc.) to increase the expression level of sirtuin.

Claims (10)

A sirtuin expression level enhancer containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan. The sirtuin expression level enhancer according to claim 1, containing at least one selected from the group consisting of euglena, paramylon, and processed paramylon. The sirtuin expression level enhancer according to claim 1 or 2, which contains the euglena and the euglena is Euglena gracilis. The sirtuin expression level enhancer according to claim 1 or 2, which contains the Euglena and is the Euglena gracilis EOD-1 strain (Accession No. FERM BP-11530). The sirtuin expression level enhancer according to claim 1, which contains the paramylon, and the paramylon is derived from Euglena gracilis. The sirtuin expression level enhancer according to claim 1, which contains the paramylon, and the paramylon is derived from the Euglena gracilis EOD-1 strain (accession number FERM BP-11530). The sirtuin expression level increasing agent according to any one of claims 1 to 6, which is a food composition, a food additive, a cosmetic, a cosmetic additive, or a medicine. The sirtuin expression level enhancer according to any one of claims 1 to 7, which is an oral composition or a composition for external use. Life extension, anti-aging, wrinkle suppression, spot suppression, dementia prevention and/or improvement, sleep improvement, gray hair suppression, antioxidant, fatigue improvement, muscle enhancement, mitochondrial enhancement, energy metabolism improvement, anti-arteriosclerosis, anti-diabetes, anti The sirtuin expression level enhancer according to any one of claims 1 to 8, which is used for at least one selected from the group consisting of inflammation and antiobesity. Containing at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β1,3-glucan,
Life extension, anti-aging, wrinkle suppression, spot suppression, dementia prevention and/or improvement, sleep improvement, gray hair suppression, antioxidant, fatigue improvement, muscle enhancement, mitochondrial enhancement, energy metabolism improvement, anti-arteriosclerosis, anti-diabetes, anti A composition for use in at least one selected from the group consisting of inflammation and antiobesity.