Autophagy activators

Abstract

To provide an autophagy activator containing an Agaricus mushroom extract as an active ingredient.SOLUTION: The present invention provides an autophagy activator containing an Agaricus mushroom extract as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an autophagy activator containing an Agaricus blazei extract as an active ingredient.

Background Art

[0002] Autophagy, which is translated as autophagy in Japanese, is an intracellular degradation mechanism in which a part of the cytoplasm is surrounded by an isolation membrane to form a membrane structure called an autophagosome, and this autophagosome fuses with a lysosome to become an autolysosome, decomposing waste products and unnecessary proteins, etc., and is widely recognized. Autophagy plays an important role in physiological or pathological functions in the living body, and it is known that a decrease in autophagy activity is involved in the onset of various diseases.

[0003] It is known that autophagy activity decreases with aging, and activating autophagy is considered important for maintaining and promoting health. Currently, as methods for activating autophagy, methods using urolithin (Patent Document 1), methods using quercetin glycoside (Patent Document 2), methods using fermented garlic (Patent Document 3), methods using β - cryptoxanthin (Patent Document 4), etc. have been disclosed, but none of them have sufficient effects.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of this invention is to provide an autophagy activator containing Agaricus mushroom extract as an active ingredient. [Means for solving the problem]

[0006] The present invention provides an autophagy activator containing Agaricus mushroom extract as an active ingredient. [Effects of the Invention]

[0007] The autophagy activator of the present invention has the effect of activating autophagy, using Agaricus mushroom extract as an active ingredient. [Modes for carrying out the invention]

[0008] The following describes embodiments for carrying out the present invention.

[0009] The autophagy activator of the present invention is characterized by containing Agaricus mushroom extract as an active ingredient.

[0010] In the present invention, Agaricus mushrooms refer to mushrooms belonging to the genus Agaricus in the family Agaricaceae, and specific examples include Agaricus campestris, Agaricus blazei and Agaricus blasiliensis (native to Brazil), Agaricus subrufescens (native to North America), Agaricus rufotegulis (native to Europe), etc., button mushroom (A. bisporus), Agaricus arvensis (white giant agaricus or horse mushroom), Agaricus silvicola, Agaricus subrutilescens, etc. In the present invention, it is preferable to use Agaricus blazei Murill.

[0011] The Agaricus mushroom extract used in the autophagy activator of the present invention can be any of the following: fruiting body extract, mycelial extract, or mycelial culture filtrate of Agaricus mushroom. However, it is preferable to use an extract of the fruiting body and / or mycelium.

[0012] When obtaining an extract from the fruiting bodies of Agaricus mushrooms, either fresh or dried fruiting bodies may be used. For handling purposes, dried fruiting bodies are preferred in terms of preservation and extraction efficiency. As an extraction solvent for obtaining the extract from Agaricus mushroom fruiting bodies, one or more mixed solvents selected from polar solvents such as water, alcohols such as ethanol, methanol, isopropanol, isobutanol, n-hexanol, methylamyl alcohol, 2-ethylbutanol, and n-octyl alcohol, polyhydric alcohols or their derivatives such as glycerin, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, triethylene glycol, 1,3-butylene glycol, and hexylene glycol, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and methyl-n-propyl ketone, esters such as ethyl acetate and isopropyl acetate, and ethers such as ethyl ether, isopropyl ether, and n-butyl ether can be suitably used. Furthermore, polar solvents to which inorganic salts such as phosphate-buffered saline have been added, or polar solvents to which surfactants have been added, may also be used, and there are no particular limitations. Among the above extraction solvents, one solvent or a mixture of two or more solvents selected from ethanol, methanol, 1,3-butylene glycol, and water, and solvents to which inorganic salts or surfactants have been added are preferably used.

[0013] Examples of extraction methods include impregnation at room temperature, cooled, or heated; distillation methods such as steam distillation; and pressing methods that directly press raw Agaricus mushrooms to obtain the extract. These methods may be used individually or in combination of two or more.

[0014] The ratio of Agaricus fruiting body to solvent during extraction is not particularly limited, but a ratio of 0.5 to 1000 times the weight of solvent to Agaricus fruiting body is preferred, particularly 0.5 to 100 times the weight of solvent, for ease of extraction and efficiency. The extraction temperature is conveniently set at room temperature to below the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature, but is preferably in the range of 2 hours to 2 weeks.

[0015] Furthermore, the Agaricus fruiting body extract obtained in this manner can be used as is, but it may also be purified by deodorization, decolorization, concentration, etc., to the extent that the efficacy is not lost, or it may be used as a fraction using column chromatography, etc. These extracts, purified products, and fractions can be dried by removing the solvent, and can also be used in a form solubilized in a solvent such as alcohol, or in the form of an emulsion. They can also be used encapsulated in vesicles such as liposomes or microcapsules.

[0016] The obtained extract can be contained together with known additives such as stabilizers, excipients, and binders to form the autophagy activator of the present invention. The additives are not particularly limited as long as they do not impair the effects of the present invention, but starch, dextrin, powdered cellulose, crystalline cellulose, cellulose derivatives, sucrose fatty acid esters, lactose, gum arabic, mannitol, trehalose, glucose, gelatin, silicon dioxide, etc. can be used individually or in combination. Among these, the use of dextrin is preferred.

[0017] As a method for culturing Agaricus mushroom strains to obtain Agaricus mushroom mycelia, either a solid culture method or a liquid culture method commonly used for culturing basidiomycetes may be adopted, but the latter method is preferably used from the viewpoint of productivity. As the medium used for culturing Agaricus mushrooms, it is only necessary that it contains various nutrients necessary for the growth of the fungus, and a normal medium formulation may be used. That is, as the carbon source, for example, carbon sources that can be assimilated such as glucose, sucrose, maltose, starch, etc. can be used. As the nitrogen source, for example, ammonium sulfate salts, ammonium nitrate salts, urea, etc., and as natural complex nutrient sources, for example, potato extract, carrot extract, malt extract, peptone, koji extract, yeast extract, yeast powder, etc. can be used, and other trace element inorganic salts and vitamins necessary for growth are appropriately added and used.

[0018] The culture is usually carried out under aerobic conditions. For example, a shaking culture method or an aeration stirring culture method is used. Stirring during the culture may be reciprocally shaken or rotationally shaken for several minutes every 24 hours, but continuous shaking is also possible. The culture temperature is 15°C to 40°C, preferably around 20°C to 30°C. The pH of the medium is appropriately in the range of 3.0 to 9.0, and the growth is good at 4.5 to 7.0. Also, it is preferably not irradiated during the culture, but irradiation for about 11 to 14 hours a day is possible.

[0019] The number of culture days varies depending on culture conditions such as the physical environment and medium composition, but it may be any period during which the growth of the mycelia can be sufficiently observed. Usually, it is 2 to 120 days, particularly preferably 5 to 90 days, which is the time when the maximum amount of mycelia is produced.

[0020] After the culture is completed, the culture solution is centrifuged or filtered to separate the mycelia and the culture filtrate. Centrifugation can be carried out by a centrifugal operation that gives a gravitational acceleration of 100 to 5000G, preferably 800 to 30G. Also, filtration is carried out by filtering using a membrane filter with 3.5 to 200 mesh, particularly preferably 4 to 16 mesh.

[0021] When obtaining an extract from the mycelium of Agaricus mushrooms, the raw mycelium obtained from the culture solution cultured as described above can be used as it is or after drying. In terms of handling, the dried product is preferred from the viewpoints of storage stability and extraction efficiency. As the extraction solvent from the mycelium, a polar solvent is preferably used. For example, one solvent or a mixed solvent of two or more solvents selected from alcohols such as water, ethanol, methanol, isopropanol, isobutanol, n-hexanol, methyl amyl alcohol, 2-ethylbutanol, n-octyl alcohol, polyhydric alcohols such as glycerin, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, triethylene glycol, 1,3-butylene glycol, hexylene glycol or derivatives thereof can be used. Also, inorganic salts, surfactants, etc. may be added to the polar solvent for use. Among these polar solvents, one solvent or a mixed solvent of two or more solvents selected from ethanol, methanol, 1,3-butylene glycol, water, and a solvent obtained by adding inorganic salts and surfactants to these solvents is preferably used.

[0022] Furthermore, as extraction methods, examples include a method of impregnating and extracting at room temperature, under cooling or heating, a method of extracting using a distillation method such as steam distillation, a squeezing method of directly squeezing the raw Agaricus mushroom mycelium to obtain an extract, etc. Extraction is performed using these methods alone or in combination of two or more.

[0023] The ratio of the Agaricus mushroom mycelium to the solvent during extraction is not particularly limited, but a solvent in a weight multiple of 0.5 to 1000 times, particularly 0.5 to 100 times in terms of extraction operation and efficiency, relative to 1 of the Agaricus mushroom mycelium is preferred. Also, the extraction temperature is conveniently set within the range from 5°C to below the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature, etc., but is preferably within the range of 2 hours to 2 weeks.

[0024] Furthermore, the Agaricus mycelium extract and the filtered mycelial culture filtrate obtained in this manner can be used as is, but they can also be purified by fractionation, deodorization, decolorization, concentration, etc., to the extent that the effects of the present invention are not lost. The purified and fractionated products of these extracts and filtrates can be dried by removing the solvent, and can be added to topical skin preparations in the form of solubilization or suspension in a solvent such as purified water, or in the form of an emulsion. They can also be used encapsulated in vesicles such as liposomes or microcapsules.

[0025] The obtained extract and filtrate can be contained together with known additives such as stabilizers, excipients, and binders to form the autophagy activator of the present invention. The additives are not particularly limited as long as they do not impair the effects of the present invention, but starch, dextrin, powdered cellulose, crystalline cellulose, cellulose derivatives, sucrose fatty acid esters, lactose, gum arabic, mannitol, trehalose, glucose, gelatin, silicon dioxide, etc. can be used individually or in combination. Among these, dextrin is preferred.

[0026] The dosage form of the autophagy activator of the present invention is not particularly limited and may be solid, semi-solid, or liquid. [Examples]

[0027] The present invention will be specifically described below with reference to examples, but this will not limit the scope of the present invention. Unless otherwise specified, the amounts are given in mass percent.

[0028] First, the method for preparing the Agaricus mushroom extract used in the examples is shown.

[0029] [Agaricus mushroom extract] A mixed extract of Agaricus blazei murill fruiting body and mycelium, to which dextrin was added, was used as the Agaricus mushroom extract.

[0030] [Autophagy activation effect] HL60 (manufacturer: JCRB, catalog number: JCRB0085) was awakened using culture medium and incubated in a CO2 incubator (37°C, 5% CO2, humid, same below) at a cell density of 1 × 10⁶ 5 cells / mL ~ 5 × 10 5 Suspension culture was performed to achieve a cell / mL concentration. 2 x 10 in a 96-well black plate 4 Cells were seeded in differentiation medium at a concentration of cells / 100 μL and cultured for 4 days to differentiate into neutrophils. On day 3 of differentiation, half of the culture medium was replaced with fresh differentiation medium. Next, half of the culture medium was replaced with a differentiation medium containing 2× concentration Agaricus mushroom extract to make it 1× concentration, and after 16 hours of incubation, Agaricus mushroom extract treatment was performed. Two hours before the completion of Agaricus mushroom extract treatment, 1 / 5 of the differentiation medium containing 60 μM chloroquine was added to make it 10 μM, and chloroquine treatment was performed for 2 hours. Each well was replaced with 100 μL of CYTO-ID staining solution (prepared by adding 1 / 500 vol. of Cyto-ID Green detection Reagent and 1 / 1000 vol. of Hoechst33342 to 1× Assay buffer with 5% FBS), incubated in a CO2 incubator for 30 minutes, and then replaced with 100 μL of 1× Assay buffer with 5% FBS. Next, using an Operetta CLS, nine fields of view in the center of each well were imaged with a 20x objective lens, with the nuclei being represented by blue fluorescence and CYTO-ID-positive vesicles (autophagosomes) by green fluorescence. After acquiring images, high-content analysis software (Harmony 4.6) was used to detect and quantify CYTO-ID-positive vesicles in the nucleus, cell membrane, cytoplasm, and cytoplasm. The number of CYTO-ID-positive vesicles per cell was calculated by dividing the number of CYTO-ID-positive vesicles by the number of nuclei. Furthermore, the difference between the group without Agaricus mushroom extract and the positive control / Agaricus mushroom extract-treated group was tested using Dunnett's test, with the group without Agaricus mushroom extract as the control. The significance level was set at a two-sided 5%. Furthermore, relative values ​​were calculated when the autophagy activity value of the group without Agaricus mushroom extract was set to 100. The results are shown in Table 1.

[0031] [Table 1]

[0032] As shown in Table 1, it was revealed that Agaricus mushroom extract increases autophagy activity levels and exhibits an effect of activating autophagy. Therefore, the autophagy activator of the present invention exhibits an effect of activating autophagy.

Claims

[Claim 1] An autophagy activator for neutrophils containing Agaricus blazei Murill extract as an active ingredient, and also containing dextrin.

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