Composition for regulating autophagosome formation, composition for activating autophagy, composition for suppressing autophagy, and composition for extending healthy lifespan

Abstract

To provide a composition for regulating autophagosome formation, a composition for activating autophagy, a composition for suppressing autophagy, a composition for extending healthy lifespan, and a composition for extending lifespan.SOLUTION: A composition for regulating autophagosome formation contains an aqueous extract of a specific plant. The specific plant includes at least one selected from the group consisting of cinnamon, Japanese pepper, cardamom, laurel, long pepper, fennel, caraway, coriander, allspice, nutmeg, lemongrass, green Sichuan pepper, clove, black pepper, and turmeric.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for regulating autophagosome formation, a composition for activating autophagy, a composition for inhibiting autophagy, a composition for extending healthy lifespan, and a composition for extending lifespan. [Background technology]

[0002] Autophagy is a widely recognized intracellular degradation mechanism in which a portion of the cytoplasm is surrounded by an isolation membrane to form a membrane structure called an autophagosome, which then fuses with a lysosome to become an autolysosome, breaking down waste products and unnecessary proteins. In relation to autophagy, Patent Document 1 describes an autophagy activator containing an Agaricus mushroom extract as an active ingredient, and Patent Document 2 describes an autophagy activator and a health lifespan enhancer containing sorghum or a processed product of sorghum. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2023-104643 [Patent Document 2] Japanese Patent Publication No. 2023-152905 Summary of the Invention [Problem to be solved by the invention]

[0004] A first object of the present invention is to provide a composition that regulates autophagosome formation. A second object of the present invention is to provide a composition that activates autophagy. A third object of the present invention is to provide a composition that inhibits autophagy. A fourth object of the present invention is to provide a composition that extends healthy lifespan. A fifth object of the present invention is to provide a composition that extends lifespan. [Means for solving the problem]

[0005] The present inventors have found that among aqueous extracts of specific plants, there are extracts that exhibit the effect of promoting autophagosome formation and activating autophagy, and extracts that exhibit the effect of inhibiting autophagosome formation and inhibiting autophagy. The present inventors have also found that among aqueous extracts of specific plants, there are extracts that exhibit the effect of extending healthy lifespan, and there are extracts that exhibit the effect of extending lifespan, and have completed the present invention. That is, the present invention encompasses the following inventions. [1] A composition for regulating autophagosome formation, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, long pepper, fennel, caraway, coriander, allspice, nutmeg, lemongrass, green Sichuan pepper, clove, black pepper and turmeric. [2] The composition described in [1] above for promoting the formation of autophagosomes. [3] The composition described in [1] above for inhibiting autophagosome formation. [4] A composition for activating autophagy, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, fennel, caraway, coriander, allspice, lemongrass, green Sichuan pepper, clove, and turmeric. [5] A composition for inhibiting autophagy, comprising an aqueous extract of a specific plant, A composition, wherein the specific plant comprises at least one selected from the group consisting of pepper, nutmeg, and black pepper. [6] A composition for extending healthy lifespan, comprising an aqueous extract of a specific plant, A composition wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark and turmeric. [7] A life-extending composition comprising an aqueous extract of cinnamon bark. [Effects of the Invention]

[0006] According to the present invention, by using a specific plant aqueous extract, it is possible to regulate autophagosome formation and activate or inhibit autophagy. [Brief explanation of the drawings]

[0007] [Figure 1] Autophagy activity test results (samples: purified hot water extract of cinnamon bark, purified hot water extract of turmeric). [Figure 2] Results of the Drosophila climbing assay test (samples: hot water extract of cinnamon bark and hot water extract of turmeric). [Figure 3] Results of a fruit fly lifespan evaluation test (sample: hot water extract of cinnamon bark). DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention will now be described in further detail. The present invention relates to a composition for modulating autophagosome formation, which contains an aqueous extract of a specific plant. The present invention also relates to a composition for activating autophagy and a composition for inhibiting autophagy, which contain an aqueous extract of a specific plant. The present invention further relates to a composition for extending healthy lifespan and a composition for extending lifespan, which contain an aqueous extract of a specific plant. <Composition for regulating autophagosome formation> The composition for modulating autophagosome formation of the present invention contains, as an active ingredient, an aqueous extract of at least one specific plant selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, long pepper, fennel, caraway, coriander, allspice, nutmeg, lemongrass, green Sichuan pepper, clove, black pepper, and turmeric. Compositions for modulating autophagosome formation of the present invention include compositions for promoting autophagosome formation and compositions for inhibiting autophagosome formation. Among these, the composition for promoting autophagosome formation contains, as an active ingredient, an aqueous extract of at least one plant selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, fennel, caraway, coriander, allspice, lemongrass, green Sichuan pepper, clove, and turmeric, while the composition for inhibiting autophagosome formation contains, as an active ingredient, an aqueous extract of at least one plant selected from the group consisting of long pepper, nutmeg, and black pepper. <Autophagy Activation Composition> The composition for activating autophagy of the present invention contains, as an active ingredient, at least one aqueous extract selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, fennel, caraway, coriander, allspice, lemongrass, green Sichuan pepper, clove, and turmeric. <Autophagy-inhibiting composition> The composition for activating autophagy of the present invention contains, as an active ingredient, an aqueous extract of at least one species selected from the group consisting of long pepper, nutmeg, and black pepper.

[0009] <Specific plants> "Specific plants" as used herein refer to cinnamon, Japanese pepper, cardamom, laurel, long pepper, fennel, caraway, coriander, allspice, nutmeg, lemongrass, green Sichuan pepper, cloves, black pepper, and turmeric. "Cinnamon" refers to plants of the genus Cinnamomum in the family Lauraceae. "Cardamom" refers to plants of the genus Elettaria or Amomum in the family Zingiberaceae. "Laurel" refers to plants of the genus Laurus in the family Lauraceae. "Long pepper" refers to plants of the genus Piper in the family Piperaceae. "Fennel" refers to plants of the genus Foeniculum in the family Apiaceae. "Caraway" refers to plants of the genus Carum in the family Apiaceae. "Coriander" refers to plants of the genus Coriandrum in the family Apiaceae. "Allspice" refers to plants of the genus Pimenta in the family Myrtaceae. "Nutmeg" refers to a plant of the genus Myristica in the family Myristicaceae. "Lemongrass" refers to a plant of the genus Cymbopogon in the family Poaceae. "Clove" refers to a plant of the genus Syzygium in the family Myrtaceae. "Turmeric" refers to a plant of the genus Curcuma in the family Zingiberaceae. "Black pepper" refers to a plant of the genus Piper in the family Piperaceae. "Japanese pepper" refers to a plant of the species piperitum in the genus Zanthoxylum in the family Rutaceae. "Green pepper" refers to a plant of the species bungeanum in the genus Zanthoxylum in the family Rutaceae. <Aqueous extract> The term "aqueous extract" as used herein refers to components obtained by extracting the above-mentioned specific plants using an aqueous solvent. The aqueous solvent may be water or a mixture of water and a water-miscible organic solvent. The water-miscible organic solvent may be a polar solvent that is liquid at room temperature and pressure and has a certain degree of water solubility. Examples of water-miscible organic solvents include, but are not limited to, lower aliphatic alcohols such as methanol and ethanol, and ethyl acetate. The method for the extraction treatment is not particularly limited, and may be, for example, solvent extraction, steam distillation extraction, supercritical or subcritical extraction, etc. The solvent extraction method is not limited in any way as long as it is a common method used to extract plant components using a solvent, and during extraction, treatments such as heating, freezing, immersion, filtration, shaking, and stirring may be used individually or in combination. The aqueous extract may be an extract obtained by adding an aqueous solvent and heating, or an extract obtained without heating. It may be a hot water extract obtained by heat treatment using water as the aqueous solvent, or an aqueous extract obtained by heat treatment at a temperature below a certain level or without heat treatment. The temperature at which the heat treatment is performed may be about 85°C or higher, or about 50°C or higher. A hot water extract refers to an extract obtained by heat treatment at a temperature of about 50°C or higher. The obtained aqueous extract may be further purified. The purification method is not particularly limited, and may be, for example, a purification method using a column or a purification method using liquid-liquid extraction. Examples of solvents used for purification include, but are not limited to, water, lower aliphatic alcohols such as methanol and ethanol, acetonitrile, and ethyl acetate.

[0010] Examples of the parts of a specific plant that can be extracted include, but are not limited to, leaves, fruits, seeds, stems, buds, bark, and peel. The particular plant used for extraction may be processed or unprocessed, for example, dried and / or crushed. The amount of the aqueous extract of the specific plant contained is not particularly limited as long as it exerts an autophagosome formation-regulating effect, but may be, for example, a concentration of about 50 μg / mL to about 800 μg / mL in terms of dry mass of the aqueous extract of the specific plant relative to the total mass of the composition, such as about 50 μg / mL, about 100 μg / mL, about 200 μg / mL, about 400 μg / mL, or about 800 μg / mL.

[0011] <Regulation of autophagosome formation, activation of autophagy, inhibition of autophagy> As described above, in this specification, "regulating autophagosome formation" or "regulating autophagosome formation" refers to regulating autophagosome formation in a direction that promotes or inhibits it. Autophagosome formation can be confirmed, for example, by flow cytometry using a fluorescent compound (e.g., DAPGreen) that is incorporated into the autophagosomal membrane as it forms. When the median fluorescence intensity (MFI) of stained cells measured by flow cytometry exceeds the median fluorescence intensity of the control, it is determined that autophagosome formation has been promoted and autophagy has been activated. When the median fluorescence intensity of stained cells is lower than the median fluorescence intensity of the control, it is determined that autophagosome formation has been inhibited and autophagy has been inhibited. This is explained in more detail below.

[0012] Screening for substances that exhibit an autophagosome formation regulating effect and an autophagy activating or inhibiting effect is performed as follows. (1) Preparation of specific plant aqueous extracts A predetermined amount of each specific plant is weighed, and an extraction solvent is added to extract it. Purification may also be carried out if necessary. (2) Preparation of chloroquine-containing medium A chloroquine-containing medium is prepared, which is added to inhibit the fusion of formed autophagosomes with lysosomes to form autolysosomes. (3)Cell culture The cells are cultured in a basal medium. The cell culture is carried out on the day before the aqueous extract of the specific plant obtained in (1) is added. (4) Preparation of specific plant aqueous extract solution The aqueous extract of the specific plant is weighed, dissolved in a chloroquine-containing medium to a predetermined concentration, and diluted to prepare a solution of the aqueous extract of the specific plant. (5) Sample preparation A sample is prepared by adding a solution of the aqueous extract of a specific plant to the cells, and a control is prepared by adding a medium containing chloroquine instead of the aqueous extract of a specific plant. (6) Flow cytometer measurement For each sample and control, one with and one without fluorescent staining are prepared, and flow cytometer measurements are performed to obtain the median fluorescence intensity for each. (7) Analysis of measurement results The background-subtracted median fluorescence intensity of the sample and the background-subtracted median fluorescence intensity of the control were calculated. The relative median fluorescence intensity of each sample was calculated by dividing the background-subtracted median fluorescence intensity of each sample by the background-subtracted median fluorescence intensity of the control. If the relative median fluorescence intensity of a sample is statistically significantly greater than 1, the sample is determined to "promote autophagosome formation" or "activate autophagy." If the relative median fluorescence intensity of a sample is statistically significantly less than 1, the sample is determined to "inhibit autophagosome formation" or "inhibit autophagy." The aqueous extract of a specific plant for promoting autophagosome formation and activating autophagy is an aqueous extract of at least one specific plant selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, fennel, caraway, coriander, allspice, lemongrass, green Sichuan pepper, clove, and turmeric. In one embodiment, the aqueous extract of a specific plant for promoting autophagosome formation and activating autophagy is an extract of any combination of these specific plants, or any combination of extracts of these specific plants. In one embodiment, the aqueous extract of a specific plant for promoting autophagosome formation and activating autophagy is an aqueous extract of cinnamon bark, preferably a hot water extract. In one embodiment, the aqueous extract of a specific plant for inhibiting autophagosome formation and autophagy is an aqueous extract of nutmeg, long pepper, black pepper, or a combination thereof. In one embodiment, the aqueous extract of a specific plant for inhibiting autophagosome formation and autophagy is preferably a hot water extract.

[0013] Autophagy activation may also be assessed using the "relative autophagy activity value." The "relative autophagy activity value" is calculated from the autophagy flux. The autophagy flux can be determined by calculating the proportion of autophagosomes that have reached degradation, the final stage of autophagy. The proportion that has reached degradation can be calculated, for example, using the tfLC3 assay provided by AutoPhagyGO, Inc. Specifically, a fusion protein consisting of the fluorescent protein GFP, which is easily degraded in lysosomes, and the fluorescent protein RFP, which is resistant to degradation, linked to the autophagosome-localized protein LC3 is expressed in HeLa-Kyoto human cultured cells, and the intracellular GFP and RFP fluorescence intensities are measured to calculate the "autophagy activity value" represented by the following formula 1. [Formula 1] Autophagy activity value = (GFP fluorescence intensity) / (RFP fluorescence intensity) The "relative autophagy activity value" is expressed by the following formula 2 and is calculated for each sample by dividing the "autophagy activity value" of the sample by the "autophagy activity value" of the growth medium. [Formula 2] Relative autophagy activity value = (autophagy activity value of sample) / (autophagy activity value of growth medium) Autophagy is determined to be activated when the relative value of autophagy activity in the sample is lower than the relative value of autophagy activity in the control.

[0014] <Effects on extending healthy lifespan and life expectancy> As used herein, "healthy lifespan" refers to the period during which one can live without daily life being restricted by health problems. Autophagy is known to have a lifespan extension effect through the metabolism of intracellular substances, etc. The compositions of the present invention that have the effect of promoting autophagosome formation or the effect of activating autophagy can extend healthy lifespan through the metabolism of intracellular substances, etc., due to the activation of autophagy. In the present invention, the effect of extending healthy lifespan can be evaluated by determining whether healthy lifespan is extended in a climbing assay test using Drosophila. In addition, in the present invention, the effect of extending lifespan can be evaluated by determining whether lifespan is extended in a lifespan evaluation test using Drosophila. The Drosophila climbing assay test utilizes the anti-geotaxis of Drosophila, which allows them to climb rearing vials, and is used as a quantitative method for evaluating age-dependent changes in movement, as an indicator of cognitive function and muscle strength (for example, Akagi Kazutaka, "Research aiming to elucidate the molecular mechanism of lifespan extension through dietary restriction and develop methods to prevent malnutrition," Geriatrics Research and Development Fund 2020 General Research Report).

[0015] <Composition> In the present invention, the "composition" is not particularly limited, but may be a pharmaceutical composition, a quasi-drug, a supplement, or a food (general food, food for specific dietary uses such as food for medical patients, or food with health claims such as food for specified health uses, food with nutrient functions, and food with functional claims), or a material to be incorporated into these. The composition may further contain any pharmaceutically or food-acceptable excipient or additive, and / or food ingredient, commonly used in the art, as long as it does not impair the object of the present invention.

[0016] The present invention will be specifically described below with reference to examples. [Example]

[0017] Example 1: Measurement of the ability of hot water extracts from specific plants to regulate autophagosome formation 1.Material (cell) Undifferentiated SH-SY5Y cells were used. (Prepared Reagents) basal medium The following reagents were mixed to prepare a basal medium. TIFF2025161795000001.tif50158 *After purchase, the product was inactivated (56°C in a water bath for 30 minutes). PBS The solution was prepared by dissolving one tablet of Phosphate Buffered Saline (PBS), pH 7.4 (Takara Bio, T9181) in 100 mL of ultrapure water. 0.1% trypsin-EDTA This solution was prepared by adding 6 mL of PBS to 4 mL of 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (containing phenol red) (Fujifilm Wako Pure Chemical Industries, Ltd., 209-16941). HBSS This was prepared by diluting 10x HBSS(-) (phenol red-free) (Fujifilm Wako Pure Chemical Industries, 082-09865) 10 times with sterile water.

[0018] Plants used Umbelliferae (Italian parsley, caraway, cumin, coriander, dill, fennel) Asteraceae (Tarragonaceae) Fabaceae (metchi seeds, metchi leaves) Lamiaceae (sage, thyme, basil, rosemary) Lauraceae (Cinnamon, Laurel) Myristicaceae (nutmeg) Myrtaceae (allspice, cloves) Piperaceae (Piper longum, black pepper) Grass (Lemongrass) Rutaceae (green pepper, Sichuan pepper, kaffir lime, Japanese pepper) Star anise Solanaceae (Red Pepper) Zingiberaceae (cardamom, ginger, turmeric)

[0019] 2. Sample Preparation (a) Preparation of freeze-dried hot water extracts of plants (1) 0.6 g of dry powder from each plant was weighed into a 50 mL centrifuge tube and 30 mL of ultrapure water was added. (2) The mixture was heated in a water bath (85°C, 30 minutes, shaking at 110 / min). During the heating process, the mixture was stirred with a vortex every 30 minutes. (3) The mixture was centrifuged (2,380 xg, 10 minutes), and the supernatant was filtered through filter paper (ADVANTEC, 5A). (4) The filtrate was frozen in a deep freezer and then subjected to a freeze-dryer to produce a freeze-dried product. (5) Store at -20°C until use.

[0020] (a) Preparation of chloroquine-containing medium (hereinafter referred to as CQ-containing medium) DMSO-containing medium was prepared by adding 1 / 1000 volume of DMSO (Nacalai Tesque, 09659-14) to the basal medium. Chloroquine diphosphate (Tokyo Chemical Industry C2301-25G, hereafter referred to as CQ) was added to the DMSO-containing medium to prepare a 25 μM CQ solution.

[0021] (c) Cell culture (U-1) Incubation before sample addition (Day 1) Add 200 μL of SH-SY5Y cell suspension (6 × 10 cells) in basal medium to a 48-well plate. 4 / well) and cultured overnight (37°C, 5% CO2) to allow the cells to adhere to the plate. (Woo 2) Washing (Day 2) After removing the culture supernatant, 200 μL / well of basal medium was added.

[0022] (d) Preparation of hot water plant extract solution samples (E-1) An appropriate amount of each freeze-dried hot water extract of a plant was weighed and dissolved in a CQ-containing medium so that the concentration of the specific plant hot water extract became 10 mg / mL. (D-2) The hot water plant extract solution was centrifuged (3000 xg, 20°C, 5 minutes), and the supernatant was collected. (E-3) The centrifuged supernatant was diluted with CQ-containing medium to prepare specific plant hot water extract solution samples at multiple concentrations (e.g., 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL).

[0023] (E) Addition of specimen (E-1) After removing the culture supernatant, each sample solution was added at 200 μL / well. To the control wells, 200 μL / well of CQ-containing medium was added instead of the sample solution. (O-2) Incubated overnight (approximately 16 hours) at 37°C and 5% CO2.

[0024] 3. Measurement Flow cytometer measurement (day 3) (a) Fluorescent staining (preparing stained and unstained wells for each specimen) (1) After removing the culture supernatant, 200 μL of basal medium was added to each well. After the addition, 100 μL of DAPGreen (DOJINDO, 340-09291) diluted to 0.1 μM in basal medium was added to each well and incubated for 30 minutes at 37°C and 5% CO2. 100 μL of DMSO-containing medium was added to each well to be stained, and the wells were incubated for 30 minutes at 37°C and 5% CO2. (2) The culture supernatant was removed from the wells, and 200 μL of basal medium was added to each well. The same procedure was repeated once more. (3) After removing the culture supernatant, 200 μL / well of PBS was added. (4) After removing the culture supernatant, 100 μL / well of 0.1% trypsin-EDTA was added to detach the cells. 300 μL / well of basal medium was added, and the detached cells were collected in a 1.5 mL tube. The cells were stored on ice until further treatment. (5) The collected cell suspension was centrifuged (600 xg, 4°C, 3 minutes), and 300 µL of the supernatant was removed. (6) 275 μL of HBSS was added to each tube to suspend the cells, and then the cells were subjected to a flow cytometer (CytoFlex, Beckman Coulter) and analyzed using software (CytExpert 2.4). (7) A cell population was selected based on the forward scattered light and side scattered light data, and the median fluorescence intensity of each particle in the cell population (excitation 488 nm, detection fluorescence 525 nm) was determined as the median fluorescence intensity of the sample. (8) For each sample, a total of six median fluorescence intensities were obtained: three stained wells and three unstained wells.

[0025] 4.Analysis (1) The median fluorescence intensity of the three unstained wells of each sample was calculated and subtracted from the median fluorescence intensity of the three stained wells of each sample to obtain the median fluorescence intensity of the stained sample with background subtracted. (2) Similarly, the average median fluorescence intensity of the control after background subtraction was set to 1, and the median fluorescence intensity of each stained sample after background subtraction was divided by the median fluorescence intensity of the control to express the value relative to the control (hereinafter referred to as the "relative median fluorescence intensity"). (3) Based on the relative values, a t-test was performed on the control and each sample to calculate the p-value.

[0026] 5.Results Below are listed hot water extracts of specific plants that demonstrated the ability to regulate autophagosome formation.

[0027] [Table 1]

[0028] As shown in Table 1, hot water extracts of allspice, Japanese pepper, cardamom, laurel, lemongrass, green Sichuan pepper, clove, cinnamon bark, coriander, turmeric, fennel, and caraway were found to promote autophagosome formation and activate autophagy. On the other hand, nutmeg, long pepper, and black pepper were found to inhibit autophagosome formation and suppress autophagy.

[0029] Example 2: Measurement of specific plant aqueous extracts by tfLC3 assay 1.Material (cell) We used HeLa-Kyoto human cultured cells stably expressing tfLC3 (tandem-fluorescent LC3; Kimura et al., 2007 ). (Prepared Reagents) Growth medium DMEM (Sigma D6429) growth medium supplemented with 10% FBS, penicillin-streptomycin solution (Sigma P4333; 1 / 100 volume), and L-glutamine solution (Sigma G7513; 1 / 100 volume) was used. (sample) The hot water extracts of cinnamon bark and turmeric, which were found to have autophagy-activating properties in Example 1, were purified and used. For cinnamon bark, the hot water extract of cinnamon bark was purified using a column with acetonitrile. For turmeric, Turmeric Extract AKY-1860 (manufactured by Inabata Aromatics Co., Ltd., containing a hot water extract of turmeric) was used.

[0030] 2.Cell culture (a) Cell seeding HeLa-Kyoto cells stably expressing tfLC3 were seeded into a 96-well plate containing 100 μL / well of growth medium and cultured for 24 hours (37°C, 5% CO ). (a) Addition of specimen (i-1) The purified hot water extracts of cinnamon bark and turmeric were dissolved in DMSO (Nacalai Tesque 09659-14) to a concentration 1,000 times higher than the concentration of the purified hot water extracts contained in the sample to be measured (e.g., 50 μg / mL, 200 μg / mL), to prepare a sample solution. (B-2) Each sample solution was diluted 500 times with the growth medium to prepare spiked samples. DMSO was also added to the growth medium and diluted 500 times to prepare a control. (i-3) 100 μL of each sample and control was added to each well and cultured for 24 hours.

[0031] 3. Measurement and Analysis (c) Cell fixation and staining (C-1) After 24 hours of culture, the cells were washed twice with PBS (Nacalai Tesque 14249-24). (C-2) Cells were fixed with 4% paraformaldehyde (Nacalai Tesque, 09154-85) containing Hoechst 33342 (Dojindo Chemical H342), and then washed twice with PBS. (d) Image capture and analysis (A-1) Fluorescence images were taken using a confocal image cytometer CQ1 (Yokogawa Electric Corporation). Cells were automatically identified in the images using software (CellPathfinder), and the integrated intracellular GFP and RFP fluorescence signals were quantified to calculate the GFP / RFP fluorescence intensity ratio for each well. (Bhargava HK, Tabata K, Byck JM, Hamasaki M, Farrell DP, Anishchenko I, DiMaio F, Im YJ, Yoshimori T, Hurley JH. 2020. Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex. Proc Natl Acad Sci USA 117:17003-17010.; Shoemaker CJ, Huang TQ, Weir NR, Polyakov NJ, Schultz SW, Denic V. 2019. CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor. PLoS Biol 17:e2007044.; Tabata K, Imai K, Fukuda K, Yamamoto K, Kunugi H, Fujita T, Kaminishi T, Tischer C, Neumann B, Reither S, Verissimo F, Pepperkok R, Yoshimori T, Hamasaki M. 2024. Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy. Nat Commun 15:7194.; Teranishi H, Tabata K, Saeki M, Umemoto T, Hatta T, Otomo T, Yamamoto K, Natsume T, Yoshimori T, Hamasaki M. 2022.Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in the initiation of lysophagy. Cell Rep 40:111349.) This fluorescence intensity ratio was used to determine the autophagy activity of each sample and control. (A-2) The autophagy activity values ​​of each sample and control well were divided by the autophagy activity value of a well cultured with growth medium alone to calculate the relative autophagy activity value of each sample and control. (E-3) The relative autophagy activity values ​​of the control and each sample were subjected to a t-test to calculate the p-value.

[0032] 4.Results The relative autophagy activity values ​​of the purified products of the cinnamon bark hot water extract and turmeric hot water extract, as well as the control, are shown in Figure 1. As shown in Figure 1, the relative autophagy activity values ​​of the purified cinnamon bark hot water extract and the purified turmeric hot water extract were significantly lower than that of the control (p<0.05), and the autophagy activation function was also confirmed in the tfLC3 assay.

[0033] Example 3: Drosophila lifespan evaluation test, climbing assay test 1. Drosophila Preparation (A) Drosophila used in the test <Drosophila strains> The Canton-S strain was used. <Drosophila breeding> (1) Approximately 20 flies were placed in a plastic vial containing the basal medium listed below and sealed with a sponge stopper. (2) The flies were reared in an incubator (25°C, 12-hour light / dark cycle). The next day, the flies were removed from the vials, leaving only the medium on which the eggs had been planted. (3) Adult Drosophila were obtained by incubation under the same conditions as in (2). <Collection and age of male Drosophila> Drosophila on the day they emerged from pupae (day 0) were anesthetized with carbon dioxide gas and only males were selected. 25 male flies were collected per vial containing basal medium, plugged with a sponge stopper, and reared in an incubator (25°C, 12-hour light / dark cycle) until the start of the experiment.

[0034] 2. Culture Medium Preparation (1) Basal medium (breeding feed) The fly food, preservative for fly food, and acid mixture for fly food were prepared using the tables in Reference 1 (“Phenotypic analysis of a transgenic Drosophila model of Alzheimer's amyloid b toxicity”, Sekiya et.al., STAR Protocols, 2021 Apr 29;2(2):100501). (2) Supplemented medium (test feed) (i) The concentrations of each component, except for agar, were the same as in the basal medium to prepare a suspension (yeast-containing suspension). (ii) The cinnamon hot water extract (lyophilized product) was dissolved in a suspension containing yeast to a concentration of 50 mg / mL (hereinafter referred to as "cinnamon hot water extract solution"). (iii) The cinnamon hot water extract solution was diluted with a yeast suspension to a concentration of 5 mg / mL to prepare a cinnamon hot water extract solution sample. (iii) To the basal medium (after preparation, the agar had solidified), 50 μL / vial of a yeast suspension (for comparison) or a cinnamon bark hot water extract solution sample was added. (iv) The tubes were left unstoppered, covered with paper towels, and dried overnight in a dark place. After drying, they were sealed with sponge stoppers and stored in a dark place until use. When a hot water extract of a specific plant other than cinnamon bark (for example, turmeric) was used, the supplemented medium was prepared in the same manner as in the case of the cinnamon bark hot water extract.

[0035] 3. Preparation for lifespan evaluation (survival) test and climbing assay test (1) Twenty-five male flies were transferred without anesthesia into each vial containing the following supplemented medium and then sealed with a sponge stopper. (2) Five vials (125 mice per group) were prepared for each test group and the mice were kept in an incubator (25°C, 12-hour light / dark cycle) during the test. The lifespan evaluation (survival) test and the climbing assay test were conducted independently as separate tests.

[0036] 4. Changing the Drosophila medium during each test Every 2 to 3 days, the Drosophila were transferred without anesthesia to a vial containing fresh supplemented medium, and the vial was sealed with a sponge stopper, and the medium was changed.

[0037] 5. Climbing Assay Test (1) Climbing assays were performed at weekly intervals starting from the fifth week of the study. The climbing assay method was based on the method described in Reference 1, with some slight modifications (the underlined parts are improvements). (2) On the day of the climbing assay, flies were transferred to empty vials and plugged with sponge stoppers. (3) To acclimate the flies to the environment, place the vials upright. 10 minutes or more Left it alone. (4) The vial was gently tapped against a foam rubber pad to cause the flies to fall to the bottom of the vial. (5) In front of a piece of construction paper with lines marked at a certain height The vial was quickly transferred to a digital camera and photographed 10 seconds later. (6) The number of flies that climbed to the top part of each vial was counted and divided by the total number of surviving flies in the same vial to calculate the percentage of flies that climbed to the top part. Climbing assays were performed five times for each vial, and the average of the five percentages of flies that climbed to the top part was calculated and used as the measurement value for each vial. Since there were five vials in each group, five measurements were obtained for each group. The measurements of each group were compared with those of the control group using a t-test. 6. Lifespan evaluation (survival) test (1) This was carried out with reference to Reference 1. (2) When the medium was changed, the date and the number of dead flies were recorded. (3) The experiment was continued until all fruit flies were extinct. After the experiment was completed, the control group and the test group were compared using the Kaplan-Meier method. The data were analyzed excluding escaped flies.

[0038] 7. Results and Discussion (1) Climbing assay test results The results of the Drosophila climbing assay test are shown in Figure 1. The percentage of Drosophila climbing to the top part in the test group bred in a medium supplemented with a hot water extract of cinnamon bark was 45%, and the percentage of Drosophila climbing to the top part in the test group bred in a medium supplemented with a hot water extract of turmeric was 34%, both of which showed favorable climbing assay scores compared to the control (16%). This indicates that the hot water extract of cinnamon bark and the hot water extract of turmeric are effective in extending healthy lifespan. (2) Life evaluation test results The results of the Drosophila survival test are shown in Figure 2. Compared to the control, the survival rate of the test group of flies bred in the medium containing the hot water extract of cinnamon bark showed a statistically significant improvement, demonstrating that the hot water extract of cinnamon bark has the effect of extending lifespan.

Claims

1. A composition for regulating autophagosome formation, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, long pepper, fennel, caraway, coriander, allspice, nutmeg, lemongrass, green Sichuan pepper, clove, black pepper and turmeric.

2. The composition of claim 1 for promoting the formation of autophagosomes.

3. The composition of claim 1 for inhibiting autophagosome formation.

4. A composition for activating autophagy, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark, Japanese pepper, cardamom, laurel, fennel, caraway, coriander, allspice, lemongrass, green Sichuan pepper, clove, and turmeric.

5. A composition for inhibiting autophagy, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of pepper, nutmeg, and black pepper.

6. A composition for extending healthy lifespan, comprising an aqueous extract of a specific plant, The composition, wherein the specific plant comprises at least one selected from the group consisting of cinnamon bark and turmeric.

7. A life-extending composition comprising an aqueous extract of cinnamon bark.

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