Autophagy activator

Abstract

To provide an autophagy activator which has superior availability of raw materials, autophagy-activating effect, and safety.SOLUTION: An autophagy activator comprises, as an active ingredient, at least one selected from shochu lees, a lactic acid fermentation product of shochu lees, and extracts thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an autophagy activator.

Background Art

[0002] Autophagy is one of the mechanisms in cells to degrade intracellular proteins. It is involved in maintaining the homeostasis of the living body by preventing the accumulation of abnormal proteins in cells, recycling proteins when protein synthesis is excessive or the nutritional environment deteriorates, and eliminating pathogenic microorganisms that have invaded the cytoplasm. Diseases that are considered to be caused by a decrease in autophagy function include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, cardiovascular diseases such as myocardial infarction, cardiomyopathy, and atherosclerosis, muscle loss diseases such as muscular dystrophy, osteoarthritis, and sarcopenia, liver function disorders such as alcoholic fatty liver, liver cirrhosis, and acute liver failure, lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pulmonary hypertension, and kidney diseases such as acute kidney injury, diabetic nephropathy, and glomerulonephritis (Non-Patent Document 1).

[0003] As autophagy activators, plant extracts such as sage, heparin-like substances, components such as glycyrrhizinate, agrimol B, Agaricus mushroom extract, and Morus alba have been reported (Patent Documents 1 to 5).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Literature

[0005]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, the effects of the autophagy activators reported so far are not fully satisfactory, and components that are even more excellent in terms of availability of raw materials, autophagy activation effect, safety, etc. are desired. Therefore, an object of the present invention is to provide an autophagy activator that is excellent in terms of availability of raw materials, autophagy activation effect, safety, etc.

Means for Solving the Problems

[0007] Therefore, the present inventor has focused on shochu lees and its lactic acid fermented product, and has examined various physiological activities. As a result, quite unexpectedly, it has been found that these have an excellent autophagy activation effect, and the present invention has been completed.

[0008] That is, the present invention provides the following inventions [1] to [8]. [1] An autophagy activator comprising, as an active ingredient, one or more selected from shochu lees, lactic acid-fermented shochu lees, and extracts thereof. [2] The autophagy activator according to [1], wherein the active ingredient is one or more selected from a shochu lees extract and a lactic acid-fermented shochu lees extract. [3] The autophagy activator according to [1], wherein the active ingredient is one or more selected from an agmatine-containing fraction having a molecular weight of 3,000 or less and a protein-containing fraction having a molecular weight of 10,000 or more in a shochu lees extract or a lactic acid-fermented shochu lees extract. [4] An autophagy activator according to [1], wherein the autophagy activation action is an autophagy activation action via phosphorylation by mTOR kinase. [5] A food composition for autophagy activation or a cosmetic composition for autophagy activation, containing one or more selected from shochu lees, lactic acid-fermented shochu lees, and their extracts. [6] The food composition for autophagy activation or the cosmetic composition for autophagy activation according to [5], wherein the active ingredient is one or more selected from shochu lees extract and lactic acid-fermented shochu lees extract. [7] The food composition for autophagy activation or the cosmetic composition for autophagy activation according to [5], wherein the active ingredient is one or more selected from the agmatine-containing fraction with a molecular weight of 3,000 or less and the protein-containing fraction with a molecular weight of 10,000 or more in shochu lees extract or lactic acid-fermented shochu lees extract. [8] The food composition for autophagy activation or the cosmetic composition for autophagy activation according to [5], wherein the autophagy activation action is an autophagy activation action via phosphorylation by mTOR kinase.

Effect of the Invention

[0009] Shochu lees have been either conventionally discarded or only used to the extent of being used as animal feed or for agricultural land restoration, etc., and other uses have been desired from the perspective of SDGs, etc. However, with the present invention, new uses can be achieved. In addition, the activation of autophagy is effective for the prevention or treatment of various diseases as described above, and the autophagy activator of the present invention can be widely used as pharmaceuticals, foods, and cosmetics.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] One embodiment of the present invention is an autophagy activator containing as an active ingredient one or more selected from sake lees, sake lees lactic acid fermented products, and their extracts. Another embodiment of the present invention is a food composition for autophagy activation or a cosmetic composition for autophagy activation containing one or more selected from sake lees, sake lees lactic acid fermented products, and their extracts. That is, the active ingredients of the medicament, food composition, and cosmetic composition of the present invention are one or more selected from sake lees, sake lees lactic acid fermented products, and their extracts.

[0012] To produce sake, first, koji mold is grown on steamed rice, wheat, etc. (original raw materials) to make koji. Koji, water, and yeast are added to a tank or vat for the first charging and fermented to make the first mash. The main raw material (added raw material: sweet potato, wheat, buckwheat, brown sugar, etc.) and water are added to the first mash for the second charging and further fermented to make the second mash. Next, the second mash is distilled to obtain sake. Here, the distillation residue of the second mash is sake lees. This sake lees contains the yeast fermentation products of the original raw materials and the added raw materials. Also, the components contained differ depending on the type of added raw material. The sake lees may be dried and used as sake lees powder. As described above, this sake lees has only been used conventionally for animal feed, agricultural land reduction, methane fermentation, etc., and cannot be said to be fully and effectively utilized.

[0013] The shochu lees lactic acid fermented product is obtained by adding lactic acid bacteria to the shochu lees and subjecting it to lactic acid fermentation. As the lactic acid bacteria used here, lactic acid bacteria commercially available for food fermentation can be used, for example, Lactis lactic acid bacteria, Gaseri bacteria, Labre bacteria, K-2 lactic acid bacteria, etc. In addition, as the genus of lactic acid bacteria, Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, Bifidobacterium, etc. can be mentioned. Specifically, water may be added to the shochu lees as necessary, lactic acid bacteria are added, the pH is adjusted to a range suitable for the growth of lactic acid bacteria (for example, pH 2 to 4.5), and fermentation may be carried out at a temperature in the range of 30°C to 35°C for 2 to 5 days. The shochu lees lactic acid fermented product may also be dried and used as a powder.

[0014] Examples of the extract of shochu lees or shochu lees lactic acid fermented product include extracts with water, ethanol, or a water-ethanol mixture. Examples of the extraction method include the method of immersing in these liquids for extraction and the Soxhlet extraction method.

[0015] As shown in the following examples, shochu lees, shochu lees lactic acid fermented product, and their extracts exhibit excellent autophagy activation effects at concentrations that do not cause cytotoxicity. Therefore, shochu lees, shochu lees lactic acid fermented product, and their extracts are useful as active ingredients of autophagy activators, food compositions for autophagy activation, and cosmetic compositions for autophagy activation. In addition, if the autophagy activator, food composition for autophagy activation, or cosmetic composition for autophagy activation of the present invention is ingested or applied to the skin, prevention or improvement of symptoms of the aforementioned neurodegenerative diseases, cardiovascular diseases, muscle loss diseases, liver dysfunction, lung diseases, kidney diseases, etc. can be expected.

[0016] The active ingredient of the autophagy activator of the present invention is one or more selected from shochu lees, lactic acid fermented shochu lees, and their extracts as described above. The present inventors examined which components in this extract are particularly effective. The extract was fractionated into fractions with a molecular weight of 3,000 or less, fractions with a molecular weight of 3,000 to 10,000, and fractions with a molecular weight of 10,000 or more, and these fractions were examined. As a result, agmatine was contained in the fraction with a molecular weight of 3,000 or less, and it was confirmed that agmatine itself has an autophagy activation effect. In addition, a considerable amount of protein was contained in all fractions, and it was confirmed that the fraction with a molecular weight of 10,000 or more particularly has a strong autophagy activation effect. Therefore, among shochu lees, lactic acid fermented shochu lees, and their extracts, the fraction with a molecular weight of 3,000 or less containing agmatine and the fraction containing proteins with a molecular weight of 10,000 or more are particularly useful as autophagy activators. In addition, the protein in the fraction containing the protein did not show a decrease in autophagy activation effect by heat treatment, nor did it show a decrease in autophagy activation effect by Proteinase K treatment.

[0017] The present inventors also examined the mechanism of action of autophagy activation of shochu lees, lactic acid fermented shochu lees, and their extracts. As a result, it was confirmed that these autophagy activation effects are exerted at least via mTORC1.

[0018] The dosage form of the autophagy activator of the present invention is not particularly limited, and it may be in any of solid, semi-solid, or liquid form. Regarding the application form of the autophagy activator of the present invention, it may be applied in any form such as oral, transdermal, enteral, intravenous, pulmonary, subcutaneous, transmucosal, intramuscular, etc., and may be appropriately set according to the diseases and symptoms to be prevented or treated.

[0019] When the autophagy activator of the present invention is in the form of a pharmaceutical, the content of the active ingredient in the pharmaceutical may be appropriately set according to the type of the pharmaceutical, the dosage of the active ingredient per day, etc. For example, in the case of an oral pharmaceutical, the active ingredient may be 0.1 to 100% by mass, preferably 5 to 90% by mass, more preferably 10 to 80% by mass in total.

[0020] The food composition for activating autophagy of the present invention may be prepared in a desired form by using the active ingredient as it is or in combination with other food materials and additive components. Examples of food and drink products include, in addition to general food and drink products, health functional foods (including foods for specified health use, foods with nutritional functions, and foods with functional claims), foods for patients, and the like. The form of these food and drink products is not particularly limited, but specifically includes beverages such as tea beverages, nutritional drinks, fruit juice beverages, carbonated beverages, lactic acid beverages, and alcoholic beverages; supplements such as capsule preparations (soft capsule preparations, hard capsule preparations), tablets, granules, powders, jelly preparations, and liposome preparations; and confectionery such as gummies, candies, and jelly. Among these food and drink products, supplements are preferably mentioned.

[0021] The cosmetic composition for activating autophagy of the present invention may be prepared in a desired form by using the active ingredient as it is or in combination with other additives, etc. Specific examples of cosmetics include cream preparations, lotion preparations, gel preparations, emulsion preparations, liquid preparations, ointment preparations, pack preparations, and the like.

Examples

[0022] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0023] Production Example 1 (Production Example of Shochu Lees) Wash the rice with water and soak it, then drain the water. Steam the rice that has absorbed an appropriate amount of moisture up to the core with steam. Cool the steamed rice with ventilation and adjust it to 35 - 46°C, and evenly attach the spores of koji. Cultivate it in a device for individual cultivation of special koji for about two days so that the mycelium of koji grows not only on the surface of the rice but also inside the rice. Add water and yeast to the grown koji, and first ferment only the koji for about 5 to 7 days for alcohol fermentation. Divide the fermented product with sufficient growth of yeast into small portions in another fermentation container, and add steamed and crushed sweet potatoes and water. Here, the amount of sweet potatoes was 4 to 5 times the weight of the rice. The fermentation period after adding the sweet potatoes is 1 to 2 weeks as a standard, and it is finished into a fermented product with about 14 - 15% alcohol. When the fermentation is completed, transfer it to a batch distillation machine (single - type distillation machine) and perform steam distillation. When the alcohol concentration of the distillate from the distillation machine reaches about 10%, end the distillation, and distillation residue remains in the distillation kettle. This distillation residue is discharged as "shochu lees" (a high - temperature liquid that has been boiled for about two and a half hours).

[0024] Production Example 2 (Production Example of Shochu Lees Powder) Put the "shochu lees" discharged from the distillery into a dedicated pit. Send it from the dedicated pit to a continuous centrifuge for dehydration and separation. Dry this dehydrated product with a twin - shaft paddle dryer. Pass this dried product through a mill with rotating protrusions to make the particle size below a certain level and suck - convey it to a dry powder pit. The moisture of the dried product was 12% or less.

[0025] Production Example 3 (Production of Lactic Acid Fermentation Product of Shochu Lees) Add twice the amount of water and a small amount of glucose to the shochu lees powder obtained in Production Example 2, and sterilize it at 90 - 95°C for 1 hour. Add lactic acid bacteria (Weizmannia coagulans) and ferment at 35°C for 3 days (during this period, the pH decreased from 4.6 to 4.1). Heat it at 85°C for 30 minutes or more to stop the fermentation, and vacuum - dry it at 60°C for 48 hours. Then, coarsely pulverize it to a particle size of about 10 mm - 30 mm and powder it with an atomizer (yield from shochu lees is about 95%).

[0026] 1.1 Test Samples and Reagents For the shochu lees, shochu lees lactic acid fermented product, their powders, and extracts used in the tests, those prepared in the above Production Examples and the following Preparation Examples were used. Among the reagents used, Torin1 was purchased from Merk Millipore. Bafilomycin A1 was purchased from Fujifilm Wako Pure Chemical Corporation (Wako).

[0027] 1.2 Preparation of Samples and Molecular Weight Fractionation For the shochu lees aqueous extract or lactic acid fermented product aqueous extract used in the cell tests, 1 g of shochu lees powder or lactic acid fermented product powder was mixed with 10 mL of ultrapure water, or 4 g of shochu lees powder or lactic acid fermented product powder was mixed with 40 mL of ultrapure water, and extraction was carried out by shaking at 4°C for 24 hours. Then, centrifugation was performed at 10,000×g for 10 minutes, and the operation of replacing the supernatant into a new tube was repeated 3 times, followed by filtration using a 20 mL Terumo syringe (TERUMO, SS-20ESZ) or a 50 mL Terumo syringe (TERUMO, SS-50ESZ) and a 0.22 μm Low Protein Binding Durapore (PVDF) Membrane (Merk Millipore, SLGVR33RB). The obtained aqueous extracts were used as the shochu lees aqueous extract and the lactic acid fermented product aqueous extract in the experiment. The molecular weight fractionation used in the cell tests was performed using the aqueous extract. Amicon TM Ultra-4 centrifugal filter unit (Merck Millipore, UFC801008) or Amicon TM Ultra-15 centrifugal filter unit (Merck Millipore, UFC910008) with a 10 kDa ultrafiltration filter and a Microsep Advance centrifugal filtration device (PALL, MCP003C41) with a 3 kDa ultrafiltration filter were used. Amicon TMFor the 10 kDa ultrafiltration filter of the Ultra-4 centrifugal filter device, first, about 4 mL of sterilized water was added to remove the influence of glycerin in the membrane, and centrifugation was performed at 7,500×g for about 15 minutes for pre-washing. When performing centrifugation, the membrane panel was oriented upward. After confirming that the sterilized water had drained from the filter, the sterilized water was removed with a pipette, then 4 mL of the sample was added and centrifugation was performed again at 7,500×g for 15 minutes. Thereafter, the extract remaining in the filter was used as a polymer solution with a MW of 10,000<, and the extract that had passed through the filter was further fractionated with a 3 kDa filter. Amicon TM For the 10 kDa ultrafiltration filter of the Ultra-15 centrifugal filter device (Merck Millipore), the flow of fractionation was the same, but the liquid volume of the sample and ultrapure water was 12 mL, and centrifugation was performed at 5,000×g for 25 minutes. The same was true for the 3 kDa ultrafiltration filter of the Microsep Advance centrifugal filtration device. Amicon TM The flow and centrifugation time were the same as those of the Ultra-4 centrifugal filter device. The extract remaining in the filter was used as a medium molecule solution with 3,000<MW<10,000, and the extract that had passed through the filter was used as a low molecule solution with MW<3,000. All extracts were sterilized and used in the experiment. In metabolome analysis, as a sample for CE-TOFMS (capillary electrophoresis-time of flight mass spectrometer), about 10 mg of shochu lees powder or lactic acid fermented product powder was mixed with 600 μL of methanol solution containing 50 μM of internal standard substance, and crushed using a crusher under cooling (1500 rpm, 120 seconds × 3 times). After crushing, 600 μL of Milli-Q water was added, stirred, and centrifuged (2300×g, 4°C, 120 minutes). After centrifugation, the supernatant was transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa). This was centrifuged (9100×g, 4°C, 120 minutes) and subjected to ultrafiltration treatment. The filtrate was dried, dissolved again in Milli-Q water, and used for measurement. As a sample for LC-TOFMS (liquid chromatography-time of flight mass spectrometer), about 15 mg of shochu lees powder or lactic acid fermented product powder was mixed with 1000 μL of 1% formic acid-acetonitrile solution containing 5 μM of internal standard substance, and crushed using a crusher under cooling (1500 rpm, 120 seconds × 3 times). 334 μL of Milli-Q water was added thereto, and further crushed (1500 rpm, 120 seconds × 1 time). This was centrifuged (2300×g, 4°C, 120 minutes), the supernatant was collected, and then transferred to an ultrafiltration tube (NANOSEP 3K OMEGA, PALL). This was centrifuged (9100×g, 4°C, 120 minutes) and subjected to ultrafiltration treatment. Next, phospholipids were removed using solid phase extraction. The filtrate was dried, dissolved in 50% isopropanol aqueous solution (v / v), and used for measurement.

[0028] 1.3 Cell culture HeLa, a human cervical cancer cell line, was cultured in Dulbecco's Modified Eagle Medium (DMEM) (Wako, 044 -29765) containing 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, F7524) and 1% Penicillin-Streptomycin (Wako, 168-23191) at 37°C in a 5% CO2 environment. Also, cells prepared by introducing the GFP-LC3-RFP-LC3ΔG probe into HeLa cells were used. The human colon cancer cell line Caco-2 was cultured in DMEM (Wako, 044-29765) containing 20% FBS (Sigma-Aldrich, F7524), 1% Penicillin-Streptomycin (Wako, 168-23191), and 1% Non-essential Amino Acids Solution (NEAA) (Wako, 139-15651) at 37°C in a 5% CO2 environment. Cells prepared by introducing the GFP-LC3-RFP probe into Caco-2 cells were also used. The human epidermal keratinocyte cell line HaCaT was cultured in DMEM (Wako, 044-29765) containing 10% FBS (Sigma-Aldrich, F7524) and 1% Penicillin-Streptomycin (Wako, 098-06465) at 37°C in a 5% CO2 environment. Cells prepared by introducing the GFP-LC3-RFP probe into HaCaT cells were also used. Additionally, cells with the Atg9 gene deleted were used.

[0029] 1.4 Analysis of autophagy activity (FACS) Cells expressing the already established GFP-LC3-RFP-LC3ΔG or GFP-LC3-RFP probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured for 24 hours. After that, the test component was added and the cells were cultured for another 24 hours. Then, the medium was aspirated, 600 μL of 1×D-PBS (Wako, 048-29805) was added, aspirated again, and 200 μL of 0.25% Trypsin (Wako, 201-16945) was added per well and incubated at 37°C in a 5% CO2 environment for about 5 minutes. In the case of HaCaT cells, TrypLE TM Express Enzyme (Gibco TM,12604021) It was incubated at 200 μL for about 10 minutes. Then, 600 μL of phenol red-free D-MEM containing 0.1% Sodium Azide (Wako, 195-11092) (Wako, 040-30095) was added per well to collect the cells. Then, the fluorescence intensity was measured using a Spectral Cell Analyzer SA3800 (SONY Corporation). The GFP / RFP ratio was determined based on the obtained GFP and RFP values.

[0030] 1.5 Cell activation and toxicity test (WST-8·MTT assay) In the WST-8 assay, cells were seeded at 1.0×10 4 cells / well in a 96-well plate, cultured for 24 hours, then treated with the test component and further cultured for 24 hours. Then, the medium in the 96-well plate was replaced with a medium containing Cell Counting Kit-8 (Dojindo, CK04). This medium was prepared by adding 1 mL of Cell Counting Kit-8 to 10 mL of medium and added at 100 μL per well. It was incubated at 37 °C in a 5% CO2 environment for about 1 hour. To remove the influence of the sample on the absorbance value, only the supernatant was transferred to a new 96-well plate and Multiskan FC absorbance microplate reader (Thermo Fisher Scientific, 51119050) was used to measure the absorbance at 450 nm. In the MTT assay, cells were seeded at 1.0×10 4Cells were seeded at cells / well, cultured for 24 hours, then treated with the test component and further cultured for 24 hours. Thereafter, the medium in the 96-well plate was replaced with a medium containing MTT powder. The medium containing MTT powder was prepared by adding 1 mL of a liquid obtained by mixing 150 mg of MTT powder (DOJINDO, 341-01823) and 30 mL of 1×PBS to 10 mL of the medium, and 100 μL was added per well. It was incubated at 37°C in a 5% CO2 environment for about one and a half hours. After aspiration, 200 μL of DMSO (Wako, 043-29355) was added to dissolve the formazan and stirred. Then, the absorbance at 535 nm was measured.

[0031] 1.6 BCA method Precision Plus Protein Dual Color Standards (BIO RAD, 1610374) and samples for creating a standard curve were added to a 96-well plate. For the standard, 200 μL of a solution prepared by mixing Reagent A and Reagent B included in the Protein Assay BCA Kit (Wako, 297-73101) at a ratio of 50 to 1 and 5 μL of Precision Plus Protein Dual Color Standards (BIO RAD, 1610374) were added for each protein concentration. For the sample, 200 μL of a solution prepared by mixing Reagent A and Reagent B at a ratio of 50 to 1 and 5 μL of the aqueous extract of the sample were added. Thereafter, it was reacted for 30 minutes in a light-shielded environment at 37°C. After 30 minutes, Multiskan The absorbance at 562 nm was measured using an FC absorbance microplate reader (Thermo Fisher Scientific, 51119050).

[0032] 1.7 Western Blotting Cells were plated at 1.0×10 in a 12-well plate 5 cells / well or 3.0×10 in a 6-well plate 5Cells were seeded at a density of cells / well and cultured for 24 hours. After that, the test compound was added and the cells were cultured for an additional 24 hours. Then, the cells were washed with 1×D-PBS (Wako, 048-29805) and lysed with Lysis Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing a protease inhibitor (complete EDTA-free protease inhibitor). The lysate was centrifuged at 4°C, 15,000×g for 15 minutes, and the supernatant was collected. The protein concentration of the supernatant was measured using the Protein Assay BCA Kit (Wako, 294-73101), and then the Lysis Buffer was added to adjust the protein concentration of each sample to be equal. Then, the proteins were separated by SDS-PAGE and transferred to a 0.2-μm PVDF membrane using the Trans-Blot Turbo Transfer Pack (Bio-Rad, 1704156). After transfer, blocking was performed for approximately 1 hour using a solution prepared by dissolving skim milk powder (Wako, 190-12865) in TBST buffer (500 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.1% Tween 20) to a concentration of 5%. The target protein was detected using specific antibodies. The primary antibodies used were Phospho-p70 S6 Kinase (T389) (D5U1O) Rabbit mAb (97596), β-actin antibody [C4] (41185), and 4E-BP1 (53H11) Rabbit mAb (42235), which were purchased from Cell Signaling Technology. The secondary antibodies used were Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch, 111-035-003) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-035-003), which were purchased from Millipore. The detection reagent was SuperSignal TMWest Pico PLUS Chemiluminescent Substrate (Thermo Fisher Science, 34580) or SuperSignal TM West Dura Extended Duration Substrate (Thermo Fisher Science, 37071) was used. Detection and quantification of protein bands were performed with FUSION SOLO S (VILBER LOURMAT).

[0033] 1.8 Gel staining For CBB staining, after electrophoresis, the gel was stained with EzStain AQua (ATTO, 2332370) for about 1 hour. After staining, the gel was immersed in ultrapure water for decolorization, and images were taken using FUSION SOLO S (VILBER LOURMAT) and UV / White Converter Plate (VILBER LOURMAT). For silver staining, after electrophoresis, the gel was stained with Silver Staining II Kit Wako (Wako, 291 - 50301) and methanol (Wako, 137 - 01823) and acetic acid (Wako, 017 - 00251). After staining, images were taken using FUSION SOLO S (VlLBER LOURMAT) and UV / White Converter Plate (VlLBER LOURMAT).

[0034] 1.9 Phenol-chloroform extraction method and ethanol precipitation 200 μL each of TE Saturated Phenol (Wako, 319-90093) and chloroform (Wako, 038-02606) were mixed and then added to 400 μL of the sample extract, followed by inverting and mixing. Thereafter, centrifugation was performed at 4°C, 12,000×rpm for 5 minutes, and the aqueous layer was recovered. An equal volume of chloroform was added to the recovered aqueous layer, followed by inverting and mixing, and then centrifugation was performed again at 4°C, 12,000×rpm for 5 minutes, and the aqueous layer was recovered. Next, to remove the chloroform layer by ethanol precipitation, ethanol (99.5) (Wako, 057-00451) was added to the recovered aqueous layer to make it 70% of the total volume, and then centrifugation was performed again at 4°C, 12,000×rpm for 5 minutes. Thereafter, the aqueous layer was removed, ethanol (99.5) (Wako, 057-00451) at 70% was added to the precipitate, and centrifugation was performed at 4°C, 12,000×rpm for 5 minutes. The aqueous layer was removed, and Kimwipe S-200 (62011) purchased from Nippon Paper Crecia Co., Ltd. was covered on the tube and dried in an environment at 55°C for about 1 hour. 200 μL of ultrapure water was added to the dried product, and the dissolved product was used in the experiment.

[0035] 1.10 Metabolome analysis Metabolome analysis was performed using CE-TOFMS and LC-TOFMS. The metabolome analysis was outsourced to Human Metabolome Technologies, Inc. The detected peaks were automatically extracted using the MasterHands ver.2.19.0.2 (developed by Keio University), an automatic integration software, for peaks with a signal / noise (S / N) ratio of 3 or more, and mass-to-charge ratio (m / z), peak area value, migration time (MT) for CE-TOFMS, and retention time (RT) for LC-TOFMS were obtained. The peak area value was converted to a relative area value based on the peak area value of the internal standard substance. For the examined peaks, peak matching and alignment were performed among the samples based on the values of m / z and MT or RT, and metabolites in the samples corresponding to each peak were searched.

[0036] 1.11 Statistical analysis The data in Figures 1-10, 14, 19, and 20 were statistically analyzed by Student's t-test ( * P < 0.05, ** P < 0.01, *** P < 0.001). The data in Figures 12 and 13 were statistically analyzed by Dunnett's test after one-way ANOVA ( * P < 0.05, *** P < 0.001, vs control). The data in Figures 11, 15, 18, and 21 were statistically analyzed by Tukey's test after one-way ANOVA ( * P < 0.05, ** P < 0.01, *** P < 0.001).

[0037] Test Example 1 (Test on the cell viability of HeLa cells) HeLa cells were seeded in a 96-well plate at 1.0×10 4 cells / well and pre-cultured in DMEM medium for 24 hours. Then, the test component was added to the DMEM medium and cultured for 24 hours. Subsequently, viable cells were measured by the WST-8 method. The test component was diluted with DMEM to 0.1%, 1%, 5%, 10%, and 20% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water and added (in the case of 1%, 40 μL of the extract was added to 4 mL of DMEM). As a result, the cell viability was significantly increased at 0.1% to 10% of shochu lees (Figure 1). Also, the cell viability was significantly increased at 0.1%, 10%, and 20% of shochu lactic acid fermented product (Figure 2).

[0038] Test Example 2 (Autophagy activation effect (HeLa cells)) HeLa GFP-LC3-RFP-LC3ΔG cells were seeded in a 12-well plate at 1.0×10 5Cells were seeded at cells / well, cultured in DMEM medium for 24 hours, then the test component was added to the DMEM and cultured for another 24 hours. After that, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). The test component was diluted with DMEM to 5% and 10% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water and then added (in the case of 5%, 200 μL of the extract was added to 4 mL of DMEM). As a result, autophagy was significantly activated at 5% to 10% of shochu lees (Figure 3). Also, autophagy was significantly activated at 10% of the shochu lees lactic acid fermented product (Figure 4).

[0039] Test Example 3 (Test on the cell viability of Caco-2 cells and HaCaT cells) Caco-2 cells or HaCaT cells were seeded at 1.0×10 4 cells / well in a 96-well plate, pre-cultured in DMEM medium for 24 hours, then the test component was added to the DMEM and cultured for 24 hours, and then evaluated by the WST-8 method. The test component was diluted with DMEM to 1%, 5%, and 10% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water and then added (in the case of 1%, 40 μL of the extract was added to 4 mL of DMEM). As a result, the values increased significantly in Caco-2 cells and HaCaT cells at 1% to 10% of shochu lees (Figure 5a, b). Also, the values increased significantly in Caco-2 cells at 5% to 10% of the shochu lees lactic acid fermented product and in HaCaT cells at 1% to 10% of it (Figure 6a, b).

[0040] Test Example 4 (Autophagy activation effect (Caco-2 cells and HaCaT cells)) Caco-2 GFP-LC3-RFP cells or HaCaT GFP-LC3-RFP cells were seeded at 1.0×10 5Cells were seeded at cells / well, pre-cultured in DMEM medium for 24 hours, then the test component was added to DMEM and cultured for 24 hours. Thereafter, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). The test component was diluted with DMEM to 1%, 5%, and 10% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water and added (in the case of 1%, 40 μL of the extract was added to 4 mL of DMEM). As a result, autophagy was significantly activated in Caco-2 cells and HaCaT cells at 1% to 10% of shochu lees (Figs. 7a, b). Also, autophagy was significantly activated in Caco-2 cells and HaCaT cells at 1% to 10% of shochu lees lactic acid fermented product (Figs. 8a, b).

[0041] Test Example 5 (Examination of mTORC1 dependence) Caco-2 cells were seeded at 1.0×10 5 cells / well in a 12-well plate or 3.0×10 5 cells / well in a 6-well plate, pre-cultured in DMEM medium for 24 hours, then the test component was added to DMEM and cultured for 24 hours. Thereafter, the cells were collected, and the phosphorylation levels of various proteins (p-4EBP1 / 4EBP1, p-p70 S6K / Actin) were measured by Western blotting. The test component was diluted with DMEM to 5% and 10% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water and added (in the case of 5%, 200 μL of the extract was added to 4 mL of DMEM). As a result, at 10% of shochu lees, the phosphorylation levels of 4EBP1 and p70 S6K were significantly decreased (Figs. 9a, b). Also, at 5% to 10% of shochu lees lactic acid fermented product, the phosphorylation level of 4EBP1 was significantly decreased, and at 10%, the phosphorylation level of p70 S6K was significantly decreased (Figs. 10a, b).

[0042] Test Example 6 (Examination of mTORC1 dependence using Torin1) Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5 cells / well, pre-cultured in DMEM medium for 24 hours, then Torin1, the test component, Torin1 + the test component were added to DMEM and cultured for 24 hours. Thereafter, the cells were detached and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). The test component was added by diluting with DMEM to 5% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder or shochu lactic acid fermented product powder to 10 mL of ultrapure water (in the case of 5%, 200 μL of the extract was added to 4 mL of DMEM). As a result, compared with the Torin1 group, Torin1 + shochu lees or Torin1 + shochu lees lactic acid fermented product significantly activated autophagy (Figs. 11a, b).

[0043] Test Example 7 (Autophagy activation effect of each fraction of the test component) The test component (shochu lees water extract) was fractionated by ultrafiltration into fractions with a molecular weight of 3,000 or less, 3,000 to 10,000, and 10,000 or more. Thereafter, ultrapure water was added and adjusted to the ratio in the test component water extract. Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5 cells / well, pre-cultured in DMEM medium for 24 hours, then each fraction of the test component was added to DMEM and cultured for 24 hours. Thereafter, the cells were detached and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). As a result, fractions of shochu lees with a molecular weight of 3,000 or less or 10,000 or more significantly activated autophagy in Caco-2 cells (Fig. 12). Also, all fractions of shochu lees lactic acid fermented product significantly activated autophagy in Caco-2 cells (Fig. 13).

[0044] Test Example 8 (Search for the activating component in the low molecular weight fraction) From the measurements by CE-TOFMS in metabolome analysis, a peak of 289 (cation 156, anion 133) was detected. Also, from the measurements by LC-TOFMS, a peak of 119 (positive 82, negative 37) was detected. As a result of proceeding with the collation of metabolites for these peaks, it was found that agmatine (molecular weight: 130.195) was contained. Therefore, the autophagy activation effect of agmatine contained in the fraction with a molecular weight of 3,000 or less was examined. Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5 cells / well and pre-cultured in DMEM medium for 24 hours. Then, agmatine with a final concentration of 5.7 mM was added to DMEM and cultured for 24 hours. After that, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). As a result, the addition of agmatine significantly activated autophagy in Caco-2 cells (Figure 14).

[0045] Test Example 9 (Measurement of the protein amount contained in each fraction of the test component) The test component (shochu lees aqueous extract) was fractionated by ultrafiltration into fractions with a molecular weight of 3,000 or less, 3,000 to 10,000, and 10,000 or more. Then, ultrapure water was added and adjusted to the ratio in the test component aqueous extract. After that, the protein amount in each fraction was measured by the BCA method. As a result, the protein amount was large in the order of the fraction with a molecular weight of 10,000 or more, 3,000 or less, and 3,000 to 10,000 (Figure 15). Also, a correlation was recognized between the protein amount and autophagy activity (Figure 16).

[0046] Test Example 10 (Effect of the protein-removed test component on autophagy activation effect) The test component (shochu lees aqueous extract) was deproteinized by phenol-chloroform extraction and ethanol precipitation (Figure 17). Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5Cells were seeded at cells / well, pre-cultured in DMEM medium for 24 hours, then the test component or the test component with the protein removed was added to DMEM and cultured for 24 hours. Thereafter, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). As a result, the autophagy activation effect of shochu lees was suppressed by protein removal (Figure 18).

[0047] Test Example 11 (Effect of heat treatment of test component on autophagy activation effect) The test component (shochu lees aqueous extract) was heated at 100 °C for 30 minutes. Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5 cells / well, pre-cultured in DMEM medium for 24 hours, then the non-heated or heated test component was added to DMEM and cultured for 24 hours. Thereafter, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). As a result, the autophagy activation effect of shochu lees did not change depending on the presence or absence of heating (Figure 19).

[0048] Test Example 12 (Effect of Proteinase K treatment of test component on autophagy activation effect) 1 μg / μL of Proteinase K was added to 50 μg / μL of the freeze-dried sample of shochu lees aqueous extract, for a total of 4 μL, and reacted at 55 °C for 2 days, then separated by polyacrylamide gel electrophoresis, and then silver staining was performed. As a result, the protein of shochu lees could not be completely decomposed by Proteinase K (Figure 20). Also, Caco-2 GFP-LC3-RFP cells were seeded in a 12-well plate at 1.0×10 5 cells / well, pre-cultured in DMEM medium for 24 hours, then the test component untreated or treated with Proteinase K was added to DMEM and cultured for 24 hours. Thereafter, the cells were detached, and the autophagy activity was measured by flow cytometry (judged by the GFP / RFP relative ratio). As a result, the autophagy activation effect of shochu lees did not change regardless of the presence or absence of Proteinase K treatment (Figure 20).

[0049] Test Example 13 (Protective effect against oxidative stress via autophagy by the test component) Wild-type or Atg9 KO HaCaT cells were seeded in a 96-well plate at 1.0×10 4 cells / well and pre-cultured in DMEM medium for 24 hours. Then, the test component was added to DMEM and cultured for 24 hours. Further, H2O2 was added, and after 24 hours, viable cells were measured by MTT assay. The test component was diluted with DMEM to 0.1% using a water extract (100 g / L) prepared by adding 1 g of shochu lees powder to 10 mL of ultrapure water and added (in the case of 0.1%, 4 μL of the extract was added to 4 mL of DMEM). As a result, in the 0.1% shochu lees + H2O2 addition group, the number of cells was significantly higher than that in the H2O2 addition group, and a protective effect was confirmed. On the other hand, in Atg9 KO cells, the effect was canceled (Figure 21a, b).

Claims

1. An autophagy activator comprising, as an active ingredient, one or more selected from shochu lees, lactic acid fermentation products of shochu lees, and extracts thereof.

2. The autophagy activator according to claim 1, wherein the active ingredient is one or more selected from a shochu lees extract and an extract of a lactic acid fermentation product of shochu lees.

3. 2. The autophagy activator according to claim 1, wherein the active ingredient is one or more selected from an agmatine-containing fraction having a molecular weight of 3,000 or less and a protein-containing fraction having a molecular weight of 10,000 or more in a shochu lees extract or a lactic acid fermentation product of shochu lees extract.

4. The autophagy activator according to claim 1, wherein the autophagy activating effect is an autophagy activating effect mediated by mTOR kinase.

5. A food composition or cosmetic composition for activating autophagy, comprising one or more selected from shochu lees, lactic acid fermentation products of shochu lees, and extracts thereof.

6. 6. The food composition or cosmetic composition for activating autophagy according to claim 5, wherein the active ingredient is one or more selected from a shochu lees extract and an extract of a lactic acid fermentation product of shochu lees.

7. 6. The food composition or cosmetic composition for activating autophagy according to claim 5, wherein the active ingredient is one or more selected from an agmatine-containing fraction having a molecular weight of 3,000 or less and a protein-containing fraction having a molecular weight of 10,000 or more in a shochu lees extract or a lactic acid fermentation product of shochu lees extract.

8. The food composition or cosmetic composition for activating autophagy according to claim 5, wherein the autophagy activating effect is an autophagy activating effect mediated by phosphorylation by mTOR kinase.

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