Autophagy activator

Abstract

To provide an autophagy activator that is capable of accelerating autophagy activity and is highly safe.SOLUTION: An autophagy activator according to the present invention contains a koji mold fermentation product of tea leaves or an extract of a koji mold fermentation product of tea leaves.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an autophagy activator containing a koji mold fermentation product of tea leaves or an extract thereof. [Background technology]

[0002] Autophagy is a system in eukaryotic cells that breaks down and recycles unnecessary proteins and organelles. Autophagy not only breaks down proteins and other substances to obtain nutritional sources such as amino acids, but also plays an important role in maintaining cellular homeostasis by purifying the interior of the cell, such as by removing damaged organelles and promoting the turnover of old cells.

[0003] In recent years, as a result of the progress of research on autophagy, it has become clear that autophagy failure is a major cause of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, kidney diseases, and the like (see Patent Documents 1 and 2). According to this, the inhibition of autophagy activity causes the accumulation of harmful proteins and abnormal intracellular organelles in cells, which is the cause of the development of these diseases. In addition to the diseases mentioned above, it is known that the inhibition or decrease of autophagy activity is involved in various diseases such as lifestyle-related diseases and carcinogenesis, and is also a cause of the onset of aging-related diseases.

[0004] On the other hand, in recent years, the value of traditional Japanese koji-fermented foods such as sake, shochu, amazake, miso, and soy sauce using koji has been rediscovered, and koji is becoming more popular. Koji is made by steaming rice, rice bran, wheat, or beans to grow koji mold. For example, rice koji grown with koji mold is called rice koji. It is known that koji mold has the function of producing many types of enzymes that enable the production of fermented foods, but research into new functions of koji mold and development of uses based on the functions are being conducted beyond this field. For example, Patent Document 3 reports that koji mold fermentation products fermented with koji mold have the effect of improving male infertility, and Patent Document 4 reports that koji mold fermentation products fermented with koji mold have the effect of promoting hair growth and hair regrowth. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2023 / 276828 [Patent Document 2] International Publication No. 2022 / 054927 [Patent Document 3] International Publication No. 2020 / 008821 [Patent Document 4] International Publication No. 2020 / 226022 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, a decrease or inhibition of autophagy activity can cause the onset of various diseases, so there is a demand for autophagy activators that can enhance autophagy activity in order to prevent and treat the onset of these diseases and achieve healthy longevity. Furthermore, there is a demand for highly safe autophagy activators that can be safely consumed on a daily basis as medicines or functional foods.

[0007] Furthermore, Patent Documents 3 and 4 each describe koji mold fermentation products obtained by fermenting grains or tea leaves with koji mold. However, no studies have been conducted to date on the use of these koji mold fermentation products to enhance autophagy activity, and the effectiveness of these products has been unknown.

[0008] Therefore, the present invention has been made in consideration of the above-mentioned points, and an object of the present invention is to provide an autophagy activator that can enhance autophagy activity and is also highly safe. [Means for solving the problem]

[0009] In order to solve the above problems, the autophagy activator of the present invention contains a koji mold fermentation product of tea leaves or an extract of the koji mold fermentation product of tea leaves. The koji mold fermentation product and extract have an excellent effect of enhancing autophagy activity and are also useful for treating, preventing, or ameliorating diseases caused by the inhibition of autophagy activity. In addition, the koji mold fermentation product of tea leaves is highly safe because it is obtained from tea leaves and koji mold.

[0010] In addition, the above-mentioned koji mold is preferably yellow koji mold or black koji mold. As a result, a koji mold that is excellent in enhancing autophagy activity is selected as the koji mold that produces the koji mold fermentation product, which is the active ingredient of the autophagy activator of the present invention. In addition, since yellow koji mold and black koji mold are koji molds used in the production of fermented foods, the koji mold fermentation products obtained from these are highly safe and can be ingested on a daily basis.

[0011] Furthermore, the koji mold in the koji mold fermentation product is a live bacteria, which allows the production of an autophagy activator that is excellent in enhancing autophagy activity.

[0012] It is also preferred that the extract of the koji mold fermentation product in the present invention is a water extract or a hydroalcoholic extract of the koji mold fermentation product, which allows an autophagy activator having excellent autophagy activating effects to be obtained safely, easily, and at low cost.

[0013] Furthermore, the method for producing an autophagy activator of the present invention includes a step of seeding koji mold on tea leaves or a fermentation raw material derived from tea leaves, and a step of fermenting the fermentation raw material with the koji mold to obtain a koji mold fermentation product, thereby obtaining an autophagy activator that has an excellent effect of enhancing autophagy activity and is also useful for treating, preventing, or ameliorating diseases caused by suppression of autophagy activity.

[0014] In the method for producing an autophagy activator of the present invention, the above-mentioned koji mold is also preferably yellow koji mold or black koji mold. This allows for the selection of a suitable koji mold that produces a koji mold fermentation product, which is the active ingredient of the autophagy activator of the present invention. Furthermore, since yellow koji mold and black koji mold are koji molds used in the production of fermented foods, an autophagy activator that is highly safe and can be ingested on a daily basis can be obtained.

[0015] Furthermore, the method for producing an autophagy activator of the present invention preferably includes a step of extracting the obtained koji mold fermentation product with water or aqueous alcohol to obtain an extract of the koji mold fermentation product, which makes it possible to safely, easily, and at low cost to obtain an autophagy activator having excellent autophagy activating effects.

[0016] Furthermore, in the method for producing an autophagy activator of the present invention, the koji mold in the koji mold fermentation product is a live bacteria, which makes it possible to produce an autophagy activator that is excellent in enhancing autophagy activity. Effect of the Invention

[0017] According to the present invention, an autophagy activator having the following excellent effects can be provided. (1) It is excellent at enhancing autophagy activity. (2) It can be used to treat, prevent, or ameliorate diseases caused by the inhibition or decrease of autophagy activity. (3) It is highly safe because its active ingredients are tea leaves, commonly known as "tea," and the fermentation product of koji mold, commonly known as a fermented food. (4) By simply extracting the koji mold fermentation product with water or aqueous alcohol, a liquid or beverage-type autophagy activator can be obtained. [Brief description of the drawings]

[0018] [Figure 1] 1 is a flow chart illustrating a schematic diagram of a method for producing an Aspergillus fermentation product of tea leaves and an extract thereof according to an embodiment of the present invention. [Diagram 2] 1 is a graph showing the results of a tfLC3 assay evaluating the effect of enhancing autophagy activity in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] First, with reference to FIG. 1, a method for producing the Aspergillus fermentation product P1 of tea leaves, which is contained in the autophagy activator of the present invention, will be described.

[0020] As shown in FIG. 1, a method for producing a koji mold fermentation product P1 of tea leaves according to an embodiment of the present invention is broadly composed of step S0 of preparing tea leaves as the fermentation raw material, step S1 of adding water to the tea leaves to allow them to absorb it, step S2 of steaming the tea leaves, step S3 of seeding the steamed tea leaves with koji mold, and step S4 of fermentation.

[0021] (Preparing tea leaves) First, the step S0 of preparing tea leaves as the fermentation raw material shown in FIG. 1 will be described. As the fermentation raw material in the present invention, tea leaves from the tea plant (Camellia sinensis) can be used. The tea leaves may include not only tea leaves but also stems and branches harvested from the tea plant. The tea leaves used as the raw material for the koji mold fermentation product of the present invention are preferably raw tea leaves as they are harvested, but may also be tea leaves refrigerated after harvesting, tea leaves heat-treated after harvesting, tea leaves oxidized and fermented with an oxidizing enzyme contained in the tea leaves after harvesting, or tea obtained through a tea manufacturing process. In addition, tea residues remaining after brewing these green teas, oolong teas, black teas, etc. may also be used. In addition, in order to increase the efficiency of the steaming process in the subsequent process and the cultivation of the koji mold, the tea leaves and tea stems may be crushed to a certain size so that they are not too large.

[0022] (Water absorption treatment) Next, the water absorption treatment step S1 will be described. In this step, water is added to the tea leaves or the tea leaves are soaked in water to absorb water. The amount of water absorption is not particularly limited, but specifically, the moisture content of the tea leaves is adjusted to 20 to 60%, preferably 30 to 50%.

[0023] (Steaming treatment) Next, the steaming process S2 will be described. The tea leaves absorbed in the above process are placed in a steamer and covered, and the steamer is heated to expose the tea leaves to steam and heat them. The steaming process time is preferably about 30 to 120 minutes, more preferably about 45 to 90 minutes, and particularly preferably about 60 minutes. By carrying out this process, various bacteria in the raw material are sterilized, and in the subsequent fermentation process, fermentation is carried out with koji mold as the dominant species. In addition, when raw tea leaves are used as the fermentation raw material, various bacteria in the tea leaves are sterilized by carrying out this process, and the oxidizing enzymes contained in the tea leaves are inactivated, so that in the subsequent process, koji mold can be efficiently propagated on the tea leaves. In addition to the steamer, it is also possible to carry out the steaming process using a steaming device that can introduce steam into the device. After the steaming process, the tea leaves are taken out of the steamer, spread evenly on a table, and cooled to about 30 to 40°C. This makes it possible to seed the koji mold on the tea leaves.

[0024] (Seeding of Aspergillus oryzae) Next, step S3 of seeding (inoculating) the koji mold on the tea leaves will be described. In this step, the koji mold is seeded on the tea leaves that have been cooled after the above-mentioned steaming step. In the present invention, the koji mold refers to a microorganism that is mainly used in producing koji fermented foods, and specifically includes yellow koji mold, black koji mold, and white koji mold. Among these, yellow koji mold refers to a group of molds of the genus Aspergillus that form yellow or yellow-green conidia, and is a microorganism that is mainly used in the production of sake, miso, soy sauce, and the like. Specifically, it includes, but is not limited to, Aspergillus oryzae, Aspergillus oryzae val Kawachi (Kawachi yellow koji mold), Aspergillus sojae, and the like. Furthermore, black koji mold refers to a group of molds of the genus Aspergillus that form black or black-brown conidia (a type of asexual spores) that are used in the production of distilled alcoholic beverages such as awamori in Okinawa and potato shochu in Kagoshima. Specific examples of the white koji mold include, but are not limited to, Aspergillus ryukyuensis (Aspergillus awamori), Aspergillus ryukyuensis val kawachi (Kawachi black koji mold), Aspergillus saitoi, Aspergillus inui, Aspergillus usamii, Aspergillus aureus, etc. In addition, the white koji mold refers to a group of molds of the genus Aspergillus that form white-ochre conidia, and specific examples thereof include Aspergillus luchuensis mut. kawachii. In the present invention, it is preferable to use yellow koji mold or black koji mold, and it is more preferable to use yellow koji mold, from the viewpoint of excellent effects of enhancing autophagy activity. Among these, the yellow koji molds Aspergillus oryzae var. kawachii, Aspergillus oryzae, or Aspergillus sojae, the black koji molds Aspergillus luchuensis var. kawachii, Aspergillus luchuensis, or Aspergillus inuii, and combinations thereof are preferably used, with Aspergillus oryzae var. kawachii being particularly preferred.The seeding of these koji molds can be carried out, for example, by mixing the koji mold with tea leaves placed in a sterilized bag and stirring. It is preferable to seed the koji mold with its spores, and it is preferable to add and seed the koji mold so that there are 500,000 or more spores, preferably 1 million or more spores per 1 g (dry weight) of the fermentation raw material. For example, if 1 g of seed koji contains 2 billion spores, about 0.5 g (0.05 w / w%) of seed koji should be added per 1 kg of tea leaves. After seeding the koji mold on the tea leaves, it is preferable to stir well to disperse the koji mold over the entire tea leaves.

[0025] (fermentation) Next, the step S4 of culturing the koji mold seeded on the tea leaves and fermenting them will be described. In this step, the koji mold is propagated on the tea leaves seeded with the koji mold. First, the tea leaves to which the koji mold has been added are placed in a culture chamber kept at about 30°C. As time passes, the fermentation progresses and the temperature of the tea leaves rises, but since koji mold generally has difficulty growing at temperatures above 40°C, the temperature is lowered by ventilation and the amount of ventilation is adjusted so that the temperature of the tea leaves is maintained at 30°C to 42°C, preferably 30°C to 40°C. The fermentation temperature is appropriately adjusted depending on the type of koji mold used for fermentation. Specifically, when yellow koji mold is selected as the koji mold, for example, the temperature of the tea leaves is adjusted to 30°C to 35°C by adjusting the room temperature of the fermentation chamber for 12 to 30 hours from the start of cultivation. Thereafter, the room temperature and the like are similarly adjusted to a slightly higher temperature of 35°C to 40°C, and the koji mold fermentation product P1 is completed when the koji mold is cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably about 40 to 60 hours from the start of culture. On the other hand, when black koji mold or white koji mold is selected as the koji mold, for example, the temperature of the tea leaves is adjusted to 35°C to 40°C by adjusting the room temperature of the fermentation room for 12 to 30 hours from the start of culture. Thereafter, the room temperature and the like are similarly adjusted to a slightly lower temperature of 30°C to 35°C, and the koji mold fermentation product P1 is completed when the koji mold is cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably about 40 to 60 hours from the start of culture.

[0026] The above-mentioned manufacturing steps S1 to S4 of the koji mold fermented product P1 can also be performed using a machine (such as a drum-type automatic koji-making device) that can perform the washing of tea leaves, water absorption, steaming, seeding with koji mold, and fermentation (koji production) within the same device.

[0027] (Koji mold fermentation product) The obtained koji mold fermentation product P1 is the so-called "koji" of tea leaves in which koji mold has proliferated on tea leaves, and the koji mold is contained in the koji mold fermentation product P1 as a live bacterium (including in the form of spores). This koji mold fermentation product P1 has an excellent autophagy activating effect and is useful for treating, preventing, or improving diseases caused by the inhibition of autophagy activity. The koji mold fermentation product P1 obtained by the above-mentioned process contains a certain amount of moisture, but the moisture can be removed by natural drying or low-temperature dehumidification drying so that the koji mold is not killed. It is also possible to pulverize the koji mold fermentation product P1 from which the moisture has been removed into a powder or granules. By reducing the moisture content of the koji mold fermentation product P1, the activity and growth of the koji mold in the koji mold fermentation product P1 can be suppressed during storage, and further, by storing it in a refrigerator, the koji mold fermentation product P1 can be stored for a long period of time with the koji mold still alive. Even if the moisture has been removed, the autophagy activating effect of the koji mold fermentation product P1 can be effectively maintained.

[0028] Next, a method for producing the extract P2 of a koji mold fermentation product contained in the autophagy activator of the present invention will be described. As shown in Figure 1, the method for producing the extract P2 of a koji mold fermentation product according to an embodiment of the present invention mainly comprises step S5 of adding an extraction solvent to the koji mold fermentation product P1 obtained by steps S1 to S4 described above to extract it.

[0029] (extraction) The extraction step S5 will be described in detail below. In this extraction step S5, an extraction solvent is added to the koji mold fermentation product P1 to perform an extraction process, and an extract P2 of the koji mold fermentation product is obtained. The extraction solvent is not particularly limited as long as it can extract a component having an effect of enhancing autophagy activity from the koji mold fermentation product P1, and examples of the extraction solvent include polar protic solvents such as water and alcohols, polar aprotic solvents such as dimethyl sulfoxide and acetone, and non-polar solvents such as hexane. Among these, water or alcohols, and water-containing alcohols obtained by combining these, are preferably used as the extraction solvent from the viewpoints of extraction efficiency, safety to the human body, and ease of handling. Among these, examples of alcohol include ethanol, 1,3-butylene glycol, isopropanol, propylene glycol, and glycerin, and it is also possible to use one or more of these in combination. Furthermore, as the above-mentioned ethanol, in addition to purified ethanol, it is also possible to use a fermentation liquid obtained by alcohol fermentation. Specifically, brewed alcohol or distilled alcohol obtained by distilling it can be used, and among these, distilled alcohol can be preferably used. The alcohol concentration of the aqueous alcohol is preferably 1 to 50 v / v%, more preferably 3 to 30 v / v%, and particularly preferably 5 to 15 v / v%. Note that the extraction solvent may contain other components such as a pH adjuster, a preservative, an extraction aid, and the like, in addition to the above-mentioned water, alcohols, or aqueous alcohol, as long as the extraction of useful components from the koji mold fermentation product P1 is not hindered.

[0030] As a method for extracting the koji mold fermentation product P1, it is preferable to add the koji mold fermentation product P1 to an extraction solvent, soak it, and extract it. When the koji mold fermentation product P1 is dried to a water content of less than 10%, it is preferable to add 1 g to 300 g of it, more preferably 5 g to 150 g, and particularly preferably 10 g to 100 g, per 1 L of extraction solvent. As for extraction conditions, in order to maintain the activity of various proteins, enzymes, and the like contained in the koji mold fermentation product P1, the extraction temperature is preferably about 0 to 30°C, more preferably about 2 to 20°C, and particularly preferably 4 to 10°C. The extraction time is preferably about 2 hours to 5 days, more preferably about 6 hours to 3 days, and particularly preferably about 12 hours to 1.5 days.

[0031] After the above-mentioned extraction treatment, the residue is removed by decantation, centrifugation, filtration, etc. to obtain extract P2 of the koji mold fermentation product. The extract P2 of the koji mold fermentation product of the present invention includes not only the extract P2 of the koji mold fermentation product itself obtained in extraction step S5, but also a concentrated liquid obtained by subjecting extract P2 to a concentration treatment such as vacuum concentration, and a solid or powdered form obtained by subjecting extract P2 of the koji mold fermentation product to a drying treatment such as freeze-drying.

[0032] In the present invention, activation of autophagy refers to enhancing autophagy activity in cells, and means that autophagy activity is enhanced compared to a control in which the autophagy activator of the present invention is not added or administered. The enhancing effect of this autophagy activity can be evaluated, for example, by the tfLC3 assay performed in the Examples described below, without particular limitation. More specifically, the GFP / RFP ratio in the tfLC3 assay is preferably less than 1.00, more preferably 0.90 or less, and even more preferably 0.80 or less.

[0033] The autophagy activator of the present invention contains the above-mentioned Koji mold fermented product P1 of tea leaves fermented with Koji mold or the extract P2 of the Koji mold fermented product of tea leaves as an active ingredient, and has an effect of enhancing autophagy activity. Therefore, the autophagy activator containing the Koji mold fermented product or the extract of the Koji mold fermented product of the present invention can be used as a medicine, quasi-drug, or food for treating, preventing, or improving diseases caused by the inhibition or decrease of autophagy activity. Among these, foods also include supplements, health foods, functional foods, and foods for specified health uses. Since the autophagy activator of the present invention contains the Koji mold fermented product used in the production of fermented foods as an active ingredient, it is highly safe and can be used easily and safely regardless of age, sex, chronic illness, etc.

[0034] The dosage of the autophagy activator of the present invention cannot be generally defined because it varies depending on the target preventive, ameliorative or therapeutic effect, the administration method, the type of disease, etc., but the usual daily oral dosage is about 10 to 1000 mg / kg body weight, preferably about 20 to 500 mg / kg body weight, and more preferably about 50 to 300 mg / kg body weight as the Koji mold fermentation product P1, which may be administered in 1 to 3 divided doses from the start of administration. Also, the daily oral dosage is about 10 to 1000 mg / kg body weight, preferably about 20 to 500 mg / kg body weight, and more preferably about 50 to 300 mg / kg body weight as the Koji mold fermentation product extract P2, which may be administered in 1 to 3 divided doses from the start of administration.

[0035] The autophagy activator of the present invention can be prepared in various forms by conventional methods. In this case, it can be formulated using additives that are accepted as pharmaceutical additives, such as carriers and excipients for normal formulations. In addition, in order to improve the bioavailability and stability of the koji mold fermentation product and extract thereof according to the present invention, a drug delivery system including formulation techniques such as microcapsules, micronization, and inclusion with cyclodextrin, etc., can also be used.

[0036] Furthermore, the autophagy activator can be used in the form of tablets, granules, capsules, or liquid for internal use, but is preferably used in a form suitable for absorption from the digestive tract. Conventional formulation techniques can also be used to provide a preparation in a desired form for reasons of distribution, storage, and the like.

[0037] In addition, the autophagy activator of the present invention can be used as a food composition in any form, such as a supplement form such as a tablet, capsule, granule, or syrup, a beverage, a confectionery such as candy, gum, or chocolate, bread, rice porridge, cereal, noodles, jelly, soup, dairy products, or seasonings. When used as a food composition in this way, it is also possible to combine various other active ingredients and nutrients such as vitamins, minerals, or amino acids, within a range that does not affect the efficacy of the active ingredient of the present invention. Foods developed from the food composition of the present invention include supplements, health foods, functional foods, and foods for specified health uses. In addition, the daily intake of the food composition of the present invention is preferably about 10 to 1000 mg / kg body weight, more preferably about 20 to 500 mg / kg body weight, and further preferably about 50 to 300 mg / kg body weight, as the above-mentioned koji mold fermentation product P1. Furthermore, the amount of extract P2 from the koji mold fermentation product is preferably about 10 to 1000 mg / kg body weight, more preferably about 20 to 500 mg / kg body weight, and even more preferably about 50 to 300 mg / kg body weight.

[0038] EXAMPLES Next, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples and comparative examples. EXAMPLES

[0039] [Example 1] 1. Preparation of Aspergillus fermented tea leaves In this example, raw tea leaves picked from tea plants were used as the fermentation raw material. Water was added to the tea leaves so that the moisture content was about 50%, and the tea leaves were subjected to a water absorption treatment, and then the leaves were placed in a pressurized steam kettle and steamed. The steaming treatment conditions were 115°C for 60 minutes, and the steaming treatment was performed twice with one cooling in between. After that, the tea leaves were cooled to 30°C, and then seed koji of black koji mold (Aspergillus luchuensis var. kawachii) (product name: Kawachi Genichiro Shoten Kawachikin G-type black koji, 2 billion spores / 1g of seed koji) was added to the tea leaves at 0.1% by weight of the weight of the tea leaves, which are the fermentation raw material, and mixed, and koji was made for 3 days. The koji-making temperature was adjusted so that the temperature of the tea leaves was 35°C to 40°C for 24 hours after the addition of the seed koji, and then for the next 48 hours, the temperature of the tea leaves was adjusted slightly lower, to 30°C to 35°C. The black koji mold fermentation product obtained after three days of koji-making was then subjected to a low-temperature drying process to a moisture content of 8%, yielding a black koji mold fermentation product of tea leaves.

[0040] A white koji mold fermentation product of tea leaves was obtained in the same manner as described above, except that a white koji mold (Aspergillus luchuensis mut. kawachii) seed koji (product name: Kawachikin White Koji, Kawachi Genichiro Shoten, 2 billion spores / 1 g seed koji) was used instead of the black koji mold seed koji used in the preparation of the black koji mold fermentation product of tea leaves described above.

[0041] Similarly, instead of the black koji mold seed koji used in the preparation of the black koji mold fermentation product of tea leaves described above, a yellow koji mold (Aspergillus oryzae var. kawachii) seed koji (product name: Kawachikin Yellow Koji, Kawachi Genichiro Shoten, 2 billion spores / 1 g of seed koji) was used, and the koji-making temperature was adjusted so that the temperature of the tea leaves was 30°C to 35°C for 24 hours after the addition of the seed koji, and then adjusted slightly higher to 35°C to 40°C for the following 48 hours, to obtain a yellow koji mold fermentation product of tea leaves in the same manner as described above.

[0042] For comparison, raw tea leaves were treated in the same manner as in the case of using Aspergillus oryzae, except that no Aspergillus oryzae was added (seeded), to obtain tea leaf samples.

[0043] [Example 2] 2. Preparation of extract from Aspergillus fermented tea leaves In the tfLC3 assay for evaluating the enhancing effect of autophagy activity, which will be described in detail in Example 3, an extract of the Aspergillus fermentation product of tea leaves obtained in Example 1 was prepared as a test sample to be added to the culture medium of HeLa-tfLC3 cells. In the tfLC3 assay in Example 3, the amount added to the culture medium of HeLa-tfLC3 cells is set to 0.1%, so it is necessary to prepare a test sample with a concentration 1000 times higher than the final concentration in the culture medium. Therefore, each extract sample was prepared by concentrating the extract obtained by extraction with a solvent using an evaporator.

[0044] Specifically, in this example, distilled water was used as the extraction solvent. The koji mold fermentation product of tea leaves obtained in Example 1 was crushed with a mill, and 100 g of the koji mold fermentation product was added to 2000 mL of distilled water. This was left to stand in a refrigerator at 4°C for 19 hours for extraction treatment. After 19 hours, it was filtered using No. 2 filter paper, and the filtrate was collected. The collected filtrate was concentrated using an evaporator to obtain an extract of the koji mold fermentation product of tea leaves. The results are shown in Table 1 below. Note that the koji mold fermentation product concentration (w / w%) in Table 1 below indicates the amount of koji mold fermentation product relative to a mixture of distilled water as a solvent and the koji mold fermentation product.

[0045] [Table 1]

[0046] [Comparative Example 1] 3. Preparation of rice fermentation product In this comparative example, a rice koji mold fermentation product, known as "rice koji", was prepared using the same steps and methods as in Example 1, except that raw rice was used as the fermentation raw material instead of the raw tea leaves used in Example 1. The same three types of koji mold as in Example 1 were used for fermentation, and a black koji mold rice fermentation product, a white koji mold rice fermentation product, and a yellow koji mold rice fermentation product were obtained. In addition, for comparison, raw rice was processed using the same steps and methods as above to obtain rice samples, except that no koji mold was added (seeded).

[0047] [Comparative Example 2] 4. Preparation of extract from rice fermentation product In this comparative example, distilled water was also used as the extraction solvent. The rice koji mold fermentation product obtained in Comparative Example 1 was crushed with a mill, and 100 g of the koji mold fermentation product was added to 1000 mL of distilled water. This was left to stand in a refrigerator at 4°C for 19 hours for extraction treatment. After 19 hours, it was filtered through No. 2 filter paper and the filtrate was collected. The collected filtrate was concentrated using an evaporator to obtain an extract of the rice koji mold fermentation product. The results are shown in Table 2 below. Note that the koji mold fermentation product concentration (w / w%) in Table 2 below indicates the amount of koji mold fermentation product relative to a mixture of distilled water as the solvent and the koji mold fermentation product.

[0048] [Table 2]

[0049] [Example 3] 5. Evaluation of enhancement of autophagy activity A test was carried out to evaluate the enhancing effect of autophagy activity for each extract sample prepared in the above-mentioned Example 2 and Comparative Example 2. In detail, a tfLC3 assay was carried out to evaluate autophagy activity using a tfLC3 fluorescent probe in which a red fluorescent protein mRFP (RFP: Red Fluorescent Protein) and a green fluorescent protein EGFP (GFP: Green Fluorescent Protein) are tandemly fused to the amino terminus of LC3 (MAP1LC3: Microtubule-associated protein 1 light chain 3), an autophagosome marker protein (see Autophagy, Vol. 3, pp. 452-460). The fluorescence of the GFP signal (green) of tfLC3 is attenuated in the environment inside the lysosome, whereas the RFP signal (red) is stable even in the same environment. In the tfLC3 assay, when autophagosomes mature into autolysosomes, they turn red due to GFP quenching. By taking advantage of this property of tfLC3, the GFP / RFP ratio in cells can be analyzed to determine whether a sample added to the culture medium has the ability to activate autophagy.

[0050] Specifically, HeLa cells (HeLa-tfLC3 cells) stably expressing tfLC3 were established, and the HeLa-tfLC3 cells were seeded in a 96-well plate (PerkinElmer product, product number: 6055308, PhenoPlate (registered trademark) 96-well microplates) at 3000 cells / well, and cultured for 24 hours in a 5% CO2 incubator set at 37°C. As the growth medium, DMEM medium (Sigma-Aldrich product, product number: D6546) prepared by adding 10% FBS and 4 mM L-glutamine was used. After 24 hours, the growth medium was removed from each well and replaced with a growth medium containing 0.1% of each extract sample. After replacement, the cells were cultured for 24 hours in a 5% CO2 incubator set at 37°C. As negative controls, we also tested a solvent control (0.1% distilled water, 0.1% DMSO) containing only the solvent used for sample extraction and dissolution, and a blank control (Blank) containing only growth medium. Torin1, an mTOR inhibitor that induces autophagy by blocking signals necessary for cell growth and proliferation and simulating a state of cell starvation, was selected as a positive control substance, and Torin1 dissolved in DMSO to a specified concentration was tested as a positive control. After 24 hours, the cells were washed and fixed with paraformaldehyde, and observed and analyzed under a fluorescent microscope. The test was performed with N=5 for each test group and positive control group, and N=15 for the negative control group.

[0051] In the tfLC3 assay, when autophagosomes mature into autolysosomes, GFP is quenched but RFP is maintained, so the GFP / RFP value decreases when autophagy activity is enhanced. Therefore, the GFP / RFP value measured in the solvent control (0.1% distilled water, 0.1% DMSO) containing only the solvent used for sample extraction and dissolution was set to 1, and the ratio of the GFP / RFP value measured in the test section containing each sample was calculated. The results are shown in Figure 2 and Table 3 below.

[0052] [Table 3]

[0053] According to these results, all of the tea leaves fermented with Aspergillus oryzae had a GFP / RFP ratio of less than 1, indicating an effect of enhancing autophagy activity. On the other hand, even though they were the same Aspergillus oryzae fermented products, all of the rice Aspergillus oryzae fermented products had a GFP / RFP ratio of about 1, which was almost the same value as the negative control, indicating no effect on autophagy activity. Among the Aspergillus oryzae fermented products of tea leaves, the GFP / RFP ratio of Aspergillus oryzae fermented product of tea leaves was particularly significant at 0.759, indicating a greater enhancement of autophagy activity than the positive control 10 nM Torin1. In addition, the GFP / RFP ratio of Aspergillus oryzae fermented product of tea leaves was also found to be 0.874, indicating a high reduction rate. These results indicate that the Aspergillus oryzae fermented product of tea leaves according to the present invention can be used as an autophagy activator that enhances autophagy activity.

[0054] The present invention is not limited to the above-described embodiments or examples, and the technical scope of the present invention includes various modified forms of design within the scope that does not deviate from the gist of the invention described in the claims.

Claims

1. An autophagy activator characterized by containing a koji mold fermentation product of tea leaves or an extract of the koji mold fermentation product of tea leaves.

2. The autophagy activator according to claim 1, characterized in that the koji mold is Aspergillus oryzae.

3. The autophagy activator according to claim 1 or 2, characterized in that the koji mold in the koji mold fermentation product is a live koji mold.

4. The autophagy activator according to claim 1 or 2, characterized in that the extract of the koji mold fermentation product is a water extract or a hydroalcoholic extract of the koji mold fermentation product.

5. A method for producing an autophagy activator, comprising the steps of seeding koji mold on tea leaves or a fermentation raw material derived from tea leaves, and fermenting the fermentation raw material with the koji mold to obtain a koji mold fermentation product.

6. The method for producing an autophagy activator according to claim 5, characterized in that the koji mold is Aspergillus oryzae or Aspergillus niger.

7. The method for producing an autophagy activator according to claim 5 or 6, further comprising a step of extracting the koji mold fermentation product with water or aqueous alcohol to obtain an extract of the koji mold fermentation product.

8. The method for producing an autophagy activator according to claim 7, wherein the koji mold in the koji mold fermentation product is a live koji mold.

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