Autophagy activator

Abstract

Provided is an autophagy activator that is capable of accelerating autophagy activity and is highly safe.　This autophagy activator contains a koji mold fermentation product of tea leaves or an extract of a koji mold fermentation product of tea leaves.

Description

Autophagy activators

[0001] The present invention relates to an autophagy activator containing a tea leaf fermentation product with koji mold or an extract thereof.

[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 nutrients 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] Recent advances in research on autophagy have revealed that autophagy deficiency is a major cause of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as kidney diseases (see Patent Documents 1 and 2). According to these findings, inhibition of autophagy activity leads to the intracellular accumulation of harmful proteins and abnormal organelles, which causes these diseases. In addition to the diseases mentioned above, inhibition or reduction of autophagy activity is known to be involved in various diseases such as lifestyle-related diseases and carcinogenesis, as well as the onset of aging-related diseases.

[0004] Meanwhile, in recent years, the value of traditional Japanese koji-fermented foods, such as sake, shochu, amazake, miso, and soy sauce, has been rediscovered, leading to increased popularity of koji. Koji is made by steaming rice, rice bran, barley, or beans to grow koji mold. For example, koji mold grown on rice is called rice koji. While koji mold is known to produce a variety of enzymes that enable the production of fermented foods, research into new functions of koji mold and the development of applications based on these functions are progressing beyond this field. For example, Patent Document 3 reports that koji mold fermentation products have the effect of improving male infertility, and Patent Document 4 reports that koji mold fermentation products have the effect of promoting hair growth and hair restoration.

[0005] International Publication No. WO 2023 / 276828 International Publication No. WO 2022 / 054927 International Publication No. WO 2020 / 008821 International Publication No. WO 2020 / 226022

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

[0007] Furthermore, Patent Documents 3 and 4 each describe koji mold fermentation products obtained by fermenting grains and tea leaves with koji mold. However, the use of these koji mold fermentation products to enhance autophagy activity has not been investigated to date, and their effectiveness has been unknown.

[0008] Therefore, the present invention has been made in view 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.

[0009] To solve the above problems, the autophagy activator of the present invention contains a koji mold-fermented product of tea leaves or an extract of the koji mold-fermented product of tea leaves. The koji mold-fermented product and extract have excellent autophagy activity enhancing properties and are useful for treating, preventing, or ameliorating diseases caused by the inhibition of autophagy activity. Furthermore, the koji mold-fermented product of tea leaves is highly safe because it is obtained from tea leaves and koji mold.

[0010] Furthermore, the koji mold is preferably yellow koji mold or black koji mold. This allows for the selection of koji molds that have excellent autophagy-enhancing properties to produce the koji mold fermentation product, which is the active ingredient of the autophagy activator of the present invention. Furthermore, because yellow koji mold and black koji mold are used in the production of fermented foods, the koji mold fermentation products obtained from these molds are highly safe and can be consumed on a daily basis.

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

[0012] It is also preferred that the extract of the koji mold fermentation product of the present invention is a water extract or a hydroalcoholic extract of the koji mold fermentation product, which allows an autophagy activator with 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 the steps of inoculating tea leaves or tea-leaf-derived fermentation raw materials with koji mold and fermenting the fermentation raw materials with the koji mold to obtain a koji mold fermentation product. This produces an autophagy activator that has an excellent autophagy-enhancing effect and is also useful for treating, preventing, or ameliorating diseases caused by the inhibition of autophagy activity.

[0014] In the method for producing an autophagy activator of the present invention, the koji mold is preferably yellow koji mold or black koji mold. This allows for the selection of a koji mold that is suitable for producing a koji mold fermentation product, which is the active ingredient of the autophagy activator of the present invention. Furthermore, because 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 taken on a daily basis can be obtained.

[0015] The method for producing an autophagy activator of the present invention preferably also 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 allows for the safe, easy, and low-cost production of an autophagy activator with 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 koji mold, which allows the production of an autophagy activator that is excellent at enhancing autophagy activity.

[0017] The present invention provides an autophagy activator with the following excellent effects: (1) It has excellent effects in enhancing autophagy activity. (2) It can be used to treat, prevent, or improve diseases caused by the suppression or decrease of autophagy activity. (3) It is highly safe because its active ingredients are tea leaves, commonly known as "tea," and a product fermented by koji mold, a popular fermented food. (4) A liquid or beverage-type autophagy activator can also be obtained simply by extracting the koji mold fermentation product with water or aqueous alcohol.

[0018] 1 is a flow chart showing an outline of a method for producing an Aspergillus oryzae fermentation product of tea leaves and an extract thereof according to an embodiment of the present invention. 2 is a graph showing the results of a tfLC3 assay for evaluating the enhancing effect of autophagy activity in Example 3.

[0019] First, with reference to FIG. 1 , a method for producing the koji mold-fermented tea leaf product P1 contained in the autophagy activator of the present invention will be described.

[0020] As shown in FIG. 1 , the method for producing a koji mold fermented 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 carrying out fermentation.

[0021] (Preparation of Tea Leaves) First, step S0 of preparing tea leaves as the fermentation raw material shown in FIG. 1 will be described. Tea leaves from the tea plant (Camellia sinensis) can be used as the fermentation raw material in the present invention. 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-fermented product of the present invention are preferably fresh tea leaves as they are harvested. However, they may also be tea leaves refrigerated after harvesting, tea leaves heat-treated after harvesting, tea leaves oxidized and fermented with the oxidizing enzymes contained in the tea leaves after harvesting, or tea obtained through a tea-making process. Tea residues remaining after brewing green tea, oolong tea, black tea, or the like can also be used. Furthermore, to improve the efficiency of the subsequent steaming process and koji mold cultivation, the tea leaves or 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 treatment step S2 will be described. The tea leaves absorbed in the above-mentioned step are placed in a steamer, the lid is closed, and the steamer is heated to expose the tea leaves to steam and heat them. The steaming treatment time is preferably approximately 30 to 120 minutes, more preferably approximately 45 to 90 minutes, and particularly preferably approximately 60 minutes. By performing this step, undesirable bacteria in the raw materials are sterilized, and in the subsequent fermentation step, fermentation is carried out with koji mold as the dominant species. Furthermore, when raw tea leaves are used as the fermentation raw material, this step sterilizes undesirable bacteria in the tea leaves and inactivates the oxidizing enzymes contained in the tea leaves, allowing koji mold to efficiently propagate on the tea leaves in the subsequent step. In addition to a steamer, steaming treatment can also be carried out using a steaming device capable of introducing steam into the device. After steaming, the tea leaves are removed from the steamer, spread evenly on a table, and cooled to approximately 30 to 40°C. This allows koji mold to be seeded on the tea leaves.

[0024] (Seeding of Koji Mold) Next, step S3 of seeding (inoculating) koji mold onto tea leaves will be described. In this step, koji mold is seeded onto tea leaves that have been cooled after the steaming step described above. In the present invention, koji mold refers to a microorganism primarily used in producing koji-fermented foods, and specific examples include 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 are primarily used in the production of sake, miso, soy sauce, and the like. Specific examples include, but are not limited to, Aspergillus oryzae, Aspergillus oryzae var. Kawachi (Kawachi yellow koji mold), and Aspergillus sojae. Furthermore, black koji mold refers to a group of molds of the genus Aspergillus that form black or dark brown conidia (a type of asexual spore) and are used in the production of distilled spirits such as awamori in Okinawa and sweet potato shochu in Kagoshima. Specific examples include, but are not limited to, Aspergillus ryukyuensis (Aspergillus awamori), Aspergillus ryukyuensis val. kawachi (Kawachi black koji mold), Aspergillus saitoi, Aspergillus inui, Aspergillus usami, and Aspergillus aureus. White koji mold refers to a group of fungi of the genus Aspergillus that form white-ochre conidia, and specific examples include Aspergillus kawachii (Aspergillus luchuensis mut. kawachii). In the present invention, yellow koji mold or black koji mold is preferably used, with yellow koji mold being more preferred, due to its superior effect in enhancing autophagy activity.Among these, yellow koji molds such as Aspergillus oryzae var. kawachii, Aspergillus oryzae, or Aspergillus sojae, black koji molds such as Aspergillus luchuensis var. kawachii, Aspergillus luchuensis, or Aspergillus inuii, and combinations thereof are preferably used, with Aspergillus oryzae var. kawachii being particularly preferred. 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 preferred to sow koji mold spores, and to add and sow koji mold spores so that there are 500,000 or more, preferably 1,000,000 or more, per 1 g (dry weight) of fermentation raw material. For example, if 1 g of seed koji contains 2 billion spores, then about 0.5 g (0.05 w / w%) of seed koji should be added to 1 kg of tea leaves. After seeding the koji mold on the tea leaves, it is preferable to thoroughly stir the mixture to disperse the koji mold throughout the tea leaves.

[0025] (Fermentation) Next, we will explain step S4, which involves culturing koji mold seeded on tea leaves and fermenting them. In this step, the koji mold is propagated on the seeded tea leaves. First, the tea leaves to which the koji mold has been added are placed in a culture chamber maintained at around 30°C. As fermentation progresses and the temperature of the tea leaves rises over time, koji mold generally has difficulty growing above 40°C. Therefore, the temperature is lowered by ventilation, and the ventilation volume is adjusted so that the temperature of the tea leaves remains between 30°C and 42°C, preferably between 30°C and 40°C. The fermentation temperature is adjusted appropriately 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 for 12 to 30 hours after the start of cultivation by adjusting the room temperature of the fermentation chamber, for example. Thereafter, the room temperature and other conditions are similarly adjusted to a slightly higher temperature of 35°C to 40°C, and the koji mold fermentation product P1 is considered complete when the koji mold has been cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably 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 chamber, for 12 to 30 hours from the start of culture. Thereafter, the room temperature and other conditions are similarly adjusted to a slightly lower temperature of 30°C to 35°C, and the koji mold fermentation product P1 is considered complete when the koji mold has been cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably 40 to 60 hours from the start of culture.

[0026] The above-described steps S1 to S4 of producing the koji mold fermented product P1 can also be performed using a machine (for example, 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 so-called "koji" of tea leaves, in which koji mold has proliferated on tea leaves. The koji mold is contained in the koji mold fermentation product P1 as viable bacteria (including spores). This koji mold fermentation product P1 has an excellent autophagy activation effect and is useful for treating, preventing, or ameliorating diseases caused by the inhibition of autophagy activity. The koji mold fermentation product P1 obtained by the above-described process contains a certain amount of moisture. However, the moisture can be removed by natural drying or low-temperature dehumidification drying to prevent the koji mold from dying. The koji mold fermentation product P1 from which the moisture has been removed can also be pulverized into powder or granules. Reducing the moisture content of the koji mold fermentation product P1 allows the koji mold activity and growth in the koji mold fermentation product P1 to be suppressed during storage. Furthermore, refrigerated storage enables the koji mold fermentation product P1 to be stored for long periods while the koji mold remains viable. The autophagy activation effect of the koji mold fermentation product P1 can be effectively maintained even after the moisture has been removed.

[0028] Next, we will explain the method for producing the koji mold fermentation product extract P2 contained in the autophagy activator of the present invention. As shown in Figure 1, the method for producing the koji mold fermentation product extract P2 according to an embodiment of the present invention generally comprises step S5, in which an extraction solvent is added 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, and an extraction treatment is performed to obtain an extract P2 of the koji mold fermentation product. The extraction solvent is not particularly limited as long as it can extract components that enhance autophagy activity from the koji mold fermentation product P1. Examples of the extraction solvent include polar protic solvents such as water and alcohols, polar aprotic solvents such as dimethyl sulfoxide and acetone, and nonpolar solvents such as hexane. Of these, water or alcohols, and aqueous alcohols formed by combining these, are preferred as extraction solvents from the standpoints of extraction efficiency, safety for the human body, and ease of handling. Examples of alcohols include ethanol, 1,3-butylene glycol, isopropanol, propylene glycol, and glycerin, and these can be used alone or in combination. Furthermore, in addition to purified ethanol, fermentation liquid obtained by alcoholic fermentation can also be used as the ethanol. Specifically, brewed alcoholic beverages and distilled alcoholic beverages obtained by distilling brewed alcoholic beverages can be used, and distilled alcoholic beverages are preferred. 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%. In addition to the water, alcohols, or aqueous alcohol described above, the extraction solvent may also contain other components such as a pH adjuster, a preservative, or an extraction aid, as long as the components do not interfere with the extraction of useful components from the koji mold fermentation product P1.

[0030] A preferred method for extracting the koji mold fermentation product P1 is to add the koji mold fermentation product P1 to an extraction solvent and allow it to soak and extract. When the koji mold fermentation product P1 is a dried product with a moisture content of less than 10%, 1 g to 300 g of this is preferably added to 1 L of extraction solvent, more preferably 5 g to 150 g, and particularly preferably 10 g to 100 g. Furthermore, in terms of extraction conditions, in order to maintain the activity of the 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. Furthermore, 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. Examples of extract P2 of the koji mold fermentation product of the present invention include extract P2 itself obtained in extraction step S5, a concentrated liquid obtained by subjecting extract P2 to a concentration treatment such as vacuum concentration, and extract P2 of the koji mold fermentation product obtained by subjecting it to a drying treatment such as freeze-drying to form a solid or powder.

[0032] In the present invention, "activating 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. This enhancing effect on 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 as an active ingredient the koji mold fermentation product P1 of tea leaves fermented with koji mold or the extract P2 of the koji mold fermentation product of tea leaves, and has the effect of enhancing autophagy activity. Therefore, the autophagy activator containing the koji mold fermentation product or the extract of the koji mold fermentation product of the present invention can be used as a pharmaceutical, quasi-drug, or food for treating, preventing, or ameliorating diseases caused by the suppression or decrease of autophagy activity. Examples of such foods include supplements, health foods, functional foods, and foods for specified health uses. Because the autophagy activator of the present invention contains as an active ingredient a koji mold fermentation product used in the production of fermented foods, it is highly safe and can be used easily and with confidence regardless of age, gender, or chronic illnesses.

[0034] The dosage of the autophagy activator of the present invention cannot be generally defined because it varies depending on factors such as the desired preventive, ameliorative, or therapeutic effect, the method of administration, and the type of disease. However, a typical daily oral dosage is approximately 10 to 1,000 mg / kg body weight, preferably approximately 20 to 500 mg / kg body weight, and more preferably approximately 50 to 300 mg / kg body weight, of the koji mold fermentation product P1, which may be administered in one to three divided doses from the start of administration. Furthermore, the daily oral dosage is approximately 10 to 1,000 mg / kg body weight, preferably approximately 20 to 500 mg / kg body weight, and more preferably approximately 50 to 300 mg / kg body weight, of the koji mold fermentation product extract P2, which may be administered in one to three divided doses from the start of administration.

[0035] The autophagy activators of the present invention can be prepared in various forms using conventional methods. In this case, formulations can be made using additives accepted as pharmaceutical additives, such as carriers and excipients typically used in pharmaceutical preparations. Furthermore, to improve the bioavailability and stability of the koji mold fermentation product and extract thereof of the present invention, drug delivery systems, including formulation techniques such as microencapsulation, micronization, and inclusion with cyclodextrin, can also be used.

[0036] Furthermore, the autophagy activator can be used in the form of tablets, granules, capsules, or oral liquid preparations, but is preferably used in a form suitable for absorption from the gastrointestinal tract. Furthermore, conventional formulation techniques can be used to provide a formulation in a desired form for reasons such as distribution and storage stability.

[0037] Furthermore, the autophagy activator of the present invention can be used as a food composition in any form, including supplements such as tablets, capsules, granules, and syrups; beverages; confectioneries such as candy, gum, and chocolate; bread; rice porridge; cereals; noodles; jellies; soups; dairy products; and seasonings. When used as a food composition, it is possible to combine various other active ingredients and nutrients such as vitamins, minerals, and amino acids, as long as the efficacy of the active ingredient of the present invention is not affected. Foods developed from the food composition of the present invention include supplements, health foods, functional foods, and foods for specified health uses. The daily intake of the food composition of the present invention is preferably about 10 to 1,000 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, of the koji mold fermented product P1. 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] 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.

[0039] Example 1 1. Preparation of Koji Mold Fermented Tea Leaves In this example, fresh tea leaves harvested from tea plants were used as the fermentation raw material. Water was added to the tea leaves to bring their moisture content to approximately 50%, and the tea leaves were then subjected to a water absorption treatment. They were then placed in a pressurized steamer and steamed. The steaming conditions were 115°C for 60 minutes, with two separate steaming treatments, each with a cooling interval. The tea leaves were then cooled to 30°C, and a koji starter made from black koji mold (Aspergillus luchuensis var. kawachii) (product name: Kawachi Genichiro Shoten, Kawachikin G-type black koji, 2 billion spores per gram of koji starter) was added to the tea leaves at a concentration of 0.1% by weight of the tea leaves, and the mixture was allowed to ferment 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 adjusted slightly lower for the next 48 hours to 30° C. to 35° C. The black koji mold fermentation product obtained after three days of koji-making was then subjected to low-temperature drying treatment 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: Kawachi Genichiro Shoten Kawachikin White Koji, 2 billion spores / 1 g of 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, a yellow koji mold fermentation product of tea leaves was obtained in the same manner as described above, except that starter koji of yellow koji mold (Aspergillus oryzae var. kawachii) (product name: Kawachi Kin Yellow Koji, manufactured by Kawachi Genichiro Shoten, 2 billion spores / 1 g of starter koji) was used instead of the black koji mold starter koji used in the preparation of the black koji mold fermentation product of tea leaves described above, 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 starter koji, and then adjusted slightly higher for the subsequent 48 hours to 35°C to 40°C.

[0042] For comparison, raw tea leaves were treated in the same manner as in the case of using yellow koji mold, except that no koji mold was added (seeded), to obtain a tea leaf sample.

[0043] Example 2 2. Preparation of Extract of Aspergillus oryzae Fermented Tea Leaves An extract of Aspergillus oryzae fermented 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 for evaluating the effect of enhancing autophagy activity, as described in detail in Example 3. In the tfLC3 assay in Example 3, the amount added to the culture medium of HeLa-tfLC3 cells was set at 0.1%, so it was necessary to prepare a test sample at a concentration 1000 times the final concentration in the culture medium. Therefore, each extract sample was prepared by first extracting with a solvent and concentrating the extract obtained 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 using 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. After 19 hours, the mixture was filtered using No. 2 filter paper, and the filtrate was recovered. The recovered 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 the mixture of distilled water as the solvent and the koji mold fermentation product.

[0045]

[0046] Comparative Example 1 3. Preparation of Koji Mold Fermented Rice In this comparative example, a koji mold fermented rice 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 fermented rice product, a white koji mold fermented rice product, and a yellow koji mold fermented rice product were obtained. For comparison, raw rice was treated using the same steps and methods as above, except that no koji mold was added (seeded), to obtain rice samples.

[0047] Comparative Example 2 4. Preparation of an extract of koji mold fermented rice In this comparative example, distilled water was also used as the extraction solvent. The koji mold fermented rice product obtained in Comparative Example 1 was crushed using a mill, and 100 g of the koji mold fermented product was added to 1,000 mL of distilled water. This was left to stand in a refrigerator at 4°C for 19 hours for extraction. After 19 hours, the mixture was filtered through No. 2 filter paper, and the filtrate was recovered. The recovered filtrate was concentrated using an evaporator to obtain an extract of koji mold fermented rice. The results are shown in Table 2 below. Note that the koji mold fermented product concentration (w / w%) in Table 2 below indicates the amount of koji mold fermented product relative to the mixture of distilled water as the solvent and the koji mold fermented product.

[0048]

[0049] Example 3 5. Evaluation of the effect of enhancing autophagy activity Tests were conducted to evaluate the effect of enhancing autophagy activity on each of the extract samples prepared in Example 2 and Comparative Example 2 described above. Specifically, a tfLC3 assay was performed to evaluate autophagy activity using a tfLC3 fluorescent probe in which a red fluorescent protein (mRFP: Red Fluorescent Protein) and a green fluorescent protein (eGFP: Green Fluorescent Protein) are tandemly fused to the amino terminus of the autophagosome marker protein LC3 (MAP1LC3: Microtubule-associated protein 1 light chain 3) (see Autophagy, Vol. 3, pp. 452-460). The fluorescence of the GFP signal (green) of tfLC3 is attenuated in the lysosomal environment, whereas the RFP signal (red) remains stable even in the same environment. In the tfLC3 assay, the property of tfLC3 is that when autophagosomes mature into autolysosomes, they turn red due to quenching of GFP. By analyzing the GFP / RFP ratio within the cells, it is possible to determine whether a sample added to the culture medium has the ability to activate autophagy.

[0050] Specifically, HeLa cells stably expressing tfLC3 (HeLa-tfLC3 cells) were established, and these HeLa-tfLC3 cells were seeded at 3,000 cells / well in a 96-well plate (PerkinElmer, product code: 6055308, PhenoPlate (registered trademark) 96-well microplates). The cells were then incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 24 hours. The growth medium used was DMEM medium (Sigma-Aldrich, product number: D6546) supplemented with 10% FBS and 4 mM L-glutamine. After 24 hours, the growth medium was removed from each well and replaced with growth medium containing 0.1% of each extract sample. After replacement, the cells were incubated in a 5% CO atmosphere at 37°C. ２ The cells were cultured in an incubator for 24 hours. As negative controls, 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 growth medium alone were also tested. Additionally, Torin1, an mTOR inhibitor that induces autophagy by blocking signals necessary for cell growth and proliferation and mimicking a state of cell starvation, was selected as a positive control substance. Torin1 dissolved in DMSO to a predetermined concentration was tested as a positive control. After 24 hours, the cells were washed, fixed with paraformaldehyde, and observed and analyzed under a fluorescence microscope. N = 5 was used for each test group and positive control group, and N = 15 for the negative control group.

[0051] In the tfLC3 assay, GFP is quenched as autophagosomes mature into autolysosomes, but RFP is maintained; therefore, 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), which contained only the solvent used for sample extraction and dissolution, was set to 1, and the ratio of the GFP / RFP values ​​measured in the test sections to which each sample was added was calculated. The results are shown in Figure 2 and Table 3 below.

[0052]

[0053] These results showed that all of the koji-fermented tea leaves had a GFP / RFP ratio of less than 1, demonstrating an effect of enhancing autophagy activity. On the other hand, even though they were koji-fermented products, all of the koji-fermented rice products had a GFP / RFP ratio of approximately 1, roughly the same as the negative control, demonstrating no effect on autophagy activity. Among the koji-fermented tea leaves, the GFP / RFP ratio of the yellow koji-fermented tea leaves was particularly significant, at 0.759, demonstrating a greater enhancement of autophagy activity than the positive control (10 nM Torin1). Furthermore, the GFP / RFP ratio of the black koji-fermented tea leaves also showed a significant reduction of 0.874. These results demonstrate that the koji-fermented tea leaves of the present invention can be used as an autophagy activator to enhance autophagy activity.

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

[0055] The present invention provides an autophagy activator comprising a koji mold fermentation product of tea leaves or an extract thereof, and is therefore useful in a wide range of industries, including functional foods, health foods, pharmaceuticals, and medical care.

Claims

1. An autophagy activator comprising 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 described in claim 5, characterized in that the koji mold is Aspergillus oryzae.

7. A method for producing an autophagy activator as described in 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, characterized in that the koji mold in the koji mold fermentation product is a live koji mold.

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