Autophagy derivative

Abstract

To provide a novel autophagy derivative.SOLUTION: The autophagy derivative contains Equisetum arvense L. and / or Ligustrum Lucidum Aiton.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to autophagy inducers and the like. [Background technology]

[0002] Autophagy is a general term for a system that breaks down cytoplasmic components including proteins and parts of the cytoplasm, such as intracellular organelles such as mitochondria. Autophagy plays a role in supplying amino acids by breaking down proteins under starvation conditions. Autophagy is a series of processes in which an isolation membrane generated from the rough endoplasmic reticulum and mitochondrial contact site in the cell takes up a substrate, forms a double-membrane structure called an autophagosome, fuses with a lysosome, and becomes a single-membrane autophagolysosome, in which decomposition enzymes are supplied and the internal substrate is broken down. Autophagy is classified into macroautophagy, microautophagy, and chaperone-mediated autophagy, but macroautophagy is usually referred to as autophagy. Thus, efforts are underway to discover ingredients that are effective in inducing autophagy. For example, Patent Document 1 discloses an autophagy derivative containing a quercetin glycoside, and Patent Document 2 discloses an autophagy inducer containing fulvic acid. There is a need for novel autophagy inducers that are different from these autophagy inducers. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2021-104968 A [Patent Document 2] Patent Publication No. 2022-108114 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present disclosure is to provide a novel autophagy inducer. [Means for solving the problem]

[0005] As a result of extensive investigations aimed at solving the above problems, the inventors have discovered that horsetail and / or Ligustrum lucidum are useful for inducing autophagy, and have completed the present disclosure.

[0006] That is, the present disclosure is as follows. Section 1. An autophagy inducer containing horsetail and / or Ligustrum lucidum. Section 2. Item 2. The autophagy inducer according to Item 1, wherein the horsetail is a horsetail extract. Section 3. Item 3. The autophagy inducer according to Item 1 or 2, wherein the Ligustrum lucidum is a Ligustrum lucidum extract. Section 4. Item 4. The autophagy inducer according to any one of Items 1 to 3, wherein the autophagy inducer contains both Equisetum arvense and Ligustrum lucidum. Section 5. Item 5. The autophagy inducer according to any one of Items 2 to 4, wherein the horsetail extract is extracted from ethanol or a mixed solution of ethanol and water. Section 6. Item 6. The autophagy inducer according to any one of Items 3 to 5, wherein the Ligustrum lucidum extract is extracted from ethanol or a mixed solution of ethanol and water. Section 7. Item 7. A food or drink composition comprising the autophagy inducer according to any one of Items 1 to 6. Section 8. Item 7. A cosmetic preparation comprising the autophagy inducer according to any one of items 1 to 6. Section 9. Item 7. A pharmaceutical comprising the autophagy inducer according to any one of Items 1 to 6. Section 10. Use of Horsetail and / or Ligustrum lucidum for inducing autophagy. Effect of the Invention

[0007] According to the present disclosure, a novel autophagy inducer can be provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The autophagy inducer of the present disclosure contains horsetail and / or Ligustrum lucidum. The autophagy inducer of the present disclosure may contain either horsetail or Ligustrum lucidum, or may contain both horsetail and Ligustrum lucidum. The autophagy inducer of the present disclosure preferably contains both horsetail and Ligustrum lucidum.

[0009] Horsetail (Equisetum arvense L.) is a plant of the genus Equisetum, order Equisetales, and family Equisetaceae. The vegetative stem is generally called "horsetail" and the spore stem "tsukushi" (horsetail, first-leaf vegetable).

[0010] Horsetail contains equisetonin, a type of saponin, flavonoids (equisetrin, isoquercitonin, luteolin-5-monoglucoside, apigenin, luteolin, kaempferol, etc.), fats, amino acids, vitamin C, silicon, manganese, potassium, sulfur, magnesium, tannins, and alkaloids (nicotine, palustrine, palustrin, etc.).

[0011] In this specification, the term "Equisetum arvense" refers to the plant body or a part thereof of the Equisetum arvense plant, unless otherwise specified. "A part thereof" includes organs or parts thereof (leaves, rhizomes, vegetative stems, sporangium (horsetail), sporangium, sporangium, or fragments thereof), cultured cells, callus, protoplasts, transformed plant cells, transformed plants, etc. For the autophagy inducer of the present disclosure, the whole plant body of Equisetum arvense may be used, or any one or more organs may be used. From the viewpoint of expecting a high effect, it is preferable to use vegetative stems or sporangium stems. Vegetative stems and sporangium stems may also be used.

[0012] The horsetail in the present specification may be a horsetail extract obtained by extracting the horsetail with an extraction solvent. The horsetail as the extraction raw material may be raw, dried, roasted, etc. When drying, it may be naturally dried, dried under heating conditions, or frozen and dried. Before being subjected to the extraction process, pretreatment such as cutting and crushing may be performed so that extraction is performed efficiently. It is preferable to use a horsetail extract for the autophagy derivative of the present disclosure.

[0013] The horsetail extract used in the present disclosure can be prepared by a conventional method, for example, by soaking horsetail in an extraction solvent. The extraction solvent is not particularly limited, and may be, for example, one or more selected from the group consisting of water, alcohols (monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol; dihydric alcohols such as ethanediol, propylene glycol, 1,3-butanediol, and 1,4-butanediol; and trihydric alcohols such as glycerin), ketones (acetone, methyl ketone, and the like), ethers (diethyl ether, tetrahydrofuran, and the like), esters such as ethyl acetate, and hexane. The alcohols are preferably monohydric alcohols or dihydric alcohols. The carbon number of the alcohols is preferably about 1 to 5. Furthermore, the solvent is more preferably water and / or ethanol, and particularly preferably ethanol and a water-ethanol mixed solution.

[0014] The conditions for ethanol or extraction can be appropriately designed by a person skilled in the art, but for example, the horsetail can be immersed in the solvent for a period of 1 hour to several months at room temperature or under heating. The ratio of raw horsetail to solvent can be appropriately designed by a person skilled in the art, but for example, the amount of solvent per 1 part by mass of raw horsetail may be an amount that allows the raw material to be sufficiently immersed, and is preferably 0.5 parts by mass or more, more preferably 1 part by mass or more, and more preferably 2 parts by mass or more. The upper limit of the amount of solvent can be determined from an economical point of view, and the amount of solvent per 1 part by mass of raw material can be, for example, 100 parts by mass or less, preferably 50 parts by mass or less, and more preferably 25 parts by mass or less. The extraction temperature and period can also be appropriately designed by a person skilled in the art, and the extraction can be performed by immersing in the solvent for a period of 1 hour to several months at room temperature or under heating.

[0015] Glossy Privet (Ligustrum lucidum Aiton) is an evergreen tall tree of the genus Ligustrum in the Oleaceae family. Glossy Privet is mainly cultivated in China (such as Zhejiang Province, Jiangsu Province, Fujian Province, Hunan Province, Jiangxi Province, Guangxi Zhuang Autonomous Region, Sichuan Province, etc.). The dried mature fruit of Glossy Privet is known as the Chinese crude drug "Fructus Ligustri Lucidi", which is said to have effects such as cardiac strengthening, diuretic, laxative, tonic, and spermatogenic effects.

[0016] In this specification, when referring to "Glossy Privet", unless otherwise specified, it refers to the whole plant body or a part thereof of the Glossy Privet plant. "A part thereof" includes organs or parts of them (leaves, roots, stems and branches, flowers, stamens, pistils, or pieces thereof), fruits (sometimes also referred to as "fruits"), seeds, cultured cells, callus, protoplasts, transformed plant cells, transformed plant bodies, etc. For the autophagy inducer of the present disclosure, the whole plant body of Glossy Privet can be used, or any one or two or more organs can be used. From the perspective of expecting high effects, it is preferable to use fruits or leaves. Fruits and leaves can also be used. In this specification, when referring to "the fruit or leaf of Glossy Privet", unless otherwise specified, it refers to any one or more selected from the group consisting of the fruit of Glossy Privet and the leaf of Glossy Privet.

[0017] For Glossy Privet in this specification, the Glossy Privet extract obtained by extracting the above-mentioned Glossy Privet with an extraction solvent can be used. Glossy Privet as the extraction raw material may be raw, or may be a dried product, a roasted product, etc. When drying, it may be natural drying, drying under heating conditions, or freeze-drying. Before subjecting it to the extraction process, pretreatment such as cutting and pulverization may be performed so that the extraction can be carried out efficiently. It is preferable to use the Glossy Privet extract for the autophagy derivative of the present disclosure. The extract of Ligustrum lucidum used in the present disclosure can be prepared by a general method. For example, it can be prepared by immersing Ligustrum lucidum in a solvent. The extraction solvent is not particularly limited, and for example, one or more selected from the group consisting of water, alcohols (monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol; dihydric alcohols such as ethanediol, propylene glycol, 1,3-butanediol, and 1,4-butanediol; trihydric alcohols such as glycerin), ketones (acetone, methyl ketone, etc.), ethers (diethyl ether, tetrahydrofuran, etc.), esters such as ethyl acetate; and hexane can be used. The alcohols are preferably monohydric alcohols or dihydric alcohols. The carbon number of the alcohols is preferably about 1 to 5. Furthermore, water and / or ethanol are more preferable as the solvent, and ethanol and a water-ethanol mixed solution are particularly preferable.

[0018] The conditions for ethanol or extraction can be appropriately designed by a person skilled in the art, and for example, Ligustrum lucidum can be immersed in a solvent at room temperature or under heating for a period of 1 hour to several months. The ratio of raw Ligustrum lucidum to solvent can be appropriately designed by a person skilled in the art, and for example, the amount of solvent per 1 part by mass of Ligustrum lucidum raw material may be an amount that allows the raw material to be sufficiently immersed, and is preferably 0.5 parts by mass or more, more preferably 1 part by mass or more, and more preferably 2 parts by mass or more. The upper limit of the amount of solvent can be determined from an economical viewpoint, and the amount of solvent per 1 part by mass of raw material can be, for example, 100 parts by mass or less, preferably 50 parts by mass or less, and more preferably 25 parts by mass or less. The temperature and period of extraction can also be appropriately designed by a person skilled in the art, and can be immersed in a solvent at room temperature or under heating for a period of 1 hour to several months.

[0019] An example of a typical method for preparing an extract of Ligustrum lucidum that can be used in the present disclosure is as follows. The raw material is crushed to an appropriate size, soaked in 5 to 20 times the amount of 25 to 75% by volume ethanol at room temperature for one week, and the solid content is removed from the soaked liquid. The soaked liquid may be concentrated or dried, and can be obtained in powder form by drying.

[0020] In this disclosure, the term "extraction" also includes distillates obtained by steam distillation. Steam distillation is a method in which steam is passed through natural raw materials, and the aroma components distilled along with the steam are condensed together with the steam. There are pressurized steam distillation, normal pressure steam distillation, reduced pressure steam distillation, and gas-liquid multistage alternating current contact distillation (spinning cone column), and any of these can be used in this disclosure. Those skilled in the art can appropriately determine the pretreatment of raw materials for steam distillation, distillation time, temperature, air pressure, etc., taking into consideration economic efficiency, etc., with reference to the results of several experiments.

[0021] In the present disclosure, unless otherwise specified, the term "extract" may refer to the liquid obtained from the extraction process itself (solids may be removed, if necessary), or the liquid may be concentrated, dried, or the like. The extract may be in any form, such as liquid, paste, gel, solid, or powder. To obtain a solid or powder form, the liquid may be subjected to a drying process and dried to a solid, or may be dried by spray drying to obtain a powder.

[0022] In the present disclosure, unless otherwise specified, the term "agent" may refer to the extract itself, or may refer to an extract and an additive, such as a diluent, stabilizer, antioxidant, preservative, etc. The agent of the present disclosure does not include existing compositions for external use on the skin.

[0023] The agent of the present disclosure may contain, as a solid content (amount equivalent to the dried matter obtained by the method shown in the examples of the present specification), preferably 0.00001% or more of Ligustrum lucidum extract, more preferably 0.0001% or more, and even more preferably 0.001% or more. Note that, when the content of a component is expressed in %, it is mass % (the ratio of the mass of the component per 100 g of the total amount) unless otherwise specified.

[0024] The ratio of horsetail and / or Ligustrum lucidum contained in the autophagy inducer of the present disclosure can be, for example, 0.0001 to 99 mass %, 0.001 to 80 mass %, or 0.01 to 70 mass %. In addition, in the present disclosure, in the case of a mixture of horsetail and Ligustrum lucidum, the content of horsetail is usually 1 to 10,000 parts by mass, preferably 10 to 1,000 parts by mass, and more preferably 50 to 150 parts by mass, per 100 parts by mass of Ligustrum lucidum.

[0025] As described above, "autophagy (function)" refers to a system in which, for example, intracellular organelles or proteins are degraded via autophagosome formation in cells. Furthermore, activation of autophagy refers to a state in which the recycling mechanism of intracellular proteins is activated by the induction or promotion of autophagy in cells, specifically, a state in which at least one of the processes of isolation membrane formation, isolation membrane extension, autophagosome formation, and fusion of autophagosomes with lysosomes is induced or promoted. According to the autophagy inducer of the present disclosure, such autophagy (function) can be induced. In this specification, induction is a concept including expression, improvement, enhancement, activation, etc. Thus, the present disclosure encompasses the use of Horsetail and / or Ligustrum lucidum to induce autophagy.

[0026] The activity of autophagy can be confirmed by a conventionally known method for measuring autophagy activity. Such a method for measuring autophagy activity includes a method for detecting an autophagy marker protein. An example of an autophagy marker protein is LC3 (rat microtubule-associated protein 1 light chain 3). There are two forms of LC3, namely, LC3-I and LC3-II. It is known that LC3 exists as LC3-I when present in the cytoplasm, and LC3-I is converted to lipid-added LC3-II during the formation of autophagosomes and is incorporated into the inner and outer membranes of autophagosomes. Therefore, autophagy activity can be confirmed by detecting LC3-I and LC3-II and measuring the conversion rate to LC3-II (Kabeya Y., Mizushima N., Ueno T., Yamamoto A., Kirisako T., Noda T., Kominami E., Ohsumi Y., Yoshimori T.: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO Journal, 19:5720-5728 (2000)).

[0027] The activity of autophagy can also be confirmed by expressing tfLC3, which is a tandem fusion of LC3 with two types of fluorescent proteins (mRFP and EGFP), in cells (Kimura S., Noda T., Yoshimori T.: Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3. Autophagy 3:452-460(2007)). According to this method, EGFP disappears in the (auto)lysosomal environment, and the lower the ratio of EGFP / mRFP in the cell, the more activated autophagy is. For example, a fluorescence microscope can be used to measure the amount of fluorescence in the cell, and analysis software can be used to calculate the amount of fluorescence.

[0028] The autophagy inducer of the present disclosure can be used, for example, as a food or drink composition, a cosmetic, a pharmaceutical, etc. The food and drink compositions include autophagy-inducing foods, health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, etc. The food and drink compositions include beverages and foods. Furthermore, the autophagy inducer of the present disclosure also encompasses the meaning of an additive that imparts an autophagy-inducing effect.

[0029] The autophagy inducer of the present disclosure may contain only the active ingredients horsetail and / or Ligustrum lucidum, or may further contain other ingredients as long as they do not interfere with the effects of the present disclosure. The other components are not particularly limited, and examples thereof include pharma- ceutically acceptable components (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, etc.) and components acceptable as foods (e.g., minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, wetting agents, etc.).

[0030] The horsetail and / or Ligustrum lucidum of the present disclosure can be used in a daily dose of 1 mg to 10 g, preferably 50 mg to 8 g, more preferably 150 mg to 3 g, and even more preferably 150 mg to 1.5 g, in terms of the amount of active ingredient, for an adult weighing 60 kg. The dose can be appropriately set within the above range depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents.

[0031] The autophagy inducer of the present disclosure may be administered orally (ingested) or parenterally. As long as the daily amount of the active ingredient is within the above-mentioned range, the autophagy inducer of the present disclosure may be administered once a day or in multiple divided doses, such as twice or three times a day.

[0032] Since the autophagy-inducing effect is more pronounced, the autophagy inducer of the present disclosure is preferably orally administered once a day at an amount equivalent to 150 mg to 1.5 g of active ingredient per adult weighing 60 kg.

[0033] The autophagy inducer of the present disclosure may be in any form, such as a solid (solid composition), liquid, or paste. The form of the autophagy inducer of the present disclosure may be, for example, a plain tablet, a sugar-coated tablet, a granule, a powder, a tablet, or a capsule (hard capsule, soft capsule, seamless capsule, etc.). The autophagy inducer of the present disclosure can be prepared, for example, by mixing a triterpene compound as an active ingredient with other ingredients as necessary and molding the mixture into the above-mentioned shape.

[0034] The autophagy inducer of the present disclosure can be used as a medicine, quasi-drug, food, or cosmetic itself, or added to a medicine, quasi-drug, food, or cosmetic. It is preferable that the food emphasizes its tertiary function (physical conditioning function, i.e., autophagy induction function, etc.). The functions of "food" are classified into primary function (nutritional function to maintain life), secondary function (taste and sensory function including taste, aroma, texture, etc., to enjoy meals: sensory function), and tertiary function (function to repair abnormalities in the physiological functions of the living body: bioregulation function). In recent years, particularly from the standpoint of disease and aging prevention, attention has been focused on the tertiary functions of "food," such as boosting immunity, preventing diseases such as high blood pressure, obesity, and diabetes, recovering from fatigue, and preventing aging by suppressing the production of active oxygen. Examples of foods that emphasize their tertiary functions include health foods, functional foods, dietary supplements, supplements, and foods for specified health uses.

[0035] A pharmaceutical, quasi-drug, or food comprising the autophagy inducer of the present disclosure, or a pharmaceutical, quasi-drug, or food containing the autophagy inducer of the present disclosure may be for use in inducing autophagy, and may be labeled as "inducing autophagy," or the like.

[0036] The content of the autophagy inducer of the present disclosure in drugs, quasi-drugs, and foods may be set appropriately depending on the type of drug, quasi-drug, or food, etc., so that the amount of active ingredient ingested per day falls within the above-mentioned range.

[0037] When the autophagy inducer of the present disclosure is used as a food itself or added to a food, the form of the food is not particularly limited, and may be, for example, beverages (coffee, juice, tea drinks, soft drinks such as jelly drinks, milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, etc.), spreads (custard cream, etc.), pastes (fruit paste, etc.), Western confectionery (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, etc.), Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.), frozen desserts (ice cream, popsicles, sorbet, etc.), foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.), or seasonings (dressing, furikake, umami seasonings, soup bases, etc.).

[0038] When the autophagy inducer of the present disclosure is used as a food whose tertiary function is emphasized (e.g., health food, functional food (functional food), dietary supplement, supplement, or food for specified health uses) itself, or added to a food whose tertiary function is emphasized, the form of the food whose tertiary function is emphasized may be, in addition to the food forms described above, for example, plain tablets, sugar-coated tablets, granules, powder, tablets, capsules (hard capsules, soft capsules, seamless capsules, etc.), etc.

[0039] The food and beverage composition of the present disclosure contains the above-mentioned autophagy inducer. The autophagy inducer can be used as is in the above-mentioned food and beverage compositions, or, if necessary, excipients, vitamins, minerals, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, fragrances, stabilizers, preservatives, sustained-release regulators, surfactants, gloss agents, solubilizers, humectants, etc. can be blended therein.

[0040] The food and drink composition includes any food and drink that can be ingested by mammals (including humans). Examples of the food and drink composition include dairy products; fermented foods (yogurt, cheese, etc.); beverages (coffee, juice, cocoa, tea drinks, sports drinks, soft drinks such as nutritional drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, fruit liquor, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery (chocolate, donuts, pies, cream puffs, gum, gummies, jellies, candies, cookies, cakes, puddings, biscuits, etc.); ice creams (ice cream, popsicles, sorbets, etc.); foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, ham, sausage, bacon, etc.); seasonings (dressing, furikake, umami seasoning, soup base, miso, soy sauce, sauce, ketchup, oyster sauce, etc.).

[0041] The method for producing the food and drink composition is not particularly limited, and any known method can be used as appropriate.

[0042] The dosage unit form for use as a supplement is not particularly limited and can be selected appropriately, and examples include tablets, capsules, granules, liquids, powders, and the like.

[0043] The intake amount of the food and drink composition can be appropriately determined depending on various conditions of the consumer, such as weight, age, sex, symptoms, etc.

[0044] The pharmaceutical or quasi-drug product of the present disclosure contains the autophagy inducer described above. The autophagy inducer may be used alone in the pharmaceutical or quasi-drug product, or may be used in combination with other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

[0045] When the autophagy inducer of the present disclosure is used as a drug or quasi-drug itself, or added to a drug or quasi-drug, the form of the drug or quasi-drug is not particularly limited and may be, for example, a plain tablet, sugar-coated tablet, granule, powder, tablet, capsule (hard capsule, soft capsule, seamless capsule, etc.), etc.

[0046] Specifically, when the autophagy inducer of the present disclosure is prepared as a pharmaceutical, the autophagy inducer can be prepared together with a non-toxic carrier, diluent, or excipient acceptable for pharmaceuticals into the form of a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsule, pill, powder (dispersed medicine), fine granules, granules, liquid, suspension, emulsion, syrup, paste, injectable solution (including cases where the agent is mixed with distilled water or an infusion such as an amino acid infusion or electrolyte infusion at the time of use to prepare a liquid), or the like to form a pharmaceutical formulation.

[0047] The method of administering the pharmaceutical agent is not particularly limited, and can be, for example, transdermal administration, oral administration, intraarterial administration, intravenous administration, buccal administration, rectal administration, enteral administration, or the like.

[0048] The dosage of a pharmaceutical agent can be appropriately determined depending on various conditions such as the patient's weight, age, sex, symptoms, etc.

[0049] The method for producing a drug, quasi-drug, or food to which the autophagy inducer of the present disclosure has been added is not particularly limited, and can be any known method as appropriate. For example, the drug, quasi-drug, or food used for the above-mentioned purposes can be obtained by mixing the autophagy inducer of the present disclosure with an intermediate product or a final product in the manufacturing process of the drug, quasi-drug, or food.

[0050] The present disclosure is effective against diseases associated with autophagy, such as type 2 diabetes, arteriosclerosis, infectious diseases, nephropathy, heart failure, various inflammations, Crohn's disease, metabolic syndrome, muscle atrophy / myopathy, anemia, neurodegenerative diseases, and cancer.

[0051] The cosmetic preparation of the present disclosure contains the above-mentioned autophagy inducer. In addition to the autophagy inducer, the cosmetic preparation may contain, as necessary, appropriate ingredients that are typically used in cosmetics, such as whitening agents, moisturizers, antioxidants, oily components, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, and various skin nutrients.

[0052] The cosmetic materials include all cosmetic materials that are applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, and the like of animals (including humans).

[0053] The cosmetic preparation may take a wide variety of formulations, such as aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-layer systems, and water-oil-powder three-layer systems.

[0054] The use of the cosmetic is also arbitrary. For example, basic cosmetics include face wash, lotion, milky lotion, cream, gel, essence, beauty serum, pack, mask, etc. For example, makeup cosmetics include foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc. For example, nail cosmetics include nail polish, base coat, top coat, nail polish remover, etc. Other uses include face wash, (paste or liquid) dentifrice, mouthwash, massage agent, cleansing agent, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling agent, hair tonic agent, hair growth agent, antiperspirant, bath agent, etc.

[0055] The autophagy inducer of the present disclosure is applicable to mammals, including humans (preferably humans). In addition, the pharmaceuticals and cosmetics in this disclosure also include quasi-drugs. EXAMPLES

[0056] The present disclosure will be described more specifically below with reference to examples, but the technical scope of the present disclosure is not limited to these examples.

[0057] Manufacturing Example 1 Horsetail was powdered, and 40 mg of the powder was mixed with dimethyl sulfoxide (DMSO) (40 ml) to obtain Horsetail extract 1 (40 ml).

[0058] Example 1 The horsetail extract 1 prepared at the appropriate time was used and diluted with phosphate buffered saline (Nacalai Tesque, Inc.) to a final concentration of 40 μg / ml.

[0059] Production Example 2 (Horsetail Extract 2) Horsetail was powdered, and 40 mg of the powder was mixed with ethanol (40 ml) to obtain Horsetail extract 2 (40 ml).

[0060] Examples 2 to 7 The horsetail extract 2 prepared at the appropriate time was used and diluted with phosphate buffered saline (Nacalai Tesque, Inc.) to the final concentrations shown in Table 1 below.

[0061] Production Example 3 (Ligustrum lucidum extract 1) Ligustrum lucidum was powdered, and 40 mg of the powder was mixed with dimethyl sulfoxide (DMSO) (40 ml) to obtain Ligustrum lucidum extract 1 (40 ml).

[0062] Example 8 The Ligustrum lucidum extract 1 prepared at an appropriate time was used and diluted with phosphate buffered saline (Nacalai Tesque, Inc.) to a final concentration of 40 μg / ml.

[0063] Production Example 4 (Ligustrum lucidum extract 2) Ligustrum lucidum was powdered, and 40 mg of the powder was mixed with ethanol (40 ml) to obtain Ligustrum lucidum extract 2 (40 ml).

[0064] Examples 9 to 14 The Ligustrum lucidum extract 2 prepared at an appropriate time was used and diluted with phosphate buffered saline (manufactured by Nacalai Tesque, Inc.) to the final concentrations shown in Table 1 below.

[0065] Examples 15 to 19 The horsetail extract 2 obtained in Production Example 2 above and the Ligustrum lucidum extract 2 obtained in Production Example 4 above were used and diluted with phosphate buffered saline (manufactured by Nacalai Tesque, Inc.) to the final concentrations shown in Table 1 below.

[0066] Comparative Examples 1 to 3 Torin1 (Chemscene LLC) was used as a positive control and diluted with phosphate-buffered saline (Nacalai Tesque, Inc.) to a final concentration of 6.0 ng / ml (Comparative Example 1), 3.0 ng / ml (Comparative Example 2), or 1.5 ng / ml (Comparative Example 3).

[0067] Comparative Examples 4 to 6 As a positive control, trans-resveratrol (Tokyo Chemical Industry Co., Ltd.) was used and diluted with phosphate-buffered saline (Nacalai Tesque, Inc.) to a final concentration of 5.7 μg / ml (Comparative Example 4), 2.85 μg / ml (Comparative Example 5), or 1.6 μg / ml (Comparative Example 6).

[0068] Reference example 1 For the blank test, a blank sample of water only was prepared without using the above samples containing horsetail or Ligustrum lucidum.

[0069] Test Example Autophagy activity assessment According to the method of Bhargava et al. (Bhargava KH et al.: Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex. Proceedings of the National Academy of Sciences of the United States of America 117:17003-17010(2020)), a tfLC3 stable cell line of cervical epithelioid carcinoma HeLa cells (JCRB cell bank: JCRB9004) was obtained using a retrovirus. The obtained cells were suspended in growth medium (DMEM medium (catalog number D6546; Sigma-Alddrich) supplemented with 10% FBS and 4 mM L-glutamine) to a cell concentration of 25000 cells / mL, and 100 μL was dispensed into each well of a 96-well plate (Cell Carrier 96 Ultra Black 96well, Clear Bottom, with Lid; PerkinElmer). The 96-well plate was placed in a CO2 incubator and cultured at 37°C under 5% CO2 + 95% air for 24 hours.

[0070] To the samples of the present disclosure of Examples 1 to 19 and the comparative samples of Comparative Examples 1 to 6, phosphate-buffered saline (PBS; manufactured by Nacalai Tesque, Inc.) was added as a solvent to prepare diluted solutions of the samples. Each of the obtained sample solutions was added to the cell culture medium in each well so as to obtain the final concentration shown in Table 1. The wells were then placed in a CO2 incubator and cultured for 24 hours under conditions of 37°C and 5% CO2 + 95% air (n=3). Furthermore, at this time, as a negative control (blank), cells cultured in a growth medium without the addition of the sample solution were cultured in a different well from the above under the same conditions as above (n=9).

[0071] After incubation, the culture medium was removed from each well, and the wells were washed twice by adding and removing phosphate-buffered saline (PBS). Then, PBS containing 4% paraformaldehyde (Nacalai Tesque) containing Hoechst dye (Hoechst33342; Dojindo Laboratories) was added to each well, and the wells were left to stand for 20 minutes to immobilize the cells.

[0072] The supernatant in each well was removed, and the wells were washed twice with PBS. The cells were photographed using a confocal image cytometer (CQ1; Yokogawa Electric Corporation), and image data including information on the fluorescence intensity of EGFP and mRFP were obtained. In measuring the fluorescence intensity of EGFP, the detection bandpass filter used was BP525 / 50, and the excitation wavelength was 488 nm. In measuring the fluorescence intensity of mRFP, the detection bandpass filter used was BP617 / 73, and the excitation wavelength was 561 nm. The ratio of EGFP fluorescence intensity to mRFP fluorescence intensity (EGFP / mRFP value) was calculated from the obtained cell image data using image analysis software (CellPathfinder; Yokogawa Electric Corporation).

[0073] Table 1 shows the autophagy effect as the EGFP / mRFP values ​​in Examples 1 to 19 and Comparative Examples 1 to 6, expressed as relative values ​​when the EGFP / mRFP value of the negative control was set to 1. Note that a lower EGFP / mRFP value indicates a more activated autophagy.

[0074] [Table 1]

[0075] result As shown in Table 1, in Examples 1 to 7 (Horsetail), Examples 8 to 14 (Ligustrum lucidum), and Examples 15 to 19 (mixture of Horsetail and Ligustrum lucidum), the EGFP / mRFP value was lower than 1, indicating enhanced autophagy activity. On the other hand, the autophagy inducers in Comparative Examples 1 to 6 are conventionally known autophagy inducers, but it was found that the autophagy inducers of the present disclosure have autophagy effects that are equivalent to or superior to those of the autophagy inducers in Comparative Examples 1 to 6.

Claims

1. An autophagy inducer containing horsetail and / or Ligustrum lucidum.

2. The autophagy inducer according to claim 1 , wherein the horsetail is a horsetail extract.

3. The autophagy inducer of claim 1 , wherein the Ligustrum lucidum is a Ligustrum lucidum extract.

4. The autophagy inducer of claim 1 , which contains both Equisetum arvense and Ligustrum lucidum.

5. The autophagy inducer according to claim 2 , wherein the horsetail extract is extracted from ethanol or a mixed solution of ethanol and water.

6. The autophagy inducer according to claim 3 , wherein the Ligustrum lucidum extract is extracted from ethanol or a mixed solution of ethanol and water.

7. A food or drink composition comprising the autophagy inducer according to any one of claims 1 to 6.

8. A cosmetic preparation comprising the autophagy inducer according to any one of claims 1 to 6.

9. A pharmaceutical comprising the autophagy inducer according to any one of claims 1 to 6.

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