Alternative autophagy activators

Abstract

An alternative autophagy activator containing at least one selected from the group consisting of coffee extract and / or coffee grounds extract, black pepper extract, Pinus densiflora extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

Description

Technical Field

[0001] This invention relates to an alternative autophagy activator. Furthermore, this invention also relates to food or beverage compositions for activating alternative autophagy. Background Technology

[0002] Autophagy is known as an intracellular purification / recycling system found in all eukaryotes, including yeast and plants, and has been the subject of research in recent years.

[0003] By studying the molecular mechanism of autophagy, more than 30 molecules related to autophagy were identified. Among these molecules, Atg5, Atg7, and LC3 are particularly related to autophagy and are essential molecules for its function.

[0004] However, a novel type of autophagy has recently been discovered that does not require these molecules and originates from the Golgi apparatus and endosomes, and is regulated by molecules such as Rab9 (Non-Patent Literature 1). This type of autophagy is distinct from existing autophagy that depends on Atg5, Atg7, LC3, etc. (sometimes referred to simply as Atg5-dependent autophagy) and is called alternative autophagy.

[0005] In recent years, it has been established that the reduced activity of alternative autophagy leads to decreased cell and tissue function. Non-patent literature 2 discloses that enteritis is exacerbated in mice with Trim31, a protein associated with alternative autophagy, which is missing.

[0006] In addition, Patent Document 1 discloses alternative autophagy inducers and anticancer agents with benzothiophene compounds as active ingredients. Patent Document 2 discloses an inhibitor of photoinflammatory inflammation with alternative autophagy inducers as active ingredients.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: International Publication No. 2013 / 118842

[0010] Patent Document 2: International Publication No. 2020 / 091070

[0011] Non-patent literature

[0012] Non-patent literature 1: Nishida et al., Nature, 2009, 641, 654-658

[0013] Non-Patent Document 2: Hiroichi Nibe et al., Research Results Report (funded by research grants), 2021, Research Topic / Region Code 19K17426 Summary of the Invention

[0014] The problem the invention aims to solve

[0015] The problem to be solved by this invention is to provide a novel component that can activate alternative autophagy.

[0016] Solution for solving the problem

[0017] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract or apple polyphenol extract can activate alternative autophagy, thereby completing the present invention.

[0018] The present invention is as follows. [1]

[0020] An alternative autophagy activator comprising at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract. [2]

[0022] According to the alternative autophagy activator described in [1], at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3. [3]

[0024] The alternative autophagy activator according to [1] or [2] is used for at least one of the following purposes: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells. [4]

[0026] A food or beverage composition for activating alternative autophagy, comprising at least one selected from the group consisting of coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract. [5]

[0028] A food or beverage composition comprising at least one of the following: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells, wherein the composition contains at least one of the following: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract. [6]

[0030] The food or beverage composition according to [5] exhibits, through alternative autophagy activation selected from at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells. [7]

[0032] The food or beverage composition according to [5] or [6], wherein at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, independently of Atg5, Atg7 and / or LC3, promotes the fusion of lysosomal proteins with autophagosomes, thereby exhibiting at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan and improving germ cell quality.

[0033] The present invention may be as described below.

[0034] [1A]

[0035] An alternative autophagy activator comprising at least one selected from the group consisting of dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK).

[0036] [2A]

[0037] According to the alternative autophagy activator described in [1A], at least one selected from the group consisting of dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK) activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3.

[0038] [3A]

[0039] The alternative autophagy activator according to [1A] or [2A] is used for at least one purpose selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0040] [4A]

[0041] A food or beverage composition for activating alternative autophagy, comprising at least one selected from the group consisting of dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK).

[0042] [5A]

[0043] A food or beverage composition comprising at least one of the following groups: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells, wherein the composition contains at least one of the following groups: dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK).

[0044] [6A]

[0045] The food or beverage composition according to [5A] exhibits, through alternative autophagy activation selected from at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells, by means of alternative autophagy activation selected from at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0046] [7A]

[0047] The food or beverage composition according to [5A] or [6A], wherein at least one of the group consisting of dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK) independently of Atg5, Atg7 and / or LC3, by promoting the fusion of lysosomal proteins with autophagosomes, thereby exhibiting at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0048] In addition, in the above [1] to [7] methods, the coffee grounds extract may contain DC and / or DK, and the DC and DK may be derived from the coffee grounds extract.

[0049] [A1]

[0050] A method for activating alternative autophagy, which uses at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract.

[0051] [A2]

[0052] According to the method described in [A1], alternative autophagy is activated by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3, using at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract.

[0053] [A3]

[0054] According to the method described in [A1] or [A2], wherein alternative autophagy is activated in order to select at least one of the following groups: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan, and improving the quality of germ cells.

[0055] [A4]

[0056] A method for activating alternative autophagy, comprising a food or beverage composition containing at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

[0057] [A5]

[0058] A method using a food or beverage composition comprising at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, thereby achieving at least one selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, extending healthy lifespan, and improving the quality of reproductive cells.

[0059] [A6]

[0060] According to the method described in [A5], by using a food or beverage composition containing at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, alternative autophagy is activated, thereby resulting in at least one selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, extending healthy lifespan, and improving the quality of germ cells.

[0061] [A7]

[0062] According to the method described in [A5] or [A6], a food or beverage composition containing at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract is used to promote the fusion of lysosomal proteins with autophagosomes, independently of Atg5, Atg7, and / or LC3, thereby achieving at least one selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, extending healthy lifespan, and improving the quality of germ cells.

[0063] [B1]

[0064] Use of at least one of the following groups: coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract in activating alternative autophagy.

[0065] [B2]

[0066] According to the use described in [B1], it is used to activate alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3.

[0067] [B3]

[0068] According to the uses described in [B1] or [B2], it is used to activate alternative autophagy for at least one of the uses selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0069] [B4]

[0070] Use of a food or beverage composition containing at least one selected from the group consisting of coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract for activating alternative autophagy.

[0071] [B5]

[0072] The use of a food or beverage composition containing at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, for the purpose of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells.

[0073] [B6]

[0074] According to the use described in [B5], it is used to achieve, through alternative autophagy activation selected from at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan and improving the quality of germ cells.

[0075] [B7]

[0076] According to the uses described in [B5] or [B6], it is used to promote the fusion of lysosomal proteins with autophagosomes by promoting at least one of the following groups: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, without dependence on Atg5, Atg7, and / or LC3, thereby achieving the following effects: regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0077] [C1]

[0078] Use of at least one of the following groups: coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract in the manufacture of an alternative autophagy activator.

[0079] [C2]

[0080] According to the use described in [C1], it is used to manufacture an alternative autophagy activator that activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3.

[0081] [C3]

[0082] According to the use described in [C1] or [C2], it is used to manufacture an alternative autophagy activator, said alternative autophagy activator activating alternative autophagy for at least one use selected from the group consisting of adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0083] [C4]

[0084] Use of at least one of the following groups: coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract in the manufacture of a food or beverage composition for activating alternative autophagy.

[0085] [C5]

[0086] The use of at least one of the following groups: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, in the manufacture of a food or beverage composition for the purpose of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells.

[0087] [C6]

[0088] According to the use described in [C5], it is used to manufacture food or beverage compositions, said food or beverage compositions exhibiting at least one of the following effects by alternative autophagy activation selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract: regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0089] [C7]

[0090] According to the use described in [C5] or [C6], it is used to manufacture a food or beverage composition, said food or beverage composition exhibiting, independently of Atg5, Atg7 and / or LC3, at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, through promoting the fusion of lysosomal proteins with autophagosomes, thereby exhibiting at least one of the following effects: regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0091] [D1]

[0092] Coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract are used to activate alternative autophagy.

[0093] [D2]

[0094] According to [D1], coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract are used to activate alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3.

[0095] [D3]

[0096] According to [D1] or [D2], the coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract are used to activate alternative autophagy for at least one of the following purposes: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0097] [D4]

[0098] A food or beverage composition containing at least one selected from the group consisting of coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, for activating alternative autophagy.

[0099] [D5]

[0100] A food or beverage composition comprising at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, for use in regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells.

[0101] [D6]

[0102] The food or beverage composition according to [D5] is used to achieve, through alternative autophagy activation selected from at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, at least one of the group consisting of regulating gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan, and improving the quality of germ cells.

[0103] [D7]

[0104] The food or beverage composition according to [D5] or [D6] is used to cause at least one of the following groups to produce, independently of Atg5, Atg7 and / or LC3, a product by promoting the fusion of lysosomal proteins with autophagosomes, thereby achieving, at least one of the following groups: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, enhancing motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan, and improving the quality of germ cells.

[0105] In the above [A1] to [D7], a food or beverage composition containing at least one of the group consisting of dehydrocaffeine (DC) and dehydrocaffeoyl alcohol (DK) as its active ingredient may be used instead of the prescribed extract, and the coffee residue extract may also contain DC and / or DK.

[0106] Furthermore, in this invention, particularly in [A1] to [D7] above, it can be a therapeutic invention or a non-therapeutic invention. That is, it can be an invention of a therapeutic or non-therapeutic method, an invention of a therapeutic or non-therapeutic use, or an invention of a therapeutic or non-therapeutic product.

[0107] Furthermore, in this invention, "non-therapeutic invention" refers to an invention that does not involve medical procedures when applied to or ingested by a subject; that is, it refers to an invention that does not involve methods of performing surgery, treatment, or diagnosis on a subject, including a person. Depending on the circumstances, the subject may also include non-human animals. More specifically, "non-therapeutic invention" refers to an invention that does not involve methods of performing surgery, treatment, or diagnosis on a person by a doctor or a person receiving instructions from a doctor.

[0108] As a "non-therapeutic invention," examples include inventions aimed at improving health or for cosmetic purposes. In addition, a "non-therapeutic invention" can also be a "preventive invention" as long as it does not fall under the category of "therapeutic inventions."

[0109] The effects of the invention

[0110] According to the present invention, novel ingredients capable of activating alternative autophagy can be provided. Attached Figure Description

[0111] Figure 1 This indicates the results obtained from evaluating alternative autophagy activity.

[0112] Figure 2 This indicates the results obtained from evaluating alternative autophagy activity. Detailed Implementation

[0113] Hereinafter, embodiments of the present invention (hereinafter referred to as "this embodiment") will be described, but the scope of protection of the present invention is not limited by the following embodiments.

[0114] One embodiment of the present invention relates to an alternative autophagy activator containing at least one selected from the group consisting of coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract.

[0115] In this specification, an extract refers to a composition containing a specific component separated from a raw material, and food or beverage compositions containing extracts are distinguished from the raw material itself.

[0116] In this embodiment, any method or apparatus known to those skilled in the art can be used in the preparation of the extract (including fractions). Examples of preparation methods include solvent extraction, supercritical fluid extraction, steam distillation, or pressing. The extract obtained by pressing (which may be fractions (including pressed material)) can be further processed into extracts (including fractions). In the preparation of the extract (including fractions), during solvent extraction, a solvent can be appropriately used depending on the raw material and the specific component desired. Examples of solvents include water, acetic acid, ethanol, methanol, acetone, hexane, benzene, or diethyl ether. A solvent containing an organic solvent is preferred as the solvent used for extraction from coffee residue.

[0117] The resulting extract (including fractions) can be in any form, such as liquid, solid, or powder. When in liquid form, it can be a liquid in the solvent used for extraction, or a liquid concentrate obtained through concentration, for example. Alternatively, it can be a suspension from which a portion of the extract (including fractions) precipitates during the concentration process.

[0118] Liquid extracts (including fractions) can also be freeze-dried or spray-dried directly or together with excipients to produce solid extracts (including fractions).

[0119] In this specification, "coffee" refers to the seeds (coffee beans) of plants belonging to the genus *Coffea*. In this embodiment, only one species of *Coffea* may be used, or two or more species may be used together.

[0120] In this instruction manual, "black pepper" refers to the plant *Pipernigrum*, a vine belonging to the genus *Piper*.

[0121] In this manual, "red pine" refers to the evergreen coniferous tree belonging to the genus Pinus, scientifically known as Pinus densiflora.

[0122] In this instruction manual, "juniper berries" refers to the fruit of a coniferous tree belonging to the genus Juniper, specifically Juniper communis.

[0123] In this specification, "turmeric" refers to perennial herbs belonging to the genus *Curcuma*, sometimes also simply called turmeric. Examples include *C. longa*, *C. aromatica*, *C. xanthorrhiza*, and *C. zedoaria*. *C. longa* is preferred. In this embodiment, only one of these *Curcuma* species may be used, or two or more may be used in combination.

[0124] In this specification, "apple" refers to the fruit of a deciduous tree belonging to the genus *Malus*. In this embodiment, only one species of *Malus* may be used, or two or more species may be used together.

[0125] In this specification, coffee grounds extract refers to components separated by extraction from coffee grounds after extraction with water or the like, and coffee extract refers to components separated by extraction from coffee beans (such as ground coffee beans, hereinafter the same).

[0126] Coffee grounds extract can be, for example, an ethanol or acetone extract of the grounds obtained by extracting coffee beans with water or the like (also known as coffee extraction grounds). The concentration of the solvent used for extraction is not particularly limited and can be 50-100%, 70-100%, 70-99.5%, or 80-99.5%. Alternatively, coffee grounds extract can be, for example, the pressed residue of the grounds obtained by extracting coffee beans with water or the like (coffee grounds pressed), or coffee oil derived from coffee extraction grounds. The alcohol-soluble components of the coffee grounds pressed can also be used as coffee grounds extract. Examples of alcohols include methanol and ethanol.

[0127] Coffee extract and / or coffee grounds extract may be a mixture of coffee extract and coffee grounds extract.

[0128] Coffee beans can be green beans (including fermented beans, the same applies below), roasted beans, or beans that contain both green and roasted beans. Additionally, coffee beans can be made by grinding green coffee beans directly or by grinding roasted beans into powder. Coffee beans can consist of one type of coffee bean or two or more types of beans.

[0129] Coffee extracts or coffee grounds extracts may contain dehydrocaffeine (DC) and / or dehydrocatechol (DK). Furthermore, in this specification, inventions relating to coffee extracts or coffee grounds extracts can also be understood as inventions that replace coffee extracts or coffee grounds extracts with dehydrocaffeine (DC) and / or dehydrocatechol (DK).

[0130] The structure of DC is shown below.

[0131]

[0132] The structure of DK is shown below.

[0133]

[0134] There are no particular limitations on the method for obtaining a coffee grounds extract containing DC and / or DK. The following described method for obtaining a coffee grounds extract containing DC and / or DK may also be used as a method for obtaining a coffee grounds extract.

[0135] The preferred extraction solvent for obtaining coffee residue is water, and the extraction temperature can be, for example, above 70°C or at a low temperature of around 5~40°C.

[0136] The coffee beans used are preferably roasted coffee beans. The roasting conditions can be appropriately determined according to the type of coffee beans, for example, roasting at a temperature of 200°C or above for 8 minutes or more. Alternatively, commercially available coffee beans that have been roasted (preferably dark roast) (or their powdered form) can be used.

[0137] The obtained coffee grounds can be directly used to prepare coffee grounds extract. From the viewpoint of obtaining coffee grounds extract containing DC and / or DK at higher concentrations, the coffee grounds can be further dried. Drying methods can include forced air drying, hot air drying, vacuum drying, freeze drying, etc.

[0138] After obtaining coffee grounds, a coffee grounds extract can be prepared using methods and apparatus known to those skilled in the art, such as solvent extraction, where an organic solvent can be used as the solvent. The resulting coffee grounds extract preferably contains DC and / or DK.

[0139] Coffee residue extracts containing DC and / or DK can be specifically manufactured as follows.

[0140] Arabica coffee beans are roasted at 220-260°C for at least 8 minutes. The roasted beans are then ground using a coffee grinder and drip-filtered with water at 75-95°C to obtain coffee grounds. The grounds are dried at below 100°C for 5-20 hours until the moisture content is below 10%. 70-95% (v / v) ethanol is added to the dried grounds, and the mixture is stirred at 40-60°C for 1-6 hours. The mixture is then filtered through filter paper, and the filtrate is collected. The remaining coffee grounds on the filter paper are processed in the same manner, and the filtrate is collected. This process is repeated 1-3 times. The collected filtrates are combined and concentrated using an evaporator until the ethanol is removed, yielding a coffee grounds extract containing DC and / or DK.

[0141] DC and DK can be separated using coffee grounds or other raw materials, or they can be commercially available.

[0142] There are no particular limitations on the method for separating DC and DK from coffee grounds. For example, chromatography can be used to separate them from extracts or presses of coffee grounds. In the case of separation by chromatography, the mobile phase can be a solvent such as water, acetonitrile, or alcohol. A single solvent or a mixture of two or more solvents can be used, and elution can also be carried out in a mobile phase under gradient conditions.

[0143] DC and DK can, for example, separate coffee grounds as a raw material, as shown below.

[0144] Coffee grounds with a moisture content of less than 10% are obtained using the method described above. The obtained coffee grounds are heated to 80-100°C in a roasting kettle and pressed 1-3 times to recover the oil. The recovered oil is then filtered to obtain coffee grounds pressed oil.

[0145] Add 2-6 times the amount of hexane to coffee grounds pressed oil and mix. Then add 1-3 times the amount of methanol to the coffee grounds pressed oil and mix. After standing for a period of time to confirm separation into two layers, recover the methanol layer. Add an equal amount of methanol to the recovered methanol layer and mix. Repeat the above operation 1-3 times to obtain a methanol layer. Add 1 / 5-1 / 15 of the amount of distilled water to this methanol layer and mix. After standing and separation, recover the methanol layer. Concentrate and dry the recovered methanol layer to obtain a dried product. Perform fractionation by reversed-phase chromatography using ultrapure water, acetonitrile, and isopropanol as the mobile phase. Elute from ultrapure water-acetonitrile (95:5) to 100% acetonitrile, then flow into isopropanol. Concentrate and dry the eluted fractions to obtain a dry solid. Apply the dry solid to HPLC to separate DC and DK fractions. Ultrapure water and acetonitrile were used in the mobile phase, and the ultrapure water-acetonitrile (60:40) mixture was flowed to separate and recover the eluent from each peak. The components of each peak were identified, and the eluents identified as DC and DK were used as DC and DK separation solutions, respectively.

[0146] In this specification, black pepper extract, red pine extract, juniper berry extract, or turmeric extract refer to components that are extracted and separated from black pepper, red pine, juniper berries, or turmeric as raw materials.

[0147] The raw materials in each extract can be the plant itself, the dried plant material, or a substance made by cutting or crushing the plant itself or the dried plant material, or it can be in powder form.

[0148] In this specification, the extracts may be described, for example, as follows.

[0149] The extract of red pine can be an ethanol extract of red pine or an ethanol extract of red pine powder. There is no particular limitation on the concentration of ethanol used for extraction; it can be 50-100%, 70-100%, 70-99.5%, or 80-99.5%.

[0150] Juniper berry extract can be an ethanol extract of juniper berries or an ethanol extract of juniper berry powder. There is no particular limitation on the concentration of ethanol used for extraction; it can be 50-100%, 70-100%, 70-99.5%, or 80-99.5%.

[0151] Apple polyphenol extracts can be fractions containing apple polyphenols (apple polyphenol fractions derived from apples).

[0152] Black pepper extract can be an ethanol extract of black pepper or an ethanol extract of black pepper powder. There is no particular limitation on the concentration of ethanol used for extraction; it can be 50-100%, 70-100%, 70-99.5%, or 80-99.5%. Black pepper extract may contain piperine as the main component.

[0153] In this embodiment, the coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract can be commercially available products.

[0154] As a black pepper extract or turmeric extract, commercially available products with piperine or curcumin as the main components can be used, for example. Alternatively, the prepared substance can be mixed with commercially available products and used as an extract (including fractions).

[0155] Specifically, turmeric extracts can be exemplified by products containing three compounds (curcuminoids): curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), and standardized with over 95% curcumin. Products derived from dried turmeric (Curcuma longa) rhizomes can also be considered.

[0156] Apple polyphenol extracts can be obtained using commercially available products with apple polyphenols as the main ingredient.

[0157] In this embodiment, the alternative autophagy activator may contain any amount of at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, as long as it can exert an alternative autophagy activating effect. Furthermore, it may also contain other active ingredients, provided that its function is not impaired. These other active ingredients may be other components known as alternative autophagy activators, or other components that are unknown or do not possess alternative autophagy activating capabilities.

[0158] Alternative autophagy activators may contain only one of the following: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract; or they may contain only two or more.

[0159] The alternative autophagy activator in this embodiment can activate alternative autophagy by comprising at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract.

[0160] In this embodiment, the activation of alternative autophagy can, for example, induce or promote the breakdown of intracellular molecules such as proteins, organelles, or extracellular invading bacteria, viruses, etc., independently of Atg5, Atg7, and / or LC3. The activation of alternative autophagy can also be induced independently of Atg5, Atg7, and / or LC3 by the fusion of lysosomal proteins with autophagosomes. That is, the activation of alternative autophagy can be induced independently of at least one molecule selected from the group consisting of Atg5, Atg7, and LC3 by the fusion of lysosomal proteins with autophagosomes, or it can be induced independently of all molecules of Atg5, Atg7, and LC3 by the fusion of lysosomal proteins with autophagosomes.

[0161] In this embodiment, the alternative autophagy activator may be an alternative autophagy activator selected from at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, which activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes without depending on Atg5, Atg7 and / or LC3, or without depending on at least one molecule selected from the group consisting of Atg5, Atg7 and LC3.

[0162] Furthermore, in this embodiment, the activation of alternative autophagy can also induce or promote the fusion of lysosomal proteins with autophagosomes, and induce or promote the breakdown of intracellular proteins and other molecules, organelles, and extracellular invading bacteria and viruses, without depending on autophagy-related molecules including Atg5, Atg7, and LC3. That is, the molecular mechanisms of autophagy are not involved in the activation of alternative autophagy in this embodiment.

[0163] In this embodiment, the alternative autophagy activator can be an alternative autophagy activator that does not activate autophagy.

[0164] By activating alternative autophagy, intracellular substances are broken down and reused, facilitating cellular metabolism. Furthermore, it can prevent the accumulation of abnormal proteins and maintain immune function by eliminating external invaders. Thus, through the activation of alternative autophagy, functions such as maintaining homeostasis and eliminating abnormal molecules are improved, thereby improving the state of all cells and organs in the body. Therefore, it can prevent various diseases and infections and improve their symptoms. Specifically, by activating alternative autophagy, the following effects are achieved: regulating gastrointestinal condition, maintaining intestinal function, rejuvenating the gut, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan, and / or improving the quality of reproductive cells.

[0165] As one embodiment of the present invention, an alternative autophagy activator is provided, which is used for at least one purpose selected from the group consisting of adjusting gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells. The alternative autophagy activator contains at least one of the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

[0166] Furthermore, alternative autophagy can also be induced independently of Atg5, Atg7, and / or LC3 by the fusion of lysosomal proteins with autophagosomes. Therefore, as an embodiment of the present invention, an alternative autophagy activator is provided, which is used for at least one of the following uses: adjusting gastrointestinal state, maintaining intestinal function, intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving germ cell quality. Specifically, at least one of the following extracts selected from coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract activates alternative autophagy independently of Atg5, Atg7, and / or LC3 by promoting the fusion of lysosomal proteins with autophagosomes.

[0167] Specifically, the alternative autophagy activator in this embodiment, or the food or beverage composition in this embodiment, can activate alternative autophagy by comprising at least one of the extracts, and can be used for the purposes listed in Table 1.

[0168] [Table 1]

[0169]

[0170] In this embodiment, the alternative autophagy activator can be a fusion promoter of lysosomal proteins and autophagosomes, an alternative autophagy decomposition enhancer, or an intracellular metabolism promoter.

[0171] Activation of alternative autophagy can be evaluated using methods known to those skilled in the art. Examples include evaluating the alternative autophagy activity at the fusion of autophagosomes and lysosomes, evaluating using fluorescence intensity, and evaluating by electron microscopy. Furthermore, the determination of alternative autophagy activity can be achieved by measuring the gene expression levels or protein expression levels of alternative autophagy-associated proteomes. Specifically, referring to Patent Document 1, evaluation can be performed using cells expressing lysosomal proteins with attached fluorescent proteins, or cells lacking Atg5, Atg7, and / or LC3. Specifically, the fluorescence intensity generated when alternative autophagy is induced and lysosomal proteins aggregate can be used as an indicator for evaluation. The generated fluorescence intensity can also be quantified for evaluation. Additionally, referring to Patent Document 1, compared to when the alternative autophagy activator of this embodiment is not provided, when the alternative autophagy activator of this embodiment is provided, a high fluorescence intensity caused by lysosomal protein aggregation can be evaluated as indicating that alternative autophagy is activated. Additionally, for example, when the fluorescence intensity increases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% or more, it can be evaluated as alternative autophagy being activated. Preferably, when the increase is 30% or more, it can be evaluated as alternative autophagy being activated.

[0172] Alternative autophagy activators can function, for example, by being applied directly or indirectly to body organs or cells.

[0173] The dosage form of the alternative to autophagy activator can be any form as long as it can perform its function. For example, it can be administered in tablets, capsules, granules, fine granules, powders, liquids, syrups, chewable tablets, oral tablets, ointments, gels, creams, injections, sublingual tablets, inhalers, or suppositories. To manufacture these dosage forms, conventionally known additives can be used without particular limitation. For example, excipients, binders, diluents, disintegrants, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, and / or preservatives known in the industry can be used as needed.

[0174] The dosage and frequency of application of alternative autophagy activators can be appropriately set according to the route of application and various conditions such as the age, weight and symptoms of the recipient.

[0175] The preferred recipients of the alternative autophagy activator are mammals, more preferably humans, monkeys, cats, pigs, horses, cattle, mice, rats, guinea pigs, dogs, and rabbits, and even more preferably humans.

[0176] As one embodiment of the present invention, food, beverage, feed, cosmetic, medicine or quasi-medicine, etc., are provided, which contain the alternative autophagy activator in this embodiment.

[0177] As food, beverage, feed, cosmetic, medicine or quasi-medicine, it can be formulated into the same dosage form as those described as alternative autophagy activators.

[0178] As another embodiment of the present invention, a food or beverage composition for activating alternative autophagy is provided, comprising at least one selected from the group consisting of coffee grounds extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract.

[0179] Food or beverage compositions can be formulated into forms suitable for use, without particular limitation, such as tablets, capsules, granules, fine granules, powders, liquids, syrups, chewable tablets, oral dosage forms such as tablets, ointments, gels, creams, injections, sublingual tablets, inhalants, or suppositories. Food or beverage compositions are not particularly limited in form; for example, they can be formulated into edible forms such as solids, liquids, granules, powders, capsules, pastes, porridges, or gels. Furthermore, for example, when formulated into capsules, they can be coated with one or more layers of film as needed. To form these dosage forms and formats, conventionally known additives can be used without particular limitation; for example, excipients, binders, diluents, disintegrants, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, and / or preservatives known in the food industry can be used as needed.

[0180] As a composition for food or beverage use, there are no particular limitations, such as making health foods, functionally labeled foods, foods for specific health purposes, nutritional functional foods, health supplements, and nutritional supplements.

[0181] Compositions for use in food or beverages include, for example, food, beverages, seasonings and food additives. Specifically, food includes snacks, dairy products, breads, noodles, rice, soups and side dishes. Specifically, beverages include milk drinks, soft drinks, fruit juice drinks, carbonated drinks, sports drinks, nutritional drinks and alcoholic drinks.

[0182] In food or beverage compositions, colorants, flavorings, sweeteners, bittering agents, acidulants, preservatives, antioxidants, pH adjusters, and / or thickening and stabilizing agents may be added as needed.

[0183] Food or beverage compositions may be provided after heat sterilization, or in sealed bags or containers. Additionally, when provided, they may be labeled with at least one item selected from the group consisting of "regulating gastrointestinal condition," "maintaining intestinal function," "rejuvenating the gut," "improving cognitive function," "enhancing memory," "clearing brain waste," "maintaining healthy liver function," "improving motor function," "maintaining skin hydration and / or elasticity," "improving skin blemishes, wrinkles, and / or dullness," "improving hair color, moisture, and / or shine," "extending healthy lifespan," and "improving the quality of reproductive cells."

[0184] As another embodiment of the present invention, the food or beverage composition in this embodiment may also be a food or beverage composition that activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7 and / or LC3.

[0185] The food or beverage composition of this embodiment, by comprising at least one selected from the group consisting of coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, is able to activate alternative autophagy and bring about at least one effect selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

[0186] Therefore, as an embodiment of this invention, a food or beverage composition is also provided, which is selected from at least one of the following groups: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells. The food or beverage composition contains at least one of the following groups: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

[0187] As a food or beverage composition in this embodiment, alternative autophagy activation is achieved by at least one of the following groups: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract; or by promoting the fusion of lysosomal proteins with autophagosomes independently of Atg5, Atg7, and / or LC3; or by promoting the fusion of lysosomal proteins with autophagosomes independently of Atg5, Atg7, and / or LC3. This alternative autophagy activation can exhibit effects such as regulating gastrointestinal state, maintaining intestinal function, promoting intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, extending healthy lifespan, and improving germ cell quality.

[0188] Furthermore, as a food or beverage composition in this embodiment, by comprising at least one of the extracts, it is possible to activate alternative autophagy, and the food or beverage composition containing at least one of the extracts respectively brings about the effects described in Table 1 above. Therefore, as in this embodiment, a food or beverage composition is also provided, which contains at least one of the following extracts that brings about the effects described in Table 1 above: coffee residue extract, coffee extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

[0189] Food or beverage compositions, when ingested orally, can exert their alternative autophagy activation effect.

[0190] Example

[0191] The following specific examples further illustrate the present invention. It should be noted that the present invention is not limited thereto.

[0192] (1) Preparation of samples for the assay of alternative autophagy activity

[0193] (Black pepper extract)

[0194] The following ingredients were used: pepper extract (piperine, HJK-3458) from Inabatake Spices, piperine (Bioperine) from Sabinsa Japan Corporation, black pepper extract powder (95%) from VIDYA JAPAN KK, black pepper extract powder (GIH-95) from GOA International, and black pepper ethanol extract extracted by our company.

[0195] (Red pine extract)

[0196] The whole, dried red pine was pulverized into powder. 3.0 g of the powder was mixed with 30 mL of 99.5% ethanol and stirred at room temperature for 30 minutes to extract the product. After removing the residue using conventional methods, the product was dried to obtain 0.116 g of the dried product.

[0197] (Juniper berry extract)

[0198] Whole, dried juniper berries were pulverized into powder. 3.0 g of the powder was then mixed with 30 mL of 99.5% ethanol and stirred at room temperature for 30 minutes to extract the berries. After removing the residue using conventional methods, the berries were dried to obtain 0.948 g of the dried product.

[0199] (Curcuma extract)

[0200] Uses the registered trademark "Curcumin C3 Complex" from Sabinsa Japan Corporation.

[0201] (Coffee grounds and pressed product)

[0202] A blend of Arabica and Robusta coffee varieties was used to dry the residue from the extraction of roasted (ground) coffee beans with hot water in a dryer at 80°C for 30 hours, resulting in coffee extraction residue with a moisture content of 3.8%.

[0203] 10 kg of coffee grounds were heated to 100°C in a roasting kettle and then pressed using an oil press (H-54, HANDEROIL MACHINERY CO.) to obtain oil. This process was repeated three times, recovering a total of 700 g of oil. The recovered oil was then filtered using No. 2 filter paper, yielding a final yield of 425 g of coffee oil (coffee grounds pressed).

[0204] (Grading of coffee grounds pressed product)

[0205] Weigh 5.0 g of coffee oil into a 100 mL separatory funnel, add 20 mL of hexane, and mix thoroughly. Then add 10 mL of methanol and mix. After visually confirming separation into two layers, recover the methanol layer. Add another 5 mL of methanol, mix, and recover the methanol layer again. Repeat this process twice. Place the recovered methanol layer into a 100 mL separatory funnel and add 0.5 mL of distilled water to mix. Since the hexane layer has separated, recover the methanol layer into a 50 mL round-bottom flask. Dry the methanol layer to obtain 0.42 g of dried product.

[0206] (80v / v% ethanol extract of coffee grounds)

[0207] Add 900 mL of 80 v / v ethanol to 100 g of coffee grounds. Extract in a 30°C bath with a stirrer for 1 hour, then filter using a Buchner funnel lined with No. 2 filter paper. Collect the permeate and add it to a round-bottom flask for vacuum concentration to remove the ethanol, yielding a concentrated solution. Freeze the concentrated solution at -80°C, and then freeze-dry it using a freeze dryer.

[0208] (99.5% v / v ethanol extraction of coffee grounds)

[0209] Add 900 mL of 99.5% v / v ethanol to 100 g of coffee extraction residue. Extract in a constant temperature bath at 30°C with a stirrer for 1 hour, then filter using a Buchner funnel lined with No. 2 filter paper. Collect the permeate and add it to a round-bottom flask for concentration under reduced pressure to remove the ethanol, yielding a concentrated solution.

[0210] (Acetone extract from coffee grounds)

[0211] Add 800 mL of acetone to 200 g of coffee grounds. Extract in a constant temperature bath at 30°C with a stirrer for 1 hour, then filter using a Buchner funnel lined with No. 2 filter paper. Collect the permeate and add it to a round-bottom flask for vacuum concentration to remove the acetone, yielding a concentrated solution.

[0212] (Dehydrocaffeine (DC) and dehydrocatechol (DK))

[0213] A blend of Arabica and Robusta coffee varieties is used to dry the residue produced during hot water extraction of roasted (ground) coffee beans at 80°C for 16 hours, resulting in coffee extraction residue with a moisture content of less than 10%.

[0214] The dried coffee grounds were heated to 100°C in a roasting kettle and then pressed using an oil press (H-54, HANDER OIL MACHINERY CO.). This process was repeated three times, recovering a total of 700g of oil. The recovered oil was then filtered using No. 2 filter paper, yielding a final yield of 425g of coffee grounds pressed oil.

[0215] Add 5g of coffee grounds pressed oil to a separatory funnel, add 4 times the amount of hexane and mix, then add 2 times the amount of methanol and mix. After standing for a period of time to confirm separation into two layers, recover the methanol layer. Add an equal amount of methanol to the recovered methanol layer and mix. Repeat the above operation twice to obtain a methanol layer. Add 1 / 10 the amount of distilled water to this methanol layer and mix, let stand and separate, then recover the methanol layer. Concentrate and dry the recovered methanol layer to obtain 0.42g of dried product. Fractionate this dried product using a medium-pressure preparative liquid chromatograph (Yamazen Corporation EPCLC-W-Prep 2XY) under the following conditions. The column used was a UNIVERSAL Premium ODS-SM 30μm 120A, 2L size (3x20cm, 51g) (Yamazen Corporation). The mobile phase was ultrapure water, acetonitrile, and isopropanol. A linear gradient elution was performed from ultrapure water-acetonitrile (95:5) to 100% acetonitrile at a flow rate of 20 mL / min for 40 min, followed by isopropanol flow for 12 min, fractionating in 10 mL increments. The fractions eluted between 37 and 45 min were concentrated and dried. DC and DK were separated from the dried solids using a preparative HPLC system (Shimadzu Prominence) under the following conditions. The column used was a SunFire 5μm (19x150 mm) (Waters). The mobile phase was ultrapure water and acetonitrile. The column temperature was 30℃ and the flow rate was 17 mL / min. The ultrapure water-acetonitrile (60:40) was flowed in for 160 minutes. The peak eluted from 116 min to 126 min (component A) and the peak eluted from 128 min to 141 min (component B) were separated and recovered, and 5 mg and 14 mg of concentrated dry solids of components A and B were obtained, respectively.

[0216] NMR and mass spectrometry analyses were performed on components A and B separated above. NMR measurements were performed using an AVANCENEO 400MHz instrument (Bruker), and mass spectrometry analyses were performed using a Vanquish Flex liquid chromatograph (Thermo Fisher Scientific) and a Thermo Fisher Scientific OrbiTrap Exploris 240 mass spectrometer (Thermo Fisher Scientific). The following data were obtained, identifying component A as dehydrocaffeine (DK) and component B as dehydrocaffeine (DC).

[0217] The concentrated dried solids of components A and B were used as our purified DK and DC, respectively. Commercially available DC was also used as a standard in the evaluation of alternative autophagy activity.

[0218] The following shows the results of DK's NMR and mass spectrometry analyses.

[0219] 1 ¹H-NMR (DMSO-d6, 400MHz, corrected using solvent): δ (ppm) 7.50 (¹H, d, J = 1.5Hz, H19), 6.45 (¹H, brs, H18), 6.29 (¹H, d, J = 10.0Hz, H2), 5.93 (¹H, d, J = 10.1Hz, H1), 5.34 (¹H, s, H15), 4.65 (¹H, brt, -OH, H1) 7), 3.99 (2H, s, H17), 2.54-2.50 (2H, overlapped, H5, 13), 2.01 (1H, d, J=10.2Hz, H14a), 1.86 (1H, m, H6a), 1.78-1.33 (9H, overlapped, H6b, 7a, 7b, 9, 11a, 11b, 12a, 12b, 14b), 0.94 (3H, s, H2O)

[0220] 13 C-NMR (DMSO-d6, 100MHz, corrected with solvent): δ (ppm) = 149.37 (C3), 147.22 (C16), 141.72 (C19), 138.60 (C1), 133.60 (C15), 121.43 (C4), 115.03 (C2), 109.05 (C18), 59.05 (C17), 48.62 (C8), 44.43 (C14), 43.72 (C5), 41.23 (C10), 40.36 (C13), 40.00 (C9), 37.26 (C7), 24.86 (C12), 20.62 (C6), 19.02 (C11), 15.18 (C20)

[0221] ESI MS (m / z) C 20 H 23 O([M+H-H2O)) + Theoretical value 279.1743, measured value 279.1742; C 20 H 25 O2 ([M+H)) + Theoretical value: 297.1849; Measured value: 297.1847

[0222] The following shows the results of DC's NMR and mass spectrometry analyses.

[0223] 1¹H-NMR (DMSO-d6, 400MHz, corrected using solvent): δ (ppm) = 7.39 (¹H, d, J = 1.4Hz, H19), 6.29 (¹H, d, J = 1.7Hz, H18), 5.32 (¹H, s, H15), 4.62 (¹H, brt, -OH, H17), 3.99 (2H, brs, H17), 2.57–2.55 (2H, m, H2), 2.21 (¹H, dd, J = 1 2.5, 1.9Hz, H5), 2.11 (1H, d, J=10.2Hz, H14a), 2.03 (1H, dt, J=12.8, 4.2Hz, H1a), 1.79-1.33 (10H, overlap, H6a, 6b, 7a, 7b, 11a, 11b, 12, 14b), 1.24-1.17 (1H, m, H1b), 1.14 (1H, d, J=8.4Hz, H9), 0.79 (3H, s, H2O)

[0224] 13 C10-NMR (DMSO-d6, 100MHz, corrected with solvent): δ (ppm) = 148.00 (C3), 147.04 (C16), 140.84 (C19), 133.97 (C15), 119.88 (C4), 108.50 (C18), 59.08 (C17), 48.24 (C8), 44.48 (C14), 43.62 (C9), 43.47 (C5), 40.48 (C13), 38.02 (C10), 37.81 (C7), 35.17 (C1), 25.01 (C12), 21.56 (C6), 20.20 (C2), 18.95 (C11), 12.93 (C20)

[0225] ESI MS (m / z) C 20 H 25 O([M+H-H2O)) + Theoretical value 281.1900, measured value 281.1899; C 20 H 27 O2 ([M+H)) + Theoretical value: 299.2006; Measured value: 299.2005

[0226] (High-content residue extracts of DC and DK)

[0227] 200g (dry weight) of the coffee extract residue obtained above, with a moisture content adjusted to 2.5%, was mixed with 70v / v% ethanol to make a total volume of 1L. The mixture was stirred at 250rpm for 2 hours at 50°C using a three-in-one motor (BL-600, manufactured by Shin-To Science Co., Ltd.). After 2 hours of extraction, the mixture was filtered using No. 2 filter paper, and the filtrate and the extract residue on the filter paper were recovered. For the recovered extract residue on the filter paper, 70v / v% EtOH was added to make a total volume of 1L, and the mixture was extracted again at 50°C for 1 hour while stirring. After 1 hour of extraction, the mixture was filtered using No. 2 filter paper, and the filtrate was recovered. The first and second filtrates were combined and concentrated using an evaporator until EtOH was removed. The concentrate was then freeze-dried. 7.9g of the freeze-dried extract was obtained. This extract was used as a high-content residue extract for DC and DK.

[0228] (Apple polyphenol extract)

[0229] Apple Phenon (registered trademark) sold by BGG Japan (Made in China).

[0230] (2) Evaluation of alternative autophagy activity

[0231] The medium used was DMEM (containing 4.5 g / L glucose) containing 10% FBS (Sigma Aldrich), 1 mM sodium pyruvate solution (NACALAI TESQUE, INC.), 1 mM HEPES buffer (NACALAI TESQUE, INC.), 0.1 mM non-essential amino acid solution (FUJIFILM WakoPure Chemical Corporation), 0.1 mM MEM vitamin solution (Invitrogen), 100 U / mL penicillin-100 μg / mL streptomycin (NACALAI TESQUE, INC.), and 0.05 mM 2-mercaptoethanol (Bio-Rad).

[0232] ATG5-KO / GFP-Lamp1 was prepared with reference to Patent Document 1 (International Publication No. 2013 / 118842).

[0233] Immortality was achieved by introducing a gene expressing the SV40T antigen into mouse embryonic fibroblasts (MEFs) established from ATG5-deficient C57 / B6J mice at 14.5 days of viviparous life, using the method described in Mizushima, N. et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657-668 (2001). This was accomplished by introducing a retrovirus expressing the SV40T antigen via electroporation using the Nucleofector system (Amaxa). Furthermore, a retrovirus expressing the Lamp1-GFP fusion protein (also known as "GFP-Lamp1") was introduced into plat E cells (used as retroviral packaging cells) prepared by introducing a retrovirus prepared from the ATG5-KO / GFP-Lamp1 gene.

[0234] ATG5-KO / GFP-Lamp1 was used at 4.0 x 10⁻⁶. 4 30 μL of cells / mL was seeded into each well of a 384-well Greiner plate and incubated for 24 hours at 37°C and 5% CO2. Then, prepared coffee grounds extract, coffee grounds pressed extract, dehydrocatechol (DC), dehydrocatechol (DK), high-content DC and DK grounds extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract were added to each well. After 24 hours, 12 μL of 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako Pure Chemical Corporation) was added to each well, and the mixture was incubated at room temperature for 15 minutes. After washing the cultures with HBSS (FUJIFILM Wako Pure Chemical Corporation), 30 μL of HBSS (prepared by diluting Hoechst (Invitrogen) 2000 times) was added to each well, and the mixture was incubated at room temperature for 15 minutes.

[0235] Then, microscopic images were obtained using a confocal quantitative cell imaging system CQ1 (Yokogawa Electric). Referring to Patent Document 1, the cumulative fluorescence intensity of GFP-Lamp1 fluorescent particles per cell was output from the microscopic images. This value was divided by the vehicle group and used as an indicator to evaluate alternative autophagy activity. The results are shown in Table 2 and... Figure 1 The results of extracts containing high concentrations of DC, DK, DC, and DK residues are presented in... Figure 2 .

[0236] It should be noted that the same experiment was also conducted on the available extracts for the extract disclosed in Patent Document 2, and the results showed that it did not have a sufficient effect on activating alternative autophagy (the results were not disclosed).

[0237] [Table 2]

[0238]

[0239] According to Table 2 and Figure 1 and Figure 2 The results confirmed that coffee grounds extract, coffee grounds press, dehydrocatechol (DC), dehydrocatechol (DK), high-content DC and DK grounds extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract have the function of activating alternative autophagy.

[0240] <Example>

[0241] Table 3 shows the results of the determination of the changes in DC and DK content in the coffee extract residue obtained above under various drying conditions. The content of DC and DK in the extract obtained by extracting the freeze-dried residue with 75 v / v% ethanol solution was set as 100%, and the DC+DK content (%) is shown.

[0242] [Table 3]

[0243]

Claims

1. An alternative autophagy activator comprising at least one selected from the group consisting of coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

2. The alternative autophagy activator according to claim 1, wherein, At least one of the following groups consisting of coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract activates alternative autophagy by promoting the fusion of lysosomal proteins with autophagosomes, independent of Atg5, Atg7, and / or LC3.

3. The alternative autophagy activator according to claim 1 or 2, used for at least one purpose selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, rejuvenating the intestine, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin spots, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of germ cells.

4. A food or beverage composition for activating alternative autophagy, comprising at least one selected from the group consisting of coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

5. A food or beverage composition comprising at least one of the following groups: adjusting gastrointestinal state, maintaining intestinal function, rejuvenating the intestines, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, prolonging healthy lifespan, and improving the quality of reproductive cells, wherein the composition contains at least one of the following groups: coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract.

6. The food or beverage composition according to claim 5, wherein, By activating alternative autophagy with at least one of the following groups, namely coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract and apple polyphenol extract, the product exhibits effects of at least one of the following groups: regulating gastrointestinal state, maintaining intestinal function, promoting intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles and / or dullness, improving hair color, moisture and / or shine, extending healthy lifespan, and improving germ cell quality.

7. The food or beverage composition according to claim 5 or 6, wherein, The enzyme, selected from at least one of the groups consisting of coffee extract and / or coffee residue extract, black pepper extract, red pine extract, juniper berry extract, turmeric extract, and apple polyphenol extract, exhibits at least one effect selected from the group consisting of regulating gastrointestinal state, maintaining intestinal function, promoting intestinal rejuvenation, improving cognitive function, enhancing memory, clearing brain waste, maintaining healthy liver function, improving motor function, maintaining skin hydration and / or elasticity, improving skin blemishes, wrinkles, and / or dullness, improving hair color, moisture, and / or shine, extending healthy lifespan, and improving germ cell quality, without relying on Atg5, Atg7, and / or LC3.

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