Nicotinamide adenine dinucleotide production promoter and method for producing NAD production promoter

Abstract

The purpose of the present disclosure is to provide a nicotinamide adenine dinucleotide (NAD) production promoter derived from a novel material, and a method for producing the NAD production promoter. The technology disclosed herein relates to an NAD production promoter and is characterized by containing a Euglena-derived substance, which is a component derived from microalgae belonging to the genus Euglena. It is confirmed that the NAD production promoter has an NAD-production-promoting action on a living animal body. Because NAD production promoter uses microalgae belonging to Euglena as a material, the NAD production promoter can be stably supplied by means of culturing.

Description

Nicotinamide adenine dinucleotide production promoter and method for producing NAD production promoter

[0001] The technology disclosed herein relates to a production promoter that promotes the production of nicotinamide adenine dinucleotide (NAD) and a method for producing the NAD production promoter. This application claims priority based on Japanese Patent Application No. 2023-133432 filed on August 18, 2023, the entire contents of which are incorporated herein by reference.

[0002] NAD is an important biomolecule involved in energy metabolism as a coenzyme that catalyzes redox reactions, as well as in intracellular functions such as DNA damage response and gene expression regulation.

[0003] For example, the salvage pathway, which is one of the NAD biosynthetic pathways, is maintained by an enzyme called nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is the rate-limiting enzyme in the NAD biosynthesis system, synthesizing nicotinamide mononucleotide (NMN), an intermediate of NAD, from nicotinamide. As shown in Non-Patent Document 1, the activity of NAMPT is known to decrease with age. Furthermore, as shown in Non-Patent Document 2, the amount of NAD in the body is also known to decrease with age.

[0004] On the other hand, Non-Patent Document 3 suggests that exogenous administration of NMN or the like to living mice may increase the amount of NAD in the body, thereby improving age-related decline in biological functions and associated age-related diseases. Furthermore, in recent years, development of substances (NAD production promoters) that increase the amount of NAD in living animals has been progressing. For example, Patent Document 1 discloses an NAD production promoter containing placenta extract and collagen peptide. Furthermore, Patent Document 2 discloses an NAD production promoter containing Roman chamomile extract, chamomile extract, parsley extract, and kudzu extract as active ingredients.

[0005] Japanese Patent Application Publication No. 2022-35271 Japanese Patent Application Publication No. 2022-129527

[0006] Nampt Expression Decreases Age-Related Sensence in Rat Bone Marrow Mesenchymal Stem Cells by Targeting Sirt1. , Ma, et al, PLoS One, 2017;12(1):e0170930Age-Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue. , Massudi, et al. , PLoS One, 2012;7(7):e42357Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. , Mills, et al. , Cell Metab. , 2016 Dec 13;24(6), 795-806

[0007] As described above, NAD production promoters extracted from various materials have been proposed in recent years. The present inventors have been studying novel materials that have the effect of promoting NAD production. The technology disclosed herein was developed based on such studies and aims to provide an NAD production promoter derived from a novel material and a method for producing the same.

[0008] In creating a novel NAD production promoter, the present inventors focused on microalgae. Microalgae have the advantage of being easily propagated through cultivation. Therefore, if an NAD production promoter derived from microalgae could be realized, it would contribute to the stable supply of NAD production promoters. As a result of extensive research by the present inventors, it was found that an extract of microalgae belonging to the genus Euglena has an NAD production promoting effect in living animals. The NAD production promoter disclosed herein was made based on this discovery.

[0009] That is, the nicotinamide adenine dinucleotide (NAD) production promoter disclosed herein is characterized by containing a Euglena-derived substance, which is a component derived from microalgae belonging to the genus Euglena. Experiments conducted by the present inventors have confirmed that this NAD production promoter has an NAD production-promoting effect in living animals. Furthermore, because this NAD production promoter is made from microalgae belonging to the genus Euglena, it can be stably supplied by culturing.

[0010] In one preferred embodiment, the NAD production promoter disclosed herein further contains NAD.

[0011] In one preferred embodiment, the NAD production promoter disclosed herein further comprises an NAD precursor.

[0012] In one preferred embodiment of the NAD production promoter disclosed herein, the NAD precursor is nicotinic acid or nicotinamide.

[0013] In a preferred embodiment of the NAD production enhancer disclosed herein, the NAD production enhancer is used in a food composition, a food additive, a feed, a cosmetic, a cosmetic additive, or a medicine.

[0014] Another aspect of the technology disclosed herein provides a method for producing an NAD production promoter. The technology disclosed herein includes a culture step of culturing microalgae belonging to the genus Euglena in a medium, a recovery step of recovering the microalgae from the medium after the culture, and a Euglena-derived substance production step of producing a Euglena-derived substance from the microalgae recovered in the recovery step. This realizes a method for providing an NAD production promoter containing a substance derived from microalgae belonging to the genus Euglena.

[0015] In a preferred embodiment of the production method disclosed herein, the culturing step is carried out in a medium supplied with an NAD precursor.

[0016] In a preferred embodiment of the production method disclosed herein, the NAD precursor includes nicotinic acid or nicotinamide.

[0017] FIG. 1 is a flow chart schematically showing a method for producing an NAD production promoter according to one embodiment. FIG. 2 is a graph showing the trends in nicotinic acid concentration and NAD concentration in Euglena when Euglena is cultured in a medium supplemented with nicotinic acid in the culture step. FIG. 3 is a graph showing the trends in nicotinamide concentration and NAD concentration in Euglena when Euglena is cultured in a medium supplemented with nicotinamide in the culture step. FIG. 4 is a graph showing the results of an evaluation of the effect of a Euglena-derived substance on NAD levels in ARPE-19 cells. FIG. 5 is a graph showing the results of an evaluation of the effect of a Chlorella-derived substance on NAD levels in ARPE-19 cells. FIG. 6 is a graph showing the results of an evaluation of the effect of a Euglena-derived substance on NAD levels in NIH / 3T3 cells. FIG. 7 is a graph showing the results of an evaluation of the effect of an NAD precursor on NAD levels in NIH / 3T3 cells.

[0018] Hereinafter, embodiments of the technology disclosed herein will be described. It should be noted that matters not mentioned in this specification but necessary for implementing the technology disclosed herein can be understood as design matters for a person skilled in the art based on the prior art in the relevant field. The technology disclosed herein can be implemented based on the contents disclosed in this specification and common technical knowledge in the relevant field. In this specification, a numerical range expressed as "A to B" includes A and B, and also encompasses the meaning of "preferably greater than A" and "preferably smaller than B."

[0019] As used herein, "NAD" refers to NAD ＋ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), NADP ＋ (oxidized nicotinamide adenine dinucleotide phosphate) and NADPH (reduced nicotinamide adenine dinucleotide phosphate). ＋ Furthermore, in vivo, NAD ＋When the 2'-hydroxy group of the adenine ribose moiety of NADP is phosphorylated, it can become NADP (nicotinamide adenine dinucleotide phosphate). NADP is a coenzyme that catalyzes many oxidation-reduction reactions in the body, including photosynthesis. Similarly, NADP can also be converted into NADP in the body. ＋ , NADPH can take two states.

[0020] Furthermore, the term "NAD production-promoting activity" as used herein refers to the activity of promoting NAD production in biological cells. This "NAD production-promoting activity" refers to the confirmed increase in NAD amount as a result of adding the NAD production promoter of the present disclosure to biological cells, regardless of which part of the NAD production cycle in the biological cells is promoted. The term "NAD production-promoting activity" as used herein refers to the activity of increasing the amount of NAD in biological cells by 10% or more (preferably 25% or more, more preferably 50% or more, and particularly preferably 100% or more) compared to the amount of NAD in the biological cells before culture when cultured for 24 hours after application of the NAD production promoter. The "NAD concentration in living animal tissue" as used herein can be measured by conventional methods in the technical field to which the technology disclosed herein pertains, such as by cycling assay. Such measurement methods can be applied to biological cells, Euglena, and NAD production promoters.

[0021] <NAD production promoter> The NAD production promoter according to this embodiment contains a substance derived from microalgae belonging to the genus Euglena (hereinafter referred to as a "Euglena-derived substance") as an active ingredient. The components of the NAD production promoter according to this embodiment will be described below.

[0022] (Euglena-Derived Substances) Euglena-derived substances are components derived from the cells of microalgae of the genus Euglena. As used herein, "microalgae belonging to the genus Euglena (hereinafter also referred to as "Euglena")" generally encompasses microalgae that belong to the biological genus Euglena, as well as variants and mutant strains thereof. Examples of mutant strains include gene mutant strains and mutant strains obtained by recombination, transduction, transformation, etc. Microalgae belonging to the genus Euglena are not particularly limited, and examples thereof include Euglena gracilis, Euglena acus, Euglena agilis, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena granulata, Euglena gymnodiniales, Euglena intermedia, and Euglena longa. Examples of suitable Euglena species include Euglena longa, Euglena mutabilis, Euglena proxima, Euglena viridis, Euglena vermiformis, Euglena pride, Euglena oxyuris, and Euglena spirogyra, and preferably Euglena gracilis and mutants thereof can be used.

[0023] The method for obtaining Euglena used in the technology disclosed herein can be any known method and is not particularly limited. For example, Euglena may be collected from the natural world, such as a pond, purchased commercially, or obtained through a conservation institution or depository. Furthermore, Euglena obtained by subculture may be used, or mutant strains may be produced by known methods. From the viewpoint of stable supply of Euglena-derived substances, it is preferable to use Euglena obtained by culture.

[0024] The form of the Euglena-derived substance in the technology disclosed herein is not particularly limited, and various forms can be employed. For example, the NAD production promoter disclosed herein can use Euglena (Euglena algae bodies) that have not been subjected to any particular extraction treatment as the Euglena-derived substance. The Euglena algae bodies may be in a raw state or in a dried state. Dried Euglena algae bodies can be obtained by conventionally known methods such as freeze-drying and spray-drying. When such Euglena algae bodies are used, the content of the Euglena-derived substance in the NAD production promoter is preferably 50 wt% or more (preferably 55 wt% or more, more preferably 60 wt% or more, and particularly preferably 65 wt% or more). On the other hand, the upper limit of the Euglena algae body content is preferably 100 wt% or less (preferably 95 wt% or less, more preferably 90 wt% or less, and particularly preferably 80 wt% or less). This allows the NAD production-promoting effect to be optimally exhibited.

[0025] Meanwhile, Euglena-derived substances can also be produced by extracting intracellular components from Euglena algae. There are no particular limitations on the method for obtaining such Euglena extract, and various methods can be employed. For example, intracellular components can be extracted by disrupting the Euglena cell membrane and then performing centrifugation or filtration. In this embodiment, disrupting the Euglena cell membrane exposes the Euglena intracellular components. This makes it easier for the Euglena-derived substance to be more favorably absorbed by the subject. Examples of methods for disrupting the Euglena cell membrane include crushing, ultrasonication, enzymatic methods, and treatment with a high-pressure emulsifier. When using Euglena extracts produced by these methods, it is preferable that the content of Euglena extract in the NAD production promoter be 10 wt% or more (preferably 15 wt% or more, more preferably 20 wt% or more, and particularly preferably 25 wt% or more). On the other hand, the upper limit of the Euglena extract content is preferably 95 wt % or less (preferably 90 wt % or less, more preferably 85 wt % or less, and particularly preferably 75 wt % or less), which allows the NAD production-promoting effect to be optimally exhibited.

[0026] Another method for obtaining Euglena-derived substances is solvent extraction. Typically, water-soluble components in Euglena can be obtained by extracting Euglena algae bodies with an aqueous solvent. Water is preferably used as the aqueous solvent used as the extraction solvent. That is, it is preferable to use water-soluble components obtained by extracting Euglena algae bodies with water as the Euglena-derived substance. The water used for extraction may contain impurities as long as the effects of the technology disclosed herein are not significantly impaired. Examples of water used for extraction include, but are not limited to, tap water, distilled water, pure water, and ultrapure water. Furthermore, the extraction solvent may contain solvents other than water as long as the effects of the invention are not significantly impaired. For example, but not limited to, the aqueous solvent may contain alcohols as a solvent other than water. Examples of such alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol. These solvents may be used alone or in combination. When water-soluble components of Euglena are used, the content of Euglena extract in the NAD production promoter is preferably 0.05 wt% or more (preferably 0.1 wt% or more, more preferably 0.5 wt% or more, and particularly preferably 1 wt% or more). On the other hand, the upper limit of the content of water-soluble components in Euglena is preferably 25 wt% or less (preferably 20 wt% or less, more preferably 15 wt% or less, and particularly preferably 10 wt% or less). This allows the NAD production-promoting effect to be optimally exhibited.

[0027] When a Euglena-derived substance is obtained by solvent extraction, the pH of the extraction solvent used is not particularly limited as long as it does not significantly impair the effects of the technology disclosed herein. For example, the pH is preferably 4 to 10 (preferably 5 to 9, more preferably 6 to 8).

[0028] The Euglena-derived substance may be used in the form extracted with an extraction solvent (extract), or the extract may be subjected to treatment such as concentration or dilution as necessary. The extract may also be used as a dried product by subjecting it to treatment such as concentration to dryness, spray drying, or freeze drying.

[0029] (NAD) The NAD production promoter according to this embodiment may contain, in addition to the Euglena-derived NAD production-promoting component described above, NAD, which is a product of the NAD biosynthesis pathway. This directly increases the amount of NAD in the living body of the animal to which the agent is administered. The NAD may be NAD mixed into the Euglena-derived substance or separately purified NAD.

[0030] When raw Euglena algae are used, the NAD concentration in the NAD production promoter is preferably 250 nmol / gFW or more (preferably 275 nmol / gFW or more, more preferably 300 nmol / gFW or more). On the other hand, the upper limit of the NAD concentration is not particularly limited and may be 15,000 nmol / gFW or less, or 14,250 nmol / gFW or less.

[0031] When dried Euglena algae cells are used, the NAD concentration in the NAD production promoter is preferably 500 nmol / gDW or more (preferably 550 nmol / gDW or more, more preferably 600 nmol / gDW or more). On the other hand, the upper limit of the NAD concentration is not particularly limited and may be 30,000 nmol / gDW or less, or 28,500 nmol / gDW or less.

[0032] When a Euglena extract obtained by extracting intracellular components from Euglena algae is used, the NAD concentration in the NAD production promoter is preferably 200 μM or higher (preferably 300 μM or higher, more preferably 400 μM or higher). On the other hand, the upper limit of the NAD concentration is not particularly limited and may be 5,700 μM or lower, or 5,400 μM or lower.

[0033] When a water-soluble component of Euglena is used, the NAD concentration in the NAD production promoter is preferably 0.5 μM or higher (preferably 1 μM or higher, more preferably 5 μM or higher). On the other hand, the upper limit of the NAD concentration is not particularly limited and may be 750 μM or lower, or may be 600 μM or lower.

[0034] (NAD Precursor) The NAD production promoter according to this embodiment preferably contains a Euglena-derived substance obtained from Euglena cultured in the presence of an NAD precursor. As will be described in detail later, experiments have confirmed that the Euglena-derived substance obtained in the presence of an NAD precursor has a superior NAD production-promoting effect. While not intended to limit the technology disclosed herein, it is speculated that this effect occurs because the NAD precursor activates the NAD biosynthetic pathway in Euglena, resulting in the production of a large amount of NAD production-promoting components. Furthermore, the Euglena-derived substance obtained in the presence of the NAD precursor contains unused NAD precursor. Furthermore, known NAD precursors in the NAD biosynthetic pathway can be used as the NAD precursor. Suitable examples of such NAD precursors include nicotinic acid and nicotinamide. The above description is not intended to prohibit the separate addition of an NAD precursor to the NAD production promoter. Even when an NAD precursor is added separately, NAD production in the living body of the animal to which it is administered can be promoted to some extent.

[0035] (Carrier) The NAD production promoter according to this embodiment preferably contains a carrier that is pharmaceutically (medically) acceptable in terms of safety. The NAD production promoter is preferably a mixture of the Euglena-derived substance and a carrier so that the NAD production-promoting effect is appropriately exerted. For example, when obtaining a liquid NAD production promoter, an aqueous dispersion medium such as tap water, purified water, distilled water, water for injection, phosphate-buffered saline (PBS), or physiological saline is preferably used as the carrier. Other examples of liquid carriers include an aqueous solution of an alcohol (such as ethanol) at an appropriate concentration, a polyhydric alcohol, or a liquid oil. When obtaining a solid NAD production promoter, an excipient such as lactose, starch, dextrin, or sorbitol is preferably used as the carrier. Other examples of solid carriers include a binder, a disintegrant, or the like. However, the NAD production promoter disclosed herein may also include embodiments that do not contain the above-mentioned carrier. For example, when dried Euglena algae bodies are added to a food composition or when intracellular components are used in an extracted state, it is not necessary to add a carrier.

[0036] (Other Components) The NAD production promoter of this embodiment may contain other components depending on the intended use, as long as the NAD production promoting effect is not significantly impaired. The other components can be selected depending on the form and application of the NAD production promoter, and are not particularly limited as long as they are components that can be used in food compositions, food additives, feed, cosmetics, cosmetic additives, medicines, etc. Non-limiting examples of other components include preservatives, emulsifiers, thickeners, colorants, flavorings, seasonings, pH adjusters, antiseptics, stabilizers, bases, carriers, solvents, dispersants, excipients, binders, disintegrants, lubricants, chelating agents, surfactants, antioxidants, humectants, thickeners, vitamins, etc.

[0037] The NAD production promoter disclosed herein contains a Euglena-derived substance as an active ingredient. Although the detailed mechanism of the NAD production-promoting effect, such as the direct components, is unclear, experiments have confirmed that the Euglena-derived substance promotes NAD production in living animals when it comes into contact with the animal. Furthermore, because the NAD production promoter disclosed herein is made from microalgae belonging to the genus Euglena, it can be stably supplied by culturing.

[0038] In this specification, "biological cells" refers to cells of an organism. In addition, in this specification, "organism" refers to a target (administration target) to which the NAD production promoter is administered, and includes organisms other than Euglena. In this specification, the "administration target" is not particularly limited, and examples include animals in general. Specific administration targets include, for example, humans and non-human animals, preferably humans and non-human mammals, and more preferably humans. Examples of non-human animals include mammals such as dogs, cats, hamsters, mice, rats, rabbits, horses, cows, pigs, sheep, goats, and monkeys; birds such as chickens, quails, and ducks; and fish such as carp, medaka, rainbow trout, and yellowtail.

[0039] (Uses) The NAD production promoter according to this embodiment contains a Euglena-derived substance as an active ingredient and can therefore be used in a wide range of industrial fields targeting both humans and non-human animals. The NAD production promoter according to this embodiment can be suitably used, for example, in food compositions, food additives, feed, cosmetics, cosmetic additives, and medicines.

[0040] As described above, the NAD production promoter according to this embodiment promotes NAD production in living animals. Therefore, the NAD production promoter according to this embodiment can be used to prevent or improve declines in biological functions caused by a decrease in NAD levels and associated diseases. Examples of such declines include insulin resistance, mitochondrial function, and renal function. In addition to the declines in biological functions described above, specific examples of declines in biological functions include skin aging (e.g., wrinkles, age spots, freckles, sagging skin, dryness, rough skin, etc.), declines in motor function (e.g., bone function, muscle mass, etc.), declines in brain function (e.g., learning ability, memory, etc.), gray hair, decreased physical strength, decreased bone mass (including teeth), decreased eyesight, and decreased immune function. Examples of such diseases include metabolic diseases such as diabetes, dyslipidemia, and skeletal muscle atrophy; neurological diseases such as Alzheimer's; kidney diseases such as chronic kidney disease and diabetic nephropathy; and cancer.

[0041] Food compositions in which the NAD production promoter of this embodiment can be used include, but are not limited to, beverages, confectioneries, breads, processed seafood products, processed livestock products, dairy products, processed grain products, processed vegetables or fruits, soups, seasonings, oils and fats, noodles, etc., and the NAD production promoter can also be used as an additive (food additive) for these foods. In addition to the above, food compositions also include nutritional supplements (supplements, nutritional drinks, etc.), nutritional supplement beverages, nutritionally functional foods, foods for specified health uses, functional food and beverages, etc.

[0042] The NAD production promoter of this embodiment can also achieve the NAD production promoting effect in animals other than humans. Therefore, the NAD production promoter disclosed herein can also be suitably applied to feed. Examples of feed include, but are not limited to, livestock feed, pet feed (pet food), and nutritional supplements for pet animals. The form of the feed is not particularly limited, and may be a powder, a liquid, or a solid such as a pellet.

[0043] When the NAD production promoter of this embodiment is used as a pharmaceutical, it may be applied internally or externally, and the form is not limited thereto. Furthermore, when used as a pharmaceutical, it may be administered orally or parenterally. Furthermore, when the NAD production promoter of this embodiment is used as a pharmaceutical, its application is not limited to humans, and it may also be an animal medicine for animals other than humans. Pharmaceutical forms include, but are not limited to, internal use, external use, injections, and the like. Examples of the internal use preparation include capsules, tablets, pills, granules, fine granules, sugar-coated tablets, chewable tablets, lozenges, emulsions, suspensions, syrups, and dry syrups. Examples of the external use preparation include poultices, plasters, tapes, liquids, gels, eye drops, nasal drops, creams, ointments, suppositories, lotions, and aerosols.

[0044] Cosmetics in which the NAD production promoter of this embodiment can be used include, for example, skin care cosmetics such as lotions, serums, emulsions, creams, gels, packs, sunscreens, facial cleansers, cleansing agents, and bath cosmetics, and makeup cosmetics such as makeup bases, concealers, foundations, lipsticks, blushers, eyeliners, and eye shadows, and can also be used as an additive (cosmetic additive) for these cosmetics.

[0045] The application amount of the NAD production promoter according to this embodiment is not particularly limited as long as it is an amount that can exhibit the desired effect, and may vary depending on, for example, the subject (human or animal), body weight, administration schedule, and form of the NAD production promoter. When the subject is a human, the daily intake amount per adult is not particularly limited, but may be, for example, 10 mg or more (preferably 100 mg or more, more preferably 300 mg or more) in terms of Euglena-derived substances (dried Euglena algae bodies). On the other hand, the daily intake amount per adult may be, for example, 100,000 mg or less (preferably 10,000 mg or less, more preferably 5,000 mg or less) in terms of Euglena-derived substances (dried Euglena algae bodies). Furthermore, the above application amount does not need to be administered once a day; it may be administered in two or more divided doses so that the daily amount falls within the above range.

[0046] The NAD production promoter of this embodiment is preferably diluted appropriately before use, taking into consideration various factors such as the target of application, use, and other ingredients, so as to enhance the NAD production-promoting effect in the living body of the animal to which it is administered. As described above, the content of the Euglena -derived substance may vary depending on the form of the Euglena -derived substance (e.g., extraction procedure). When diluting the NAD production promoter disclosed herein for use, it is preferable to take this into consideration. Furthermore, when diluting the NAD production promoter, the liquid used for dilution is not particularly limited, as long as it does not significantly impair the effects of the technology disclosed herein. Examples of such liquids for dilution include liquids that can be used as carriers as described above (such as tap water, purified water, distilled water, water for injection, phosphate-buffered saline (PBS), physiological saline, alcoholic aqueous solutions, polyhydric alcohols, and liquid oils).

[0047] As an example, the dilution amount when dried Euglena algae bodies are used as the Euglena-derived substance and the drink is intended for use as a nutritional drink will be described. In this case, the NAD production promoter is added to the nutritional drink so that the concentration of the Euglena-derived substance (dried Euglena algae bodies) is 0.0001 wt% or more (preferably 0.001 wt% or more, more preferably 0.005 wt% or more) when the total volume of the nutritional drink is 100 wt%. This allows the NAD production-promoting effect to be preferably achieved in the subject to whom it is administered. Meanwhile, the upper limit of the concentration of the Euglena-derived substance (dried Euglena algae bodies) in the nutritional drink is not particularly limited, but may be 50 wt% or less or 25 wt% or less.

[0048] Furthermore, the NAD production promoter of the present embodiment may be administered in combination with a conventionally known NAD production promoter (e.g., NMN, nicotinamide riboside (NR)), thereby more suitably improving the NAD production promoting effect in the living body of the animal to which the NAD production promoter is administered.

[0049] The effect of promoting NAD production may vary depending on various factors such as the subject, the subject's weight, the subject's health condition, other additives, etc. Therefore, the content of the Euglena-derived substance in each of the above-mentioned uses is an example of the content under specific conditions and is not intended to limit the uses of the NAD production promoter disclosed herein.

[0050] <Production Method> The method for producing the NAD production promoter disclosed herein will be described with reference to FIG. 1. FIG. 1 is a flow chart schematically illustrating a method for producing an NAD production promoter according to one embodiment. As shown in FIG. 1, the method for producing an NAD production promoter according to this embodiment includes, for example, a culture step S101, a recovery step S102, and a Euglena-derived substance production step S103. This production method can typically be carried out in the order described above, but the order is not limited thereto. Furthermore, the production method disclosed herein may further include other steps at any stage.

[0051] (Culturing Step S101) In the culturing step S101, microalgae belonging to the genus Euglena are cultured. Various Euglena culturing methods can be used in the culturing step S101. Note that the culturing conditions do not necessarily need to be constant in the culturing step S101, and the culturing conditions may be changed as appropriate during the culturing step S101. Note that the Euglena used in this embodiment can be the ones described above. Therefore, a redundant description will be omitted here.

[0052] In the culturing step S101, the culture may be performed under any of autotrophic, mixotrophic, or heterotrophic conditions, without any particular limitation. From the viewpoint of Euglena growth, it is preferable to perform the culture under mixotrophic or heterotrophic conditions. In this specification, "autotrophic conditions" refers to an environment in which an organism grows without absorbing an external organic carbon source. Growth environments in the presence of inorganic salts such as minerals, vitamins, and the like are also encompassed within the term "autotrophic conditions." In this specification, "mixotrophic conditions" refers to an environment in which an organism grows while absorbing an external organic carbon source and carbon dioxide as carbon sources. In other words, these conditions refer to conditions in which photosynthesis occurs while utilizing an external carbon source. In this specification, "heterotrophic conditions" refers to an environment in which an organism grows while absorbing an organic carbon source as a carbon source, without photosynthesis. In this specification, "organic carbon source" refers to an exogenous organic substance (e.g., sugars) that can serve as a carbon source necessary for the growth of an organism. In addition, in this specification, "light" refers to light including visible light having wavelengths used by microalgae for photosynthesis.

[0053] The medium used in the technology disclosed herein can be appropriately selected based on known media typically used for culturing Euglena, depending on the type of Euglena and the culture environment. The medium is not limited to, but may be, for example, Cramer and Myers medium (CM medium) when culturing step S101 under autotrophic conditions. When culturing step S101 under mixotrophic or heterotrophic conditions, for example, Koren and Hunter medium (KH medium), Hunter medium, modified Bristol's medium (MBM medium), or the like may be used. An organic carbon source may be mixed into the medium as needed. Examples of the organic carbon source include glucose, molasses, ethanol, and acetic acid. Furthermore, inorganic salts such as minerals, vitamins, and the like may be mixed into the medium. From the viewpoint of Euglena growth, it is preferable to culture the Euglena in a medium containing an organic carbon source.

[0054] The pH of the medium used here can be appropriately selected depending on the type of Euglena and the culture environment. For example, it is pH 3 to 10, preferably pH 3.5 to 9, and more preferably pH 3.5 to 8. A buffer solution for adjusting the pH may also be added to the medium as appropriate. Known buffer solutions can be used, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium phosphate buffer, and potassium phosphate buffer.

[0055] The light conditions in the culturing step S101 are not particularly limited, and the culture may be performed under light irradiation from a light source (bright conditions) or without irradiation (dark conditions). Because Euglena can perform photosynthesis, the culturing step S101 is preferably performed under light conditions. When the culturing step S101 is performed using a light source, the light source is not particularly limited as long as it includes the wavelengths used by Euglena for photosynthesis. For example, a light source that emits white light having a wavelength range of 380 nm to 800 nm is used. The light source may be either natural light or artificial light; for example, sunlight, fluorescent lamps, LED (Light Emitting Diode) lamps, incandescent lamps, halogen lamps, or HID (High Intensity Discharge) lamps are preferably used. The light source may be continuous or discontinuous, alternating between light and dark periods. In the case of discontinuous irradiation, the cycle of light and dark periods is not particularly limited as long as it does not interfere with the cultivation of Euglena, and can be appropriately selected depending on the type of Euglena and the cultivation environment.

[0056] In the technology disclosed herein, the light intensity of the light source is expressed as photon flux density (μmol photons m －２ ・s －１ The photon flux density can be measured using a photon meter or the like. When the culturing step S101 is performed under bright conditions, the light intensity of the light source is set to approximately 5 to 2,000 μmol photons m , taking into account the amount of light energy required for culturing Euglena and its resistance to strong light stress. －２ ・s －１ is preferred, and is approximately 30 to 1,000 μmol photons m－２ ・s －１ When the culture step S101 is performed under dark conditions, for example, 5 μmol photons m －２ ・s －１ The light intensity may be less than 1000 .mu.m.

[0057] The culture temperature in the culture step S101 can be appropriately selected depending on the type of Euglena and the culture environment, and is not particularly limited as long as it does not significantly inhibit the culture of Euglena. The culture temperature is, for example, 15°C to 40°C, preferably 20°C to 37°C, and more preferably 25°C to 35°C.

[0058] The culture device is not particularly limited, and known culture devices such as open pond type, raceway type, and tubular photobioreactors may be used, or laboratory-scale culture devices such as tube culture and flask culture may be used. The culture method may be static culture, shaking culture, or agitation culture, but shaking culture and agitation culture are preferred from the viewpoint of Euglena proliferation. Shaking culture may be performed by reciprocal shaking or rotary shaking. Agitation culture may be performed by agitation using a stirring device or aeration agitation.

[0059] The culturing step S101 may be performed under anaerobic or aerobic conditions, and is not particularly limited. From the viewpoint of ensuring a carbon source for photosynthesis by Euglena, the culturing step S101 is preferably performed in the presence of carbon dioxide. Microalgae of the genus Euglena can typically use carbon dioxide present in the atmosphere as a carbon source, but a carbon dioxide source may also be additionally supplied to the medium. When culturing microalgae of the genus Euglena under autotrophic or mixotrophic conditions, it is preferable to additionally supply carbon dioxide to the medium. The carbon dioxide source is not particularly limited, and any conventionally known source can be used without limitation. For example, a carbon dioxide-containing gas (e.g., carbon dioxide gas), sodium bicarbonate, acetic acid, etc. may be used. Furthermore, the medium may be stirred as needed to suitably supply carbon dioxide to the medium.

[0060] The culturing step S101 is preferably carried out in a medium supplied with an NAD precursor. Experiments by the present inventors have confirmed that this enables the production of an NAD production promoter with particularly excellent NAD production-promoting effects. Although the detailed mechanism is unclear, it is presumed that the amount of NAD production-promoting component produced in Euglena cells increases when Euglena takes up an NAD precursor from the outside during culture. The NAD precursor may be supplied to the medium before or after adding Euglena to the medium, or may be added simultaneously with Euglena. The NAD precursor may be added not only once, but also in multiple batches.

[0061] Nicotinic acid or nicotinamide is preferably used as the NAD precursor used in the culture step S101. Experiments have confirmed that Euglena-derived substances obtained from Euglena cultured in a medium containing nicotinic acid or nicotinamide as the NAD precursor have a superior effect of promoting NAD production in living animals. The concentration of nicotinic acid or nicotinamide in the culture medium is, for example, 1 μM or more, preferably 10 μM or more, and more preferably 100 μM or more. On the other hand, when nicotinic acid or nicotinamide is supplied before or simultaneously with the addition of Euglena, the concentration of nicotinic acid or nicotinamide in the culture medium is preferably 100 μM or less from the viewpoint of Euglena growth. When nicotinic acid or nicotinamide is supplied after the addition of Euglena, the concentration of nicotinic acid or nicotinamide in the culture medium is preferably 5,000 μM or less, more preferably 1,000 μM or less from the viewpoint of Euglena culture efficiency.

[0062] Although not particularly limited, in the culturing step S101, Euglena is preferably cultured until the NAD concentration in the Euglena algae reaches 500 nmol / gFW or more (preferably 750 nmol / gFW or more, more preferably 1,000 mol / gFW or more). This is presumably because the production of a sufficient amount of NAD increases the NAD production-promoting component in the Euglena. Therefore, the NAD concentration of Euglena can be used as one of the indicators for producing a high-performance NAD production promoter. On the other hand, the upper limit of the NAD concentration in the Euglena algae is not particularly limited, but may be, for example, 15,000 nmol / gFW or less or 10,000 nmol / gFW or less from the viewpoint of Euglena production efficiency.

[0063] As shown in FIGS. 2 and 3 , Euglena cells cultured under appropriate conditions contain 500 nmol / g FW to 15,000 nmol / g FW of NAD. Although detailed explanations are omitted, experiments have confirmed that the use of Euglena cells with such NAD concentrations enables the production of high-performance NAD production promoters. FIG. 2 is a graph showing the trends in nicotinic acid concentration and NAD concentration in Euglena when Euglena is cultured in a medium supplemented with nicotinic acid as an NAD precursor in the culture step S101. FIG. 3 is a graph showing the trends in nicotinamide concentration and NAD concentration in Euglena when Euglena is cultured in a medium supplemented with nicotinamide as an NAD precursor in the culture step S101. However, these graphs do not necessarily imply that nicotinic acid or nicotinamide is necessary for the production of high-performance NAD production promoters. 2 and 3 show the results of examining the tendency of NAD concentration in Euglena algae cultured under appropriate conditions.

[0064] The culture time in the culture step S101 is not particularly limited, as it may vary depending on the culture environment, the Euglena used, etc. The culture time in the culture step S101 may be, for example, 1 hour or more and 15 days or less. When an NAD precursor is used in the culture step S101, from the viewpoint of producing a sufficient amount of NAD production-promoting component, it is preferable to continue the culture for 1 hour or more after adding the NAD precursor, more preferably 6 hours or more, even more preferably 12 hours or more, and particularly preferably 24 hours (1 day) or more. On the other hand, the upper limit of the culture time after adding the NAD precursor is not particularly limited, and may be 15 days or less, or may be 10 days or less.

[0065] (Recovery Step S102) In the recovery step S102, microalgae (Euglena) are recovered from the medium that has been subjected to the culture step S101. Any known method can be used to recover Euglena. For example, Euglena may be recovered by centrifuging the culture solution using a centrifuge or the like and then removing the supernatant. Euglena may also be recovered by filtering the culture solution using a filter with a pore size smaller than the Euglena in the culture solution. However, these are merely examples, and the recovery method is not limited to these.

[0066] The culture solution after Euglena collection may be used to culture new microalgae. However, it is not necessary to directly use the culture solution after microalgae collection for culturing new microalgae. For example, deficient components such as inorganic salts and vitamins, or a buffer solution may be appropriately added to the culture solution after microalgae collection. Furthermore, the culture solution after microalgae collection may be additionally mixed with a separately prepared culture solution.

[0067] (Euglena-derived substance production step S103) In the Euglena-derived substance production step S103, a Euglena-derived substance is produced from the Euglena collected in the collection step S102. As a method for producing a Euglena-derived substance, various methods relating to techniques for extracting intracellular components of microalgae can be used without limitation, depending on the form of the Euglena-derived substance, as long as the NAD production-promoting effect of the Euglena-derived substance is not significantly impaired. An example of the Euglena-derived substance production step S103 will be described below, but the method is not limited to the one described below.

[0068] In some preferred embodiments, Euglena-derived substances can be obtained by solvent extraction using an aqueous solvent. This allows water-soluble components in Euglena to be extracted as Euglena-derived substances. In the case of extraction using an aqueous solvent, for example, Euglena and the aqueous solvent are placed in a container and immersed. The state of the Euglena is not particularly limited as long as it allows extraction of the Euglena-derived substance. For example, the algae may be dried, or the Euglena cell membrane may be disrupted by crushing, ultrasonication, enzymatic methods, or the like. The immersion may be performed in a stationary state, but shaking or stirring is preferred. This allows efficient contact between the solvent and Euglena, thereby enabling efficient extraction. The immersion time in the Euglena-derived substance production step S103 is not particularly limited as long as the Euglena-derived substance can be extracted, and can be adjusted depending on, for example, the state of the Euglena and the solvent used. The type and pH of the extraction solvent used for aqueous solvent extraction have already been described above, and therefore, a repeated explanation will be omitted here.

[0069] In the case of extraction using an aqueous solvent, the extraction temperature is not particularly limited as long as it does not significantly impair the NAD production-promoting effect of the Euglena-derived substance, but is preferably 1°C or higher (preferably 5°C or higher, more preferably 10°C or higher, and even more preferably 20°C or higher). On the other hand, the extraction temperature is preferably 120°C or lower (preferably 100°C or lower, more preferably 80°C or lower, and even more preferably 50°C or lower). A Euglena-derived substance extracted using an extraction solvent at a temperature within this range can preferably exhibit the NAD production-promoting effect.

[0070] Here, methods for extracting water-soluble components from Euglena have been described as Euglena-derived substances. However, the above description is merely illustrative. For example, when the Euglena-derived substance is to be an "intracellular component of Euglena," it is preferable to carry out a treatment such as a crushing method, an ultrasonic method, or an enzymatic method, as described above. Such treatments can destroy the cell membrane of Euglena and allow the intracellular component of Euglena to be extracted.

[0071] The extract obtained after the extraction treatment can be filtered or centrifuged as needed to remove solid matter, thereby obtaining a Euglena-derived substance. The extract may be further purified. Examples of purification methods include partition extraction, chromatography, and activated carbon treatment. Two or more of the above treatments may be combined.

[0072] The extract may be subjected to treatments such as concentration and dilution as necessary to obtain a Euglena-derived substance. Alternatively, the extract may be subjected to treatments such as concentration to dryness, spray drying, and freeze drying to obtain a Euglena-derived substance.

[0073] When Euglena algae are used as a Euglena-derived substance, the Euglena algae are preferably subjected to a process such as freeze-drying or spray-drying, which allows for the production of dried Euglena algae. Alternatively, the NAD production promoter may be prepared by mixing Euglena algae with a carrier or other components.

[0074] [Test Examples] Several examples of the technology disclosed herein are described below, but are not intended to limit the technology to these examples. In all cases, three independent cultures (i.e., n = 3) were performed. The values ​​in Figures 4 to 7 represent the mean ± standard deviation (n = 3). Furthermore, Welch's t-test was performed for Studies 1 to 4 to compare the results with those of the FK866-treated group (*: p < 0.05).

[0075] [Study 1: Study of the Effect of Euglena-Derived Substances on Promoting NAD Production in Human Cells] Here, a human retinal pigment epithelial cell line (ARPE-19 cells) was used as the human cells to study the effect of Euglena-derived substances on promoting NAD production in human cells. <Preparation of Microalgae> (Example 1) Euglena gracilis NIES-48 strain (hereinafter referred to as "Euglena") was prepared as microalgae of the genus Euglena. CM medium (pH 3.5) was prepared as the medium. Euglena was added to the CM medium at an initial concentration of OD ６８０ The culture was carried out by adding 40 to 50 μmol photons m using a three-wavelength daylight fluorescent lamp as the light source. －２ ・s －１ The culture medium was then continuously irradiated for 24 hours at a light intensity of 100 rpm at 30° C. for 7 days with shaking. In this way, the culture medium according to Example 1 was obtained.

[0076] (Example 2) In Example 2, the same Euglena and CM medium as in Example 1 were prepared. In Example 2, culture was carried out for 6 days under the same conditions as in Example 1. After 6 days, nicotinic acid as an NAD precursor was added to the medium of Example 2 to a concentration of 1,000 μM, and culture was continued for another day under the same conditions. Except for this, the Euglena of Example 2 was cultured in the same manner as in Example 1. In this way, the culture solution of Example 2 was obtained.

[0077] <Recovery and Extraction of Culture Fluid> After the above-described culture, the culture fluids of Examples 1 and 2 were centrifuged using a swinging bucket rotor (500 × g, 4°C, 3 minutes) to precipitate Euglena cells in the culture fluid. After centrifugation, the recovered Euglena cells were washed with phosphate buffer and pure water. Subsequently, the Euglena cells were finally recovered by precipitating them by centrifugation (15,000 rpm, 4°C, 10 minutes). The finally recovered Euglena cells were frozen at −80°C and further freeze-dried. Pure water was added to the freeze-dried Euglena cells so that the cell concentration was 100 mg / mL (equivalent to dry cells), and the cells were uniformly dispersed by ultrasonication for 60 seconds. The supernatant obtained by centrifugation (15,000 rpm, 4°C, 10 minutes) was then collected and filtered through a 0.22 μm filter to obtain the Euglena extracts (Euglena-derived substances) of Examples 1 and 2. The obtained Euglena extract was used as a sample to be added to ARPE-19 cells and as a sample for NAD quantification, as described below.

[0078] <Addition of Euglena extract to human cells> First, ARPE-19 cells (human retinal pigment epithelial cell line, ATCC, CRL-2302) were prepared as human cells. ARPE-19 cells were cultured at a cell concentration of 1.2 × 10 ５ The cells were seeded in DMEM / F-12 medium (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) (Thermo Fisher Scientific or Fujifilm Wako Pure Chemical Industries, Ltd., containing 10% FBS (Fetal Bovine Serum)) to give 100 cells / φ60 mm dish, and incubated at 37°C and 5% CO ２ The cells were cultured for 3 days under reduced pressure. After 3 days of culture, the medium was replaced with 5 mL of serum-free DMEM / F-12 medium and cultured overnight. In this way, a medium for evaluation of ARPE-19 cells (referred to as ARPE-19 cell medium) was prepared.

[0079] In this test, an inhibitor of the biosynthetic pathway from nicotinamide to NMN (NAMPT inhibitor) was used to facilitate confirmation of the NAD production-promoting effect in each example. Specifically, 5 μL of the NAMPT inhibitor (FK866) (10 μM) was added to the ARPE-19 cell culture medium prepared above to a final concentration of 10 nM, and 50 μL of the Euglena extracts according to Examples 1 and 2 (100 mg / mL in terms of dry cells) were added to a final concentration of 1,000 μg / mL. As a positive control, an ARPE-19 cell culture medium was prepared by adding 5 μL of FK866 and 50 μL of NMN (nicotinamide mononucleotide) to a final concentration of 10 μM (NMN-treated group). As a negative control, 5 μL of FK866 (10 μM) and 50 μL of distilled water instead of the microalgae extract were added to the ARPE-19 cell culture medium (FK866-treated group). As an untreated group, 5 μL of DMSO and 50 μL of distilled water were added to the ARPE-19 cell culture medium without adding FK866. These were incubated at 37°C and 5% CO ２ After standing for 24 hours under the conditions described above, the cells were recovered and frozen at -80°C to prepare frozen samples for evaluation.

[0080] <Quantification of NAD Amount in ARPE-19 Cells> The NAD amount in ARPE-19 cells was determined by NAD ＋ (oxidized form) was quantified and the calculated value was taken as the amount of NAD.

[0081] (1) NAD ＋ Extraction of (oxidized) 250 μL of 0.2 M HCl was added to the tube containing the frozen sample for evaluation, and ultrasonic treatment (Duty cycle: Constant, Output control: 2) was performed for 5 seconds using a Sonifier 250 (manufactured by Branson). The tube was then immersed in boiling water for 1 minute and then cooled on ice. After centrifugation (15,000 rpm, 10 minutes, 4°C), 150 μL of the supernatant in the tube was collected and transferred to a new 1.5 mL plastic tube. The collected supernatant was diluted with 0.2 M NaH ２ P.O. ４15 μL of 0.2 M NaOH (pH 5.6) and 120 μL of 0.2 M NaOH were added and stirred with a vortex mixer to obtain the NAD of ARPE-19 cells. ＋ was extracted and used as a sample for NAD quantification.

[0082] (2) Quantification of NAD Amount The NAD amount in this example was quantified by cycling assay. Specifically, 15 μL of 100% ethanol, 100 μL of 0.1 M HEPES-KOH (pH 7.5), 2.5 μL of 1.2 mM 2,6-Dichlorophenolindophenol (DCPIP), 10 μL of 20 mM 1-Methoxy-5-methylphenazinium methylsulfate (1-MethoxyPMS), 17.5 μL of autoclaved distilled water, and 35 μL of a sample for NAD quantification were mixed. To this mixture, 10 μL of 1,770 unit / mL alcohol dehydrogenase was added and mixed by pipetting. The time course of absorbance at a wavelength of 595 nm was then measured using a spectrophotometer. A calibration curve for NAD quantification was also prepared by performing similar measurements using a standard solution instead of the NAD quantification sample. The amount of NAD was calculated using the prepared calibration curve. The results of the evaluation of the effect on NAD amount in ARPE-19 cells are shown in FIG. 4.

[0083] <Quantification of NAD Amount in Euglena Extract> The Euglena extract obtained above was used as a sample for NAD quantification. The amount of NAD in the Euglena extract was quantified and calculated using the NAD quantification method described above, and this was used as the NAD amount in the Euglena extract. The NAD concentration (μM) (mean value ± standard deviation, n = 3) in the Euglena extract (100 mg / mL in terms of dry cells) of each of Examples 1 and 2 is shown in parentheses below the graph of each example in Figure 4.

[0084] As shown in Figure 4, an increase in NAD levels in ARPE-19 cells was observed in Example 1, in which a Euglena extract (a Euglena-derived substance) was added. Furthermore, in Example 2, in which a Euglena-derived substance cultured in a medium supplemented with nicotinic acid was used as an NAD precursor, even more favorable results were obtained regarding the increase in NAD levels in ARPE-19 cells. This demonstrates that the Euglena-derived substance has an effect of promoting NAD production in ARPE-19 cells, which are human cells. Furthermore, it was found that when Euglena cultured in a medium supplemented with nicotinic acid was used as the Euglena-derived substance, an even more favorable effect of promoting NAD production in ARPE-19 cells was obtained.

[0085] [Study 2: Examination of the effect of chlorella-derived substances on promoting NAD production in human cells] Here, the effect of chlorella-derived substances on promoting NAD production in human cells was examined using ARPE-19 cells.

[0086] <Preparation of Microalgae> (Example 3) First, Chlorella vulgaris NIES-2170 strain (hereinafter referred to as "Chlorella") was prepared as a microalgae of the genus Chlorella. As a medium, Bold's Basal Medium (BBM medium), which is a medium conventionally used for culturing Chlorella, was prepared. Chlorella was added to the BBM medium at an initial concentration of OD ７５０ The culture was carried out by adding 40 to 50 μmol photons m using a three-wavelength daylight fluorescent lamp as the light source. －２ ・s －１ The culture medium was then continuously irradiated for 24 hours at a light intensity of 100 rpm at 30° C. for 7 days with shaking. In this way, the culture medium according to Example 3 was obtained.

[0087] (Example 4) In Example 4, the same chlorella and BBM medium as in Example 3 were prepared. In Example 4, culture was carried out for 6 days under the same conditions as in Example 3. After 6 days, nicotinamide as an NAD precursor was added to the medium of Example 4 to a concentration of 100 μM, and then culture was carried out for another day under the same conditions. Except for this, the chlorella of Example 4 was cultured in the same manner as in Example 3. In this way, a culture solution of Example 4 was obtained.

[0088] <Recovery and Extraction of Culture Fluid> After the above-described culture, the culture fluids of Examples 3 and 4 were centrifuged using an angle rotor (8,000 rpm, 4°C, 10 minutes) to precipitate Chlorella cells in the culture fluid. After centrifugation, the recovered Chlorella cells were washed with phosphate buffer and pure water. The Chlorella cells were then precipitated by centrifugation (15,000 rpm, 4°C, 10 minutes) to finally recover the Chlorella cells. The Chlorella extracts (Chlorella -derived substances) of Examples 3 and 4 (100 mg / mL in terms of dry cells) were obtained from the finally recovered Chlorella cells using the same method as in Study 1. The obtained Chlorella extracts were used as samples to be added to ARPE-19 cells and as samples for NAD quantification, as shown below.

[0089] <Addition of Chlorella extract to human cells> First, an ARPE-19 cell culture medium was prepared using the same method as in Study 1. 50 μL of the Chlorella extract (100 mg / mL in terms of dry cells) according to Examples 3 and 4 was added to the above-prepared ARPE-19 cell culture medium to give a final concentration of 1,000 μg / mL. Furthermore, using the same method as in Study 1, an untreated group, an FK866-treated group, and an NMN-treated group were each prepared. These were incubated at 37°C and 5% CO ２ After standing for 24 hours under the conditions described above, the cells were recovered and frozen at -80°C to prepare frozen samples for evaluation.

[0090] <Extraction and Quantification of NAD in ARPE-19 Cells> NAD in ARPE-19 cells was extracted from the above-mentioned frozen samples for evaluation, and the amount of NAD was quantified and calculated by the same method as in Study 1. The results are shown in Figure 5.

[0091] <Quantification of NAD Amount in Chlorella> The chlorella extract obtained above was used as a sample for NAD quantification to determine the amount of NAD in the chlorella extract. The amount of NAD in the chlorella extract was calculated by the same method as the method for quantifying the amount of NAD in the Euglena extract described above. The NAD concentration (μM) (mean ± standard deviation, n = 3) in the chlorella extract (100 mg / mL in dry cell equivalent) for each of Examples 3 and 4 is shown in parentheses below the graph for each example in Figure 5.

[0092] As shown in FIG. 5, in Examples 3 and 4 in which Chlorella extract was added instead of Euglena extract, no increase in NAD amount was observed in ARPE-19 cells.

[0093] [Study 3: Study on the Effect of Promoting NAD Production in Mouse Cells] Here, a mouse embryonic fibroblast cell line (NIH / 3T3) was used as the mouse cells to study the effect of Euglena-derived substances on promoting NAD production in mouse cells.

[0094] <Preparation of Microalgae> (Example 5) In Example 5, Euglena was cultured under the same conditions as in Example 1. As a result, a culture solution according to Example 5 was obtained.

[0095] (Example 6) In Example 6, Euglena was cultured under the same conditions as in Example 2. As a result, a culture medium according to Example 6 was obtained.

[0096] In Example 7, Euglena was cultured under the same conditions as in Example 2, except that nicotinamide was added to a concentration of 1,000 μM instead of nicotinic acid as the NAD precursor. Thus, a culture medium according to Example 7 was obtained.

[0097] <Collection and extraction of culture fluid> Euglena extracts (Euglena-derived substances) (100 mg / mL in terms of dry cells) were obtained from the culture fluids of Examples 5 to 7 that had undergone the above-described culture in the same manner as in Example 1.

[0098] <Addition of microalgae extract to mouse cells> NIH / 3T3 cells (mouse embryonic fibroblast cell line, ECACC, catalog number 93061524) were added to the mouse cells at a cell concentration of 3.0 × 10 ４The cells were seeded in DMEM (Dulbecco's Modified Eagle Medium) (Thermo Fisher Scientific, low glucose, containing 10% CS (bovine calf serum)) at 37°C and 5% CO ２ The cells were cultured under reduced pressure for 3 days. The medium was then replaced with fresh DMEM. In this way, a medium for evaluation of NIH / 3T3 cells (referred to as NIH / 3T3 cell medium) was prepared.

[0099] To the NIH / 3T3 cell culture medium prepared above, 5 μL of FK866 (10 μM) was added to a final concentration of 10 nM, and 50 μL of each of the Euglena extracts (100 mg / mL in terms of dry cells) from Examples 5 to 7 was added to a final concentration of 1,000 μg / mL. As a positive control, NIH / 3T3 cell culture medium was prepared by adding 5 μL of FK866 and 50 μL of NMN (nicotinamide mononucleotide) to a final concentration of 100 μM (NMN-treated group). As a negative control, NIH / 3T3 cell culture medium was prepared by adding 5 μL of FK866 (10 μM) and 50 μL of distilled water instead of the Euglena extract (FK866-treated group). As an untreated group, NIH / 3T3 cell culture medium was prepared by adding 5 μL of DMSO and 50 μL of distilled water without adding FK866. These were incubated at 37°C and 5% CO ２ After standing for 24 hours under the conditions described above, the cells were recovered and frozen at -80°C to prepare frozen samples for evaluation.

[0100] <Extraction and quantification of NAD in NIH / 3T3 cells> For the above-mentioned frozen samples for evaluation, NAD in NIH / 3T3 cells was extracted and the amount of NAD was quantified and calculated using the same method as the method for extracting and quantifying NAD in ARPE-19 cells in Study 1. The results are shown in Figure 6.

[0101] <Quantification of NAD Amount in Euglena Extract> The NAD amount in the Euglena extracts of Examples 5 to 7 was quantified and calculated using the same method as in Study 1. The NAD concentration (μM) (mean ± standard deviation, n = 3) in each Euglena extract (100 mg / mL in dry cell equivalent) of Examples 5 to 7 is shown in parentheses below the graph of each example in Figure 6.

[0102] As shown in FIG. 6 , among Examples 5 to 7 in which a Euglena-derived substance was added, Examples 6 and 7, which used Euglena cultured in a medium containing nicotinic acid or nicotinamide, an NAD precursor, as a Euglena-derived substance, showed favorable results in terms of increasing the amount of NAD in NIH / 3T3 cells.

[0103] [Study 4: Study of the effect of adding NAD precursors on NAD concentration in mouse cells] Here, as a reference example, we studied the effect of adding only NAD precursors to NIH / 3T3 cell culture medium (i.e., when no microalgae extract was added) on NAD concentration.

[0104] <Addition of NAD precursor to mouse cells> First, an NIH / 3T3 cell culture medium was prepared in the same manner as in Study 2. To the above-prepared NIH / 3T3 cell culture medium, 5 μL of FK866 (10 μM) was added to make the concentration 10 nM, and 50 μL of nicotinamide or nicotinic acid as an NAD precursor to make the concentration 100 μM were added to prepare a nicotinamide-treated group and a nicotinic acid-treated group, respectively. Furthermore, an untreated group, an FK866-treated group, and an NMN-treated group were prepared in the same manner as in Study 2. These were incubated at 37°C, 5% CO ２ After standing for 24 hours under the conditions described above, the cells were recovered and frozen at -80°C to prepare frozen samples for evaluation.

[0105] <Extraction and Quantification of NAD in NIH / 3T3 Cells> NAD was extracted from the above-described frozen samples for evaluation and the amount of NAD was quantified in the same manner as in Study Example 3. The results are shown in FIG.

[0106] As shown in FIG. 7, no increase in the amount of NAD in the NIH / 3T3 cells was observed in the nicotinamide-treated group and the nicotinic acid-treated group, in which nicotinamide or nicotinic acid was added alone.

[0107] As described above, specific aspects of the technology disclosed herein include those described in the following items. Item 1: A nicotinamide adenine dinucleotide (NAD) production promoter containing a Euglena-derived substance, which is a component derived from microalgae belonging to the genus Euglena. Item 2: The NAD production promoter according to Item 1, further comprising NAD. Item 3: The NAD production promoter according to Item 1 or 2, further comprising an NAD precursor. Item 4: The NAD production promoter according to Item 3, which comprises nicotinic acid or nicotinamide as the NAD precursor. Item 5: The NAD production promoter according to any one of Items 1 to 4, which is used in a food composition, a food additive, a feed, a cosmetic, a cosmetic additive, or a medicine. Item 6: A method for producing a nicotinamide adenine dinucleotide (NAD) production promoter, comprising: a culturing step of culturing microalgae belonging to the genus Euglena in a medium; a recovering step of recovering the microalgae from the medium after the culturing; and a Euglena-derived substance producing step of producing a Euglena-derived substance from the microalgae recovered in the recovering step. Item 7: A method for producing the NAD production promoter according to Item 6, wherein the culturing step is carried out in a medium supplied with an NAD precursor. Item 8: A method for producing the NAD production promoter according to Item 7, wherein the NAD precursor includes nicotinic acid or nicotinamide.

[0108] Although the embodiments of the technology disclosed herein have been described in detail above, these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and alterations of the above-described embodiments.

[0109] S101 Cultivation step S102 Recovery step S103 Euglena-derived substance production step

Claims

1. A nicotinamide adenine dinucleotide (NAD) production promoter containing a Euglena-derived substance, which is a component derived from microalgae belonging to the Euglena genus.

2. The NAD production promoter according to claim 1, further comprising NAD.

3. The NAD production promoter according to claim 1 or 2, further comprising an NAD precursor.

4. The NAD production promoter according to claim 3, comprising nicotinic acid or nicotinamide as the NAD precursor.

5. The NAD production promoter according to claim 1 or 2, which is used in a food composition, a food additive, a feed, a cosmetic, a cosmetic additive, or a medicine.

6. A method for producing a nicotinamide adenine dinucleotide (NAD) production promoter, comprising: a culture step of culturing microalgae belonging to the genus Euglena in a culture medium; a recovery step of recovering the microalgae from the culture medium after the culture; and a Euglena-derived substance production step of producing a Euglena-derived substance from the microalgae recovered in the recovery step.

7. A method for producing an NAD production promoter as described in claim 6, wherein the culture step is carried out in a medium supplied with an NAD precursor.

8. The method for producing an NAD production promoter according to claim 7, wherein the NAD precursor is nicotinic acid or nicotinamide.

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