ATP production promoters, oral anti-inflammatory agents, and food and beverages.

Abstract

To provide an ATP production promoter or an anti-inflammatory agent which efficiently exerts ATP production promoting activity and anti-inflammatory activity, or a food / drink product containing the ATP production promoter or the anti-inflammatory agent.SOLUTION: Provided are an ATP production promoter or an anti-inflammatory agent which contains amaranthus seeds, and a food / drink product including them. It is preferable that the ATP production promoter or the anti-inflammatory agent includes an extract of amaranthus seeds. It is also preferable that the ATP production promoter or the anti-inflammatory agent contains an ethanol extract as an extract of amaranthus seeds.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an ATP production promoter and an anti-inflammatory agent.

[0002] ATP (adenosine triphosphate) is widely distributed in the cells of all living organisms and plays an important role as an energy source for in vivo reactions in eukaryotes in particular. ATP functions as an important energy source in various in vivo reactions such as, for example, carbohydrate metabolism, muscle contraction, active transport, and biosynthesis. ATP, which plays an important role in the energy metabolism of living organisms, may cause a decrease in cell functions such as cell growth, metabolism, and repair, and may induce aging and ultimately cell death due to a decrease in its production ability. In order to increase the functions of deteriorated cells and promote cell division, it is important to supply the cells with the energy necessary for division. Therefore, if ATP production can be promoted, ATP can be supplied to cells as an energy source necessary for cell functions and cell division, and as a result, an effect of activating functions such as cell growth, metabolism, and repair can be expected.

[0003] In addition, inflammation is a biological defense reaction induced when a living body is subjected to some harmful stimulus, and induces tissue reactions such as vasodilation, increased vascular permeability, leukocyte migration, and connective tissue proliferation, and inflammatory symptoms such as erythema, fever, swelling, and pain appear. Chronic inflammation causes various diseases and can also contribute to carcinogenesis. Therefore, the prevention, treatment, and symptom relief of inflammation are major issues, and many anti-inflammatory agents have been developed.

[0004] Patent Document 1 describes an anti-inflammatory agent containing an extract of amaranth leaves. However, the extract of amaranth seeds is not described in this document.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] The present invention aims to provide a safe and effective novel ATP production promoter or anti-inflammatory agent. [Means for solving the problem]

[0007] The present invention provides an ATP production promoter containing amaranth seeds. The present invention also provides an anti-inflammatory agent containing amaranth seeds. The present invention also provides food and beverages containing the aforementioned ATP production promoter or anti-inflammatory agent. [Effects of the Invention]

[0008] The ATP production promoter of the present invention can effectively promote intracellular ATP production. Furthermore, the anti-inflammatory agent of the present invention exhibits excellent anti-inflammatory effects. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a graph showing the ATP production rates of the examples and comparative examples. [Figure 2] Figure 2 is a graph showing the NO production rates of the examples and comparative examples. [Figure 3] Figure 3 is a graph showing the ATP production rates of the examples and comparative examples when water contact treatment was performed under multiple conditions with different pH levels. [Figure 4] Figure 4 is a graph showing the ATP production rates of the examples and comparative examples when amaranth seeds were subjected to heat treatment and / or water contact treatment. [Modes for carrying out the invention]

[0010] Preferred embodiments of the present invention are described below. Unless otherwise specified, the term "agent of the present invention" includes both an ATP production promoter and an anti-inflammatory agent. The agent of the present invention is preferably an oral preparation. Furthermore, the ATP production promoter and anti-inflammatory agent of this embodiment contain amaranth seeds as active ingredients.

[0011] Amaranth is a general term for plants belonging to the genus Amaranthus in the family Amaranthaceae. The "amaranth" used in this invention can be any plant belonging to the Amaranthaceae family, genus Amaranthus: Amaranthus tricolor L. subsp. maugostanus, (Amaranthus maugostanus L.), but other species of the same genus can also be used, such as Amaranthus tricolor ssp. tricolor, Amaranthus caudatus L., Amaranthus cruentus L., Amaranthus hybridus, (Amaranthus patrus Bertol), Amaranthus retroflexus L., Amaranthus spinosus L., and Amaranthus hypocondriacus. Other subgenera of the Amaranthus genus, such as Amaranthus gangeticus L., Amaranthus lividus L., and Amaranthus gracilis Dest., can also be used, but Amaranthus caudatus L. is particularly preferred. As for the seeds, any seeds harvested at the time when they will be harvested as grain after ripening, and that have not yet germinated, are acceptable.

[0012] The agent of the present invention requires amaranth seeds as an essential component. The agent of the present invention may contain other parts of amaranth besides the seeds, but it is preferable that the amaranth raw material mainly consists of amaranth seeds. "Mainly consisting of amaranth seeds" preferably means that the proportion of amaranth seeds in the amaranth raw material is 50% by mass or more, and more preferably 70% by mass or more. In the present invention, only amaranth seeds may be used as the amaranth component.

[0013] The agent of the present invention may contain amaranth seeds as they are, or it may contain processed products such as crushed seeds, seed extracts, or heated seed products. The extracts referred to here include solvent extracts. For example, if the amaranth seeds are used as an extract, the amaranth seeds to be used in the extraction process may or may not be crushed beforehand. Furthermore, the water contact treatment and heat treatment described later may or may not be applied beforehand.

[0014] In the present invention, it is preferable to use amaranth seeds that have been in contact with an aqueous solution with a pH of 2.5 to 8, or that have been heated, because this provides an excellent ATP production promoting effect. In the present invention, an extract of amaranth seeds can be used as amaranth seeds that have been in contact with an aqueous solution with a pH of 2.5 to 8, or that have been heated. It is also possible to perform both the treatment of contact with an aqueous solution with a pH of 2.5 to 8 (hereinafter also referred to as "water contact treatment") and the heat treatment.

[0015] When water contact treatment is performed, that is, when amaranth seeds are brought into contact with an aqueous solution with a pH of 2.5 to 8, the ATP production promoting effect can be further enhanced, with a pH of 3 to 7 being particularly preferred, and a pH of 4 to 7 being particularly preferred. An aqueous solution with a pH of 2.5 to 8 can be prepared by mixing water with a pH adjusting agent such as hydrochloric acid, organic acid, sodium hydroxide, sodium carbonate, or sodium bicarbonate. Note that the pH referred to here should be the pH at the temperature of the aqueous solution when it comes into contact with the amaranth seeds.

[0016] Here, the water contact treatment includes, in addition to immersing amaranth seeds in an aqueous solution of a predetermined pH, treating such as spraying the aqueous solution or contacting the seeds in a state where the aqueous solution is flowing. However, adopting the treatment of immersing the seeds in the aqueous solution is advantageous in that it can uniformly and easily bring the amaranth seeds into contact with the aqueous solution in a short time.

[0017] As described above, when performing the water contact treatment, the amaranth seeds are brought into contact with an aqueous solution having a pH of 2.5 or more and 8 or less. At this time, the amaranth seeds to be contacted may be heated or unheated. The usage amount of the above aqueous solution is preferably usually 2 parts by mass or more, more preferably 3.5 parts by mass or more, still more preferably 5 parts by mass or more, with respect to 1 part by mass of the seeds (amaranth). Also, usually 100 parts by mass or less is preferable, more preferably 50 parts by mass or less, and still more preferably 25 parts by mass or less.

[0018] Also, the contact time is preferably 10 minutes or more and preferably 48 hours or less. More preferably, it is 1 hour or more and 24 hours or less, and still more preferably 1 hour or more and 12 hours or less.

[0019] The temperature of the water to be contacted is not particularly limited, but for example, 25 to 100 °C is preferably cited. When performing a heat treatment as the water contact treatment, the temperature of the aqueous solution may be the preferable heating temperature described later.

[0020] As described above, after solid-liquid separation, the treated product obtained by the water contact treatment can be dried and used in the extraction step described later. Preferably, freeze-drying is performed on the solid obtained by solid-liquid separation of the treated product obtained by the water contact treatment, and it is preferable in terms of extraction efficiency to use the obtained freeze-dried product in the extraction step described later. Note that centrifugation, filtration, etc. can be adopted for solid-liquid separation.

[0021] Also, as described above, in the present invention, as the amaranth seeds, those subjected to a heat treatment can also be suitably used as the agent of the present invention. In the present invention, when heated amaranth seeds are used, the heating temperature is preferably 50 to 200°C, and more preferably 60 to 125°C, as it is excellent in promoting ATP production. Furthermore, the heating conditions for amaranth seeds may include heating them while immersed in an aqueous solution such as water, steam heating using steam, heating under normal pressure, or heating under pressure. Furthermore, when the heating temperature is set as described above, the heating time is preferably 1 minute to 24 hours, and more preferably 10 minutes to 12 hours. Furthermore, when using heated amaranth seeds, the heated amaranth seeds can be subjected to the extraction process described later, and in that case, solid-liquid separation and drying processes similar to the post-treatment after the water contact treatment described above can be performed.

[0022] (extraction process) Solvents used for solvent extraction of amaranth seeds include water and organic solvents. Generally, polar and nonpolar organic solvents are known. Examples include water; alcohols such as methanol, ethanol, propanol, and butanol (preferably lower alcohols with 1 to 4 carbon atoms); polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, and cyclohexane; aromatic hydrocarbons such as benzene and toluene; halogenated hydrocarbons such as dichloromethane, chloroform, and dichloroethane, as well as supercritical carbon dioxide.

[0023] In the present invention, polar solvents are preferred, and in particular, at least one selected from water, alcohol, and polyhydric alcohol is preferred, water and alcohol are more preferred, a mixed solvent of water and alcohol or an alcohol is particularly preferred, and ethanol is most preferred.

[0024] If the solvent is a mixed solvent of water and alcohol, or an alcohol, it is preferable that the proportion of alcohol in the extraction solvent be 30% by mass or more, and more preferably 50% by mass or more. In this specification, ethanol extracts include not only extracts from pure ethanol with a concentration of 99.5% or higher, but also extracts from water and ethanol mixed solvents where ethanol is the main component (60% by mass or more in the solvent, and may be 70% by mass or more, 80% by mass or more, or 90% by mass or more).

[0025] The amount of the above-mentioned extraction solvent used is not particularly limited, but is usually 1 part by mass or more, more preferably 2 parts by mass or more, even more preferably 3.5 parts by mass or more, per 1 part by mass of the seeds (amaranth), and is usually 100 parts by mass or less, more preferably 50 parts by mass or less, and even more preferably 10 parts by mass or less.

[0026] Furthermore, the extraction temperature is usually preferably 0°C or higher, more preferably 10°C or higher, even more preferably 20°C or higher, and usually preferably 100°C or lower, more preferably 70°C or lower, and even more preferably 50°C or lower.

[0027] Furthermore, the extraction time is preferably 10 minutes or more, and preferably 24 hours or less. More preferably 1 hour or more and 10 hours or less, and even more preferably 2 hours or more and 5 hours or less.

[0028] The extract obtained in the extraction process can be used as is, or after dilution, concentration, solvent removal, etc., and prepared as needed in powder or paste form. Solvent removal can be carried out by known methods such as vacuum distillation, vacuum / vacuum drying, freeze-drying, and spray drying. The extract may contain the solvent, or only the solvent may be removed to obtain an aqueous solution.

[0029] Amaranth seeds may be subjected to enzymatic hydrolysis or fermentation and subsequent extraction (fermentation extraction), but in the ATP production promoter and anti-inflammatory agent of the present invention, it is not necessary to use enzymatic hydrolysates or fermented extracts as amaranth seeds.

[0030] The agent of the present invention can be used as a pharmaceutical, quasi-drug, or food / beverage for animals, including humans, or for the manufacture of such products. The agent of the present invention may be directly administered to or ingested by animals, including humans, as a pharmaceutical, quasi-drug, or food / beverage, or it may be added to or blended with food / beverage or animal feed such as pet food to be used as food / beverage or animal feed for promoting ATP production or as an anti-inflammatory agent. In the latter case, the method of adding or blending amaranth seeds into food / beverage or animal feed is not particularly limited. For example, amaranth seeds may be directly blended as a raw material before the manufacture of food / beverage or animal feed, added during the manufacturing process of food / beverage or animal feed, or added to the manufactured food / beverage or animal feed. The aforementioned "food and beverages" refers to anything that can be consumed by humans as food, and includes not only general food and beverages, including so-called health foods, but also health functional foods such as Foods for Specified Health Uses and Foods with Nutrient Function Claims as defined by the Ministry of Health, Labour and Welfare's Health Functional Foods System, as well as supplements, etc. The aforementioned "animal feed" refers to substances given as food to animals other than humans (animals raised by humans), such as livestock, poultry, and farmed fish. Examples include livestock feed and pet food.

[0031] When the agent of the present invention is used as a pharmaceutical or quasi-drug, it may contain amaranth seeds as the active ingredient alone, or it may further contain a pharmaceutically acceptable carrier, or it may contain other active ingredients or pharmacological components to the extent that the ATP production promoting effect or anti-inflammatory effect of amaranth seeds is not impaired. Examples of such carriers include excipients, coating agents, binders, bulking agents, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, humectants, thickeners, activity enhancers, bactericides, flavoring agents, and odor-masking agents.

[0032] When the agent of the present invention is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form. The dosage form may be oral or parenteral. For example, oral dosage forms include solid forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid forms such as elixirs, syrups, and suspensions. Parenteral dosage forms include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, and patches. Of these, oral dosage forms are preferred. In the agent of the present invention, the amount of amaranth seeds is arbitrary as long as it can serve as an active ingredient, and amaranth seeds may account for 30% or more by mass, 50% or more by mass, or 70% or more by mass of the solid content in the agent. Here, "solid content" refers to the total amount excluding the solvent, and the solvent may include the various solvents listed above as extraction solvents or organic solvents commonly used as solvents.

[0033] When the agent of the present invention is used as a food or beverage, it may contain amaranth seeds as the active ingredient alone, or it may further contain various additives used in the manufacture of food and beverages, to the extent that the ATP production promoting or anti-inflammatory effect of amaranth seeds is not impaired. Examples of such additives include various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, flavoring components, acidulants such as organic acids, stabilizers, flavors, colorants, fragrances, and the like.

[0034] Examples of food and beverage products that use it include oral medications (gum, candy, etc.), processed seafood products such as kamaboko and chikuwa, livestock products such as sausages and ham, bread, Western-style sweets, Japanese-style sweets, noodles such as fresh noodles, Chinese noodles, boiled noodles, and soba, seasonings such as sauces, soy sauce, dips, sugar, honey, powdered syrup, and starch syrup, spices such as curry powder, mustard powder, and pepper powder, jams, marmalades, chocolate spreads, pickles, side dishes, furikake, or processed vegetables and fruits such as canned and bottled vegetables and fruits, dairy products such as cheese, butter, and yogurt, beverages such as miso soup, soups, fruit juices, vegetable juices, whey drinks, soft drinks, and alcoholic beverages, and other general food and beverage products such as health foods.

[0035] The present invention includes a package comprising a packaging body and the aforementioned ATP production promoting or anti-inflammatory agent of the present invention contained in the packaging body, or a food or animal feed containing the same. The packaging can contain the agent, food or beverage, or animal feed of the present invention, and can be printed with information on its ingredients, etc., and its form and material are not particularly limited. Examples of the form of the packaging include a box or a bag. Examples of the material of the packaging include paper, plastic, woven fabric, metal, etc. The packaging clearly displays various information, such as the content of amaranth seeds in the agent of the present invention, food or beverage, or animal feed contained in the packaging. The method of presenting information in such packaging is not particularly limited, and for example, 1) it may be printed on the outer or inner surface of the packaging, 2) the ATP production promoting use or anti-inflammatory use may be printed on a printing medium such as printing paper enclosed together with the food or beverage or animal feed inside the packaging, or 3) an internet URL may be written on the packaging or the printing medium enclosed therein, and the information may be presented by accessing that URL.

[0036] This invention effectively enhances intracellular ATP production by using amaranth seeds. This makes it possible to prevent or improve diseases or conditions that can be prevented or improved by promoting intracellular ATP production. The ATP production promoter of this invention and the food and beverages containing it can prevent or improve diseases, conditions, or symptoms caused by decreased ATP production. For example, the present invention includes a method for preventing or improving diseases or conditions caused by decreased ATP production, comprising the step of administering amaranth seeds to a subject suffering from a disease or condition caused by decreased ATP production, preferably for non-medical purposes such as improving athletic performance or muscle recovery. The present invention also includes the use of amaranth seeds as a preventive or ameliorative (therapeutic) agent or food / beverage for diseases or conditions caused by decreased ATP production, and the use of amaranth seeds for manufacturing preventive or ameliorative (therapeutic) agents or food / beverages for diseases or conditions (symptoms) caused by decreased ATP production. The present invention also includes food / beverages, their manufacture, and use aimed at promoting ATP production for purposes such as improving athletic performance or muscle recovery.

[0037] For example, as shown in the examples described later, the present invention can effectively promote ATP production in muscle cells such as striated muscle cells. For this reason, the present invention can be suitably used for promoting energy production in muscle cells, activating muscle tissue, activating mitochondria, and the like.

[0038] Furthermore, as shown in the examples described later, the present invention effectively suppresses inflammation by using amaranth seeds to inhibit NO production, an inflammatory component, by macrophages. Nitric oxide (hereinafter also referred to as "NO"), produced by nitric oxide synthase in macrophages, exhibits strong chemical reactivity and is known as a major cytotoxic factor in tissue destruction occurring in inflammatory responses and autoimmune mechanisms. Numerous animal studies have confirmed that many inflammatory conditions are improved by NO production inhibitors. As described later, the anti-inflammatory agent containing amaranth seeds of the present invention, which has excellent NO production inhibitory activity, can be suitably used to prevent or improve various inflammatory diseases. In particular, the present invention can effectively prevent or improve inflammatory diseases involving NO production by macrophages. Such diseases include arthritis (such as chronic rheumatoid arthritis), hepatitis, and inflammatory bowel disease.

[0039] Because the active ingredient of the present invention is derived from natural plants, it is highly safe and can be taken continuously for a long period of time. For example, when amaranth seeds are used as a solid extract to promote ATP production, the daily intake for an adult (weighing 60 kg) is preferably 10 mg to 2000 mg, and more preferably 100 mg to 1000 mg. When used to promote ATP production, the daily intake of amaranth seeds (in terms of the total amount of seeds) for an adult (weighing 60 kg) is preferably 200 mg to 40 g, and more preferably 2 to 20 g.

[0040] Furthermore, for example, if the amaranth seeds are in the form of a solid extract and are used for anti-inflammatory purposes, the daily intake for an adult (weighing 60 kg) is preferably 10 mg to 2000 mg, with 100 mg to 1000 mg being more preferable. Also, when used for anti-inflammatory purposes, the daily intake of amaranth seeds (in terms of the total amount of seeds) for an adult (weighing 60 kg) is preferably 200 mg to 40 g, with 2 to 20 g being more preferable. [Examples]

[0041] (1) Method for producing ethanol extract of amaranth 5 mL of ethanol was added to 1 g of crushed amaranth (scientific name A. caudatus L.) seeds, and the mixture was stirred for 2 hours at room temperature (25°C). After centrifugation for 5 minutes (3,000 rpm), the ethanol was removed by distillation under reduced pressure to obtain an ethanol extract.

[0042] (2) Evaluation of energy production promotion (ATP production rate) (2-1) Examples 1-1 and 1-2 Solution A was obtained by dissolving the ethanol extract of amaranth seeds prepared in (1) in dimethyl sulfoxide to a concentration of 400 mg / mL. 10 μL of this solution A was mixed with 10,000 μL of 2% by mass HS (horse serum)-high glucose DMEM (Dulbecco's modified Eagle medium) to obtain the culture medium sample of Example 1-1 containing 400 μg / mL of amaranth seed ethanol extract. Furthermore, 5 μL of solution A and 5 μL of dimethyl sulfoxide were mixed with 10,000 μL of 2% by mass HS (horse serum)-high glucose DMEM to obtain culture medium samples for Examples 1-2 containing 200 μg / mL of the ethanol extract of amaranth seeds mentioned above.

[0043] (2-2) Comparative Example 1 A culture medium sample for Comparative Example 1 was obtained by mixing 10 μL of dimethyl sulfoxide with 10,000 μL of 2% HS-high glucose DMEM by mass.

[0044] (2-3) Preculture C2C12 cells (mouse striated muscle-derived cells) were converted to 5.0 × 10⁶ cells in 10% FBS (fetal bovine serum)-high glucose DMEM. 4 The concentration was adjusted to cells / mL. C2C12 cells, prepared to the above concentration, were seeded at a rate of 100 μL / well in a white 96-well plate coated with 0.1% by mass gelatin aqueous solution, and cultured at 37°C under conditions of 5% by volume CO2. After 24 hours, the culture medium was changed to 2% by mass HS (horse serum)-high glucose DMEM, and the cells were incubated at 37°C and 5% by volume CO2 for 96 hours.

[0045] (2-4) Measurement The C2C12 cells cultured above were replaced with the culture medium sample from Example 1-1 or 1-2 or Comparative Example 1 at a rate of 100 μL / well, and cultured for 48 hours at 37°C and 5 volume % CO2. After 48 hours, the 96-well plate was left to stand at room temperature for 30 minutes, and an equal volume of ATP assay reagent (Cell Titer Glo 2.0 Reagent (Promega)) was added to the culture medium. The plate was then shaken at 400 rpm for 2 minutes. The experiment was performed in a 4-well sequence. After standing for 10 minutes, the luminescence intensity was measured using a plate reader.

[0046] Figure 1 shows the ATP production rate as the ratio (%) of the average luminescence intensity when the average luminescence intensity of Comparative Example 1 is set to 100. The error range in Figure 1 is the standard deviation of the luminescence intensity when the average luminescence intensity of Comparative Example 1 is set to 100. As shown in Figure 1, it was found that ATP production can be effectively promoted by using amaranth seeds.

[0047] (3) Anti-inflammatory evaluation For the anti-inflammatory test, macrophage-like cells, which are a model cell for inflammation, were used. These cells react to lipopolysaccharide (LPS), a substance that causes inflammation, and produce nitric oxide, a free radical that is also an inflammatory substance.

[0048] (3-1) Examples 2-1 and 2-2 The amaranth seed ethanol extract obtained in (1) above was dissolved in dimethyl sulfoxide to an extract concentration of 50 mg / mL. As the test solution for Example 2-1, 10000 μL of the 50 mg / mL amaranth seed ethanol extract / dimethyl sulfoxide solution obtained above was added to 10,000 μL of medium B (10% FBS-1% penicillin / streptomycin-DMEM medium) containing 0.1 μg / mL LPS (lipopolysaccharide) and mixed to prepare a test solution containing amaranth seed ethanol extract at a concentration of 500 μg / mL. Furthermore, as the test solution for Example 2-2, 50 μL of the 50 mg / mL amaranth seed ethanol extract / dimethyl sulfoxide solution obtained above and 50 μL of dimethyl sulfoxide were added to 10,000 μL of medium B containing 0.1 μg / mL LPS and mixed to prepare a test solution containing amaranth seed ethanol extract at a concentration of 250 μg / mL.

[0049] (3-2) Comparative Example 2 The test solution for Comparative Example 2 was prepared by adding 10000 μL of dimethyl sulfoxide to 10,000 μL of medium B (10% FBS - 1% penicillin / streptomycin-DMEM medium) containing 0.1 μg / mL of LPS (lipopolysaccharide) and mixing.

[0050] (3-3) Preculture Separately from the above, RAW264 cells (mouse macrophage-like cells) were cultured in 10% FBS-1% penicillin / streptomycin-DMEM medium in a 2.5 × 10⁶ culture medium. 6 The concentration was adjusted to cells / mL. RAW264 cells prepared to the above concentrations were seeded at a rate of 100 μL / well in a 96-well plate and cultured for 24 hours at 37°C with 5 volume % CO2.

[0051] (3-4) Measurement Pre-cultured RAW264 cells (mouse macrophage-like cells) were treated with the test solution of Example 2-1 or 2-2, or Comparative Example 2. Furthermore, sample blanks were prepared by adding the same culture medium as in Examples 2-1, 2-2, and Comparative Example 2, except that RAW264 cells were not included, and then performing the same procedure. After reacting overnight at 37°C and 5 vol.% CO2, the cell supernatant was separated into a separate plate. Griess reagent was added to the cell supernatant and reacted at 37°C for 20 minutes. Using a microplate reader, the absorbance (540 nm) of an azo compound formed by the reaction of NO2 ions, which are generated from the transformation of the inflammatory substance NO, with Griess reagent was measured. Absorbance was measured using a NaNO2 solution as a standard solution, and a calibration curve was created. From the calibration curve, the NO2 concentration (μmol / L) was calculated from the absorbance of each well. In this calculation, the absorbance used in the calibration curve was the absorbance of the measurement well minus the absorbance of the sample blank well. The NO production rate was calculated using the following formula. NO production rate (%) = 100 × Average NO2 concentration [Example] / Average NO2 concentration [Comparative Example 2]

[0052] The average NO production rate is shown in Figure 2. As shown in Figure 2, amaranth seeds can effectively suppress the production of NO, an inflammatory substance, in macrophage-like cells. Therefore, it is clear that amaranth seeds are effective as an anti-inflammatory agent.

[0053] Next, in (4) to (5) below, we investigated the effect of ethanol extraction conditions for amaranth on the ATP production promoting effect.

[0054] (4) Another method for producing ethanol extract of amaranth (Examples 3-1 to 3-6) Amaranth seeds were ground in a mill to obtain amaranth powder. The obtained amaranth powder was treated according to the processing conditions described below. After treatment, 5 mL of ethanol was added to 1 g of the obtained amaranth sample and stirred at room temperature for 2 hours. After centrifugation for 5 minutes (3,000 rpm), the ethanol was removed by distillation under reduced pressure to obtain an ethanol extract.

[0055] <Processing conditions> (Example 3-1) An aqueous solution adjusted to pH 3 was added to amaranth powder in a mass ratio of 20 times, and the mixture was stirred at room temperature (25°C) for 6 hours. The mixture was then centrifuged for 10 minutes (4,700 rpm), and the resulting precipitate was dewatered by decantation and then freeze-dried. The pH of the aqueous solution was adjusted using hydrochloric acid. (Example 3-2) The procedure was the same as in Example 3-1, except that an aqueous solution adjusted to pH 5 was used instead of an aqueous solution adjusted to pH 3. The pH of the aqueous solution was adjusted using hydrochloric acid. (Example 3-3) The procedure was the same as in Example 3-1, except that pure water with a pH of 7 was used instead of an aqueous solution adjusted to pH 3. (Example 3-4) The procedure was the same as in Example 3-1, except that an aqueous solution adjusted to pH 9 was used instead of an aqueous solution adjusted to pH 3. The pH of the aqueous solution was adjusted using sodium hydroxide. (Examples 3-5) Amaranth powder was autoclaved (121°C, 30 minutes), then centrifuged for 10 minutes (4,700 rpm), and the resulting precipitate was dewatered by decantation and then freeze-dried. (Example 3-6) Similar to Example 3-3, 20 times the mass of pure water (pH 7) was added to the amaranth powder and stirred for 6 hours. Then it was autoclaved (121°C, 30 minutes). Next it was centrifuged for 10 minutes (4,700 rpm), and the resulting precipitate was dewatered by decantation and then freeze-dried.

[0056] (5) Evaluation of energy production promotion (ATP production rate) (5-1) Examples 3-1 to 3-6 A solution was obtained by dissolving the ethanol extract of amaranth seeds from each example prepared in (4) in dimethyl sulfoxide to a concentration of 400 mg / mL. 10 μL of this solution was mixed with 10,000 μL of 2% by mass HS (horse serum)-high glucose DMEM (Dulbecc's modified Eagle medium) to obtain culture medium samples for Examples 3-1 to 3-6 containing 400 μg / mL of amaranth seed ethanol extract.

[0057] (5-2) Comparative Example 3 A culture medium sample for Comparative Example 3 was obtained by mixing 10 μL of dimethyl sulfoxide with differentiation medium (2% by mass HS-high glucose DMEM medium).

[0058] (5-3) Preculture C2C12 cells (mouse striated muscle-derived cells) were converted to 5.0 × 10⁶ cells in 10% FBS-high glucose DMEM. 4 The concentration was adjusted to cells / mL. C2C12 cells prepared to the above concentration were seeded at a rate of 100 μL / well in a white 96-well plate and cultured for 24 hours at 37°C with 5 volume % CO2. After 24 hours of incubation, the culture medium was changed to differentiation medium (2% HS-high glucose DMEM medium) and incubated for 96 hours at 37°C and 5% CO2 by volume.

[0059] (5-4) Measurement The C2C12 cells cultured above were replaced with 100 μL / well of the culture medium sample from one of Examples 3-1 to 3-6 or Comparative Example 3, and cultured for 48 hours at 37°C and 5 volume % CO2. After 48 hours, 100 μL of Cell Titer Glo 2.0 Reagent (Promega) was added, and the mixture was stirred for 2 minutes at 22°C and 400 rpm. The experiment was performed using a 6-component system. After standing at 25°C for 10 minutes, the luminescence intensity (ATP amount) was measured using a plate reader.

[0060] Figures 3 and 4 show the ATP production rate as the ratio (%) of the average luminescence intensity when the average luminescence intensity of Comparative Example 3 is set to 100. The error range in Figures 3 and 4 is the standard deviation of the luminescence intensity when the average luminescence intensity of Comparative Example 3 is set to 100. As shown in Figure 3, it was found that ATP production can be effectively promoted by performing ethanol extraction of amaranth seeds at a pH of 2.5 to 8. Furthermore, as shown in Figures 3 and 4, it was found that the ATP production promoting effect of the obtained ethanol extract was excellent even when water contact treatment or heat treatment was performed before ethanol extraction of amaranth seeds.

Claims

1. This product contains an ethanol extract obtained by heating or steam heating amaranth seeds in an aqueous solution with a pH of 2.5 to 7, or by immersing them in an aqueous solution, followed by ethanol extraction of the amaranth seeds at a temperature of 0°C to 100°C for 10 minutes to 24 hours. An ATP production promoter.

2. The ATP production promoter according to claim 1, wherein the daily intake for adults is 10 mg to 2000 mg.

3. Food or beverage for promoting ATP production, comprising the agent described in claim 1 or 2.

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