Composition for promoting nicotinamide adenine dinucleotide production

Abstract

To provide a novel composition that exhibits a promotion effect on production of nicotinamide adenine dinucleotide in vivo.SOLUTION: The present invention provides a composition for promoting production of nicotinamide adenine dinucleotide (NAD) in vivo, the composition containing scutellarin as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for promoting the production of nicotinamide adenine dinucleotide, and more particularly to a composition for promoting the production of nicotinamide adenine dinucleotide in a living body. [Background technology]

[0002] Nicotinamide adenine dinucleotide (NAD) is the oxidized form of NAD + Nicotinamide adenine dinucleotide is also known to play a role as a co-substrate in protein modifications such as deacetylation and ADP-ribosylation. For example, NAD + The main enzymes involved in this process are poly ADP ribose polymerases (PARPs), NAD + Glycohydrolase / ADP-ribosyl cyclase (CD38 family), sirtuin, etc. are known. All of these enzymes are involved in the production of NAD + Depending on the concentration, nicotinamide adenine dinucleotide directly or indirectly regulates various biological functions such as post-translational modification of proteins, intracellular and extracellular signal transduction, energy metabolism, and cell differentiation and proliferation. Therefore, a deficiency of nicotinamide adenine dinucleotide in the body causes a decrease in the activity of these enzymes, i.e., a decrease in each function in which these enzymes are involved.

[0003] Nicotinamide adenine dinucleotide is known to be produced in the body through three pathways: the de novo pathway using tryptophan as a starting material, the Preiss-Handler pathway using nicotinic acid, and the salvage pathway using nicotinamide (NAM). However, it is not yet clear which organs primarily use which pathway. On the other hand, since deficiency of nicotinamide adenine dinucleotide causes a decrease in each biological function as described above, it is desirable to increase the concentration of nicotinamide adenine dinucleotide in the body in order to express, maintain, or improve them.

[0004] As a method for promoting the production of nicotinamide adenine dinucleotide in vivo, for example, administration or ingestion of precursors of nicotinamide adenine dinucleotide, such as nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid riboside (NaR), and nicotinamide riboside (NR), is known. However, the production of these precursors has problems such as time and cost.

[0005] In addition, for example, apigenin inhibits CD38 activity and reduces NAD in vivo. + Non-Patent Document 1 (Carlos Escande et al., DIABETES, Vol. 62(4), APRIL 2013, p. 1084-1093) describes that quercetin increases the amount of NAD in epidermal and subcutaneous cells, and the same document also describes that quercetin inhibits CD38 activity. + The composition for increasing the concentration of intracellular NAD containing hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients is + Production enhancers are described. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2023-053658 A [Non-patent literature]

[0007] [Non-Patent Document 1] Carlos Escande et al.,DIABETES,Vol.62(4),APRIL 2013,p.1084-1093 Summary of the Invention [Problem to be solved by the invention]

[0008] Methods for promoting the production of nicotinamide adenine dinucleotide in vivo other than administering or ingesting a precursor of nicotinamide adenine dinucleotide have not yet been fully developed. In addition, the present inventors have found that, for example, even if apigenin described in Non-Patent Document 1 is administered or ingested, the effect of promoting the production of nicotinamide adenine dinucleotide in vivo is still insufficient.

[0009] The present invention has been made in consideration of such problems in the conventional technology, and aims to provide a new composition that has the effect of promoting the production of nicotinamide adenine dinucleotide in the body. [Means for solving the problem]

[0010] In order to achieve the above object, the present inventors have conducted extensive research and have found that administration of scutellarin from among many other ingredients promotes the production of nicotinamide adenine dinucleotide in the body, thereby increasing the production of NAD. + It was found that the concentration of NAD in the body (blood and tissues) increased. + The in vivo concentration of NAD was sufficiently high compared to the case of administering apigenin according to the prior art. Also, unlike the case of administering apigenin, for example, only when scutellarin was administered, NAD +Since the expression level of the NAMPT gene, which is a synthetic enzyme, and the amount of eNAMPT, which is the blood form of NAMPT, increased, it was found that the effect of promoting the production of nicotinamide adenine dinucleotide by administration of scutellarin is an effect exerted by a new attribute of scutellarin, which has a different mechanism of action from that of apigenin in the prior art, and the present invention was completed. The aspects of the present invention obtained based on such findings are as follows. [1] A composition for promoting in vivo nicotinamide adenine dinucleotide (NAD) production, comprising scutellarin as an active ingredient. [2] The composition described in [1], which is a composition for increasing the in vivo concentration of nicotinamide adenine dinucleotide (NAD). [3] The composition according to [1] or [2], which is a composition for oral administration, a composition for intraperitoneal administration, or a composition for subcutaneous administration. [4] The composition according to any one of [1] to [3], which is a food or drink composition, a pharmaceutical composition, or a quasi-drug composition. [5] The composition according to any one of [1] to [4], wherein the dosage for humans is 0.01 to 10 mg of scutellarin per kg of body weight per day. [6] Use of scutellarin to promote in vivo nicotinamide adenine dinucleotide (NAD) production. [7] Use of scutellarin to increase the in vivo concentration of nicotinamide adenine dinucleotide (NAD). [8] Use of scutellarin for the manufacture of a composition for promoting in vivo nicotinamide adenine dinucleotide (NAD) production. [9] Use of scutellarin for the manufacture of a composition for increasing the in vivo concentration of nicotinamide adenine dinucleotide (NAD).

[10] A method for promoting in vivo nicotinamide adenine dinucleotide (NAD) production, comprising administering an effective amount of scutellarin to a subject.

[11] A method for increasing in vivo levels of nicotinamide adenine dinucleotide (NAD) comprising administering an effective amount of scutellarin to a subject. Effect of the Invention

[0011] According to the present invention, it is possible to provide a novel composition that has the effect of promoting the production of nicotinamide adenine dinucleotide in the body. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows (a) a graph showing the amount of NAD+ in 1 μL of whole blood (pmol / μL, mean value ± standard error) in the Scutellarin group and the control group (Ctrl) obtained in (3-1) of Test Example 1, and (b) a graph showing the amount of NAD+ in 1 mg of liver tissue (pmol / mg tissue, mean value ± standard error). [Diagram 2] 1 is a graph showing the expression level of CD38 gene in the liver (relative mRNA expression, mean value ± standard error) in the Scutellarin group and the control group (Ctrl) obtained in Test Example 1 (3-2). [Diagram 3] 1 is a graph showing the amount of NAD+ (pmol / μL, mean value ± standard error) in 1 μL of whole blood in the Scutellarin group, the Apigenin group, and the control group (Ctrl) obtained in Test Example 2 (3-1). [Figure 4] This is a graph showing the expression level of the NAMPT gene in white fat (relative mRNA expression, mean value ± standard error) in the Scutellarin group, the Apigenin group, and the control group (Ctrl) obtained in Test Example 2 (3-2). [Diagram 5]Graphs showing (a) Western blotting images of eNAMPT and Transferrin in whole blood and (b) eNAMPT levels (Relative protein levels, mean ± standard error) in the Scutellarin group, Apigenin group, and Control group obtained in (3-3) of Test Example 2. [Figure 6] Graph showing CD38 Activity measured with the addition of each flavonoid obtained in Test Example 3. [Figure 7] Graph showing the amount of NAD+ (μg / mL, mean ± standard error) in 1 mL of whole blood in the Scutellarin group and Control group obtained in Test Example 4.

Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail according to its preferred embodiments.

[0014] <Composition for Promoting NAD Production> The present invention provides a composition for promoting the production of nicotinamide adenine dinucleotide (NAD) in vivo (hereinafter sometimes referred to as "composition for promoting NAD production" or simply "composition of the present invention") containing scutellarin as an active ingredient.

[0015] In the present invention, nicotinamide adenine dinucleotide is also abbreviated as "NAD", and can take two states: the oxidized form "NAD + " and the reduced form "NADH". The nicotinamide adenine dinucleotide (NAD) according to the present invention includes both NAD + and NADH, but the nicotinamide adenine dinucleotide that improves production in vivo by the present invention is preferably NAD + .

[0016] The active ingredient contained in the composition for promoting NAD production of the present invention is scutellarin. The present inventors have found that scutellarin has an effect of promoting the production of nicotinamide adenine dinucleotide in vivo. In the present invention, scutellarin refers to 4',5,6,7-tetrahydroxyflavone 7-glucuronide (CAS number: 27740-01-8) having a molecular weight of 462.36, and is represented by the following chemical structure. As scutellarin, for example, commercially available products may be used as appropriate.

[0017] [ka]

[0018] In the composition of the present invention, the content of scutellarin is not particularly limited, as it is appropriately adjusted depending on the form of the composition, the dosage or intake amount, the purpose and method of administration or intake, etc., and can be 0.001 to 100 mass%, 0.1 to 100 mass%, or 1 to 100 mass% relative to the total mass of the composition, but can be, for example, in the range of 1 to 90 mass%, 1 to 70 mass%, or 1 to 50 mass%.

[0019] The amount of administration or intake of the composition of the present invention (hereinafter sometimes referred to as "dose") can be appropriately determined for each individual case, taking into consideration the form of the composition, the purpose and method of administration or intake, the species, age, body weight, sex, disease, severity of symptoms, etc. of the subject. Thus, without being particularly limited, for example, the dose for humans (preferably adults) is preferably 0.01 to 10 mg, more preferably 0.1 to 10 mg, of scutellarin per kg of subject body weight per day, and can be, for example, 0.1 to 8 mg, 0.1 to 6 mg, or 0.1 to 4 mg.

[0020] The composition of the present invention may further contain other ingredients acceptable for food or pharmaceutical use in addition to scutellarin, so long as the effects of the present invention are not inhibited. The other ingredients are not particularly limited, but include, for example, water; lipids; minerals; vitamins; proteins; peptides; amino acids; organic acids; lactic acid bacteria (Lactobacillus genus, etc.); bifidobacteria; yeast; processed products of the lactic acid bacteria, bifidobacteria, and / or yeast (crushed products, heat-treated products, etc.); fermented products of the lactic acid bacteria, bifidobacteria, and / or yeast; and formulation auxiliary agents. The other ingredients may also include active ingredients other than scutellarin.

[0021] The lipid is not particularly limited, but more specific examples thereof include natural fats and oils such as soybean oil, corn oil, palm oil, perilla oil, canola oil, safflower oil, sunflower oil, sesame oil, rice oil, grape seed oil, fish oil, safflower oil, rapeseed oil, and peanut oil; and synthetic fats and oils such as medium-chain triglycerides (MCT) having about 6 to 12 carbon atoms.

[0022] The minerals are not particularly limited, but more specific examples include calcium, magnesium, sodium, potassium, phosphorus, iron, manganese, copper, zinc, iodine, zinc, selenium, chromium, and molybdenum.

[0023] The vitamins are not particularly limited, but more specific examples include vitamins A, B1, B2, B5, B6, B7, B9, B12, C, D, E, and K.

[0024] The formulation auxiliary is not particularly limited, but more specific examples thereof include solvents (such as PEG400), dispersants, emulsifiers, thickeners (such as carboxymethylcellulose (CMC)), thickening stabilizers, gelling agents, surfactants (such as Tween 80), buffers, stabilizers, excipients, binders, disintegrants, lubricants, flavorings, dissolution aids, suspending agents, coating agents, carriers (solid carriers; liquid carriers such as water, saline, and DMSO), preservatives, fragrances, colorants, and pH adjusters.

[0025] The other components may be contained alone or in combination of two or more. When these other components are contained, the content of the other components is not particularly limited and can be appropriately adjusted depending on the form of the composition, the dosage, the purpose and method of administration or ingestion, etc.

[0026] The administration form of the composition of the present invention is not particularly limited, and includes oral administration, enteral administration, intraperitoneal administration, subcutaneous administration, etc., and the composition of the present invention can be made into a composition according to each administration form. The composition of the present invention can be administered or ingested either orally or parenterally, and is preferably administered or ingested orally, intraperitoneally, or subcutaneously, and is preferably made into a composition for oral administration, a composition for intraperitoneal administration, or a composition for subcutaneous administration accordingly. In addition, the composition of the present invention can be made into, for example, a pharmaceutical composition, a quasi-drug composition, a food and drink composition, or a feed composition depending on the purpose, subject, method, dosage, etc. of administering or ingesting the composition.

[0027] The pharmaceutical composition and quasi-drug composition according to the present invention can be, for example, a preparation, the form of which is not particularly limited, and examples thereof include oral preparations such as tablets (including buccal tablets and lozenge tablets), pills, granules, powders, powders, capsules, suspensions, emulsions, syrups, and jellies; parenteral preparations such as injections, inhalants, intraperitoneal preparations, suppositories, lotions, sprays, ointments, creams, patches, and sustained-release preparations. The preparations can be produced by adding, for example, one or more of the formulation auxiliary agents to scutellarin, and further adding, as necessary, one or more of the other components, by a known method or a method similar thereto.

[0028] The form of the food and drink composition according to the present invention is not particularly limited, and examples thereof include solid forms such as bars, liquid forms such as beverages and liquid foods, pastes, semi-liquid forms, gels (jelly forms), gel-like oils and fats (semi-solid oils and fats), powders, etc. Furthermore, the food and drink compositions can be given as liquid foods, powdered liquid foods, nutritional pastes, oral and tube-fed nutritional supplements, beverages, gel foods, etc., to be ingested by oral and tube-fed patients, the elderly, infants, etc.

[0029] Examples of the food and drink composition according to the present invention include, but are not limited to, beverages (tea, carbonated beverages, cocoa, coffee, lactic acid bacteria beverages, soy milk beverages, fruit and vegetable juice beverages, soft drinks, nutritional beverages, alcoholic beverages, etc.), processed foods (chocolate, gum, gummy candy, jelly, baked goods (bread, cake, cookies, biscuits, etc.), candy, etc.), dairy products (prepared milk powder (powdered milk, etc.), preparative milk, milk beverages, fermented milk, yogurt, ice cream, cheese, cream, butter, margarine, condensed milk, etc.), seasonings (sauces, soups, dressings, mayonnaise, mayonnaise-type seasonings, cream, etc.), supplements, edible oils, and functional edible oils and fats. Such food and drink compositions can be produced, for example, by a method of blending the scutellarin according to the present invention with an existing food and drink, or its raw materials, or an intermediate product in the manufacturing process. The scutellarin blended in this case may be in the form of the pharmaceutical composition or quasi-drug composition.

[0030] Furthermore, the food and beverage composition according to the present invention may be, for example, a general food, a health food, a nutritional supplement, a health supplement, a functional food, a health functional food (e.g., a food for specified health uses, a nutritionally functional food, a food with functional claims, etc.), a food for special purposes (e.g., food for infants, food for pregnant women, food for the sick, etc.), a medical food (a food prescribed under the supervision of a doctor as defined by the U.S. Food and Drug Administration (FDA) and the Orphan Drug Act), a therapeutic food (a food that serves a therapeutic purpose and is prepared based on a menu prepared by a nutritionist or the like in accordance with a doctor's dietary prescription), or a dietary therapy food.

[0031] The feed composition according to the present invention may be the above-mentioned food and drink composition modified appropriately depending on the purpose, subject, method, dose, etc. of administration or ingestion of the feed composition.

[0032] As the composition of the present invention, it is preferably packaged (preferably enclosed) in a packaging container from after production until administration or ingestion. The packaging container is not particularly limited, and examples thereof include wrapping paper, packaging bags, films and sheets (such as strip packaging, PTP packaging, etc.), soft packs, tubes, tear packs, paper containers, cans, bottles, and capsules.

[0033] By containing scutellarin as an active ingredient, the composition of the present invention can be used for promoting the production of NAD in vivo, for example, it can be used for increasing the in vivo concentration of NAD. Therefore, the present invention also provides the use of scutellarin for promoting NAD production or increasing the in vivo concentration of NAD, and the use of scutellarin for the production of a composition for promoting NAD production or a composition for increasing the in vivo concentration of NAD. Here, "in vivo" at this time may be any of blood or tissues (for example, liver, kidney, heart, skeletal muscle, brain). Among these, when referring to "production of NAD in vivo", it mainly indicates production in the above-mentioned tissues. The composition of the present invention and the use of scutellarin of the present invention can be used for the expression, maintenance, or improvement of various functions in which NAD is directly or indirectly involved in vivo.

[0034] In the present invention, the promotion of NAD production in vivo can be confirmed, for example, by collecting whole blood or the above-mentioned tissues and measuring the NAD + concentration, and based on the subject before administration of the composition of the present invention or the scutellarin according to the present invention or the group that was not administered, the NAD + concentration has increased, etc. The NAD + concentration can be measured by an appropriately known method or a method analogous thereto. For example, it can be measured by the method using a liquid chromatograph mass spectrometer (LC-MS / MS) described in the following examples.

[0035] <Method for Promoting NAD Production> By administering the above-mentioned scutellarin to a subject, it is possible to promote the production of NAD in the subject's body. In addition, this can increase the subject's NAD concentration in the body. Therefore, the present invention also provides a method for promoting NAD production and a method for increasing NAD concentration in the body (hereinafter, sometimes collectively referred to as the "method of the present invention"), which involves administering an effective amount of scutellarin to a subject.

[0036] The method of the present invention includes a step of administering an effective amount of scutellarin according to the present invention to a subject. The subject of the method of the present invention includes humans and non-human mammals, and the non-human mammals include mice, sheep, cows, pigs, horses, monkeys, dogs, cats, rabbits, etc.

[0037] In the method of the present invention, scutellarin can be administered as it is, or more preferably in the form of the composition of the present invention, and may be administered to the subject via any administration route, either oral or parenteral, for example, oral administration, intraperitoneal administration, or subcutaneous administration is preferred. Note that in the present invention, oral administration also includes ingestion of the food and drink composition, the feed composition, and the like.

[0038] In the method of the present invention, the effective amount of scutellarin is not particularly limited, since it is appropriately determined for each individual case taking into consideration the purpose and method of administration, the species, age, body weight, sex, disease, severity of symptoms, etc. of the subject, but like the dose of the composition of the present invention described above, for example, the amount of scutellarin for humans (preferably adults) is preferably 0.01 to 10 mg, more preferably 0.1 to 10 mg, per kg of subject body weight per day, and can be, for example, 0.1 to 8 mg, 0.1 to 6 mg, or 0.1 to 4 mg. In the method of the present invention, the number of administrations can be divided into once to an appropriate number of times per day. EXAMPLES

[0039] The present invention will be described in more detail below based on test examples, but the present invention is not limited to the following examples.

[0040] <Test Example 1> (1) Drug to be administered A scutellarin solution was prepared by dissolving scutellarin (Selleck biotech) in solvents (saline: 50 v / v%, DMSO: 5 v / v%, PEG400: 40 v / v%, Tween80: 5 w / v%) to a concentration of 5 mg / mL.

[0041] (2) Subjects and administration Nineteen male C57BL / 6J mice aged 96 weeks were used as subjects for administration. Before and during the study, the mice were kept in a breeding room with a room temperature of 22±2°C, humidity of 55±15%, and a light period of 7:00 to 19:00 and a dark period of 19:00 to 7:00.

[0042] On the first day of administration, the mice were randomly divided into two groups: a control group (n=10) and a scutellarin group (n=9). The scutellarin solution prepared in (1) was subcutaneously administered at a dose of 50 mg / kg to the scutellarin group, and the same volume of the solvent used in the preparation of (1) was subcutaneously administered to the control group (Ctrl). Administration was performed once a day for 7 days.

[0043] (3) Analysis Seven days after the start of administration, all animals were dissected. They were euthanized by taking all blood from the heart under isoflurane anesthesia, and the abdomen was opened to collect the liver.

[0044] (3-1) NAD in whole blood and tissues + amount The whole blood collected in the above dissection was mixed with methanol (final concentration 80 v / v%) and left to stand on ice for 30 minutes. Then, this was centrifuged, and the resulting supernatant was dried using a centrifugal evaporator. The resulting dried matter was then dissolved in an ammonium acetate solution, and the filtrate was filtered through a 0.22 μm filter. The filtrate was subjected to a liquid chromatography mass spectrometer (LC-MS / MS) under the following conditions to analyze NAD. +The amount of NAD was measured. The liver collected in the above dissection was crushed with a bead shocker, mixed with methanol (final concentration 80 v / v%), and then left to stand on ice for 60 minutes. This was then centrifuged, and the resulting supernatant was dried using a centrifugal evaporator. The resulting dried matter was then dissolved in an ammonium acetate solution, and the filtrate was filtered through a 0.22 μm filter. The filtrate was subjected to a liquid chromatography mass spectrometer (LC-MS / MS) under the following conditions to analyze NAD + The amount was measured. (LC-MS / MS conditions) Detector: QTRAP4500 (SCIEX) HPLC system: Shimadzu Corporation Column: Hypercarb Column (particle size 3 μm, inner diameter 2.1 mm × length 100 mm; Thermo Fischer Scientific) mobile phase Solution A: 7.5 mM ammonium acetate aqueous solution containing 0.05 v / v% ammonium hydroxide Solution B: 0.05 v / v% ammonium hydroxide in acetonitrile Ratio of B solution in A solution + B solution (v / v%): Start of mobile phase flow → 1.8 min: 5%; 1.8 min → 14 min: 5 → 54%; 14 min → 14.1 min: 54 → 90%; 14.1 min → 17.1 min: 90%; 17.1 min → 17.2 min: 90 → 5%; 17.2 min → 32.2 min: 5% Column temperature: 60℃ Flow rate: 0.2mL / min Injection volume: 3μL Ionization: ESI positive mode NAD + m / z Precursor ion (Q1 Mass, (Da)): 664.2 Product ion (Q3 Mass, (Da)): 428.2.

[0045] Figure 1(a) shows the NAD in 1 μL of whole blood in each group. + (b) shows the amount of NAD per mg of liver tissue in each group. +As shown in Figure 1, the amount of NAD in the blood and tissues was significantly higher in the Scutellarin group, which received the Scutellarin solution, than in the control group (Ctrl), which did not receive the solution. + The concentration increased, with Student's t-test showing p<0.01 (** in Figure 1) or p=0.07).

[0046] (3-2) Expression of CD38 gene in tissues From the liver collected at the dissection, mRNA was extracted using an RNeasy mini kit (QIAGEN), and cDNA was synthesized using the mRNA. + The gene expression level of the CD38 gene (degrading enzyme) was measured. The GAPDH gene was used for normalization, and the relative value (relative mRNA expression, mean ± standard error) to the control group value of 1.0 was calculated to determine the gene expression level. For qPCR, a primer set purchased from Takara Bio Inc. (for CD38 gene: MA168624, for GAPDH gene: MA050371) was used.

[0047] Figure 2 shows the expression level of CD38 gene in the liver in each group (relative mRNA expression, mean ± standard error). As shown in Figure 2, the Scutellarin group, which received the Scutellarin solution, had a higher NAD than the control group (Ctrl), which did not receive the solution. + The gene expression level of CD38, a degrading enzyme, was significantly lower, with p<0.05 in Student's t-test (Figure 2, *).

[0048] Administration of scutellarin reduces NAD + It was also confirmed that administration of scutellarin decreased the expression level of the CD38 gene. CD38 is a cellular NAD +It is known that scutellarin reduces the amount of NAD in vivo by inhibiting the expression of CD38 (Claudia CS Chini et al., Biochem Biophys Res Commun., 513(2), May 28, 2019, p. 486-493). + It was suggested that the amount of

[0049] <Test Example 2> (1) Drug to be administered Scutellarin (Selleck biotech) and apigenin (Selleck biotech) were dissolved in solvent (saline: 90 v / v%, DMSO: 10 v / v%) to prepare a scutellarin solution and an apigenin solution, respectively, at 10 mg / mL.

[0050] (2) Subjects and administration Eighteen male mice of the C57BL / 6J strain, 20 weeks old, were used as subjects for administration. Before and during the test, the mice were kept in a breeding room with a room temperature of 22±2°C, humidity of 55±15%, light period of 7:00 to 19:00, and dark period of 19:00 to 7:00. Following the method described in Non-Patent Document 1, the mice were fed a high-fat diet (D12492, Research Diets) for 4 weeks to induce obesity. The high-fat diet was continued to be fed even after the start of administration.

[0051] On the first day of administration, the mice were divided into three groups (n=6 per group): control group, scutellarin group, and apigenin group, so that the average body weights were equal. The scutellarin solution prepared in (1) was intraperitoneally administered to the scutellarin group at a dose of 100 mg / kg, and the apigenin solution prepared in (1) was intraperitoneally administered to the apigenin group at a dose of 100 mg / kg. The control group (Ctrl) was intraperitoneally administered the same volume of the solvent used in the preparation in (1). Administration was performed once a day for 7 days.

[0052] (3) Analysis Seven days after the start of administration, all animals were dissected. They were euthanized by taking all blood from the heart under isoflurane anesthesia, and the abdomen was opened to collect white fat tissue.

[0053] (3-1) NAD in whole blood + amount In the same manner as in (3-1) of Test Example 1 above, the whole blood collected at the autopsy was treated and analyzed by liquid chromatography mass spectrometry (LC-MS / MS) to obtain NAD + Figure 3 shows the amount of NAD in 1 μL of whole blood in each group. + As shown in Figure 3, the Scutellarin group to which the Scutellarin solution was administered had a higher blood NAD than the control group (Ctrl) to which the Scutellarin solution was administered, and also compared to the Apigenin group (Apigenin) to which the Apigenin solution was administered. + The concentration increased (in Figure 3, "**" indicates p<0.01 by Tukey test).

[0054] (3-2) Expression of NAMPT gene in tissues From the white fat collected in the dissection, mRNA was extracted using the RNeasy Plus Universal Mini Kit (QIAGEN), and cDNA was synthesized using the mRNA. + The gene expression level of the NAMPT gene (synthetic enzyme) was measured. The GAPDH gene was used for normalization, and the relative value (relative mRNA expression, mean ± standard error) to the control group value of 1.0 was calculated to determine the gene expression level. For qPCR, a primer set purchased from Takara Bio Inc. (for NAMPT gene: MA101388, for GAPDH gene: MA050371) was used.

[0055] Figure 4 shows the expression level of the NAMPT gene in white fat in each group (relative mRNA expression, mean ± standard error). As shown in Figure 4, the Scutellarin group, which received the Scutellarin solution, showed a higher NAD than the control group (Ctrl), which did not receive the Scutellarin solution, and also compared to the Apigenin group (Apigenin), which received the Apigenin solution. + The gene expression level of the synthetic enzyme NAMPT was significantly increased (in Figure 4, "*" indicates p<0.05 by Tukey test, and "**" indicates p<0.01 by Tukey test).

[0056] (3-3) Amount of eNAMPT in the blood Plasma was separated from the whole blood collected at the dissection, and the amount of eNAMPT (the form of NAMPT in blood) was measured by Western blotting. Western blotting was performed as previously reported (Mitsukuni Yoshida et al., Cell Metabolism, 30(2), August 6, 2019, p. 329-342). Transferrin was used for normalization, and the relative values ​​(relative protein levels, mean ± standard error) were calculated to determine the amount of eNAMPT.

[0057] Figure 5 (a) shows the Western blotting images of eNAMPT and Transferrin in whole blood in each group, and (b) shows the amount of eNAMPT (relative protein levels, mean ± standard error) in each group. As shown in Figure 5, the Scutellarin group to which the Scutellarin solution was administered showed a higher NAD than the control group (Ctrl) to which the Scutellarin solution was administered, as well as the apigenin group (Apigenin) to which the apigenin solution was administered. + The amount of eNAMPT, the synthetic enzyme, was significantly increased (in Figure 5, "*" indicates p<0.05 by Tukey test, and "**" indicates p<0.01 by Tukey test).

[0058] It was confirmed that the promotion of NAD production by administration of scutellarin is superior to that by administration of apigenin of the prior art. The mechanism by which scutellarin and apigenin contribute to NAD production in vivo is currently unknown, but as shown in FIG. 4, the expression level of the NAMPT gene increased only in the scutellarin group, suggesting that scutellarin contributes to the promotion of NAD production in vivo by a mechanism different from that of apigenin of the prior art. It has been reported that most of the eNAMPT in blood is derived from white fat (Myeong Jin Yoon et al., Cell Metabolism, 21(5), May 5, 2015, p. 706-717). That is, in the present invention, the administration of scutellarin increases the expression level of the NAMPT gene in white fat, and the increase in eNAMPT also promotes the promotion of NAD production in vivo. + It was suggested that the amount of

[0059] <Test Example 3> In addition to scutellarin and apigenin, the inhibitory activity of other flavonoids against CD38 activity was measured. CD38 activity was measured using a kit (CD38 Inhibitor Screening Assay Kit (Hydrolase Activity), Bioscience). Each component was dissolved in DMSO to a final concentration of 10 μM and used as a sample for measurement, and each measurement was performed according to the protocol attached to the kit. The measured value when DMSO alone (no flavonoids added) was measured in the same manner was set to 100, and the relative value of the measured value of each sample was calculated to obtain the CD38 activity value (CD38 Activity). The components used in the measurements were scutellarin, as well as those with structural formulas relatively close to scutellarin, including apigenin, scutellarein, gastrodenol, apigetrin, gallocatechin ((+)-), fisetin, luteoloside, homoplantaginin, 2',3',4'-trihydroxy-flavone (2-D08), hematoxylin, epigallocatechin ((-)-epigallocatechin), quercetin, quercetin dihydrate, 4',5-dihydroxyflavone, and genkwanin. Their chemical structures are shown below.

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[0065] [ka]

[0066] [ka]

[0067] The CD38 activity measured in samples to which each component (flavonoid) was added is shown in Figure 6. As shown in Figure 6, the CD38 activity measured when scutellarin was added was significantly lower than many other flavonoids with similar structures, and it was confirmed that scutellarin has a particularly high inhibitory effect on CD38 activity.

[0068] <Test Example 4> (1) Drug to be administered A scutellarin solution was prepared by dissolving scutellarin (Selleck biotech) in a solvent (aqueous carboxymethylcellulose (CMC) solution, Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 5 mg / mL.

[0069] (2) Subjects and administration Twelve male C57BL / 6J mice aged 110 weeks were used as subjects for administration. Before and during the study, the mice were kept in a breeding room with a room temperature of 22±2°C, humidity of 55±15%, and a light period of 7:00 to 19:00 and a dark period of 19:00 to 7:00.

[0070] On the first day of administration, the mice were randomly divided into two groups, a control group and a scutellarin group (n=6 in each group). The scutellarin solution prepared in (1) was forcefully administered to the scutellarin group at a dose of 50 mg / kg, and the same volume of the solvent used in the preparation of (1) was forcefully administered to the control group (Ctrl). Administration was performed once a day for 21 days.

[0071] (3) Analysis Seven days after the start of administration, all animals were dissected. Under isoflurane anesthesia, all blood was collected from the heart and euthanized. In the same manner as in (3-1) of Test Example 1 above, the whole blood collected at the dissection was processed and analyzed by liquid chromatography mass spectrometer (LC-MS / MS) to determine NAD + Figure 7 shows the amount of NAD in 1 mL of whole blood in each group. + As shown in FIG. 7, even with oral administration, the scutellarin group (Scutellarin) to which the scutellarin solution was administered had a higher blood NAD than the control group (Ctrl) to which the scutellarin solution was not administered. + There was a tendency for the concentrations to be higher (p=0.09 by Student's t-test). [Industrial Applicability]

[0072] As described above, according to the present invention, it is possible to provide a new composition having an effect of promoting the production of nicotinamide adenine dinucleotide in a living body. In addition, since the composition of the present invention can be provided as a food and drink composition, it is easy to take and easy to make taking it a habit.

Claims

1. A composition for promoting in vivo nicotinamide adenine dinucleotide (NAD) production, characterized by containing scutellarin as an active ingredient.

2. The composition according to claim 1, which is a composition for increasing the in vivo concentration of nicotinamide adenine dinucleotide (NAD).

3. The composition according to claim 1 or 2, which is a composition for oral administration, a composition for intraperitoneal administration, or a composition for subcutaneous administration.

4. The composition according to claim 1 or 2, which is a food or drink composition, a pharmaceutical composition, or a quasi-drug composition.

5. 3. The composition according to claim 1 or 2, wherein the dosage for humans is 0.01 to 10 mg of scutellarin per kg of body weight per day.

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