Intracellular NAD+ production promoter

Abstract

To provide a novel intracellular NAD+ production promoter.SOLUTION: An intracellular NAD+ production promoter contains hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an intracellular NAD + production promoter.

Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is a coenzyme that mediates redox reactions in vivo and plays a central role in energy metabolism. As the biosynthesis pathway of NAD + , there are two pathways: a de novo pathway starting from tryptophan and a salvage pathway that reuses the nicotinamide ring of nicotinamide (NAM) and nicotinamide riboside (NR).

[0003] NAD + is also deeply involved in post-translational modifications of proteins (e.g., deacetylation, ADP-ribosylation, etc.) and is known to regulate various intracellular functions such as differentiation and proliferation (Patent Document 1). For example, poly ADP-ribose polymerase (PARP), an enzyme involved in the repair of single-stranded DNA damage, performs ADP-ribosylation using NAD + . Moreover, sirtuins (Sirtuin1 - 7), which play an important role in the aging control mechanism, are all NAD + -dependent deacetylase enzymes, and sirtuin activity is also controlled by the concentration of NAD + that serves as an acetyl group acceptor. Generally, it is known that an increase in sirtuin activity causes changes such as an increase in the expression of antioxidant enzyme genes or a suppression of the expression of inflammatory protein genes.

[0004] On the other hand, it has been found that the intracellular NAD + level decreases with aging and causes various aging symptoms and related diseases. For example, NAD +It is known that a decrease in levels can affect metabolic dysfunction, DNA repair disorders, inflammation, cellular senescence, reduced resistance to oxidative stress, neurodegeneration, etc. (Non-Patent Document 1). In contrast, there are also reports of cases where diseases associated with aging can be treated by promoting NAD + biosynthesis or inhibiting NAD + degradation. For example, in animal models, it has been reported that increasing intracellular NAD + levels can delay aging, restore muscle function, and provide a protective effect against metabolic diseases (Non-Patent Document 2).

[0005] In Patent Document 2, it has been reported that nicotinic acid riboside or nicotinamide riboside derivatives, and their reduced derivatives are used as precursors to increase NAD + .

[0006] In recent years, for increasing intracellular NAD + levels, intravenous drip therapy and supplements of nicotinamide mononucleotide (NMN), which is known as an intermediate in the NAD + biosynthesis pathway, are used for anti-aging purposes, and one of their effects is to suppress skin aging such as wrinkles, sagging, spots, and dullness. In addition, cosmetics containing NMN are expected to activate sirtuin by increasing NAD + in epidermal and subcutaneous cells, promote the production of natural moisturizing factor (NMF), and prevent skin aging (Patent Documents 3 and 4).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

[0008] [Non-Patent Document 1] NAD+ metabolism and its roles in cellular processes during aging,Anthony J.Covarrubias et al.,Nature Reviews Molecular Cell Biology vol.22,pp.119-141,2021 [Non-Patent Document 2] De novo NAD+ synthesis enhances mitochondrial function and improves health,Elena Katsyuba et al.,Nature vol.563,pp.354-359,2018 [Overview of the project] [Problems that the invention aims to solve]

[0009] This invention relates to novel intracellular NAD + The objective is to provide a production promoter. [Means for solving the problem]

[0010] The inventors, after diligent research to achieve the above objective, have found that hinokitiol or its salt, and nicotinic acid or its salt, are used in intracellular NAD + We discovered that it has a production-promoting effect, which led to the completion of the present invention.

[0011] One embodiment of the present invention is an intracellular NAD containing hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients. + Intracellular NAD containing nicotinic acid or a salt thereof as an active ingredient, administered simultaneously with a production promoter, hinokitiol, or a salt thereof. +A production promoter, and intracellular NAD containing hinokitiol or a salt thereof as an active ingredient, administered simultaneously with nicotinic acid or a salt thereof. + The production promoter is intracellular NAD + It has the effect of increasing concentration. Intracellular NAD in the epidermis and subcutaneous tissue of the skin. + By increasing the concentration, functions such as DNA repair and antioxidant stress can be restored, therefore, the above intracellular NAD + The production promoter is also useful as a preventive or ameliorative agent for skin aging symptoms. + By incorporating a production promoter into a cosmetic composition or topical composition, NAD in the epidermal and subcutaneous cells of the skin can be increased. + This can provide a cosmetic composition or topical composition that promotes production and is suitable for anti-aging care. In this specification, "simultaneous administration" means administering the other while one is acting, thereby allowing both to act together. Administration may be done together or at different times. Furthermore, both may be administered as single agents, or as two separate agents, each containing one of the agents. Similarly, "simultaneous addition" means adding the other while one is acting, thereby allowing both to act together. Addition may be done together or at different times. Furthermore, both may be added as single agents, or as two separate agents, each containing one of the agents.

[0012] In other words, embodiments of the present invention include the following [1] to

[13] . [1] Intracellular NAD containing hinokitiol or a salt thereof, and nicotinic acid or a salt thereof as active ingredients. + A production promoter. [2] Intracellular NAD containing nicotinic acid or a salt thereof as an active ingredient, administered simultaneously with hinokitiol or a salt thereof. + A production promoter. [3] Intracellular NAD containing hinokitiol or a salt thereof as an active ingredient, administered simultaneously with nicotinic acid or a salt thereof. + A production promoter. [4] The intracellular NAD described in [1]-[3], wherein the molar ratio of hinokitiol or its salt to nicotinic acid or its salt administered is 1-3:1-60. + A production promoter. [5] Intracellular NAD as described in [1] to [4], which is a preventive or ameliorative agent for skin aging symptoms. + A production promoter. [6] Skin aging symptoms include wrinkles, sagging, age spots, freckles, rough skin, or dry skin, and the intracellular NAD described in [5]. + A production promoter. [7][1]~[6] intracellular NAD + A cosmetic composition containing a production promoter as an active ingredient. A cosmetic product containing the cosmetic composition described in [8][7] as an active ingredient. Intracellular NADs described in [9][1]~[6] + A topical composition containing a production promoter as an active ingredient. A topical preparation containing the topical composition described in

[10] [9] as an active ingredient. Intracellular NADs described in

[11] [1]~[6] + A method for preventing or improving symptoms of skin aging, comprising the step of administering a production inhibitor to a human.

[12] Intracellular NADs as described in [1] to [6], used to prevent or improve symptoms of skin aging. + Production inhibitor.

[13] Intracellular NADs as described in [1] to [6] for manufacturing preventive or ameliorative agents for skin aging symptoms + Use of production inhibitors. [Effects of the Invention]

[0013] According to the present invention, a novel intracellular NAD + This will make it possible to provide production promoters. [Brief explanation of the drawing]

[0014] [Figure 1] This bar graph shows the amount of intracellular NAD+ when each compound that serves as a precursor for NAD+ biosynthesis is added alone or together with hinokitiol, in one embodiment of the present invention. [Modes for carrying out the invention]

[0015] Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily reproduce the present invention from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only; the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the present invention as disclosed herein. Unless otherwise specified in the embodiments and examples, methods described in standard protocol collections such as MR Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified or altered methods thereof, may be used. Furthermore, when using commercially available reagent kits or measuring instruments, unless otherwise specified, the protocols provided with them should be used.

[0016] One embodiment of the present invention is an intracellular NAD containing hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients. + Intracellular NAD containing nicotinic acid or a salt thereof as an active ingredient, administered simultaneously with a production promoter, hinokitiol, or a salt thereof. +Intracellular NAD containing hinokitiol or a salt thereof as an active ingredient, administered simultaneously with a production promoter or nicotinic acid or a salt thereof. + It is a production promoter.

[0017] <Intracellular NAD + Production promoters > Hinokitiol is a known compound, a monocyclic monoterpene with an unsaturated seven-membered ring. Nicotinic acid (NA) is a known compound that is named 3-pyridinecarboxylic acid according to IUPAC nomenclature.

[0018] The salts of hinokitiol are not particularly limited as long as they are medically, pharmacologically, or physiologically acceptable. Examples of such salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and organic base salts such as ammonium salts and trialkylamine salts. Intracellular NAD containing hinokitiol or its salts + The production promoter may contain one type of hinokitiol or a salt thereof, or two or more types of hinokitiol or salts thereof. In particular, it is preferable that the hinokitiol or salt thereof contains hinokitiol and sodium hinokitiol.

[0019] The salts of nicotinic acid are not particularly limited, as long as they are medically, pharmacologically, or physiologically acceptable. Examples of such salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and organic base salts such as ammonium salts and trialkylamine salts. Intracellular NAD containing nicotinic acid or its salts + The production promoter may contain one type of nicotinic acid or a salt thereof, or two or more types of nicotinic acid or salts thereof. In particular, it is preferable that the nicotinic acid or salt thereof contains nicotinic acid and sodium nicotinate.

[0020] Intracellular NAD +Hinokitiol and its salts used in the production of the production promoter may be extracted from natural sources, chemically synthesized, or obtained commercially. Examples of raw material plants for the hinokitiol and its salt extracts include Taiwanese cypress, Aomori cypress, and North American Western red cedar. The purity of the extract is not particularly limited and may be a highly purified product, and intracellular NAD + It may also be a crude product that does not inhibit the effect of promoting production. As a crude product of hinokitiol, for example, an intermediate substance in the purification process of extracting and purifying hinokitiol and its salt from raw plant material may be used.

[0021] Intracellular NAD + Nicotinic acid and its salts used in the production of the production accelerator may be synthesized by known methods or obtained commercially. Nicotinic acid and its salts may be used as a single compound or as a combination of two or more compounds.

[0022] Intracellular NAD containing nicotinic acid or its salts + When the production promoter is a liquid formulation, the concentration of nicotinic acid or its salt is not particularly limited and is set appropriately according to the concentration of hinokitiol or its salt used in combination, the formulation form, the method of use, etc. Also, intracellular NAD containing hinokitiol or its salt + When the production promoter is a liquid formulation, the concentration of hinokitiol or its salt is not particularly limited and is set appropriately according to the concentration of nicotinic acid or its salt used in combination, the formulation form, the method of use, etc.

[0023] Intracellular NAD containing both hinokitiol or its salt and nicotinic acid or its salt +The molar ratio of hinokitiol or its salt and nicotinic acid or its salt contained in the production promoter is not particularly limited and is set appropriately depending on the type of hinokitiol or its salt and nicotinic acid or its salt used, the formulation form, the method of use, etc. For example, the molar ratio of each active ingredient is preferably hinokitiol or its salt : nicotinic acid or its salt = 1~3:1~60, more preferably 1~3:2~30, and even more preferably 1~3:6~20.

[0024] Intracellular NAD according to this embodiment + The production promoter is NAD in cells. + As long as they do not inhibit the production-promoting effect, they may contain, for example, excipients, diluents, humectants, pH adjusters, emulsifiers, solubilizers, dispersants, thickeners, surfactants, preservatives, antioxidants, oils and fats, hydrocarbons, fatty acids, alcohols, fragrances, powders, pigments, etc.

[0025] Intracellular NAD according to this embodiment + The dosage form of the production promoter is not particularly limited and can be arbitrarily selected according to the purpose of use. For example, it may be in the form of a cream, paste, ointment, lotion, emulsion, mousse, solution, powder, pack, gel, etc.

[0026] Intracellular NAD + The production promoter is NAD in cells. + By increasing the concentration of NAD, it is possible to promote DNA repair by PARP or suppress the expression of inflammatory protein genes (Nature Reviews Molecular Cell Biology Vol.22, pp.119-141, February 2021). In other words, intracellular NAD + By increasing the concentration of [the substance], metabolic function in the skin can be improved, DNA repair can be promoted, inflammation can be suppressed, cellular senescence can be inhibited or improved, and resistance to oxidative stress can be enhanced (Nature vol.563, pp.354-359, 2018; International Publication No. 2019 / 078177). Therefore, intracellular NAD according to this embodiment +The production promoter can be suitably used as an agent to prevent or improve cell aging (particularly skin aging), a DNA repair promoter, an anti-inflammatory agent, and an oxidative stress resistance enhancer. Examples of skin aging symptoms include wrinkles, sagging, age spots, freckles, rough skin, and dry skin.

[0027] <Composition> Intracellular NAD according to this embodiment + Production promoters can be incorporated into cosmetic compositions, food compositions, reagent compositions, and pharmaceutical compositions.

[0028] One-border mechanism of intracellular NAD + Cosmetic compositions containing production promoters can be used, for example, as cosmetic compositions for improving the metabolic function of the skin, cosmetic compositions for promoting DNA repair, cosmetic compositions for suppressing or improving cellular aging, cosmetic compositions for strengthening resistance to oxidative stress, and the like. The form of cosmetics containing these cosmetic compositions as active ingredients is not particularly limited and includes, for example, lotions, creams, emulsions, ointments, oils, all-in-one gels, masks, serums, foundations, cleansers, facial washes, cleansing agents, shampoos and conditioners, eyeshadows, eyeliners, and the like. These cosmetics are preferably used as topical agents, applied to the skin.

[0029] One-border mechanism of intracellular NAD + Food compositions containing production promoters can be used, for example, as food compositions for improving metabolic function, promoting DNA repair, inhibiting or improving cellular senescence, or enhancing oxidative stress resistance. The types of foods containing the food composition as an active ingredient are not particularly limited and include, for example, general foods, fermented foods (yogurt, royal jelly, etc.), beverages (whey drinks, tea, sports drinks, fruit juices, soft drinks, etc.), confectionery (gum, jelly, candy, etc.), health foods (tablets, capsules, tablets, etc.), nutritional supplements (nutritional drinks, etc.), supplements, functional foods, foods with health claims, foods for specified health uses, pet food, animal feed, etc.

[0030] One-border mechanism of intracellular NAD + A reagent composition containing a production promoter can be used in vitro as a reagent. When this reagent is administered to a culture, such as cultured cells, cultured tissue, or cultured organ, it increases the intracellular NAD production in the culture. + This reagent produces effects such as increased production, suppression of increased intracellular reactive oxygen species concentration, improved metabolic function, accelerated DNA repair, suppression or improvement of cellular senescence, and enhanced resistance to oxidative stress. There are no particular restrictions on the method of using the reagent; for example, hinokitiol or its salt and nicotinic acid or its salt may be added simultaneously to the culture medium.

[0031] One-border mechanism of intracellular NAD + Pharmaceutical compositions containing production promoters can be used, for example, as pharmaceutical compositions for improving metabolic function, pharmaceutical compositions for promoting DNA repair, pharmaceutical compositions for inhibiting or improving cellular senescence, pharmaceutical compositions for enhancing oxidative stress resistance, and the like. A pharmaceutical composition containing the said pharmaceutical composition as an active ingredient can be administered to human or non-human animals. The form of administration is not particularly limited and may be, for example, oral or parenteral administration. There are no particular limitations on the dosage form for oral administration and examples include tablets, powders, granules, fine granules, capsules, liquids, emulsions, syrups, suspensions, lozenges, etc. There are no particular limitations on the dosage form for parenteral administration and examples include injections (intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, etc.), drip infusions, topical preparations (ointments, powders, granules, liquids, gels, emulsions, creams, lotions, mousses, jellies, sheets, sprays, etc.), suppositories, etc., but it is preferable to use it as a topical preparation.

[0032] As explained above, intracellular NAD according to this embodiment + The production promoter is particularly effective in promoting NAD production in epidermal and subcutaneous cells. + This can increase the concentration of NAD + It is useful as a preventative and corrective agent for skin aging symptoms caused by a decline in levels. Furthermore, cosmetic compositions and topical compositions containing it can be provided. [Examples]

[0033] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0034] [Test Example 1: NAD in cells] + [Evaluation of the production-promoting effect] NAD + The intracellular NAD levels when nicotinic acid (NA), nicotinic acid mononucleotide (NAMN), nicotinic acid riboside (NAR), vitamin E nicotinate (NAVE), nicotinamide (NAM), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR), which are precursor compounds for biosynthesis, are added to normal human epidermal keratinocytes (NHEK), either individually or in combination with hinokitiol.+ The production-promoting effect was evaluated using the following method.

[0035] NHEK (manufactured by Kurabo Industries Ltd.), 1.0 x 10 5 Cells were seeded in 96-well plates at a density of 1 cell / well and cultured at 37°C under 5% CO2 conditions. After 1 day, each compound (NA, NAMN, NAR, NAVE, NAM, NMN, NR) was added to each well, either alone or in combination with hinokitiol, to the final concentrations shown in Table 1 below. After culturing for 6 hours at 37°C under 5% CO2 conditions, the cells were harvested and intracellular NAD was measured using Promega's NAD / NADH-Glo(TM) Assay. + The relative amounts were measured. A SynergyHTX multi-plate reader (BioTek) was used to measure luminescence. Figure 1 shows the NAD levels in the control group NT (no compound or hinokitiol added). + Taking the amount as 100%, relative intracellular NAD + The quantities are shown on the vertical axis as bar graphs. In the graphs, except for NT, the white bars represent the relative NAD when each compound is added individually. + The graph shows the quantity, and the black bar graph represents the relative NAD when hinokitiol is added to each compound. + The quantity is shown. Also, in NT, the white bar graph represents the baseline NAD when nothing is added. + The graph shows the quantity, and the black bar graph represents the relative NAD when only hinokitiol is added. + It indicates quantity.

[0036] [Table 1]

[0037] As shown in Figure 1, when only hinokitiol was added and no other compound was added, the intracellular NAD levels were higher compared to the control group where nothing was added. + Although no significant difference in quantity was observed, when hinokitiol was added along with NA, the intracellular NAD levels were higher compared to when NA was added alone. +The amount increased, and a synergistic effect between NA and hinokitiol was observed. However, no synergistic effect with hinokitiol was observed with other nicotinic acid analogs. Thus, NAD in cells + NA and hinokitiol have a synergistic effect in promoting production.

Claims

【Request Item 1】 Intracellular NAD containing hinokitiol or its salt, and nicotinic acid or its salt as active ingredients ＋ A production promoter. 【Request Item 2】 Intracellular NAD containing nicotinic acid or a salt thereof as an active ingredient, administered simultaneously with hinokitiol or a salt thereof. ＋ A production promoter. 【Request Item 3】 Intracellular NAD containing hinokitiol or a salt thereof as an active ingredient, administered simultaneously with nicotinic acid or a salt thereof. ＋ A production promoter. 【Request Item 4】 The intracellular NAD according to any one of claims 1 to 3, wherein the molar ratio of administered hinokitiol or its salt to nicotinic acid or its salt is 1 to 3:1 to 60. ＋ A production promoter. 【Request Item 5】 An intracellular NAD according to any one of claims 1 to 4, which is a preventive or ameliorative agent for symptoms of skin aging. ＋ A production promoter. 【Request Item 6】 The intracellular NAD according to claim 5, wherein the symptoms of skin aging are wrinkles, sagging, age spots, freckles, rough skin, or dry skin. ＋ A production promoter.

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