JP2024022503A - Nampt-activating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel NAMPT-activating agent.

SOLUTION: An NAMPT-activating agent includes, as an active ingredient, one or plural components selected from the group consisting of grape seed, Silybum marianum, Panax ginseng, Houttuynia cordata, Psidium guajava, Punica granatum, HMB-Ca, Vaccinium vitis-idaea, cinnamon bark, Gardenia jasminoides fruit, mulberry leaves, D. longan, Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow, NMN, and Peucedanum japonicum.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention provides NAMPT activators.

NAD (nicotinamide adenine dinucleotide) is an important coenzyme that exists in various parts of the body and plays various roles such as energy metabolism, cell metabolism, gene expression, and DNA repair. However, since the molecular weight of NAD is large, it is difficult to be taken up even when administered from the outside, and there is a problem that it is degraded in the intestines (Non-Patent Documents 1 and 2).

Supplements and the like are on the market for ingesting NMN (β-nicotinamide mononucleotide), a precursor of NAD. However, NMN in an aqueous solution has the disadvantage of being sensitive to heat (Non-Patent Document 1), and there is a possibility that the actual effect in the body will be lower than expected.

On the other hand, if NMN can be synthesized in the body, NAD will be increased in the body, and various functions can be expected to be improved by NAD. In order to synthesize NMN, the NMN synthase NAMPT (nicotinamide phosphoribosyltransferase) plays an important role. However, NAMPT is known to decrease with aging, and it has been reported that aerobic exercise and training are effective for increasing NAMPT (Non-Patent Documents 2 and 3).

There are also attempts to use NAMPT activation/NAMPT promoters as functional foods. Patent Document 1 discloses a composition for promoting NAMPT expression that contains a hydrolyzate of a protein selected from whey protein and casein. Patent Document 2 discloses phenylephrine, trichostatin A, quercetin, etc. as NAMPT upregulating factors. A substance that has a further NAMPT activating effect, especially a substance that can exert the NAMPT activating effect at a desired site in the body even after being orally taken into the intestine, is desired.

Japanese Patent Application Publication No. 2021-126067 Japanese Patent Application Publication No. 2021-515796

JFRL News Vol.7 No.10 Oct. 2021 ISSN 2186-9138 https://www.amed.go.jp/news/release_20190614.html Physiol Rep, 7 (12), 2019, e14139, https://doi.org/10.14814/phy2.14139 Experimental Dermatology. 2019;28:551-560. DOI: 10.1111/exd.13908 PLoS ONE 14(5): e0217394.https://doi.org/10.1371/journal.pone.0217394, May 28, 2019 Wako Pure Chemical News Vol.87 No.2 (June 2019 issue), p.9-11 Yoshinori Katakura Search for anti-brain-aging foods and elucidation of the molecular basis of their functionality, Asahi Beer Science Foundation, April 2010 Fletcher Rachel S.et al. Molecular Metabolism Vol.6 No.8 Page. 819-832 (2017)

An object of the present invention is to provide a NAMPT activator.

As a result of intensive research, the present inventors have found that grape seeds, milk thistle, Korean ginseng, Hokudyami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass. It has been found that one or more components selected from the group consisting of: function as a NAMPT activator. Furthermore, the present inventors have also discovered that these substances act on skin cells via intestinal cells. These discoveries led us to complete the following inventions:
(1) 1 selected from the group consisting of grape seeds, milk thistle, ginseng, dokudyami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass A NAMPT activator containing one or more ingredients as active ingredients.
(2) The NAMPT activator according to (1), wherein the NAMPT activator activates NAMPT in skin cells via intestinal cells.
(3) The NAMPT activator according to (1) or (2), wherein the NAMPT activator activates NAMPT in skin cells when taken internally.
(4) A food composition for beautiful skin, containing the NAMPT activator according to any one of (1) to (3).
(5) 1 selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass; A food composition for beautifying skin, which contains one or more ingredients as beautifying ingredients having the function of activating NAMPT when taken orally.

NAMPT can be activated by administering the NAMPT activator of the present invention. According to the present invention, a composition and an internal medicine containing a NAMPT activator can be provided.

Figure 1 shows the amount of NAMPT expression due to promoter activity in HaCat (NAMPTp-EGFP) when Caco-2 cell supernatants to which each sample was added in Experiment 2-3 were used as a negative control (DMSO only, control). It is shown as a relative value with the case of adding 100.00. Figure 2 shows the NAMPT expression levels measured by RT-qPCR in HaCat when using Caco-2 cell supernatants added with each sample in Experiment 2-4, and when a negative control (DMSO only, control) was added. Shown as a relative value with 100.00.

NAMPT is an enzyme that converts NAM (nicotinamide) to NMN and controls the production of NAD. It is also known that the sirtuin 1/2/3 cycle and NAD cycle are regulated by the action of NAMPT. NMN is further converted to NAD by another enzyme. As mentioned above, NAD is an important coenzyme that plays various roles in various parts of the body.For example, sirtuin 1/2/3 (SIRT1/ 2/3) Plays an important role in gene activation. There are two independent types of NAMPT (intracellular iNAMPT and extracellular eNAMPT), and it has been suggested that eNAMPT may contribute to melanocyte immune regulation, cancer treatment, etc. (Non-patent Document 1) 4).

NAMPT can be activated by administering the NAMPT activator of the present invention. It has been suggested that activation of NAMPT is useful for treating and improving skin diseases and conditions, such as anti-aging, extending lifespan, and repairing damage (Non-Patent Documents 1 to 4). There are also reports that increased NAMPT expression is effective against diseases such as sarcopenia and diabetes (Non-Patent Document 8, Patent Document 1). Therefore, if it is possible to increase the expression of NAMPT at a desired site using an orally ingested NAMPT activator, it may contribute to the improvement of these diseases. In particular, the NAMPT activator of the present invention is expected to contribute to beautiful skin by activating NAMPT in skin cells via the intestinal tract when taken internally.

In the present invention, beautiful skin refers to improvement of skin condition through activation of NAMPT in skin cells. Improvements in such skin conditions include, for example, skin anti-aging, skin whitening, life extension, anti-oxidation, damage repair, and anti-inflammation. In one embodiment, improvement of skin condition also includes activation of NMN, NAD, and/or SIR1/2/3 via activation of NAMPT.

Activation of NAMPT means, for example, increasing the expression of the NAMPT gene. For example, when a NAMPT activator is added, the expression of the NAMPT gene is increased compared to a state where nothing is added (control). It can mean that. For example, a statistically significant difference with a significance level of 5% (for example, Student's t-test) may mean that the disease is promoted. Alternatively, activation of the NAMPT gene in the present invention means that when a NAMPT activator is added, the expression of the NAMPT gene is increased by, for example, 5% or more, 10% or more compared to a state in which nothing is added (control). % or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 400% or more , or can mean an increase of 500% or more. The expression level of NAMPT can be determined using any known technique, such as, but not limited to, a method of transfecting skin cells with the NAMPT promoter and determining changes in EGFP fluorescence derived from NAMPTp-EGFP, or RT-qPCR using NAMPT primers. It can be measured by a method of determining the expression level of the NAMPT gene by PCR such as

The NAMPT activator of the present invention activates NAMPT in skin cells, particularly through intestinal cells. "Activating NAMPT in skin cells via intestinal cells" means that the active ingredient of the present invention is administered to intestinal cells and absorbed by the intestinal cells, whereby the same active ingredient is directly applied to the skin cells. The active ingredient can be absorbed into the intestinal cells and reach the skin cells in a degraded or modified state, or the intestinal cells can take up the active ingredient and release other different ingredients, and the different ingredients can be absorbed into the skin cells. This refers to the final activation of NAMPT in skin cells through direct or indirect pathways such as action. An example of "activating NAMPT in skin cells via intestinal cells" includes activating NAMPT in skin cells by oral administration. "Activating NAMPT in skin cells by oral administration" means that by orally ingesting the NAMPT activator of the present invention, the active ingredient of the present invention is absorbed through the intestinal tract, and the substance taken in from the intestine. acts directly or indirectly, ultimately activating NAMPT in skin cells.

For example, even if a certain component is taken in from the intestine through internal administration, that substance does not directly reach the target cells, but other substances secreted from the intestinal cells may reach the target cells and act indirectly. For example, as described in Non-Patent Documents 5 to 7, carnosine taken from the intestine does not directly affect the brain, but activates CREB (cAMP response element binding protein) in intestinal cells, resulting in It has been reported that BDNF (brain derived neurotrophic factor) production is enhanced, and BDNF activates nerve cells and has indirect effects, such as improving brain function. Furthermore, it has been suggested that such brain-gut-related activation is mediated by exosomes. In other words, even if a component is given to the intestine, it is unknown what kind of effect that component will have on the target cell, so the test substance is given to the intestinal cells and then it is confirmed what kind of effect it has on the target cell. There is a need. Furthermore, as mentioned above, NAD has a large molecular weight, so it is difficult to take in even if administered from the outside, and NMN in an aqueous solution is sensitive to heat, so even if these substances are ingested orally, they are absorbed from the intestines and are absorbed into skin cells. It is unclear whether the effect can be exerted at the desired site. Therefore, if the NAMPT activator taken into the body by oral administration according to the present invention can be absorbed from the intestines and activate NAMPT in a desired site such as skin cells, NAMPT, NMN, NAD, and/or SIRT1/ Improvements in various functions can be expected by activating 2/3.

Such intestinal cell-mediated effects can be measured by various methods including in vivo, in vitro, ex vivo, and the like. For example, it can be determined by administering a test substance to intestinal cells and determining the activity of NAMPT in skin cells via the intestinal cells. For example, as in the Examples herein, a test substance is administered to a culture medium of intestinal cells such as Caco-2, and the supernatant is added to skin cells, or an in vitro method is used, or an oral method is used in animals such as humans. In vivo methods may also be used, such as measuring the amount of NAMPT in the skin after ingestion. Methods for evaluating interactions between intestinal epithelial cells and epidermal keratinocytes are known in the art or can be easily implemented by those skilled in the art. However, the measurement method is not limited to the above method, and any other method may be adopted. For example, a test substance is added to one side of a layer of intestinal cells such as Caco-2, and the activity of NAMPT is measured in skin cells such as HaCaT that pass through this layer and are present on the opposite side of the intestinal epithelial cells. -Can be determined by the epidermal keratinocyte interaction evaluation method.

The present invention is selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass. NAMPT activators containing one or more ingredients as active ingredients are provided. Furthermore, the present invention provides a method selected from the group consisting of grape seeds, milk thistle, ginseng, hemitanthus, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, and long life grass. Compositions containing as active ingredients one or more of the following ingredients are also provided. The NAMPT activator and/or composition of the present invention may be in the form of an oral or enteral preparation, and may be a pharmaceutical, quasi-drug, food composition, cosmetic composition, etc. It may be for.

The grape seeds used in the present invention are those of Vitis vinifera, a shrub belonging to the Ericaceae family and Vaccinium genus. Grape seeds contain proanthocyanidins. Grape seeds are known for their effects on venous insufficiency, promoting wound healing, and reducing inflammation.

The milk thistle (also known as milk thistle, Silybum marianum) used in the present invention is a biennial of the family Asteraceae, genus Milk Thistle. Whole plant or seeds can be used. Milk thistle contains silymarins such as silybin, silycristin, silydianin, and isosilybin. Milk thistle has been reported to promote protein synthesis, suppress substances that cause liver damage, and protect the liver against hepatotoxic substances.

The ginseng used in the present invention is the root of Panax ginseng (also known as Panax ginseng, C.A. Meyer), and contains saponin, sitosterol, panaxinol, and the like. It is known to prevent hypotension, anemia, arteriosclerosis, improve brain function, relieve stress, prevent infections, improve sensitivity to cold, and beautify the skin.

The Houttuynia cordata used in the present invention is a perennial plant (also known as Houttuynia cordata) belonging to the Houttuyniaceae family. Preferably, above-ground parts are used. It contains aliphatic aldehydes such as decanoyl acetaldehyde and flavonoids such as quercetin. The fresh leaves are known to have antibacterial and antibacterial effects on swellings, burns, suppuration, and other skin diseases, and the dried leaves are known to have diuretic and laxative effects.

The guava (Psidium guajava L.) used in the present invention is a tropical shrub. Preferably, the leaves are used. The polyphenols contained in the leaves are known to inhibit carbohydrate breakdown and blood sugar rise.

The pomegranate (Punica granatum) used in the present invention refers to a small deciduous tree belonging to the Lythraceae family, Pomegranate genus. Preferably, the pericarp is used. Pomegranate peel contains tannins, mannitol, inulin, pectin, isoquercitrin, etc., and is known to have effects on astringency, hemostasis, diarrhea, etc.

HMB-Ca (3-HydroxyIsovaleric Acid calcium salt, Calcium 3-Hydroxy-3-methylbutyrate Hydrate, CAS registration number 135236-72-5) used in the present invention is a derivative of 3-HydroxyIsovaleric Acid. It is a calcium salt and is known to promote muscle synthesis.

Cowberry (also known as lingonberry, Vaccinium vitis-idaea L.) used in the present invention is a small evergreen shrub belonging to the Ericaceae family, genus Vaccinium. Preferably, fruits are used. Cowberries contain organic acids, vitamin C, beta-carotene, B vitamins, as well as potassium, calcium, magnesium, and phosphorus. It is known for its effects on improving skin elasticity, whitening, preventing aging, suppressing inflammation, and improving blood flow.

The cinnamon used in the present invention is the bark of Cinnamomum cassia, a member of the Lauraceae family, and contains cinnamon aldehyde, cinnamic acid, eugenol, and the like. It is known to promote blood circulation in capillaries, warm the body, and have effects on stomach health and intestinal regulation.

The gardenia fruit used in the present invention refers to the fruit of Gardenia jasminoides, and contains crocin, crocetin, and the like. It is known to have anti-inflammatory effects, insomnia, and skin dryness.

The mulberry leaves used in the present invention are leaves of deciduous trees of the genus Morus, family Moraceae, such as Morus australis, Morus bombycis and Morus alba L.. Mulberry leaves contain deoxynojirimycin, pectin, fructose, glucose, pentozan, galactone, minerals, and chlorophyll. It is used to improve constipation, strengthen liver function, suppress fat, and prevent diabetes.

The longan (also known as longan) used in the present invention is the aril of the longan (Euphoria longana Lamarck), which belongs to the Sapindaceae family. It is also known as a herbal medicine called longan meat. Contains glucose, organic acids, vitamin A, vitamin B1, etc. It has been reported that longanchoensis has a nourishing and tonic effect, a sedative effect, and an effect on insomnia.

Ziziphus jujuba (Ziziphus jujuba Miller var. spinosa Hu ex H.F.Chow) used in the present invention is a small deciduous tree belonging to the family Buckthorn. Preference is given to using seeds. It is also known as a herbal medicine called sour jujube. It is known to have mental stabilizing effects, antiperspirant effects, insomnia, palpitations, anxiety reduction, and other effects.

NMN (Nicotinamide mononucleotide: CAS registration number 1094-61-7) used in the present invention is an intermediate metabolite in the biosynthesis of the coenzyme NAD. Many aspects of NMN's uptake and action mechanisms into the body are still unclear, and various studies are underway. etc. are known.

Peucedanum japonicum (Japanese name Peucedanum japonicum) used in the present invention is a plant belonging to the Apiaceae family, Peucedanum japonicum. Preferably, the leaves are used. Chomeiso contains polyphenols, vitamin A, vitamin B2, vitamin C, vitamin E, carotene, calcium, etc. It is also used to treat colds, asthma, kidney disease, and neuralgia, and it has also been reported to activate sirtuin 1, which is known as the longevity gene.

The NAMPT activator of the present invention contains grape seed, milk thistle, Korean ginseng, Hokutami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life as active ingredients. One or more components selected from the group consisting of grass, based on dry weight, for example, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass The content may be 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more. In some embodiments, the NAMPT activator of the present invention is grape seed, milk thistle, ginseng, hectrum, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, It may also consist of one or more ingredients selected from the group consisting of

The above-mentioned components are known substances, and in the case of compounds, they may be synthesized by known methods or commercially available products may be used. In the case of plant components, they can be easily squeezed, dried, purified, extracted, etc. by known methods, and commercially available products are easily available. It can be used raw or dried, but from the viewpoint of usability, formulation, etc., it can also be used as an extract, dried product, dry powder, raw material powder, squeezed liquid, etc. Depending on the raw material, which form to use can be selected as appropriate, and treatments such as sterilization may be performed as necessary.

When used as an extract, the extract can be extracted, for example, by solvent extraction. In the case of solvent extraction, the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are dried as necessary, further shredded or crushed as necessary, and then an aqueous extractant, water, e.g. cold water is added. , hot water, hot water at or below the boiling point, a water-containing organic solvent, an organic solvent such as ethanol, methanol, ether, 1,3-butylene glycol, etc., depending on the properties of the raw materials and the intended use of the composition. It is extracted by selectively using it at room temperature or by heating. However, the extraction method is not limited to solvent extraction, and may be any conventional method known in the art, and the extraction method and form of the extract used in the present invention may be changed as long as they do not impair the effects of the present invention. Optional. The form of the above-mentioned extract may be not only the extract itself, but also one diluted or concentrated as appropriate by a commonly used method, or a powder or lump solid obtained by drying the extract. Alternatively, the juice may be appropriately diluted or concentrated using a conventional method. The extract may be fermented or treated with an enzyme such as protease or pectinase, or may be powdered by adding dextrin, gum arabic, etc.

As an example of the water-containing organic solvent, a water-containing lower alcohol (for example, C1 to C4) such as water-containing ethanol, water-containing methanol, water-containing ether, and water-containing 1,3-butylene glycol may be used, and in that case, the water content is, for example, 0. ～10v/v%, 10～40v/v%, 20～30v/v%, 30～40v/v%, 30～50v/v%, 60～70v/v%, 50～80v/v%, 80～ It may be 99.5v/v% or the like.

Dry powder can be obtained by shredding or crushing the whole plant or various parts (leaves, flowers, roots, etc.) and then drying it, or by drying the plant and then shredding or crushing it to obtain dry powder. There is a way to get it. Alternatively, a method of shredding or pulverizing the plant, subjecting it to fermentation or treatment with an enzyme such as protease or pectinase, drying it, and further pulverizing it to a predetermined particle size as necessary can be adopted as appropriate. The dried product may be powdered by adding dextrin, gum arabic, etc.

Although the NAMPT activator of the present invention is preferably taken orally or enterally, other administration routes such as transdermal administration are not excluded. In addition, it has been reported that increasing NAMPT expression may be effective against diseases such as sarcopenia and diabetes (Non-patent Document 8, Patent Document 1, Masaki Igarashi et al. npj Aging volume 8, No. 5 (2022 )etc). Therefore, if the expression of NAMPT can be increased in a desired site such as muscle by a NAMPT activator ingested through various routes such as oral administration, it may contribute to the improvement of these diseases. When administering the NAMPT activator of the present invention by various administration routes, it is preferable to apply the active ingredient of the present invention in an amount such that the NAMPT activating effect is sufficiently exerted. The amount of the plant or its solvent extract can be appropriately determined depending on the type, purpose, form, method of use, etc.

For example, the NAMPT activator of the present invention can be included in a composition for oral or enteral ingestion, such as a food composition. The form of the NAMPT activator and composition of the present invention can be arbitrarily selected, for example, powder, liquid, solid such as tablets such as supplements, granules, granules, paste, gel, etc.

For example, when using the NAMPT activator of the present invention as an oral or enteral preparation or a food composition, the amount of grape seed, milk thistle, ginseng, or Heutomia spp. , guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longannik, Sanebuto jujube, NMN, and Chomeisou have a dry weight of about 0.00001 to 0.5% by weight, about 0.0001 to 0.05% by weight, or 0.001 It may be adjusted to approximately 0.005% by weight. Alternatively, the concentration in the intestines may be adjusted to about 0.1 to 100 μg/ml, about 0.5 to 50 μg/ml, or about 1 to 10 μg/ml.

In addition, when the NAMPT activator of the present invention is used as an oral or enteral preparation or a food composition, grape seeds, milk thistle, Korean ginseng, dokudami, guava, pomegranate, HMB-Ca, The intake amount of lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longannik, Sanebutonjujube, NMN, and Chomeiso plants or their solvent extracts is, for example, about 0.005 mg to about 0.5 g (dry weight equivalent) per day. , 0.05 mg to about 50 mg, or 0.5 mg to about 5 mg (dry weight equivalent). In addition, the frequency of intake is not limited, and may be taken once, but examples include once every two weeks, once a week, once every three days, once every two days, and once every day. It can be taken once, twice a day, three times a day, four times a day, etc. Moreover, it may be taken each time, continuously, or intermittently at intervals of several months, for example.

In the NAMPT activator and composition of the present invention, additives can be arbitrarily selected and used in combination as necessary. Excipients and the like can be included as additives. Any excipient may be used as long as it is commonly used when preparing the desired dosage form, such as starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin, and crystalline cellulose. , lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharide, emulsified oligosaccharide, and other sugars, and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients can be used alone or in combination of two or more.

Other colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. are known ones. can be selected and used as appropriate.

The present invention is selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass. A method of activating NAMPT in the skin by administering one or more ingredients, e.g. by oral or enteral route We also provide a method for The method of the present invention is a method for cosmetic purposes, and may not be a treatment by a doctor or medical professional.

Furthermore, the present invention provides for the use of grape seeds, milk thistle, ginseng, helotymus, in the manufacture of pharmaceuticals such as oral or enteral preparations for skin aging, whitening, life extension, antioxidant, damage repair, or anti-inflammation. Also provided is the use of one or more ingredients selected from the group consisting of guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, and long life grass. The invention relates to grape seeds for use in methods for skin aging, whitening, life extension, antioxidant, damage repair, or anti-inflammation by activation of NAMPT in the skin, e.g. by oral or enteral administration. one or more ingredients selected from the group consisting of , milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, and long life grass. Also provided.

Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to this.

Experiment 1: Preparation of sample Experiment 1-1: Preparation of candidate sample
Candidate samples for NAMPT activators include grape seeds, milk thistle, ginseng, dokudyami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan root, jujube, NMN, and long life grass. Prepared as shown in the table below.

A total of 31 types of candidate samples were prepared, including other naturally derived ingredients and compounds such as extracts from plants and animals. Solid samples were pulverized using a masher and dimethyl sulfoxide (Fuji Film Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as DMSO) was used as a solvent to prepare a concentration of 10 mg/mL, while liquid samples were used as they were. These samples were stored at -20°C and thawed appropriately before use.

Experiment 1-2: Preparation of control samples DMSO was used as a negative control in Experiment 2, and resveratrol (CAS registration number: 501-36-0), which is known to have a NAMPT activating effect, was added in DMSO as a positive control. The concentration was adjusted to 1 mg/mL. These samples were stored at -20°C and thawed appropriately before use.

Experiment 2: Examination of NAMPT activation effect Experiment 2-1: Culture of human colon cancer-derived cell line Caco-2 cells Human colon cancer-derived Caco-2 cells (ATCC: American Tissue Culture Collection) were incubated with inactivated 10% Fetal Bovine. Using Dulbecco's Modified Eagle Medium (DMEM) (Nissui, Tokyo, Japan) containing Serum (FBS, Life Technologies, CA, USA) (complement was inactivated by heating in a thermostat at 56°C for 30 minutes). The cells were then subcultured in a Petri dish at 37°C and 5% _CO2 . DMEM medium was prepared by dissolving 4.75 g of DMEM medium "Nissui" (Nissui Pharmaceutical, Tokyo, Japan) in 470 mL of Milli-Q water, sterilizing it by autoclaving, and adding 100 U/mL penicillin (Meiji Seika Pharma, Tokyo, Japan) 1 mL, 0.1 mg/mL streptomycin (Meiji Seika Pharma) 1 mL, 10% NaHCO ₃ (Fuji Film Wako Pure Chemical Industries) 6 mL, L-glutamine filter sterilized with a 0.22 μm filter (Toyo Roshi) The sample was prepared by adding the following to a final concentration of 4 mM.

Experiment 2-2: Culture of human epidermal keratinocytes HaCaT cells Human epidermal keratinocytes HaCaT cells (Riken Bioresource Center, Tsukuba, Japan) were cultured using the same medium, equipment, etc. as in Experiment 2-1, and under the same conditions at the same temperature. subcultured under.

Experiment 2-3: Measurement of NAMPT expression in skin cells via intestinal cells by promoter activity Experiment 2-3-1: Creation of plasmid (NAMPTp-EGFP) Gibson assembly of vector (hNAMPTp-EGFP) incorporating human NAMPT promoter It was made with Specifically, hNAMPTp/pGL4.10 incorporating the hNAMPT promoter was amplified by PCR reaction, and pEGFP was amplified by inverse PCR. Each gene was assembled using In-Fusion (registered trademark) HD (Takara) to construct hNAMPTp-EGFP. This hNAMPTp-pEGFP vector was integrated into human epidermal keratinocyte HaCaT cells (Cell Lines Services) according to the standard Gibson assembly method. Specifically, using In-Fusion (registered trademark) Snap Assembly Master Mix (Clontech Takara cellartis, manufactured by Takara Bio), an incubation reaction for 15 minutes at 50°C was carried out to (1) extract the 3'-over single strand with exonuclease; Create a hang and anneal it to the other complementary strand (overlapping region), (2) fill in the gaps between each annealed fragment with polymerase, and (3) join the nicks with DNA ligase to join the DNA together. Then, the vector was integrated into HaCat cells, and HaCaT cells containing the vector in which EGFP gene expression is controlled by the NAMPT promoter (HaCaT (NAMPTp-EGFP)) were prepared. These cells were used for the following evaluation of NAMPT expression. The HaCaT (NAMPTp-EGFP) cells created in this way were subcultured in the same manner and under the same conditions as in Experiments 2-1 and 2-2.

Experiment 2-3-2: NAMPT expression in skin cells via intestinal cells By adding the supernatant of Caco-2 cells to HaCaT (NAMPTp-EGFP) cells, the expression of NAMPT in skin cells via intestinal cells was NAMPT expression was examined. Caco-2 cells obtained from Experiment 2-1 were seeded in a 48-well plate (FALCON) at a final concentration of 3×10 ⁵ cells/well. After culturing for 24 hours at 37° C. in the presence of 5% CO ₂ , the sample prepared in Experiment 1 was added at a final concentration of 10 μM for resveratrol and 10 μg/mL for other components. Note that the same amount of DMSO was used as a control without sample. The sample was added and cultured for an additional 24 hours.

HaCaT (NAMPTp-EGFP) cells obtained in Experiment 2-3-1 were seeded in a 96-well black plate using the same medium as in Experiment 2-3 at a final concentration of 6.0 × 10 ⁵ cells/mL. Culture was carried out for 24 hours at 37°C in the presence of 5% _CO2 . Thereafter, the culture medium of HaCaT (NAMPTp-EGFP) cells was removed, and 100 μL/well of the above culture supernatant of Caco-2 cells was added, followed by further culturing for 48 hours.

Experiment 2-3-3: Measurement of promoter activity Measurement of NAMPT expression level The cell fixative solution was prepared by weighing and collecting 0.08 g/mL of paraformaldehyde (Fuji Film Wako Pure Chemical Industries), 1× PBS, and 2N NaOH. A solution of 8% paraformaldehyde was prepared by adding 5 μL/mL and stored at 4°C.
As a nuclear staining solution, Hoechst 33342 was diluted with 1×PBS to a concentration of 2 μg/mL and used in a light-shielded state. It was also prepared extemporaneously at the time of use.

After fixing the cells with the above cell fixative, nuclear staining was performed using the above nuclear staining solution, and the cell number and NAMPT promoter activity were examined using IN Cell Analyzer 2200. Specifically, after culturing the HaCaT (NAMPTp-EGFP) cells obtained in Experiment 2-4, 100 μL/well of cell fixative solution was added to the culture solution and incubated at room temperature for 15 minutes. The cell culture medium and cell fixative were removed together, and the cells were washed twice with 1× PBS, and then a nuclear staining solution was added at 100 μL/well. After standing at room temperature for 20 minutes, the nuclear staining solution was removed and the cells were washed twice with 1x PBS. PBS was added at 100 μL/well and using IN Cell Analyzer 2200, NAMPT promoter activity was determined by measuring EGFP fluorescence intensity, and cell number was determined by recognizing Hoechst33342 fluorescence. Images of the fluorescence intensity of HaCaT (NAMPTp-EGFP) were acquired using IN Cell Investigator High-content image analysis software (GE Healthcare), and the numerical information of the fluorescence intensity was analyzed. The case where H 2 O was added was set as 100, and for the liquid sample, the case where only H ₂ O was added was set as 100. Calculations were performed to determine the relative value of the fluorescence value. Statistical processing was performed using Student's t-test vs control group, and a significant difference was defined as p<0.05 (*p<0.05). Specimens with p<0.01 are indicated separately and marked as significant differences (**p<0.01).

Experiment 2-4: Measurement of NAMPT expression in skin cells via intestinal cells by RT-qPCR For some of the samples in which promoter activity was observed in Experiment 2-3, the supernatant of Caco-2 cells was collected using the following method. By adding it to HaCaT cells, NAMPT expression in skin cells via intestinal cells was investigated by RT-qPCR.
Caco-2 cells obtained in the same manner as in Experiment 2-1 were seeded in a 48-well plate at a final concentration of 3×10 ⁵ cells/well. After culturing for 24 hours at 37° C. in the presence of 5% CO ₂ , the sample prepared in Experiment 1 was added at a final concentration of 10 μM for resveratrol and 10 μg/mL for other components. As a negative control, DMSO was added to the intestinal epithelial cells at a concentration of 0.1% (v/v). The sample was added and cultured for an additional 24 hours.

After seeding the HaCaT cells obtained in Experiment 2-2 at a final concentration of 6.0 × 10 ⁵ cells/mL, the medium was removed, and 100 μL/well of the above Caco-2 cell culture supernatant was added, followed by further 24 Cultured for hours. After culturing, RNA was prepared and the expression of the NAMPT gene was verified by quantitative RT-PCR using the following primers.

The sequences of the primers used are as follows.
NAMPT forward primer: 5'-GGGTTACAAGTTGCTGCCACC-3' (SEQ ID NO: 1)
NAMPT reverse primer: 5'-GCAAACCTCCACCAGAACCG-3' (SEQ ID NO: 2)
ACTB forward primer: 5'-TGGCACCCAGCACAATGAA-3' (SEQ ID NO: 3)
ACTB reverse primer: 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (SEQ ID NO: 4)

The gene expression level of NAMPT was compared with the gene expression level of ACTB (β-actin) as an internal standard, and the gene expression level was calculated for each sample. A t-test was used to test whether each sample showed significant gene expression enhancement compared to the negative control. Each sample was tested with n=4. Relative values were calculated by setting the case where only DMSO was added as 100 and the average of the negative control results for other samples as 100. Statistical processing was performed using Student's t-test vs control group, and a significant difference was defined as p<0.05 (*p<0.05). Specimens with p<0.01 are indicated separately and marked as significant differences (**p<0.01).

result:
The results of Experiment 2-3 are shown in Figure 1. The results of Experiment 2-4 are shown in Figure 2. As shown in these figures, administration of the components of the present invention significantly increased the expression level of NAMPT compared to the negative control (DMSO only). This expression level is at a good level compared to the positive control (DMSO containing resveratrol), indicating that the component of the present invention has an excellent NAMPT activation effect. It is also suggested that this effect is obtained in skin cells via intestinal cells.

The present invention is selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon berry, gardenia fruit, mulberry leaf, longan daisy, Sanebut jujube, NMN, and long life grass. Ingestion of a NAMPT activator containing one or more ingredients as active ingredients, especially ingestion via the intestinal tract such as oral administration or enteral administration, activates NAMPT in skin cells via intestinal cells, resulting in skin improvement. It can improve skin conditions such as anti-aging, whitening, life extension, anti-oxidation, damage repair, and anti-inflammation.

Claims (5)

One or more selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, and long life grass A NAMPT activator containing the following substances as active ingredients. The NAMPT activator according to claim 1, wherein the NAMPT1 activator activates NAMPT in skin cells via intestinal cells. The NAMPT activator according to claim 1, wherein the NAMPT1 activator activates NAMPT in skin cells by internal administration. A food composition for beautiful skin, comprising the NAMPT activator according to any one of claims 1 to 3. One or more selected from the group consisting of grape seeds, milk thistle, ginseng, dokudami, guava, pomegranate, HMB-Ca, lingonberry, cinnamon bark, gardenia fruit, mulberry leaf, longan daisy, jujube, NMN, and long life grass A food composition for beautifying the skin, which contains the substance as a beautifying ingredient having the function of activating NAMPT when taken internally.

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