Cell activators

Abstract

The present invention aims to discover new functions of a composition comprising a combination of nicotinamide mononucleotide (NMN) and a food additive reducing agent derived from shells and natural zeolite, and to provide uses based on the new functions. [Solution] The cell activator contains nicotinamide mononucleotide (NMN) and a food additive reducing agent obtained by adding baked shell powder obtained by pulverizing baked shells and baked zeolite powder obtained by pulverizing baked natural zeolite to water, subjecting them to heat treatment in water, and drying them.

Description

[Technical Field]

[0001] The present invention relates to a cell activator containing nicotinamide mononucleotide (NMN) and a reducing agent derived from natural materials, which is prepared by calcining shells and natural zeolite and subjecting them to heat treatment in water. [Background technology]

[0002] The sirtuin gene, also known as the anti-aging gene or longevity gene, is known to be activated by the coenzyme nicotinamide adenine dinucleotide (NAD). However, it has been reported that when the amount of NAD in the body decreases due to aging or some other cause, the sirtuin gene becomes insufficiently activated, leading to aging. In recent years, nicotinamide mononucleotide (NMN) has attracted attention. When ingested, NMN is converted into the coenzyme NAD, which activates the sirtuin gene. Therefore, ingesting NMN can be expected to compensate for the decrease in coenzyme NAD due to aging, activate the sirtuin gene, and prevent aging.

[0003] In addition to the anti-aging effects mentioned above, research is also underway into new functions of compositions containing NMN. For example, Patent Document 1 discloses that a composition combining NMN and leucine has a muscle-building effect. Furthermore, Patent Document 2 discloses that a composition combining NMN and resveratrol has a blood uric acid-reducing effect.

[0004] On the other hand, a reducing agent derived from natural materials is known, which is obtained by heating and firing shells such as oyster shells and natural zeolite in an electric furnace under specified conditions, grinding them into a powder of 10 microns, and then adding both to water, heating them in the water, and drying them (Patent Document 3). This reducing agent is manufactured and sold as a food ingredient by Wedge Co., Ltd. under the product name "Life Ceramics Powder." [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2022-146639 [Patent Document 2] Patent No. 6545256 [Patent Document 3] Patent No. 4465687 Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need to discover new functions of NMN that contribute to everyday health and beauty by combining NMN, which has anti-aging properties, with some other material, as disclosed in Patent Documents 1 and 2. It is expected that these new functions will be used to develop functional foods and pharmaceutical compositions.

[0007] Therefore, the present invention has been made in consideration of the above points, and its purpose is to discover new functions for a composition comprising a combination of NMN and a food additive reducing agent derived from shells and natural zeolite, and to provide uses based on these new functions. [Means for solving the problem]

[0008] The present inventors conducted various assays and extensive research on compositions combining NMN with food additive reducing agents derived from shellfish and natural zeolite. As a result, the present inventors discovered that compositions combining NMN with food additive reducing agents have specific functions not found in either material alone. Based on this finding, the present invention was completed.

[0009] To solve the above problems, the cell activator of the present invention contains nicotinamide mononucleotide (NMN) and a food additive reducing agent obtained by adding a calcined shell powder obtained by pulverizing calcined shells and a calcined zeolite powder obtained by pulverizing calcined natural zeolite to water, heating the resulting mixture in water, and drying the mixture. The cell activator can activate cells and activate their functions by blending NMN and the food additive reducing agent into a composition.

[0010] Furthermore, the cell activator of the present invention preferably activates hepatocytes or fibroblasts. This allows suitable cells to be selected as the cells to be activated by the composition of the present invention, which contains NMN and a food additive reducing agent derived from shells and natural zeolite. The composition of the present invention can activate both hepatocytes and fibroblasts, thereby activating their functions.

[0011] Furthermore, in the cell activator of the present invention, the blending ratio of nicotinamide mononucleotide to the food additive reducing agent (mass of nicotinamide mononucleotide:mass of food additive reducing agent) is preferably 10:90 to 40:60. This allows for the selection of a suitable blending ratio of components that provides excellent cell activation effects in the composition of the present invention containing NMN and the food additive reducing agent.

[0012] Furthermore, the composition of the present invention for improving liver function contains the above-mentioned cell activator as an active ingredient. The composition of the present invention can activate liver cells, thereby activating their function and improving liver function.

[0013] Furthermore, the skin aging inhibitor of the present invention contains the above-mentioned cell activator as an active ingredient. The composition of the present invention activates fibroblasts, thereby activating their function and enabling the inhibition and improvement of skin aging. [Effects of the Invention]

[0014] According to the present invention, a cell activator having the following excellent effects can be provided. (1) It can activate hepatocytes and fibroblasts, thereby activating the functions of the cells. (2) It is possible to enjoy the other functions that NMN and food additive reducing agents each possess. (3) Both compounds are highly safe for the human body, so they can be taken orally and used safely as functional foods. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the cell-activating effect of the composition of the present invention on hepatocytes in Example 1. Figure 1(a) shows the test results when the composition of the present invention (Example 1) and only NMN (Comparative Example 1a) were added to hepatocytes, and Figure 1(b) shows the test results when the composition of the present invention (Example 1) and only a reducing agent (Comparative Example 1b) were added to hepatocytes. [Figure 2] 2A and 2B are graphs showing the cell-activating effect of the composition of the present invention on fibroblasts in Example 2. Fig. 2(a) shows the test results when the composition of the present invention (Example 2) and only NMN (Comparative Example 2a) were added to fibroblasts, respectively, and Fig. 2(b) shows the test results when the composition of the present invention (Example 2) and only a reducing agent (Comparative Example 2b) were added to fibroblasts, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] The cell activator of the present invention is described in detail below. The cell activator of the present invention contains nicotinamide mononucleotide (NMN) and a reducing agent for food additives derived from natural materials, which is prepared by calcining and heating in water shells and natural zeolite, as described below.

[0017] In the present invention, cell activation refers to an increase in the metabolic activity of cells, activating the function of cells, and means that the metabolic activity of cells is improved and the function of cells is activated compared to a control to which the cell activator of the present invention is not added or administered. Specifically, for example, in an MTT assay, this means that the amount of formazan produced from MTT is increased compared to a control to which the cell activator of the present invention is not added or administered, i.e., the metabolic activity of cells that reduces MTT to formazan is increased.

[0018] The cells activated by the composition of the present invention are not particularly limited, but the effect on hepatocytes and fibroblasts is remarkable. Furthermore, skin fibroblasts are preferred as fibroblasts. Although the mechanism by which the composition of the present invention activates these cells has not been elucidated, a composition combining NMN with a food additive reducing agent derived from shellfish and natural zeolite exhibits excellent cell activation effects.

[0019] Furthermore, the cell activating agent according to the present invention can be used as a liver function improver that activates liver cells, thereby activating the metabolism of liver cells and improving liver function.Furthermore, the cell activating agent according to the present invention can be used as a skin aging inhibitor that activates skin fibroblasts, thereby improving the metabolic activity of skin fibroblasts and suppressing intracellular oxidation reactions, thereby inhibiting or improving skin aging.

[0020] (nicotinamide mononucleotide) The composition of the present invention contains nicotinamide mononucleotide (NMN). NMN exists as α- and β-type optical isomers, but in the present invention, β-NMN, the β-type, is preferably used. In the present invention, this NMN, when combined with a food additive reducing agent described below, improves the metabolic activity of hepatocytes and skin fibroblasts, activating these cells. Furthermore, as described above, when NMN is taken into the body, it is converted into the coenzyme NAD, which functions to activate the sirtuin gene. Therefore, ingesting NMN can be expected to compensate for the decrease in coenzyme NAD due to aging, activate the sirtuin gene, and prevent aging.

[0021] (Food additive reducing agent) The composition of the present invention contains a food additive reducing agent obtained by adding calcined shell powder, which is obtained by pulverizing calcined shells, and calcined zeolite powder, which is obtained by pulverizing calcined natural zeolite, to water, heat-treating the mixture in water, and then drying the mixture. In the present invention, the combination of the above-mentioned NMN with the food additive reducing agent improves the metabolic activity of hepatocytes and skin fibroblasts, thereby activating these cells. A suitable food additive reducing agent is Life Ceramics Powder (a product of Wedge Corporation), which is sold as a health food ingredient.

[0022] This food additive reducing agent can be produced, for example, by the method disclosed in Patent Document 3 (Japanese Patent No. 4465687), although it is not particularly limited thereto. For example, shells such as oyster shells are placed in an electric furnace and fired for 8 to 20 hours while the temperature is gradually increased within a range of 500°C to 850°C. After firing, the fired shells are pulverized into powder to obtain fired shell powder. Meanwhile, natural zeolite is also fired in an electric furnace for 5 to 10 hours while the temperature is gradually increased within a range of 200°C to 800°C. After firing, the fired natural zeolite is pulverized into powder to obtain fired zeolite powder. A mixture of 85 to 98% fired shell powder and 2 to 15% fired zeolite powder is then added to water, heated to 90°C, and ion-exchanged in water for 5 to 48 hours while maintaining this equilibrium temperature. Thereafter, the mixture is dried to obtain the food additive reducing agent.

[0023] More specifically, shells such as oyster shells are placed in an electric furnace and calcined for 3 hours at an initial temperature of 550°C, followed by 5 hours at 700°C, and then 8 hours at 840°C. After calcination, the calcined shells are ground to a 10-micron powder to obtain calcined shell powder. Natural zeolite is also calcined in an electric furnace for 2 hours at an initial temperature of 250°C, followed by 5 hours at 750°C. After calcination, the calcined natural zeolite is ground to a 10-micron powder to obtain calcined zeolite powder. A mixture of 90% calcined shell powder and 10% calcined zeolite powder is then added to water, heated to 90°C, and ion-exchanged in water for 8 hours while maintaining this equilibrium temperature. The mixture is then dried to a water content of 3% and calcined in an electric furnace at 840°C for 5 hours to obtain the food additive reducing agent. The calcination time and temperature of the shells and natural zeolite, the mixing ratio of the calcined shell powder and the calcined zeolite, and the heating temperature and reaction time in water can be changed as appropriate depending on the type and condition of the shells and natural zeolite selected as materials.

[0024] In the composition of the present invention, the blending ratio of NMN to the food additive reducing agent (mass of NMN:mass of food additive reducing agent) is preferably 10:90 to 40:60, more preferably 15:85 to 30:70, and even more preferably 20:80 to 30:70, from the viewpoint of exerting a cell activation effect. This activates and stimulates hepatocytes and skin fibroblasts.

[0025] From the viewpoint of action and effect, the blending amount of NMN in the composition of the present invention is preferably 5 to 40% by mass of the total composition, more preferably 10 to 30% by mass, and particularly preferably 15 to 30% by mass.Furthermore, from the viewpoint of action and effect, the blending amount of the food additive reducing agent in the composition of the present invention is preferably 50 to 85% by mass of the total composition, more preferably 55 to 80% by mass, and particularly preferably 55 to 70% by mass.

[0026] The cell activating composition according to the present invention can be produced by a known method, specifically by mixing and dispersing the above-mentioned ingredients.

[0027] The composition of the present invention can be formulated into various forms using conventional methods by appropriately blending ingredients such as NMN and the food additive reducing agent, as well as ingredients commonly used in the manufacture of foods such as supplements and pharmaceuticals, such as excipients, lubricants, dispersants, disintegrants, buffers, bulking agents, preservatives, flavorings, or stabilizers. Furthermore, to improve the bioavailability and stability of the active ingredients in the composition of the present invention, drug delivery systems can be used, including formulation techniques such as microencapsulation, ultrafine powdering, and inclusion with cyclodextrin.

[0028] Furthermore, the cell activator of the present invention can be in any form, such as supplements such as tablets, capsules (hard capsules and soft capsules), granules, suspensions, and syrups; beverages such as soft drinks, fruit juices, and alcoholic beverages; confectioneries such as candy, gum, cookies, biscuits, and chocolate; bread, porridge, cereal, noodles, jellies, soups, dairy products, and seasonings. The cell activator composition of the present invention can also be combined with various other functional ingredients, amino acids, vitamins, minerals, oligosaccharides, prebiotics, or other probiotics, as long as the efficacy of the active ingredient of the present invention is not affected. The composition of the present invention is highly safe and is suitable for use as a food composition, which includes supplements, health foods, functional foods, and foods for specified health uses.

[0029] Furthermore, the daily intake of the cell activator according to the present invention will vary depending on the desired effect, age, sex, body weight, etc., but as an example, it is preferable to have 20 mg to 500 mg of NMN and 100 mg to 6 g of the food additive reducing agent, more preferably 30 mg to 300 mg of NMN and 100 mg to 3 g of the food additive reducing agent, and even more preferably 50 mg to 250 mg of NMN and 200 mg to 2 g of the food additive reducing agent.

[0030] The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to these examples. [Example]

[0031] [Example 1] 1. Examination of the cell activation effect of hepatocytes In this example, HepG2 cells, a human hepatocyte cell line, were used to examine the cell-activating effect of the composition according to the present invention, which contains NMN and a food additive reducing agent derived from shells and natural zeolite, on hepatocytes.

[0032] [Preparation of sample solution] β-Nicotinamide mononucleotide (β-NMN, product of Oriental Yeast Co., Ltd.) was mixed with a food additive reducing agent (product name: Life Ceramics Powder, product of Wedge Co., Ltd.) obtained by adding calcined seashells and calcined natural zeolite to water, heating the mixture in water, drying, and then pulverizing the mixture at a ratio of β-NMN:food additive reducing agent = 23:77. This mixture was diluted with DMEM medium to prepare sample solutions of different concentrations, as shown in Table 1 below. For comparison, sample solutions were prepared by diluting only β-NMN with DMEM medium and only the food additive reducing agent with DMEM medium, as shown in Table 1 below.

[0033] [Table 1]

[0034] The cell-activating effect of the composition of the present invention on hepatocytes was confirmed by an MTT assay, which can measure the metabolic activity of cells. The metabolic activity of cells can be measured by adding MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) to the target cells and measuring the amount of formazan, a reduction product, by absorbance. MTT was a product of Dojindo Laboratories, Inc. In this test, HepG2 cells, a human liver tumor cell line, were used as live cells. HepG2 cells were cultured in DMEM medium at 37°C in the presence of 5% CO2 and under 100% saturated water vapor conditions.

[0035] First, HepG2 cells were plated in each well of a 96-well microplate at 10 3Cells were seeded at 100 μL / well and cultured for 24 hours in a CO2 incubator. After culture, the supernatant was removed from each well and the cells were divided into Example 1, Comparative Example 1a, and Comparative Example 1b groups. 100 μL of the sample solution corresponding to each group shown in Table 1 was added to each well. After 24 hours of culture in a CO2 incubator, the supernatant was removed and the medium was replaced with fresh DMEM medium (100 μL / well) that did not contain the sample solution. 10 μL of 0.5 mg / mL MTT was then added to each well. After 4 hours of culture, the supernatant was removed, and 100 μL of solubilization solution (0.1 M HCl containing 10% SDS) was added to each well. The cells were allowed to stand overnight to solubilize the formazan crystals that had formed. The next day, the absorbance of the formazan solution at 560 nm was measured using a microplate reader (Tecan Corporation), and the relative value was calculated as the absorbance ratio (% of control) when the absorbance of the formazan solution in the solvent control test plots (test plots No. 1, No. 1a, and No. 1b) containing only DMEM medium was set at 100. The results are shown in Tables 2, 3, and Figure 1. Table 2 and Figure 1(a) show the absorbance ratio (% of control) based on the concentration of NMN added, and Table 3 and Figure 1(b) show the absorbance ratio (% of control) based on the concentration of a food additive reducing agent added.

[0036] [Table 2]

[0037] [Table 3]

[0038] The results in Figure 1 and Tables 2 and 3 show that the absorbance ratio (%), i.e., the amount of formazan produced by hepatocyte metabolism, did not change significantly when NMN alone (Comparative Example 1a) or the food additive reducing agent alone (Comparative Example 1b) was used. In contrast, the addition of the composition of the present invention, which combines NMN and the food additive reducing agent, resulted in a concentration-dependent increase in the amount of formazan, demonstrating improved metabolic activity and hepatocyte activation. For example, with the composition prepared in Example 1, the absorbance ratio reached 125% at an NMN concentration of 0.73 mg / mL and a food additive reducing agent concentration of 2.44 mg / mL, and reached 255% at an NMN concentration of 6.57 mg / mL and a food additive reducing agent concentration of 22 mg / mL. Furthermore, no change in hepatocyte cell count was observed in any test section, including the solvent control, and no tendency for an increase in hepatocyte count was observed with the addition of the composition of the present invention. In this way, it has become clear that the combination of NMN with a food additive reducing agent produces an effect of improving and activating the metabolic activity of liver cells that is not seen with either component alone.

[0039] [Example 2] 2. Examination of fibroblast cell activation In this example, normal human skin fibroblasts were used to examine the cell-activating effect on hepatocytes of the composition according to the present invention, which contains NMN and a food additive reducing agent derived from shells and natural zeolite.

[0040] Sample solutions were prepared in the same manner as in Example 1 above, with a sample solution containing only NMN designated Comparative Example 2a, and a sample solution containing only the food additive reducing agent designated Comparative Example 2b. The test to confirm the cell activation effect on fibroblasts was conducted in the same manner as in Example 1 above, except that the cell line used was changed from HepG2 cells to normal human dermal fibroblasts. The absorbance ratio (% of control) was calculated relative to the absorbance of the solvent control test plots containing only DMEM medium (test plots No. 2, No. 2a, and No. 2b), which were designated 100. The results are shown in Tables 4, 5, and Figure 2. Table 4 and Figure 2(a) show the absorbance ratio (% of control) based on the concentration of NMN added, while Table 5 and Figure 2(b) show the absorbance ratio (% of control) based on the concentration of the food additive reducing agent added.

[0041] [Table 4]

[0042] [Table 5]

[0043] The results in Figure 2 and Tables 4 and 5 show that no significant changes were observed with NMN alone (Comparative Example 2a) or the food additive reducing agent alone (Comparative Example 2b). The addition of NMM alone slightly increased the amount of formazan produced by fibroblast metabolism, but the test group in which the absorbance ratio (%) increased was at a high NMM concentration of 19.7 mg / mL. In contrast, the addition of the composition of the present invention, which combines NMN and a food additive reducing agent, significantly increased the amount of formazan in a concentration-dependent manner, improving the metabolic activity of fibroblasts and activating them. For example, with the composition prepared in Example 2, the absorbance ratio significantly increased to 148% at an NMN concentration of 0.73 mg / mL and a food additive reducing agent concentration of 2.44 mg / mL. At an NMN concentration of 6.57 mg / mL and a food additive reducing agent concentration of 22 mg / mL, the absorbance ratio reached 248%. Furthermore, no change was observed in the number of fibroblast cells in each test section, including the solvent control, and no tendency for the number of fibroblasts to increase with the addition of the composition of the present invention was observed. Thus, it was revealed that the combination of NMN and a food additive reducing agent produces an effect of improving and activating the metabolic activity of fibroblasts that is not seen with either component alone.

[0044] The present invention is not limited to the above-described embodiments or examples, and various modified design forms are also included in the technical scope within the scope that does not deviate from the gist of the invention described in the claims. [Industrial Applicability]

[0045] The cell activator of the present invention can activate cells by improving the metabolic activity of hepatocytes and fibroblasts, and can be effectively used in industries such as functional foods and pharmaceuticals.

Claims

1. Nicotinamide mononucleotide (NMN), a food additive reducing agent obtained by adding a calcined shell powder obtained by pulverizing calcined shells and a calcined zeolite powder obtained by pulverizing calcined natural zeolite to water, subjecting them to a heat treatment in water, and drying them; A cell activator comprising:

2. The cell activator according to claim 1, wherein the cells are hepatocytes or fibroblasts.

3. The cell activator according to claim 1, characterized in that the blending ratio of the nicotinamide mononucleotide to the food additive reducing agent (mass of nicotinamide mononucleotide:mass of food additive reducing agent) is 10:90 to 40:

60.

4. A liver function improver comprising the cell activator according to any one of claims 1 to 3 as an active ingredient.

5. A skin aging inhibitor comprising the cell activator according to any one of claims 1 to 3 as an active ingredient.

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