Composition for inhibiting apoptosis of natural killer cells, food and beverage, and method for inhibiting apoptosis of natural killer cells

Abstract

This invention provides a composition containing a naturally derived active ingredient that can suppress apoptosis of NK cells. [Solution] This is a composition for inhibiting apoptosis of natural killer cells, in which tea catechins are used as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a composition that can be used to suppress apoptosis of natural killer cells, a food or drink containing the composition, and a method for suppressing apoptosis of natural killer cells.

Background Art

[0002] Natural killer cells (also referred to as "NK cells") are a type of lymphocyte that are distributed in the bone marrow, spleen, peripheral lymph nodes, and peripheral blood in the body, accounting for about 10% of peripheral blood lymphocytes, and are known to play an important role in the innate immune response. Immunity includes "innate immunity" that humans are originally equipped with and "acquired immunity" in which antibodies are produced by fighting pathogens. Innate immunity is a function of attacking and eliminating foreign enemies such as viruses and bacteria, and it is known that immune cells such as NK cells are involved in this.

[0003] NK cells circulate in the body, find cells infected with viruses or cancerous cells, recognize those cells as foreign substances, express substances that promote apoptosis (cell death) of those cells, and have the function of killing the target cells. It is said that even in healthy people, thousands of cells become cancerous every day, and NK cells are involved in their removal.

[0004] When the activity of NK cells decreases, the ability to attack foreign substances that have invaded the body from the outside and cancer may decrease. It has been suggested that tea catechins have an activating effect on NK cells that are responsible for innate immunity. For example, Patent Document 1 suggests that epigallocatechin gallate has an effect of enhancing the activity of NK cells. Patent Document 2 suggests that conjugated catechins such as EGCG, ECG, and epigallocatechin have an effect of enhancing the function of natural killer (NK) cells.

Prior Art Documents

[0005] [Patent Document 1] Special Publication No. 2016-525124 [Patent Document 2] Special Publication No. 2023-533971 [Overview of the project] [Problems that the invention aims to solve]

[0006] NK cell activity refers to the cytotoxic activity of individual NK cells. Even if individual NK cells are highly active, if the total number of NK cells in the body decreases, the overall activity in the body will also decrease. Therefore, it is important not only to increase the activity of individual NK cells but also to increase the number of NK cells. To achieve this, it is effective to suppress apoptosis (programmed cell death) in individual NK cells and extend their lifespan. As mentioned above, while naturally derived active ingredients that can enhance NK cell activity were known, no naturally derived active ingredients that can suppress apoptosis (cell death) of NK cells were known.

[0007] Incidentally, NK cells recognize cells that express MHC class I antigens as self and cells that do not express them as non-self. Cancer cells and virus-infected cells often have reduced expression of MHC class I, and NK cells are activated and attack these cells upon recognizing their presence. On the other hand, in response to apoptosis induction stimuli, a decrease in mitochondrial membrane potential occurs, leading to the release of the apoptosis-inducing factor AIF, which then induces apoptosis via the activation of caspase-3 or by itself. Separately from this pathway, another known pathway involves the release of cytochrome C from mitochondria, independent of changes in membrane potential, which then induces apoptosis via the activation of caspase-3. Bcl-2 may suppress apoptosis by inhibiting the former decrease in membrane potential and the latter release of cytochrome C. Therefore, an increase in Bcl-2 expression is thought to contribute to the aforementioned sites of action and suppress apoptosis. As described above, the mechanisms that activate NK cells and the mechanisms that suppress apoptosis (cell death) of NK cells are completely different.

[0008] Therefore, the object of the present invention is to provide a composition containing an active ingredient derived from a natural product that can suppress apoptosis of NK cells, a food or beverage containing the composition, and a method for suppressing apoptosis of NK cells using the active ingredient of the composition. [Means for solving the problem]

[0009] [1] A first aspect of the present invention is a composition for inhibiting apoptosis of natural killer cells, comprising tea catechin as an active ingredient.

[0010] [2] A second aspect of the present invention is a composition for inhibiting apoptosis of natural killer cells, characterized in that, in the first aspect, the inhibition of apoptosis of natural killer cells manifests as an increase in the expression level of the Bcl2 gene.

[0011] [3] A third aspect of the present invention is a composition for inhibiting apoptosis of natural killer cells, characterized in that, in the first or second aspect, the intake concentration of the active ingredient is 0.2 mg / 1 mL or more as the total amount of tea catechins. [4] A fourth aspect of the present invention is a composition for inhibiting apoptosis of natural killer cells, characterized in that the intake period is 3 days or more in any one of the first to third aspects. [5] A fifth aspect of the present invention is a composition for inhibiting apoptosis of natural killer cells, characterized in that, in any one of the first to fourth aspects, the intake of the active ingredient is 3.5 mg / day or more and is taken continuously for 3 days or more.

[0012] [6] A sixth aspect of the present invention is a food or beverage comprising a composition for inhibiting apoptosis of natural killer cells according to any one of the first to fifth aspects. [7] A seventh aspect of the present invention is a food or beverage, characterized in that the food or beverage is a packaged beverage, in the sixth aspect.

[0013] [8] An eighth aspect of the present invention is a method for inhibiting apoptosis of natural killer cells, characterized by ingesting tea catechins. [Effects of the Invention]

[0014] The composition for inhibiting apoptosis of natural killer cells proposed in this invention contains tea catechins derived from natural products as an active ingredient. By ingesting it, it can increase the expression level of the Bcl2 gene, thereby suppressing apoptosis, extending the lifespan of NK cells, and increasing the number of viable NK cells. [Brief explanation of the drawing]

[0015] [Figure 1] The graph shows the number of NK cells in splenocytes under activation conditions for each group (control, EGCG, and GCG) as a result of Experiment 1. n=3 (biological replication), *:ρ<0.05 (Dunnet method), *Defined as CD3-NK1.1+CD49b+ cell population. [Figure 2]As a result of Experiment 2, it is a graph showing the number of NK cells in splenocytes under inactivation conditions for each of the control intake group, EGCG intake group, and GCG intake group. n = 3 (biological replicates), *: ρ < 0.05 (Dunnett's method) [Figure 3] As a result of Experiment 3, it is a graph showing the relative expression levels of the Bcl2 gene in splenic NK cells for each of the control intake group, EGCG intake group, and GCG intake group. n = 4 (technical replicates), mean ± standard deviation, significant differences between different letters (ρ < 0.05 (Games-Howell) [Figure 4] As a result of Experiment 4, it is a graph showing the relative expression levels of the Bcl2 gene in splenic NK cells under inactivation conditions for each of the control intake group, EGCG intake group, and GCG intake group. n = 4 (technical replicates), mean ± standard deviation, significant differences between different letters (ρ < 0.05 (Games-Howell)

Mode for Carrying Out the Invention

[0016] Next, the present invention will be described based on the embodiment examples. However, the present invention is not limited to the embodiments described below.

[0017] <Composition for Suppressing NK Cell Apoptosis of the Present Invention> A composition for suppressing apoptosis of natural killer cells (referred to as "composition for suppressing NK cell apoptosis of the present invention") according to an example of an embodiment of the present invention is a composition containing tea catechin as an active ingredient.

[0018] In the present invention, "containing tea catechin as an active ingredient" includes the meaning that other components other than the active ingredient, that is, components other than tea catechin, may be included as long as the action and effect of the active ingredient are not inhibited.

[0019] [[ID=*]]The composition for suppressing NK cell apoptosis of the present invention can be used to suppress apoptosis (cell death) of NK cells. Here, the suppression of NK cell apoptosis manifests as an increase in the expression level of the Bcl2 gene. Therefore, if the expression level of the Bcl2 gene can be increased, it can be evaluated that the suppression of NK cell apoptosis has been achieved.

[0020] (Tea catechins) The active ingredient of the NK cell apoptosis inhibitory composition of the present invention, tea catechin, encompasses catechins extracted from tea and catechins obtained by thermal isomerization thereof. Specifically, examples include one selected from the group consisting of catechin (C), catechin gallate (CG), gallocatechin (GC), gallocatechin gallate (GCG), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), a copolymer consisting of a combination of two or more of these, or a mixture consisting of a combination of two or more of these. In particular, those containing one or two selected from GCG and EGCG as active ingredients are preferred. Furthermore, the total amount of tea catechins (also referred to as "total tea catechin amount") refers to the sum of the tea catechin content listed above.

[0021] Among the catechins mentioned above, EC, EGC, ECG, and EGCG are found in abundance in natural products, especially tea leaves, and can therefore be obtained by extraction and purification using conventionally known methods or methods that may become publicly known in the future. On the other hand, these epimers, C, GC, CG, and GCG, are hardly present in natural plants, including tea leaves. However, they can be obtained by thermal isomerization (epimerization) of each of EC, EGC, ECG, and EGCG, or a mixture of two or more of these, through heat treatment. For example, a composition containing each of EC, EGC, ECG, and EGCG, or a mixture of two or more of these, such as a tea extract or infusion, can be adjusted to a pH of 5-6, and then heat-treated at 80°C or higher to promote thermal isomerization of catechins and increase the concentration of C, GC, CG, and GCG. By separating and purifying the product after such heat treatment, a mixture of each of C, GC, CG, and GCG, or a mixture of two or more of these, can be obtained.

[0022] For the heat treatment to cause thermal isomerization (epimerization) of catechin, it is preferable to heat the catechin-containing solution (catechin solution) to at least 80°C or higher. For example, thermal isomerization of catechin has been observed in all of the following cases: heating at 100°C for 15 minutes, 115°C for 20 minutes, 120°C for 3 to 30 minutes, 123°C for 10 minutes, 131°C for 30 seconds, and 133°C for 45 seconds. It is preferable to adjust the catechin solution to pH 5-6 before heat treatment because it has been reported that catechin hardly undergoes thermal isomerization at pH 4.5 or below (Shinichi Suematsu et al., Journal of the Japan Society for Food Science and Technology, 39, 178 (1992)).

[0023] Tea catechins are polyphenols found in large quantities in plants such as the tea plant, and are known to be present in green tea leaves at a concentration of about 8-20% by mass. The components contained in green tea leaves vary depending on the variety of tea plant, cultivation method and environment, the timing of tea leaf harvesting (first flush, second flush, etc.) and part of the plant (buds, leaves, stems), and processing conditions (steaming, rolling, drying, etc.). For example, in shaded teas such as gyokuro and tencha, the proportion of epigallocatechin gallate (EGCG) in the total amount of tea catechins is higher. From the standpoint of efficiency, it is preferable to extract active ingredients from tea leaves with an increased content of the desired tea catechins. Tea catechins can be isolated using known methods. Synthetic tea catechins may also be used.

[0024] Tea catechins may be derived from one or more types of tea selected from the group consisting of, for example, raw tea leaves, unfermented tea, semi-fermented tea, fermented tea, and post-fermented tea. In addition, catechins obtained by extracting any one of these individually, or by extracting any two or more of these, or a mixture of those obtained by extracting each of them may be used. Tea catechins can also be obtained by separating and purifying tea extracts in a way that increases their catechin content, using purification methods such as solvent extraction, resin adsorption, and filtration methods such as ultrafiltration and reverse osmosis filtration. In this case, the separation and purification of tea catechins can be carried out by conventionally known methods or methods that may become publicly known in the future. For example, it is known that tea extracts can be separated by subjecting them to reverse-phase HPLC with a mixture of water, acetonitrile, and phosphate as the mobile phase, and then applying a gradient of acetonitrile concentrations.

[0025] In addition, commercially available tea polyphenol preparations can be used as tea catechins. For example, Theafran 30A (manufactured by Ito En Co., Ltd.) is a tea polyphenol preparation obtained by hot water extraction of green tea and drying the extract to achieve a tea catechin concentration of approximately 30%, while Theafran 90S (manufactured by Ito En Co., Ltd.) is a tea polyphenol preparation obtained by hot water extraction of green tea, separating the extract using an adsorption column with water and low- and high-concentration alcohol, drying it, and achieving a tea polyphenol concentration of approximately 85 to 99.5%. Other commercially available tea polyphenol preparations include "Polyphenon" manufactured by Mitsui Norin Co., Ltd., "Sunphenon" manufactured by Taiyo Kagaku Co., Ltd., and "Sun Oolong" manufactured by Suntory Holdings Limited. Theafran 90S is preferred because it contains 90% or more by mass of tea polyphenols.

[0026] (EGCG or / and GCG) The active ingredient of the NK cell apoptosis inhibitory composition of the present invention is more preferably one or two selected from epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG) among the above-mentioned tea catechins.

[0027] EGCG is the most abundant catechin found in green tea, and is an ester of epigallocatechin and gallic acid. Furthermore, EGCG may be a pharmaceutically acceptable salt of EGCG. Examples include salts of EGCG with alkali metals such as sodium and potassium, alkaline earth metals such as calcium, and quaternary ammonium salts such as ammonium salts of EGCG. Furthermore, EGCG may be a prodrug that releases EGCG in vivo.

[0028] On the other hand, gallocatechin gallate (GCG) is a low-concentration catechin found in green tea, accounting for only 0.8% to 1.5% of the total catechin content, as it is a second-carbon isomer of epigallocatechin gallate. As mentioned earlier, GCG can be obtained by thermal isomerization (epimerization) of EGCG.

[0029] The EGCG and GCG mentioned above can be obtained by isolating and purifying them from, for example, the leaves of green tea, oolong tea, black tea, etc., or from extracts of these tea leaves. For information on purification methods, please refer to the Journal of the Science of Food and Agriculture, 22, 480-484, (1971). Alternatively, commercially available purified products can also be used.

[0030] <Composition for inhibiting NK cell apoptosis according to the present invention> In the present invention's NK cell apoptosis inhibitory composition, the content of one or two types of tea catechins, particularly EGCG and GCG, is preferably 10 to 100 parts by mass per 100 parts by mass of solid content of the present invention's NK cell apoptosis inhibitory composition, and more preferably 30 parts by mass or more or 90 parts by mass or less, of which 40 parts by mass or more or 50 parts by mass or less, and of which 70 parts by mass or more or 80 parts by mass or less.

[0031] The tea catechin in the NK cell apoptosis inhibitory composition of the present invention may be a pure product of C, CG, GCGCG, EC, ECG, EGC, or EGCG, or a mixture of two or more of these, or a composition containing tea catechin.

[0032] Examples of compositions containing the aforementioned tea catechins include green tea extracts and their purified products. A specific example is a green tea extract obtained by hot water extraction of green tea, separating the extract using an adsorption column with water and low- and high-concentration alcohol, drying it, and adjusting the tea polyphenol concentration to approximately 85-99.5% by mass. For example, "Theafran 90S (product name; manufactured by Ito En Co., Ltd.)" is a preferred example. Theafran 90S contains 60% by mass or more of tea catechins in total, with ester-type catechins accounting for 50-90% by mass of the total amount of tea catechins, EGCG accounting for 40-90% by mass of the total amount of catechins, and caffeine content accounting for 0-2% by mass of the total amount of tea catechins.

[0033] <Form> The NK cell apoptosis inhibitory composition of the present invention can be provided, for example, as an orally administered pharmaceutical, quasi-drug, health food / health beverage / food for specified health uses / functional food with pharmacological effects, nutritional supplement, food / beverage, food additive, or as a drug, feed, or feed additive for animals other than humans. For example, the NK cell apoptosis inhibitory composition of the present invention can be prepared as a quasi-drug and made more easily absorbed by providing it in the form of a beverage such as a bottled drink, or in the form of a tablet, capsule, or granule.

[0034] The present invention's NK cell apoptosis inhibitory composition can take the form of, for example, a liquid, tablet, powder, granules, sugar-coated tablet, capsule, suspension, emulsion, or pill. For example, for oral administration, it can take the form of a liquid, tablet, powder, granules, sugar-coated tablet, capsule, suspension, emulsion, or pill, while for parenteral administration, it can take the form of an injection, ampoule, rectal administration, oily suppository, or water-soluble suppository.

[0035] The NK cell apoptosis inhibitory composition of the present invention may contain additives commonly used in pharmaceuticals, quasi-drugs, and nutritional supplements, such as excipients, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring and odor-correcting agents, analgesics, and stabilizers. It may also contain non-toxic additives such as starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or its salts, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene glycol-citric acid, sodium chloride, sodium sulfite, and sodium phosphate. Furthermore, when preparing it as a quasi-drug, it can be made even easier to consume by, for example, providing it in the form of a beverage in a bottle, or in the form of a tablet, capsule, or granule.

[0036] The NK cell apoptosis inhibitory composition of the present invention may contain additives such as antioxidants, emulsifiers, preservatives, pH adjusters, flavorings, seasonings, sweeteners, acidulants, and quality stabilizers, either alone or in combination. For example, vitamin C, vitamin E, and cysteine ​​can be used as antioxidants, and it is particularly preferable to contain 0.005 to 0.05% by weight of vitamin C. Also, for example, glucose, fructose, isomerized liquid sugar, fructooligosaccharides, emulsified oligosaccharides, soybean oligosaccharides, cyclodextrin, aspartame, and monk fruit extract can be used as sweeteners.

[0037] When the NK cell apoptosis inhibitory composition of the present invention is provided as food or beverage, it can be provided as a Food for Specified Health Uses, a Food with Nutrient Function Claims, a Food with Function Claims, a so-called health food (functional food, health supplement), a soft drink, etc. However, it is not limited to these. In this case, it is also possible to label the food or beverage with information about the functional properties of tea catechins.

[0038] Preferred forms of food and beverages include, for example, candy, jelly, tablets, beverages, soups, noodles, rice crackers, Japanese sweets, frozen desserts, and baked goods. Preferably, they are packaged beverages such as fruit juices, vegetable juices, fruit and vegetable juices, tea beverages (including green tea beverages), coffee beverages, and sports drinks.

[0039] As a packaged beverage, for example, by adding the above-mentioned active ingredient or a composition containing the above-mentioned active ingredient to a currently known packaged beverage, a packaged beverage containing the NK cell apoptosis inhibitory composition of the present invention can be provided. Furthermore, by processing the tea extract to increase the concentration of the above-mentioned active ingredients, or by adding a commercially available tea polyphenol preparation, the tea beverage can be packaged and provided as a beverage containing the NK cell apoptosis inhibitory composition of the present invention.

[0040] <Method for administering the NK cell apoptosis-inhibiting composition of the present invention> The NK cell apoptosis inhibitory composition of the present invention can be administered orally or parenterally (intramuscular injection, intravenous injection, subcutaneous administration, rectal administration, transdermal administration, nasal administration, etc.), and it is preferable to use a formulation and dosage form suitable for each administration method.

[0041] <Effects> By ingesting the NK cell apoptosis-inhibiting composition of the present invention, the expression level of the Bcl2 gene can be increased, thereby suppressing apoptosis (cell death) of NK cells. As a result, the number of NK cells can be increased, and the lifespan of NK cells can be extended.

[0042] <Required intake and content> The NK cell apoptosis inhibitory composition of the present invention can be expected to produce the desired effects when taken on a daily basis. In this invention, "daily intake" means intake at a frequency of once every three days or more, preferably at a frequency of once every two days or more, and particularly preferably at a frequency of once every day or more.

[0043] The intake concentration of the active ingredient in the NK cell apoptosis inhibitory composition of the present invention is preferably 0.2 mg / 1 mL or more in total amount of tea catechins, more preferably 0.3 mg / 1 mL or more, more preferably 0.7 mg / 1 mL or more, and more preferably 1.0 mg / 1 mL or more, from the viewpoint of obtaining the desired effect through daily intake, for example, as a pharmaceutical product. However, if the amount is too high, the bitter and astringent taste characteristic of tea catechins, including GCG and EGCG, will become stronger, so the upper limit is preferably 3.0 mg / 1 mL or less in total amount of tea catechins, more preferably 2.5 mg / 1 mL or less, and more preferably 2.0 mg / 1 mL or less. On the other hand, when the NK cell apoptosis inhibitory composition of the present invention is provided in the form of food or beverages, for example, the intake concentration of the active ingredient is preferably 0.02% by mass or more, more preferably 0.03% by mass or more, more preferably 0.07% by mass or more, and even more preferably 0.1% by mass or more, as a total amount of tea catechins, from the viewpoint of obtaining the desired effect through daily intake. However, if the amount is too high, the bitter and astringent taste characteristic of tea catechins, including GCG and EGCG, will become stronger, so the upper limit is preferably 0.3% by mass or less, more preferably 0.25% by mass or less, and more preferably 0.2% by mass or less, as a total amount of tea catechins.

[0044] The duration, amount, and frequency of intake of the NK cell apoptosis inhibitory composition of the present invention are not particularly limited. As a guideline, from the viewpoint of obtaining the desired effect through daily intake, it is preferable that the intake period be 3 days or more, more preferably 5 days or more, and more preferably 7 days or more, and that it be taken continuously every day. In particular, from the perspective of obtaining the desired effect through daily intake, it is preferable to take at least 3.5 mg / day of active ingredients, for example, one or more active ingredients selected from GCG and EGCG, more preferably 5 mg / day or more, more preferably 6 mg / day or more, for at least 3 days, more preferably 5 days or more, and more preferably 7 days or more, and to take them continuously every day. The frequency of intake may be increased or decreased as needed.

[0045] <Safety> The active ingredient of the present invention's NK cell apoptosis inhibitory composition is derived from tea, which has been orally consumed by humans for many years; therefore, its safety can be guaranteed from the perspective of dietary experience.

[0046] <Explanation of terms> In this invention, when expressed as "X~Y" (where X and Y are any numbers), unless otherwise specified, it includes the meaning of "greater than or equal to X and less than or equal to Y," as well as the meaning of "preferably greater than X" or "preferably less than Y." Furthermore, when we use expressions like "greater than or equal to X" (where X is any number) or "less than or equal to Y" (where Y is any number), we also imply that "greater than X is preferable" or "less than Y is preferable." [Examples]

[0047] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0048] <Experiment 1> Three C57BL / 6 strain mice, 6-8 weeks old, were given either EGCG (Sanphenon EGCG-OP, TAIYO KAGAKU, Tokyo, Japan) or GCG (cat.# HY-N0522, MedChemExpress, NJ, USA) at a concentration of 1 mg / mL in water for three days. The control group received water. To activate NK cells, 300 μg of poly I:C (cat.# 4287, TOCRIS Bioscience, MN, USA) dissolved in 200 μL of phosphate-buffered saline (PBS) was administered intraperitoneally to mice on the second day of water intake.

[0049] Spleens were removed from control mice and mice that had been given EGCG or GCG in water for 3 days. The spleens were dispersed using the floss portion of a glass slide, and splenocytes were prepared using erythrocyte lysis buffer (RBS lysis Buffer 10X: cat.# 420301, BioLegend, CA, USA). Next, NK cell markers NK1.1 and CD49b were stained with fluorescently labeled antibodies (fluorescently labeled antibodies: NK1.1-FITC (clone S17016D) (BioLegend, CA, USA), CD49b-PE (clone DX5) (BioLegend, CA, USA)). To remove dead cells, the cells were simultaneously stained with the dead cell stain Zombie Aqua (cat.# 423101, BioLegend, CA, USA), and to distinguish T cells, with a fluorescently labeled antibody for CD3 (CD3-Pacific Blue (clone 17A2, BioLegend, CA, USA)). Stained mouse splenocytes were analyzed using a flow cytometer, CytoFlex (Beckman Coulter Japan, Osaka, Japan). The results were analyzed using the analysis software FlowJo (Tree Star, Oregon, USA). Furthermore, all experiments were conducted in accordance with the animal experimentation ethics regulations established by the Department of Animal Experimentation at Ehime Prefectural University of Health Sciences. In addition, the sex of the animals was kept consistent for each experiment.

[0050] There were no significant differences in water intake among the control group, EGCG group, and GCG group. Furthermore, no changes in health status were observed due to the changes in water intake.

[0051] [Table 1]

[0052] Compared to the control group, the number of NK cells in the spleen cells was significantly increased in both the EGCG group and the GCG group (Figure 1).

[0053] <Experiment 2> The effect of oral administration of EGCG or GCG on the number of NK cells in the spleen under deactivation conditions was investigated.

[0054] The experiment was conducted in the same manner as in Experiment 1, except that poly I:C was not administered on the second day of water intake.

[0055] Compared to the control group, both the EGCG group and the GCG group showed a significant increase in NK cell counts (Figure 2).

[0056] <Experiment 3> Analysis of Bcl2 gene expression in spleen NK cells under activation conditions induced by oral administration of EGCG or GCG.

[0057] Three C57BL / 6 strain mice, 7-8 weeks old, were administered EGCG or GCG in the same manner as in Experiment 1. Splenocytes were then prepared by activating NK cells through intraperitoneal administration of poly I:C. Next, NK cells were isolated using the MojoSort Mouse NK cell Isolation Kit (cat.# 480050, BioLegend, CA, USA) according to the manufacturer's protocol. Total RNA was extracted from isolated NK cells using RNAiso Plus (cat.# 9108, TaKaRa Bio, Shiga, Japan) according to the manufacturer's protocol. Then, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (cat.# 4368814, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Reverse transcription was performed using the Takara Thermal Cycle Dice Touch (cat.# N-3804, TaKaRa Bio, Shiga, Japan). Quantitative PCR was performed using cDNA as a template with the Thunderbird Probe qPCR Mix (cat.# QPS-101, TOYOBO, Osaka, Japan) and the Applied Biosystems QuantStudio 1 Real-Time PCR Instrument (cat.# A40425, Thermo Fisher Scientific, Waltham, MA, USA) to measure the expression levels of the Bcl2 gene and the housekeeping gene Hprt1. The relative expression level of Bcl2 to Hprt1 was calculated.

[0058] Since a significant difference in the relative expression level of Bcl2 in NK cells was determined by Welch one-way ANOVA (p<0.05), multiple comparisons using the Games-Howell method were performed as a subsequent test. As a result, relative expression levels of the Bcl2 gene were significantly increased in both the EGCG group and the GCG group compared to the control group (Figure 3). Bcl2 is known to suppress the release of cytochrome C from mitochondria and thus inhibit apoptosis. Therefore, it is hypothesized that EGCG and GCG may suppress apoptosis and increase the number of NK cells by enhancing Bcl2 expression at the gene level.

[0059] <Experiment 4> Analysis of Bcl2 gene expression levels in spleen NK cells under deactivation conditions induced by oral administration of EGCG or GCG.

[0060] Bcl2 gene expression was measured in 3 C57BL / 6 strain mice per group, 6-8 weeks old, using the same method as in Experiment 3, except that poly I:C was not administered on the second day of water intake.

[0061] Since a significant difference in the relative expression level of Bcl2 in NK cells was determined by Welch one-way ANOVA (p<0.05), multiple comparisons using the Games-Howell method were performed as a subsequent test. As a result, relative expression levels of the Bcl2 gene were significantly increased in both the EGCG group and the GCG group compared to the control group (Figure 4).

[0062] (Consideration) From the above examples and comparative examples, as well as the test results conducted by the inventors to date, it has been found that ingesting tea catechins, particularly EGCG and GCG, increases the expression level of the Bcl2 gene and suppresses apoptosis of NK cells.

[0063] In all of the above experiments, the mice were not subjected to any particular stress and were tested under normal breeding conditions, and no aged mice were used. Therefore, it can be expected that the effects can be further enhanced by regularly ingesting tea catechins, especially EGCG and GCG, which are the active ingredients of this invention.

[0064] In all of the above experiments, the combined concentration of EGCG and GCG, i.e., the total amount of tea catechins, was 1 mg / 1 mL or higher, and the effect was confirmed when consumed continuously for 3 days. Considering these results and the daily intake of tea catechins, it can be concluded that an intake concentration of 0.2 mg / 1 mL or higher is preferable. Furthermore, in Experiment 1, the animals ingested approximately 6 mg / day of tea catechins at a concentration of 1 mg / mL. Considering these results, along with the results of previous experiments conducted by the inventors, and the daily intake of tea catechins, it can be concluded that to more reliably obtain the desired effect, it is preferable to ingest 3.5 mg / day or more of tea catechins for at least 3 days.

Claims

1. A composition for inhibiting apoptosis of natural killer cells, containing tea catechins as an active ingredient.

2. The composition for inhibiting apoptosis of natural killer cells according to claim 1, characterized in that the inhibition of apoptosis of natural killer cells manifests as an increase in the expression level of the Bcl2 gene.

3. The composition for inhibiting apoptosis of natural killer cells according to claim 1, characterized in that the intake concentration of the active ingredient is 0.2 mg / 1 mL or more as the total amount of tea catechins.

4. The composition for inhibiting apoptosis of natural killer cells according to claim 1, characterized in that the intake period is three days or more.

5. The composition for inhibiting apoptosis of natural killer cells according to claim 1, characterized in that the intake of the active ingredient is 3.5 mg / day or more and is taken continuously for 3 days or more.

6. Food or beverage containing the composition for inhibiting apoptosis of natural killer cells according to any one of claims 1 to 5.

7. The food or beverage according to claim 6, characterized in that the food or beverage is a packaged beverage.

8. A method for suppressing apoptosis of natural killer cells, characterized by the intake of tea catechins.

Images