Blood EGCG level enhancer, composition containing the same, and method for enhancing blood EGCG level with increased intestinal bacteria

Abstract

To provide a novel blood EGCG level enhancer, capable of enhancing the blood concentration of epigallocatechin gallate (EGCG) upon oral ingestion of EGCG in particular among catechins.SOLUTION: The present invention provides a method for enhancing blood EGCG levels with increased intestinal bacteria, comprising cyclic oligosaccharides and epigallocatechin gallate (also referred to as "EGCG") as active ingredients.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for increasing blood EGCG levels, a composition containing the same, and a method for increasing blood EGCG levels accompanied by an increase in intestinal bacteria. [Background technology]

[0002] Tea catechins contained in green tea have been reported to have various pharmacological effects, such as cholesterol elevation suppression, antitumor effects, diarrhea virus infection inhibition, caries prevention, influenza virus infection prevention, mycoplasma infection prevention, alpha-amylase activity inhibition, blood sugar elevation suppression, colon cancer prevention, gastritis, gastric or duodenal ulcer prevention, anti-arteriosclerotic effects, active oxygen generation inhibition, and gastrin secretion inhibition. Recently, it has been recognized that green tea consumption can reduce the risk of many diseases, including cardiovascular disease. This effect is thought to be primarily due to the effects of catechins contained in green tea, particularly epigallocatechin gallate (EGCG).

[0003] However, catechins, especially EGCG, are known to have low bioavailability. For example, even if a human orally ingests 50 mg of EGCG, the peak plasma concentration after ingestion is estimated to be only 0.12 μmol / L. This is a relatively low value compared to other polyphenols. Therefore, several approaches have been proposed to improve the bioavailability of catechins.

[0004] For example, Patent Document 1 discloses that by controlling the ratio of epicatechins among non-polymer catechins in a packaged black tea beverage containing non-polymer catechins at high concentrations, it is possible to further increase the amount of non-polymer catechins transferred into the blood when the beverage is ingested, even though the concentration of non-polymer catechins is the same.

[0005] Patent Document 2 discloses that at least one selected from the group consisting of serine, aspartic acid, malic acid, capric acid, lauric acid, and grapefruit juice has the effect of promoting the absorption of polyphenol compounds.

[0006] Patent Document 3 discloses that Benifuuki extract has the effect of promoting the absorption of polyphenol compounds, and that ingesting Benifuuki extract simultaneously with catechins increases the absorbability and retention time of catechins in the body.

[0007] Patent Document 4 discloses that at least one member selected from the group consisting of succinic acid, cysteine, asparagine, isoleucine, and pinitol has the effect of promoting the absorption of polyphenol compounds.

[0008] Patent Document 5 discloses that hesperetin, lime extract, and lemon extract promote the absorption of catechins.

[0009] Patent Document 6 discloses that Kochia sieboldii extract, Shikakai extract, Mitsuba honeysuckle extract, Asiatic basil extract, and tea seed extract promote the absorption of polyphenols.

[0010] Furthermore, Non-Patent Document 1 reports that fructooligosaccharides increase the plasma concentration of EGCG. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2003-333989 A [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-247282 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-11751 [Patent Document 4] Japanese Patent Application Laid-Open No. 2011-79770 [Patent Document 5] Japanese Patent Application Laid-Open No. 2016-216440 [Patent Document 6] Japanese Patent Application Laid-Open No. 2017-109991 [Non-patent literature]

[0012] [Non-Patent Document 1] Umino, T. et al. "Fructooligosaccharides Increase in Plasma Concentration of (-)-Epigallocatechin-3-Gallate in Rats." Journal of Agricultural and Food Chemistry 2021, vol. 69, issue 49, pp. 14849-14855. Published December 6, 2021 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention aims to provide a new agent for elevating blood EGCG levels, a composition containing the same, and a method for elevating blood EGCG levels, which can increase the blood levels of catechins, particularly epigallocatechin gallate (EGCG). [Means for solving the problem]

[0014] In order to solve the above problems, the present invention proposes the following invention.

[0015] [1] An agent that increases blood EGCG levels, accompanied by an increase in intestinal bacteria, containing cyclic oligosaccharides and epigallocatechin gallate (EGCG) as active ingredients.

[0016] [2] The blood EGCG concentration elevating agent described in [1], to be taken simultaneously with meals.

[0017] [3] The agent for increasing blood EGCG concentration according to [1] or [2], wherein the cyclic oligosaccharide is β-cyclodextrin.

[0018] [4] A blood EGCG concentration elevating agent according to any one of [1] to [3], wherein the intestinal bacteria to be increased are of the Collinsella genus.

[0019] [5] A blood EGCG concentration elevating agent according to [4], wherein the intestinal bacteria of the Collinsella genus is Collinsella aerofaciens.

[0020] [6] A blood EGCG concentration elevating agent according to any one of [1] to [5], wherein the intestinal bacteria to be increased are of the genus Bacteroides.

[0021] [7] The agent for increasing blood EGCG concentration according to [6], wherein the intestinal bacterium of the Bacteroides genus is Bacteroides dorei.

[0022] [8] A blood EGCG concentration elevating agent according to any one of [1] to [7], wherein the intestinal bacteria to be increased are of the genus Lactobacillus.

[0023] [9] A blood EGCG concentration elevating agent according to [8], wherein the intestinal bacteria of the Lactobacillus genus is Lactobacillus intestinalis.

[0024]

[10] A blood EGCG concentration elevating agent according to any one of [1] to [9], wherein the intestinal bacteria to be increased are of the Turicibacter genus.

[0025]

[11] A blood EGCG concentration increasing agent according to

[10] , wherein the intestinal bacterium of the Turicibacter genus is Turicibacter sanguinis.

[0026]

[12] A blood EGCG concentration elevating agent according to any one of [1] to

[11] , wherein the intestinal bacteria to be increased are of the genus Bifidobacterium.

[0027]

[13] A composition comprising the blood EGCG concentration elevating agent described in any one of [1] to

[12] .

[0028]

[14] A method for increasing intestinal bacteria and increasing blood EGCG levels, comprising ingesting cyclic oligosaccharides and EGCG simultaneously with meals.

[0029]

[15] A method for increasing the number of intestinal bacteria and the blood EGCG concentration after two weeks of oral administration of a composition containing 0.03 to 5% by mass of cyclic oligosaccharides and 0.01 to 1.0% by mass of EGCG at a dose of 7.0 to 80.0 g per kg of body weight. [Effects of the Invention]

[0030] Oral administration of the agent for increasing blood EGCG concentration proposed by the present invention can increase intestinal bacteria and also increase blood EGCG concentration. [Brief explanation of the drawings]

[0031] [Figure 1] This graph shows the relationship between blood EGCG concentrations in rats given EGCG alone and when EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin was added to the diet in an oral intake study. The values ​​in the bar graph represent the mean ± SD. Compared to the EGCG alone group, * indicates p<0.05. [Figure 2] This graph shows the EGCG content in the cecal contents per 100 g of rat body weight when EGCG alone, EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin was added to the diet in an oral intake study in rats. The values ​​in the bar graph represent the mean ± SD. [Figure 3] This graph shows the EGCG content in feces in rats given EGCG alone, or with EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin in their diets in an oral feeding study. The values ​​in the bar graph represent the mean ± SD. * indicates p<0.05 compared to the EGCG alone group. [Figure 4]This graph shows the pH of the cecal contents in rats given EGCG alone, or with EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin in their diets in an oral feeding study. The values ​​in the bar graph represent the mean ± SD. **** indicates p<0.0001 compared to the EGCG-only group. [Figure 5] This graph shows the concentrations of lactic acid and short-chain fatty acids (SCFA) in the cecal contents of rats given EGCG alone, or with EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin in their diets in an oral feeding study. Compared to the EGCG-only group, ** indicates p<0.01, *** indicates p<0.01, and **** indicates p<0.0001. [Figure 6] This graph shows the total bacterial counts in feces in rats given EGCG alone, or with EGCG + α-cyclodextrin, EGCG + β-cyclodextrin, or EGCG + γ-cyclodextrin in their diets in an oral intake study. Compared to the EGCG-only group, *** indicates p<0.01 and **** indicates p<0.0001. [Figure 7] This is a bar graph showing the abundance ratio of taxa at the genus level in the intestinal bacteria in feces collected in the second week of feeding in an oral intake test in rats, as measured by metagenomic sequencing. [Figure 8] This is a bar graph showing the abundance ratio of taxa at the species level in fecal intestinal bacteria collected in the second week of feeding in a rat oral feeding study, as measured by metagenomic sequencing. Compared to the EGCG-only administration group, * indicates p<0.05, *** indicates p<0.01, and **** indicates p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will now be described based on embodiments, although the present invention is not limited to the embodiments described below.

[0033] <The agent for increasing blood EGCG concentration of the present invention> A blood EGCG concentration elevating agent according to one embodiment of the present invention (also referred to as the "blood EGCG concentration elevating agent of the present invention") is a blood EGCG concentration elevating agent that contains cyclic oligosaccharides and epigallocatechin gallate (abbreviated as EGCG) as active ingredients and that is accompanied by an increase in intestinal bacteria.

[0034] In the present invention, "increased blood EGCG concentration" means that the blood EGCG concentration is increased compared to when the same amount of EGCG is orally ingested alone. In addition, in the present invention, "increasing the number of intestinal bacteria" means that the number of intestinal bacteria increases compared to when the same amount of EGCG is orally ingested alone. Therefore, by orally taking the blood EGCG concentration elevating agent of the present invention, it is possible to change the composition ratio of intestinal bacterial sacs and increase the number of intestinal bacteria, as well as increase the blood EGCG concentration, compared to when the same amount of EGCG is orally taken alone. By changing the composition ratio of the intestinal flora and increasing the number of intestinal bacteria, it is possible to obtain effects such as increased production of lactic acid and short-chain fatty acids. Furthermore, by increasing the blood concentration of EGCG, it is possible to further enjoy the pharmacological effects of EGCG mentioned above.

[0035] Generally, EGCG is contained in large amounts in green tea and is often taken with meals, but in this case, the absorption rate of EGCG is significantly lower than when taken on an empty stomach. However, the blood EGCG concentration elevating agent of the present invention is particularly suitable as a blood EGCG concentration elevating agent to be taken simultaneously with meals. Even when taken simultaneously with meals, the blood EGCG concentration elevating agent of the present invention can improve the absorption efficiency of EGCG in the intestine and achieve the effect of increasing blood EGCG concentration. Here, "meals" refers to the habitual daily oral intake of food and drink to ingest nutrients necessary for life support, such as breakfast, lunch, and dinner. Examples of such foods and drink include grains, potatoes and starches, sugars and sweeteners, beans, nuts, vegetables, fruits, mushrooms, algae, seafood, meat, eggs, dairy products, oils and fats, confectioneries, beverages, seasonings and spices, and prepared foods available for sale, as well as foods listed in the 2020 edition (8th revision) of the Standard Tables of Food Composition in Japan. However, the list is not limited to these. Additionally, "simultaneous" as used herein includes not only oral ingestion together with food and drink, but also ingestion slightly before or after the oral ingestion of food and drink, such as ingestion within 5 minutes before or after the oral ingestion of food and drink.

[0036] (intestinal bacteria) Examples of intestinal bacteria that increase upon ingestion of the agent of the present invention for elevating blood EGCG levels include, but are not limited to, the genus Collinsella, including Collinsella aerofaciens, the genus Bacteroides, including Bacteroides dorei, the genus Lactobacillus, including Lactobacillus intestinalis, the genus Turicibacter, including Turicibacter sanguinis, and the genus Bifidobacterium.

[0037] (EGCG) EGCG is the catechin with the highest content among the catechins contained in green tea, and is an ester of epigallocatechin and gallic acid. EGCG may be a pharmaceutically acceptable salt of epigallocatechin gallate, such as a salt of epigallocatechin gallate with an alkali metal such as sodium or potassium or an alkaline earth metal such as calcium, or a quaternary ammonium salt such as an ammonium salt of epigallocatechin gallate. EGCG may also be a prodrug that releases epigallocatechin gallate in vivo.

[0038] (cyclic oligosaccharides) Cyclic oligosaccharides (also called "cyclodextrins" or "CDs") are made up of glucose units linked together by α-1,4 bonds, forming a ring (donut-shaped) structure. Those with six glucose units are classified as α-cyclodextrins (α-CD), those with seven glucose units as β-cyclodextrins (β-CD), and those with eight glucose units as γ-cyclodextrins (γ-CD). α-CD, β-CD, and γ-CD have different cavity sizes and differ in their digestibility by digestive enzymes. α-CD and β-CD, which are less digestible, are thought to reach the large intestine. Once CD reaches the large intestine, it is converted by intestinal bacteria into short-chain fatty acids, which lowers the intraluminal pH and inhibits the oxidative polymerization of EGCG, improving the stability of EGCG in the lumen. As a result, it is thought that EGCG uptake from the intestinal tract increases, leading to an increase in blood EGCG concentrations.

[0039] Examples of cyclic oligosaccharides that are active ingredients in the agent for increasing blood EGCG levels of the present invention include α-CD, β-CD, and γ-CD. Among these, α-CD and β-CD are preferred because they are indigestible and easily assimilated by intestinal microorganisms, and β-CD is particularly preferred. The simultaneous administration of β-CD and EGCG can increase the number of intestinal bacteria and stabilize EGCG more than other CDs. β-CD administration increases the number of intestinal bacteria, which in turn increases organic acids such as lactic acid and acetic acid in the intestine, lowering the intraluminal pH and further stabilizing EGCG. It is also thought that the formation of an EGCG inclusion complex with β-CD further stabilizes EGCG.

[0040] (Required intake and content) In the blood EGCG concentration elevating agent of the present invention, the amount of EGCG as the active ingredient is preferably 30 mg to 300 mg per day, more preferably 40 mg to 250 mg, and even more preferably 50 mg to 210 mg, from the viewpoint of the efficacy and flavor of EGCG.

[0041] In the blood EGCG concentration elevating agent of the present invention, the amount of cyclic oligosaccharide as an active ingredient is preferably 350 mg to 1500 mg per day, more preferably 380 mg to 1200 mg, and even more preferably 400 mg to 1000 mg, from the viewpoint of flavor.

[0042] In the blood EGCG concentration elevating agent of the present invention, the content of cyclic oligosaccharides is preferably 180 to 1000 parts by mass per 100 parts by mass of EGCG, from the viewpoint of the stability of EGCG in the body, and more preferably 200 to 900 parts by mass, and even more preferably 240 to 850 parts by mass.

[0043] In the blood EGCG concentration elevating agent of the present invention, the total intake or administration amount of the active ingredients, cyclic oligosaccharides and EGCG, is preferably adjusted appropriately depending on the intended use. As a guideline, a single dose of 1 mg to 10,000 mg can be envisaged, with a single dose of 5 mg or more or 1,000 mg or less, and a single dose of 10 mg or more or 500 mg or less can be envisaged. The number of times of ingestion or administration is not particularly limited. As a guideline, it can be assumed to be 1 to 3 times a day, and the number of times of ingestion may be increased or decreased as necessary.

[0044] (safety) Regarding the safety of the blood EGCG concentration elevating agent of the present invention, the active ingredients, cyclic oligosaccharides and EGCG, are components that have been orally ingested by humans for many years, and therefore, it can be said that their safety is guaranteed from the standpoint of dietary experience.

[0045] <Composition of the Present Invention> A composition according to one embodiment of the present invention (referred to as the "composition of the present invention") is a composition containing the agent for elevating blood EGCG concentration of the present invention. Oral administration of the composition of the present invention can increase the number of intestinal bacteria and also increase the blood EGCG concentration.

[0046] In the composition of the present invention, the concentration of the active ingredient EGCG is preferably 0.01% by mass or more, more preferably 0.015% by mass or more, even more preferably 0.017% by mass or more, and even more preferably 0.02% by mass or more. On the other hand, from the viewpoint that a high amount of EGCG increases the bitterness and astringency, the concentration is preferably 1.0% by mass or less, even more preferably 0.5% by mass or less, even more preferably 0.4% by mass or less, and even more preferably 0.3% by mass or less.

[0047] In the composition of the present invention, the concentration of the cyclic oligosaccharide as the active ingredient is preferably 0.03% by mass or more, more preferably 0.04% by mass or more, and even more preferably 0.05% by mass or more. On the other hand, since the incorporation of a large amount of cyclic oligosaccharide makes it difficult to sense the taste of the composition itself, the concentration is preferably 5% by mass or less, more preferably 4% by mass or less, even more preferably 3% by mass or less, and even more preferably 2.5% by mass or less.

[0048] In the composition of the present invention, the content of cyclic oligosaccharides is preferably 180 to 1000 parts by mass per 100 parts by mass of EGCG, from the viewpoint of the stability of EGCG in the body and its flavor, and more preferably 200 to 900 parts by mass, and even more preferably 240 to 850 parts by mass.

[0049] The EGCG in the composition of the present invention may be a single product consisting of EGCG or a composition containing EGCG. Examples of compositions containing EGCG include green tea extracts and purified products thereof. A specific example is a green tea extract obtained by subjecting green tea to hot water extraction, separating the extract using water and low- or high-concentration alcohol in an adsorption column, and drying the extract to a tea polyphenol concentration of approximately 85 to 99.5%. A preferred example is "Theafuran 90S (trade name; manufactured by Ito En Co., Ltd.)."

[0050] <Forms of the Agent for Elevating Blood EGCG Concentration of the Present Invention and the Composition of the Present Invention> The blood EGCG concentration elevating agent of the present invention and the composition of the present invention can be provided, for example, as an orally administered drug, quasi-drug, dietary supplement, food or drink, or the like. In this case, examples of the form include liquids, tablets, powders, granules, sugar-coated tablets, capsules, suspensions, emulsions, pills, and the like.

[0051] The blood EGCG concentration elevating agent and composition of the present invention may contain additives commonly used in pharmaceuticals, quasi-drugs, and dietary supplements, such as excipients, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavorings, soothing agents, stabilizers, etc. Non-toxic additives such as starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or a salt thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium sulfite, and sodium phosphate may also be incorporated. When prepared as a quasi-drug, it can be made easier to take by making it into a drinking form such as a bottled drink, or into a tablet, capsule, granule or other form.

[0052] When the blood EGCG concentration elevating agent and the composition of the present invention are provided as food or drink, they can be provided as a food for specified health uses, a food with nutrient function claims, a food with functional claims, a so-called health food (functional food, health supplement), a soft drink, etc. However, they are not limited to these. In this case, it is also possible to make the food or drink labeled as having the pharmacological action of EGCG.

[0053] Preferred forms of food and drink include candy, jelly, tablet candy, beverages, soup, noodles, rice crackers, Japanese sweets, frozen desserts, baked goods, etc. Preferred are packaged beverages such as fruit juice drinks, vegetable juices, fruit and vegetable juices, tea drinks (including green tea drinks), coffee drinks, and sports drinks.

[0054] <Ingestion method of the present invention> A method according to one embodiment of the present invention (referred to as the "intake method of the present invention") is a method for increasing intestinal bacteria and increasing blood EGCG levels, characterized by ingesting cyclic oligosaccharides and EGCG simultaneously with a meal. For example, by ingesting the blood EGCG level elevating agent of the present invention or the composition of the present invention simultaneously with a meal, it is possible to increase intestinal bacteria and increase blood EGCG levels.

[0055] By taking cyclic oligosaccharides and EGCG simultaneously, the stability of EGCG in the body is improved, and the absorption rate does not decrease even when taken simultaneously with meals. Therefore, EGCG can be efficiently absorbed even when taken orally simultaneously with meals, such as meals containing dietary fiber.

[0056] In the intake method of the present invention, a composition containing 0.03 to 5.0% by mass of cyclic oligosaccharides and 0.01 to 1.0% by mass of EGCG is orally taken at a rate of 7 to 80 g per kg of body weight per day, and preferably, after two weeks of continued oral intake, the number of intestinal bacteria increases and the blood EGCG concentration increases.

[0057] In the ingestion method of the present invention, the content of cyclic oligosaccharides in the composition to be ingested is preferably 0.03% by mass to 5% by mass, more preferably 0.04% by mass or more or 4% by mass or less, and even more preferably 0.05% by mass or more or 2.5% by mass or less. The EGCG content in the composition to be ingested is preferably 0.01 to 1.0% by mass, more preferably 0.015% by mass or more or 0.5% by mass or less, and even more preferably 0.017% by mass or more or 0.3% by mass or less.

[0058] In the ingestion method of the present invention, the composition to be ingested may contain other components in addition to cyclic oligosaccharides and EGCG, such as water, carbohydrates, proteins, lipids, dietary fiber, vitamins, minerals, and the like, as well as foods and food additives containing these components. However, in the ingestion method of the present invention, the content of other components in the composition to be ingested is preferably 90% by mass or less, more preferably 95% by mass or less, and even more preferably 97% by mass or less.

[0059] In the ingestion method of the present invention, the amount of intake of the above composition depends on the content ratio of cyclic oligosaccharides and EGCG, but is preferably 3 g to 100 g per kg of body weight per day, more preferably 5 g or more or 90 g or less, and even more preferably 7 g or more or 80 g or less.

[0060] In addition, in the ingestion method of the present invention, it is preferable to measure the number of intestinal bacteria and the blood EGCG concentration two weeks after the start of oral ingestion. This is because the composition of the intestinal bacterial flora gradually changes after the start of oral ingestion and stabilizes after two weeks. In addition, the number of intestinal bacteria gradually increases after the start of oral ingestion and stabilizes after two weeks. In addition, blood EGCG concentrations generally begin to rise 10 minutes or more after oral ingestion, peak 30 minutes to 1 hour later, and decrease by half 2 to 6 hours after oral ingestion. Therefore, if oral ingestion is not performed regularly, it is preferable to measure EGCG concentrations 10 minutes or more or within 6 hours after oral ingestion, and more preferably 30 minutes or more or within 1 hour. In cases where oral ingestion is performed regularly, as in the rats used in this study, more specifically, when the ingestion interval is less than 2 hours, blood EGCG concentrations remain stable as long as ingestion continues, so the timing of measurement is not particularly important.

[0061] Furthermore, if the ratio of fecal intestinal bacteria to the total population increases by 2.5% or more, especially by 5% or more, and especially by 10% or more, compared to when the same amount of EGCG is orally taken alone, it can be determined that the number of intestinal bacteria has increased at the genus level. If fecal 16S rRNA increases by 10% or more, especially by 20% or more, and especially by 50% or more, it can be determined that the total number of intestinal bacteria has increased. If the blood EGCG concentration increases by 10% or more, especially by 20% or more, and especially by 50% or more, compared to when the same amount of EGCG is orally taken alone, it can be determined that the blood EGCG concentration has increased.

[0062] <Explanation of terms> In the present invention, when the expression "X to Y" (X and Y are any numbers) is used, unless otherwise specified, it means "X or more and Y or less," as well as "preferably larger than X" or "preferably smaller than Y." Furthermore, when it is expressed as "X or more" (X is any number) or "Y or less" (Y is any number), it also includes the intention that "it is preferable that it is greater than X" or "it is preferable that it is less than Y." [Example]

[0063] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples. In the following examples, "%" indicates "% by mass" unless otherwise specified.

[0064] <Animal testing> We investigated whether the addition of cyclic oligosaccharides to the diet contributes to the stabilization of EGCG in the gastrointestinal tract in an animal model, thereby affecting the plasma concentration of EGCG.

[0065] (raw materials) The following raw materials were used in the following examples and comparative examples. High-purity EGCG extract (EGCG concentration of 94% or more by mass, "Teavigo" manufactured by DSM Nutrition Japan Co., Ltd.) α-Cyclodextrin (Kanto Chemical Co., Ltd.) β-Cyclodextrin (Kanto Chemical Co., Ltd.) γ-Cyclodextrin (Kanto Chemical Co., Ltd.)

[0066] The following solutions were used for the analysis of EGCG in plasma: β-glucuronidase from Helix pomatia (Sigma-Aldrich) Sulfatase derived from abalone innards (Sigma-Aldrich)

[0067] (Animals and Diet) Twenty-four male Wistar rats (4 weeks old) were acclimated for two days in stainless steel cages at 22°C in an automatically controlled room with a 12-hour light cycle. During the acclimation period, the rats were fed AIN93G formulated diet. The 24 mice were then divided into four groups and fed one of the following experimental diets: 0.3% EGCG diet (100% diet supplemented with 0.3% EGCG), 0.3% EGCG and 2.5% α-cyclodextrin diet (100% diet supplemented with 0.3% EGCG and 2.5% α-cyclodextrin), 0.3% EGCG and 2.5% β-cyclodextrin diet (100% diet supplemented with 0.3% EGCG and 2.5% β-cyclodextrin), or 0.3% EGCG and 2.5% γ-cyclodextrin diet (100% diet supplemented with 0.3% EGCG and 2.5% γ-cyclodextrin). The mice had free access to each experimental diet and tap water for two weeks. Feces were collected weekly. On the final day of the experiment, rats were humanely killed by inhaling high-concentration carbon dioxide, and blood was immediately collected from the intraperitoneal vein. Plasma was obtained by centrifugation at 2000 × g for 10 minutes, and then stored in plastic tubes containing 10 mg of L-ascorbic acid at -40°C. The cecum was then removed, and the cecal contents were collected and stored at -40°C until use.

[0068] (Analysis of EGCG in plasma) The EGCG concentration in plasma was analyzed as follows. 0.1 mL of thawed plasma was mixed with 0.9 mL of 100 mM acetate buffer (pH 5.0), 10 μL of β-glucuronidase solution, and 10 μL of sulfatase solution, and then incubated at 37°C for 45 min. After adding 10 μL of 20 μM ethyl gallate solution as an internal standard, the reaction mixture was directly loaded onto a polymer-based solid-phase extraction cartridge (Strata-X, 33 μm particle size, Phenomenex). The cartridge was pre-washed with 1 mL of water, 1 mL of 70% (v / v) aqueous dimethylformamide (DMF) containing 0.1% (v / v) phosphoric acid, and 1 mL of water. After washing the cartridge with 1 mL of water and 1 mL of 20% (v / v) methanol, EGCG was finally eluted with 0.7 mL of 70% (v / v) DMF containing 0.1% (v / v) phosphoric acid. After filtration through a 0.45 μm syringe filter (TORAST disk, Shimadzu GLC), 10 μL of the resulting filtrate was injected into an HPLC system equipped with an electrochemical detector (Coulochem III, ESA). The analytical column (Capcell Pak 3C18, type AQ, 150 mm length x 4.6 mm inner diameter, Shiseido Co., Ltd.) was eluted with a solvent of 50 mM sodium dihydrogen phosphate (adjusted to pH 3.5 with phosphoric acid) / acetonitrile (85 / 15, v / v) containing 0.5 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) at a flow rate of 0.8 mL / min. The temperature of the column was maintained at 40° C. The voltages applied to the analytical cell were −200 mV for electrode 1, +200 mV for electrode 2, and +250 mV for the guard cell.

[0069] (Analysis of EGCG in feces and cecal contents) The freeze-dried feces were pulverized in a grinder mill. 50 mg of the resulting powder was placed in a microtube and mixed with 1 mL of 50% (v / v) acetonitrile containing 0.1% (v / v) phosphoric acid. Stainless steel beads were added to the microtube, and the mixture was stirred for 3 minutes using a bead beater (Cell Destroyer PS1000, Biomedical Sciences Co., Ltd.) to extract EGCG. The extract was then collected in a 10 mL flask. This extraction procedure was repeated three times. After filtering through a syringe filter, the filtrate was injected into the HPLC system. 18 EGCG was separated using a column (100 mm length × 4.6 mm internal diameter, Imtakt) with 15% (v / v) acetonitrile containing 0.1% (v / v) phosphoric acid and detected with an ultraviolet detector set at 230 nm. For the measurement of cecal contents, 0.2 mL of cecal contents diluted 5-fold with distilled water was added to 0.2 mL of acetonitrile containing 0.1% (v / v) phosphoric acid, and the mixture was centrifuged at 2000 × g for 5 minutes. The resulting supernatant was transferred to a separate microtube, to which 0.3 mL of 10 mM phosphate buffer (pH 2.6) was added. The mixture was then filtered and injected into the HPLC system.

[0070] (Analysis of pH, lactic acid and SCFA in cecal contents) The pH value of the thawed cecal contents was measured directly using a portable pH meter. To measure lactate and SCFA in the cecal contents, a portion of the cecal contents was diluted 5-fold with distilled water. The lactate concentration in the sample was measured using a commercially available kit (Lactate Assay Kit-WST, Dojin Chemical). The SCFA (acetic acid, propionic acid, butyric acid) concentrations in the sample were measured by HPLC using a commercially available free short-chain and long-chain fatty acid analysis kit (YMC Co., Ltd.).

[0071] (Analysis of 16S ribosomal RNA copy number in feces) The 16S ribosomal RNA copy number in feces was measured by subjecting the ribosomal RNA region of genomic DNA to real-time PCR. Approximately 10 mg of thawed dried feces was suspended in 190 μL of phosphate buffer solution (PBS), and DNA was extracted from the fecal fluid. The control for 16S ribosomal RNA copy number correction was 10 μL (3.7 × 10 8 cells) (16S ribosomal RNA copy number: 2.34 × 10 9190 μL of PBS was added to the samples (copies), and DNA was extracted from the samples in the same manner as for the fecal fluid. The fecal fluid was mixed with 0.3 g of glass beads (0.1 mm diameter, Tomy / GB-01), 500 μL of TE-saturated phenol, 250 μL of pH 8.0 TE, and 50 μL of 10% SDS. The mixture was disrupted using a disruptor and centrifuged at 15,000 rpm for 5 minutes. The supernatant was purified by adding phenol, chloroform, and isoamyl alcohol (25:24:1). After centrifugation at 15,000 rpm for 5 minutes, the supernatant was mixed with 25 μL of 3 M sodium acetate (pH 5.4) and 275 μL of isopropanol, allowed to stand at -20°C for 10–15 minutes, and then centrifuged at 15,000 rpm for 5 minutes. The supernatant was removed, rinsed with 500 μL of 70% ethanol, air-dried, and redissolved in 100 μL of TE buffer. The DNA extract was subjected to PCR measurement using PCR primers designed to consensus sequences within the 16S ribosomal RNA genome region of common enterobacteria and a commercially available kit (see Tables 1 to 3 below).

[0072] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] (Microbial analysis) Six rats were fed a "0.3% EGCG + 3% fructooligosaccharide diet" containing 3% fructooligosaccharides instead of cyclic oligosaccharides, and were raised in the same way as the other groups. They were also treated in the same way and analyzed and compared as described below. Bacterial DNA was extracted using a fecal DNA isolation and purification kit (Norgen Biotec Co., ON, Canada) according to the manufacturer's protocol, and then diluted with Tris-EDTA buffer (pH 8.5) to prepare DNA (5 ng / μL). An amplicon library was prepared by amplifying the V3-V4 region of the 16S rRNA gene according to the 16S metagenomic sequencing library protocol (Illumina Inc., Tokyo, Japan). For the first PCR (PCR1), the master mix consisted of 2.5 μL of DNA template, 5 μL of 1 μM forward primer, 5 μL of 1 μM reverse primer, 12.5 mL of KAPA HiFi HotStart ReadyMix (2X) (Kapa Biosystems, Wilmington, MA), and sterile PCR-grade water, resulting in a final volume of 25 μL.

[0076] The PCR products were cleaned up using AMPure XP magnetic bead-based purification technology (Beckman Coulter). The second PCR (PCR2) reaction consisted of 5 μL of PCR1 product, 25 μL of KAPA HiFi HotStart ReadyMix (2X), 5 μL each of forward and reverse dual indexing primers (Nextera XT index Kit v2 Set A, Illumina KK, Tokyo, Japan), and 5 μL of sterile water for PCR.

[0077] Samples were purified again using AMPure beads (Beckman Coulter) and run on an Agilent Bioanalyzer to confirm quality before sequencing. PCR2 products were quantified using a Qubit dsDNA HS Assay Kit with a Qubit Fluorometer and pooled at a concentration of 5 ng / μL. The sample pool was denatured with 0.2 M sodium hydroxide and then sequenced for 600 cycles on an Illumina MiSeq sequencing system using the MiSeq Reagent Kit v3.

[0078] Bioinformatics sequence analysis was performed using the 16S rRNA flora analysis pipeline (16S Metagenomics version 1.1.0) provided by Illumina. Briefly, paired-end sequences were easily assembled by importing FASTQ files. Sequence quality was checked, and passing sequences were clustered into operational taxonomic units. The algorithm for this workflow used the Ribosomal Database Project Classifier described in Wang Q. et al. Taxonomic ranks were assigned by BLAST of the reference database (greengenes version 13_5), and abundance ratios in samples were calculated based on classified read counts.

[0079] (statistical analysis) Data are presented as mean ± SD. A p value of less than 0.05 was considered significant. All statistical analyses were performed using IBM SPSS Statistics Ver. 25 (IBM). To compare the EGCG + fructooligosaccharide or EGCG + cyclodextrin group with the EGCG alone group, the significance of the difference between the groups was determined by Dunnett's test.

[0080] <Result> (EGCG plasma concentration) Plasma EGCG was measured after deconjugation with β-glucuronidase and sulfatase. The mean plasma EGCG concentration in rats fed a 0.3% EGCG diet (EGCG alone) was 0.186 ± 0.0620 μM (see Figure 1). All cyclodextrin groups were effective in increasing plasma EGCG concentrations. The EGCG + 2.5% β-cyclodextrin group showed a significant increase in plasma EGCG concentration to 0.589 ± 0.354 μM (p < 0.05).

[0081] (EGCG levels in cecal contents and feces) The amount of EGCG in the cecal contents was increased by administering α-cyclodextrin or β-cyclodextrin to the diet (see Figure 2). The average fecal EGCG excretion rate was 13.8 ± 3.75% in mice fed a 0.3% EGCG diet (EGCG alone) (see Figure 3). It decreased in the groups fed α-cyclodextrin and γ-dextrin. The decrease was particularly significant in the EGCG + 2.5% γ-cyclodextrin group. On the other hand, no significant changes were observed in the β-cyclodextrin group.

[0082] (pH, lactic acid and SCFA levels in cecal contents) As shown in FIG. 4, the pH of the cecal contents in all cyclodextrin-administered groups was significantly lower than that in the EGCG-only-administered group (p<0.0001). The cumulative amounts of organic acids and lactic acid in the cecum also increased in all cyclodextrin-treated groups (see Figure 5).

[0083] (Analysis results of total bacterial count in feces) The total number of bacteria in the feces was estimated by the 16S rRNA copy number. The 16S rRNA copy number increased dramatically in all cyclodextrin-treated groups, with the β-cyclodextrin group showing a particularly significant increase of approximately 3.58-fold compared to the EGCG-only group (see Figure 6).

[0084] (Fecal microbiota composition) The microbial composition of rat feces was determined by amplicon sequencing of the 16S rRNA gene using Illumina MiSeq. At the genus level, the differences in the taxonomic composition of fecal microbiota between groups are shown in Figure 7 and Table 4. Among the top 20 genera, Lactobacillus was most abundant in the EGCG-only group. In the fructooligosaccharide, α-cyclodextrin, and γ-cyclodextrin groups, there was no change in the Lactobacillus genus, but a significant increase in Collinsella and Bacteorides genus was confirmed.In the β-cyclodextrin group, an increase in Lactobacillus genus, Bifidobacterium genus, and Turicibacter genus was confirmed in addition to Collinsella and Bacteorides genus.

[0085] [Table 4]

[0086] (Microbial analysis results (bacterial species analysis results)) The results of species-level classification are shown in Figure 8. In the fructooligosaccharide group, a significant increase in Collinsella aerofaciens and Bacteroides vulgatus was confirmed. In the α-cyclodextrin and γ-cyclodextrin groups, Collinsella aerofaciens and Bacteroides dorei increased. In the β-cyclodextrin group, Collinsella aerofaciens, Bacteroides dorei, Lactobacillus intestinalis, and Turicibacter sanguinis increased.

[0087] <Consideration> In this study, simultaneous administration of EGCG and three types of cyclodextrin increased plasma EGCG concentrations after two weeks of continuous administration. EGCG is presumed to be unstable at the pH of the general intestinal environment, and its residual rate is thought to decrease due to polymerization, etc.

[0088] In this example, simultaneous ingestion of each cyclodextrin resulted in a decrease in the pH in the cecum. This is thought to be due to a shift to an acidic pH, where EGCG is more stable. At this time, the amounts of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and lactic acid in the cecum increased, which are thought to be substances that contributed to the decrease in pH. These short-chain fatty acids are generally thought to be produced by intestinal bacteria through fermentation of dietary nutrients and their digestive products.

[0089] In this example, the intake of each cyclodextrin dramatically increased the number of bacterial 16S ribosomal RNA copies in feces and significantly altered the abundance of intestinal bacterial species. Therefore, we believe that the strong stimulation of the intestinal microflora promoted an increase in short-chain fatty acids, which in turn led to a decrease in intestinal pH, thereby stabilizing EGCG. Furthermore, because the abundance of different bacteria increased depending on the type of carbohydrate, such as fructooligosaccharides or cyclodextrin, we speculate that preferences differ depending on the genus and species of bacteria. Possible reasons for this include differences in the digestibility of each added carbohydrate by digestive enzymes and the ease of fermentation by intestinal bacteria.

[0090] Interestingly, fructooligosaccharides preferentially increased Bacteroides vulgatus, whereas all cyclodextrins preferentially increased Bacteroides dorei, revealing selectivity within the Bacteoides genus depending on the type of carbohydrate when co-administered with EGCG. Furthermore, among cyclodextrins, only β-cyclodextrin increased Lactobacillus intestinalis and Turicibacter sanguinis, resulting in different results. Furthermore, although not shown in the results, the Akkermansia genus increased in the group that received fructooligosaccharides, but no increase was observed in the three cyclodextrin groups. Although there are large individual differences in the intestinal flora due to factors such as genetics and dietary habits, this study revealed that the type of carbohydrate added together with EGCG clearly changes the bacteria that become dominant in the intestinal flora.

Claims

1. An agent for increasing the blood EGCG concentration accompanied by an increase in intestinal bacteria, comprising cyclic oligosaccharide and epigallocatechin gallate (also referred to as "EGCG") as active ingredients.

2. The agent for increasing the blood EGCG concentration according to Claim 1, which is for ingestion simultaneously with a meal.

3. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the cyclic oligosaccharide is β-cyclodextrin.

4. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the increasing intestinal bacteria belong to the genus Collinsella.

5. The agent for increasing the blood EGCG concentration according to Claim 4, wherein the intestinal bacteria of the genus Collinsella is Collinsella aerofaciens.

6. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the increasing intestinal bacteria belong to the genus Bacteroides.

7. The agent for increasing the blood EGCG concentration according to Claim 6, wherein the intestinal bacteria of the genus Bacteroides is Bacteroides dorei.

8. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the increasing intestinal bacteria belong to the genus Lactobacillus.

9. The agent for increasing the blood EGCG concentration according to Claim 8, wherein the intestinal bacteria of the genus Lactobacillus is Lactobacillus intestinalis.

10. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the increasing intestinal bacteria belong to the genus Turicibacter.

11. The agent for increasing the blood EGCG concentration according to Claim 10, wherein the intestinal bacteria of the genus Turicibacter is Turicibacter sanguinis.

12. The agent for increasing the blood EGCG concentration according to Claim 1, wherein the increasing intestinal bacteria belong to the genus Bifidobacterium.

13. A composition comprising the agent for increasing the blood EGCG concentration according to any one of Claims 1 to 12.

14. A method for increasing intestinal bacteria and increasing the blood EGCG concentration, characterized by ingesting cyclic oligosaccharide and EGCG simultaneously with a meal.

15. A method for increasing the number of intestinal bacteria and increasing the blood EGCG concentration after 2 weeks of continuous oral ingestion by orally ingesting a composition containing 0.03 to 5% by mass of cyclic oligosaccharide and 0.01 to 1.0% by mass of EGCG at 7.0 to 80.0 g per kg of body weight.

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