JP2023140745A - Composition for improving bioavailability of catechins - Google Patents

Abstract

To provide a novel composition for improving the bioavailability of catechins that can increase the rate at which catechins reach during systemic circulation when ingested orally, that is, the bioavailability.SOLUTION: Provided is a composition for improving the bioavailability of catechins, which contains fermentable carbohydrates as an active ingredient.SELECTED DRAWING: None

Description

There is an application for exception to loss of novelty.

The present invention provides a composition for improving the bioavailability of catechins and its active ingredients, which can increase the rate at which catechins reach the systemic circulation when ingested, that is, the bioavailability of catechins. Containing composition.

Tea catechins contained in green tea have the effect of suppressing cholesterol rise, antitumor effect, diarrheal virus infection inhibiting effect, caries preventive effect, influenza virus infection preventive effect, mycoplasma infection preventive effect, and α-amylase activity. Various pharmacological actions have been reported, including inhibitory action, action to suppress blood sugar rise, action to prevent colorectal cancer, action to prevent gastritis, gastric or duodenal ulcer, anti-arteriosclerotic action, action to suppress active oxygen generation, and action to suppress gastrin secretion.
Recently, green tea consumption has been recognized to reduce the risk of many chronic diseases, including cardiovascular disease. It is presumed that this effect is mainly due to the action of catechins contained in green tea, especially epigallocatechin gallate (also referred to as "EGCG"), which removes active oxygen species from the blood.

However, catechins, especially EGCG, are known to have low bioavailability. For example, even if a human orally ingests 50 mg of EGCG, the maximum 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.

For example, Patent Document 1 discloses that by controlling the ratio of epi-catechins in non-polymer catechins in a packaged black tea beverage containing non-polymer catechins at a high concentration, the same concentration of non-polymer catechins can be obtained. However, it has been disclosed that the amount of non-polymer catechins transferred into the blood when drunk can be further increased.

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 absorption of polyphenolic compounds. ing.

Patent Document 3 discloses that Benifuuki extract has the effect of promoting the absorption of polyphenol compounds, and by ingesting Benifuuki extract at the same time as catechins, the absorbability and residence time of catechins can be improved. It has been disclosed that this will increase.

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

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

Patent Document 6 discloses that a broom extract, a shikakai extract, a sycamore extract, a tsukinukisako extract, and a tea seed extract promote the absorption of polyphenols.

Japanese Patent Application Publication No. 2003-333989 JP2009-247282A Japanese Patent Application Publication No. 2010-11751 Japanese Patent Application Publication No. 2011-79770 JP2016-216440A Japanese Patent Application Publication No. 2017-109991

The present invention provides a new method for improving the bioavailability of catechins that can increase the rate at which catechins reach the systemic circulation when catechins are ingested orally, that is, the bioavailability of catechins. The present invention seeks to provide compositions and compositions.

The present invention proposes a composition for improving the bioavailability of catechins, which contains a fermentable carbohydrate as an active ingredient.
The invention also proposes compositions containing catechins and fermentable carbohydrates.
The present invention further proposes a method for improving the bioavailability of catechins, which comprises orally ingesting fermentable carbohydrates.

The composition for improving the bioavailability of catechins proposed by the present invention has a composition different from that previously disclosed, and the composition improves the bioavailability of catechins by reducing the rate at which the catechins reach the systemic fluid when catechins are ingested orally. can be increased.

This is a bar graph showing the relationship between the addition ratio of fructooligosaccharide (FOS) and the plasma concentration of EGCG when EGCG and fructooligosaccharide (FOS) were added to the diet in a rat oral intake test, and each point within the group is indicates values for individual rats, bar graph values indicate mean ± SD, **** indicates p<0.0001 compared to EGCG alone group. Fig. 2 is a diagram showing the relationship between the addition ratio of fructooligosaccharide (FOS) and cecal parameters when EGCG and fructooligosaccharide (FOS) were added to the diet in a rat oral ingestion test; (a) shows the relationship between the fecal content (b) shows the relationship with the lactic acid value in the cecal contents per animal, and (c) shows the SCFA concentration (Short-chain fatty level) in the cecal contents. The numbers in the bar graph indicate the mean value ± SD, each point within a group represents the value of an individual rat, and the horizontal line represents the mean value. Bar graph values indicate mean ± SD. Also, compared to the EGCG single administration group, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001. This figure shows the microbial composition of feces collected during the second week of feeding as measured by metagenomic sequencing in a rat oral ingestion test. (a) is a bar graph showing the abundance of taxonomic groups at the genus name level; (b) ) shows the proportion of major genera, and (c) shows the average value of alpha diversity (Shannon index). Also, compared to the EGCG single administration group, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001.

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

<Catechin bioavailability improving composition>
A composition for improving the bioavailability of catechins (referred to as "the composition for improving the bioavailability of catechins") according to an embodiment of the present invention contains fermentable carbohydrates as an active ingredient.

By orally ingesting fermentable carbohydrates, it is possible to increase the rate at which the orally ingested catechins reach the systemic circulation, that is, the bioavailability of the catechins. For example, if a fermentable carbohydrate or the present catechin bioavailability improving composition is orally ingested before, at the same time, or after the catechins are ingested, the bioavailability of the catechins can be increased.
In addition to the above-mentioned effects, the composition for improving catechin bioavailability also has the effect of suppressing autoxidation of catechins, maintaining the lumen in a low pH state, increasing the amount of lactic acid production, and inhibiting lactic acid-producing bacteria. It has any one of the activating effects or a combination of two or more of them.

(Catechins)
In the present invention, the "catechins" whose bioavailability is to be improved include (-) catechin (C), (-) catechin gallate (CG), (-) gallocatechin (GC), (-) gallocatechin gallate (GCG ), (-) epicatechin (EC), (-) epicatechin gallate (ECG), (-) epigallocatechin (EGC), (-) epigallocatechin gallate (EGCG), or It is a mixture consisting of a combination of two or more types.

Among the above catechins, "epigallocatechin gallate (EGCG)" is most preferred. That is, it is preferable that the main component of the catechins in the present composition for improving the bioavailability of catechins is EGCG.
In this case, "main component" means the component with the highest mass percentage among catechins, for example, 40% by mass or more of the active ingredient, especially 50% by mass or more, 60% by mass or more, and 70% by mass of the active ingredient. % or more, especially 80% by mass or more, especially 90% by mass or more, and even more than 95% by mass (including 100% by mass). degree

EGCG is a catechin with the highest content among the catechins contained in green tea, and is an ester of epigallocatechin and gallic acid.
Note that epigallocatechin gallate may be a pharmaceutically acceptable salt of epigallocatechin gallate. Examples include salts of epigallocatechin gallate with alkali metals such as sodium and potassium, and alkaline earth metals such as calcium, and quaternary ammonium salts such as ammonium salts of epigallocatechin gallate.
Furthermore, epigallocatechin gallate may be a prodrug that releases epigallocatechin gallate in vivo.

(fermentable carbohydrates)
In the present invention, "fermentable carbohydrates" refers to carbohydrates that undergo fermentation, and specifically includes, in addition to fructooligosaccharides, glucose, fructose, sucrose, maltose, lactose, etc. Examples include dietary fibers, oligosaccharides, and indigestible dextrins that are used as food products. These may be used alone or in combination of two or more of them.
Among these, fructooligosaccharides are preferred from the viewpoint of improving the bioavailability of catechins.

Fructooligosaccharides are inulin-type fructans and are known to have β bonds that are resistant to digestive enzymes in the upper intestinal tract.
Fructooligosaccharides are indigestible carbohydrates, and undigested fructooligosaccharides eventually reach the lower intestinal tract, where they become substrates for the production of lactic acid and short-chain fatty acids (also called "SCFA"). , tilts the intestinal lumen toward the acidic side and lowers the pH within the gastrointestinal tract. Maintaining the pH in the intestinal tract at a low level is believed to help protect EGCG from autoxidation.

The present composition for improving catechin bioavailability can be taken orally alone, separately from catechins. For example, the present catechin bioavailability improving composition may be orally ingested before catechins are orally ingested, or the present catechin bioavailability improving composition may be orally ingested at the same time as catechins being orally ingested. Alternatively, the catechin bioavailability improving composition may be orally ingested after catechins are orally ingested.

The present catechin bioavailability improving composition is preferably orally ingested at a ratio of 333 to 1667 parts by mass of the active ingredient per 100 parts by mass of catechins orally ingested, particularly at a ratio of 1000 parts by mass or more. It is even more preferable.

On the other hand, the present catechin bioavailability improving composition can also be orally ingested together with catechins.
For example, it is also possible to take it orally as a composition containing catechins and fermentable carbohydrates, which will be described later.

(Other ingredients)
The present composition for improving catechin bioavailability may contain other components in addition to fermentable carbohydrates, if necessary.

(Required intake and content)
In the composition for improving the bioavailability of catechins, the concentration of the fermentable carbohydrate, such as fructooligosaccharide, which is an active ingredient, is preferably 0.1 to 100% by mass, preferably 1% by mass or more, especially 10% by mass or more, Among these, it is preferably 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, and 90% by mass or more.

In the present composition for improving catechin bioavailability, the amount of intake or administration of the active ingredient is preferably adjusted as appropriate depending on the intended use. As a guideline, it can be assumed to be 1 mg to 10,000 mg at a time, particularly 5 mg or more or less than 1,000 mg at a time, and within that, 10 mg or more or less than 500 mg at a time.
The number of times of intake or administration is not particularly limited. As a guideline, it can be assumed to be taken 1 to 3 times a day, and the number of times of intake may be increased or decreased as necessary.

(safety)
Regarding the safety of this catechin bioavailability-improving composition, the active ingredient, fermentable carbohydrate, has been orally ingested by humans for many years, so its safety cannot be guaranteed from the perspective of dietary experience. I can say that there is.

<This composition>
This composition is a composition containing catechins and fermentable carbohydrates.
If this composition containing catechins and fermentable carbohydrates is orally ingested, the bioavailability of the catechins can be increased.
In addition to the above-mentioned effects, this composition also has the effects of suppressing autoxidation of catechins, maintaining the lumen in a low pH state, increasing lactic acid production, and activating lactic acid-producing bacteria. It has either one of these or two or more of them.

The fermentable carbohydrate in this composition is as described above.
In the present composition, the content of fermentable carbohydrates is preferably 333 to 1,667 parts by mass, and more preferably 1,000 parts by mass or more, based on 100 parts by mass of catechins.

The catechins in the present composition may be pure C, CG, GC, GCG, EC, ECG, EGC, or EGCG, or may be a mixture of two or more of these. , and may also be a composition containing catechins.
Examples of compositions containing catechins include green tea extracts and purified products thereof. As a specific example, an extract obtained by hot water extraction of green tea is separated and dried using an adsorption column using water and low and high concentration alcohol, and the tea polyphenol concentration is approximately 85 to 99.5. An example of this is a green tea extract prepared in a concentration of For example, "Tearflan 90S (trade name; manufactured by ITO EN Co., Ltd.)" is a preferable example. This Theafuran 90S has an amount of ester type catechins of 50 to 90% by mass based on the total amount of catechins, an amount of EGCG of 40 to 90% by mass of the total amount of catechins, and a caffeine content also based on the total amount of catechins. It is 0 to 2% by mass.

(form)
The present catechin bioavailability improving composition and the present composition can be provided as, for example, a drug as an orally administered agent, a quasi-drug, a nutritional supplement, a food or drink, and the like.
In this case, examples of the form include liquid preparations, tablets, powders, granules, sugar-coated tablets, capsules, suspensions, emulsions, and pills.

The catechin bioavailability improving composition and the present composition contain additives commonly used in pharmaceuticals, quasi-drugs, and nutritional supplements, such as excipients, fillers, binders, wetting agents, and disintegrants. , a surfactant, a lubricant, a dispersant, a buffer, a preservative, a solubilizing agent, a preservative, a flavoring agent, a soothing agent, a stabilizer, and the like. Also, for example, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or its salts, gum arabic, polyethylene glycol, syrup, vaseline, glycerin, ethanol, propylene glycol, citric acid, chloride. It is also possible to incorporate non-toxic additives such as sodium, sodium sulfite and sodium phosphate.
In addition, when preparing it as a quasi-drug, it can be made easier to ingest by making it into a drinking form such as a bottled drink, or a form such as a tablet, capsule, or granule.

When providing this catechin bioavailability improving composition and this composition as food or drink, foods for specified health uses, foods with nutritional function claims, foods with functional claims, so-called health foods (functional foods, health supplements), soft drinks, etc. It can be provided as water. However, it is not limited to these.
In this case, it is also possible to label the food and drink as having the pharmacological effects of catechins.

Preferred forms of food and drink include, for example, candies, jellies, tablets, drinks, soups, noodles, rice crackers, Japanese sweets, frozen desserts, and baked sweets. 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.

<Explanation of words>
In the present invention, when expressed as "X to Y" (X, Y are arbitrary numbers), unless otherwise specified, it means "more than or equal to X and less than or equal to Y", and also means "preferably greater than It also includes the meaning of "small".
In addition, when expressed as "more than or equal to X" (X is any number) or "less than or equal to Y" (Y is any number), the expression "preferably greater than X" or "preferably less than Y" should be used. It also includes intent.

The present invention will be explained in more detail below using Examples. However, the present invention is not limited to the examples.
In the following examples, "%" indicates "% by mass" unless otherwise specified.

<Animal test>
We investigated whether the addition of fructooligosaccharides in the feed contributes to the stabilization of EGCG in the gastrointestinal tract in an animal model and consequently affects the plasma concentration of EGCG.

(material)
The following raw materials were used in the following examples and comparative examples.
・High purity extract of EGCG (EGCG concentration 94% by mass or more, "Teavigo" manufactured by DSM Nutrition Japan Co., Ltd.)
・Fructooligosaccharides (1-kestose 39.7%, nystose 51.5%, 1 ^F -βfructofuranosyl-nystose 4.9%, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
・β-glucuronidase from Helix pomatia and sulfatase from abalone internal organs (manufactured by Sigma-Aldrich)

(animals and diet)
Twenty-five male Wistar rats (4 weeks old) were acclimatized for 2 days in stainless steel cages at 22°C in an automatically controlled room with a 12 hour light cycle. During the acclimatization period, rats were fed AIN93G-containing diet.
After that, the 25 animals were divided into four groups. "0.3% EGCG + 1% fructooligosaccharide feed" with addition of fructooligosaccharide, "0.3% EGCG + 3% fructooligosaccharide feed" with addition of 0.3% EGCG and 3% fructooligosaccharide to 100% feed, "0.3% EGCG + 5% fructooligosaccharide feed" in which 0.3% EGCG and 5% fructooligosaccharide were added to 100% feed was given as an experimental diet (Table S1). Each animal was given free access to the experimental food and tap water for two weeks. Feces were collected every week.
On the final day of the experiment, rats were humanely killed by inhalation of high concentrations of carbon dioxide, and blood was immediately collected from the peritoneal vein. After centrifuging at 2,000 xg for 10 minutes to obtain plasma, 10 mg of L-ascorbic acid was placed in a plastic tube and stored at -40°C. The cecum was removed and the cecal contents were also collected and stored at -40°C until use.

(Analysis of EGCG in plasma)
Analysis of EGCG concentration in plasma was performed as follows.
0.9 mL of 100 mM acetate buffer (pH 5.0), 10 μL of β-glucuronidase solution, and 10 μL of sulfatase solution were added to 0.1 mL of thawed plasma, mixed, and incubated at 37° C. for 45 minutes. After adding 10 μL of a 20 μM ethyl gallate solution as an internal standard, the reaction mixture was directly applied to a polymer-based solid phase extraction cartridge (Strata-X, 33 μm particle size, Phenomenex, Inc., CA). The cartridge was pre-cleaned 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 eluted with 0.7 mL of 70% (v/v) DMF containing 0.1% (v/v) phosphoric acid.
After filtration with a 0.45 μm syringe filter (TORAST disk, Shimadzu GLC, Ltd.), 10 μL of the obtained filtrate was injected into an HPLC system equipped with an electrochemical detector (Coulochem III, ESA, Inc., MA). did. The analytical column (Capcell Pak 3C ₁₈ type AQ, length 150 mm x inner diameter 4.6 mm, Shiseido Co., Ltd.) was used with 0.5 mM ethylenediamine-N,N,N',N'-tetraacetic acid at a flow rate of 0.8 mL/min. Elution was performed with a solvent of 50 mM sodium dihydrogen phosphate (adjusted to pH 3.5 with phosphoric acid) containing (EDTA)/acetonitrile (85/15, v/v).
The column temperature was kept at 40°C. The voltages applied to the analysis cells were −200 mV for electrode 1, +200 mV for electrode 2, and +250 mV for the guard cell.

(Analysis of EGCG in feces and cecal contents)
Freeze-dried feces were ground with a grinder mill, and 50 mg of the resulting powdered feces was placed in a microtube and mixed with 1 mL of 50% (v/v) acetonitrile containing 0.1% (v/v) phosphoric acid. . After putting stainless steel beads into the tube, EGCG was extracted by stirring for 3 minutes with a bead beater (Cell Destroyer PS1000, Biomedical Science Co., Ltd.), and the extract was collected in a 10 mL flask. This extraction operation was repeated three times.
After filtration with a syringe filter, the filtrate was injected into the HPLC system. EGCG was purified with 15% (v/v) acetonitrile containing 0.1% (v/v) phosphoric acid using a Unison UK-C ₁₈ column (100 mm length x 4.6 mm inner diameter, Imtakt Co., Kyoto, Japan). It was separated and detected with an ultraviolet detector set at 230 nm.
On the other hand, for measurement of cecal contents, fecal digest (0.2 mL) diluted 5 times was added to 0.2 mL of acetonitrile containing 0.1% (v/v) phosphoric acid, and the mixture was heated at 2000 × g After centrifugation for 5 minutes at , the resulting supernatant was filtered and injected into the HPLC system.

(Analysis of pH, lactic acid and SCFA of cecal contents)
The pH value of the thawed cecal contents was directly measured using a portable pH meter.
To measure lactate and SCFA in the cecal contents, a portion of the cecal contents was diluted 5 times with distilled water. The lactic acid concentration in the sample was measured using a commercially available kit (Lactate Assay Kit-WST, Dojindo Laboratories). SCFA (acetic acid, propionic acid, butyric acid) concentration in the sample was determined using a commercially available kit (YMC Co. Ltd., Kyoto, Japan) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. , derivatized with 2-nitrophenylhydrazine hydrochloride, and then measured by HPLC.

(Analysis of microorganisms)
Bacterial DNA was extracted using a fecal DNA isolation and purification kit (Norgen Biotec Co., ON, Canada) according to the manufacturer's protocol. Thereafter, DNA (5 ng/μL) was prepared by diluting with Tris-EDTA buffer (pH 8.5).
An amplicon library was prepared by amplifying the V3-V4 region of the 16S rRNA gene based on the 16S metagenomic sequencing library protocol (Illumina Corporation, Tokyo, Japan). For the first PCR (PCR1), the master mix was 2.5 μL of DNA template, 5 μL of forward primer (1 μm), 5 μL of reverse primer (1 μm), and 12.5 KAPA HiFi HotStart ReadyMix (2X) (Kapa Biosystems, Wil/mingtonmA) and sterile water for PCR, and the final volume was 25 μL.
PCR products were cleaned up using AMPure XP magnetic bead-based purification technology (Beckman Coulter). For the second PCR (PCR2) reaction, 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, Illu/minna KK, Tokyo, Japan), and 5 μL of sterile water for PCR.
Samples were purified again using AMPure beads (Beckman Coulter), run on an Agilent Bioanalyzer to confirm quality, and then sequenced.
PCR2 products were quantified using the Qubit dsDNA HS Assay Kit with Qubit Fluorometer and pooled at a concentration of 5 ng/μL.
After denaturing the sample pool with 0.2M sodium hydroxide, it was sequenced for 600 cycles on the Illu/minna MiSeq sequencing system using MiSeq Reagent Kit v3.
Bioinformatics sequence analysis was performed using the 16S rRNA flora analysis pipeline (16S Metagenomics version 1.1.0) provided by Illumina. Simply put, we were able to easily assemble paired-end sequences by importing FASTQ files. Sequence quality was checked and passing sequences were clustered into operational taxonomic units. The algorithm for this workflow was the Ribozamal Database Project Classifier described in Wang Q. et al.
Taxonomic ranks were assigned by BLAST of the reference database (greengenes version13_5) and Shannon index was calculated based on read counts of identified species.

(Statistical analysis)
Data are shown as mean ± SD. A P value of less than 0.05 was considered significant.
All statistical analyzes were performed using GraphPad Prism version 7 (GraphPad Software, San Diego, CA).
Normality of data was confirmed using the Shapiro-Wilk test. In order to compare the EGCG + fructooligosaccharide administration group and the EGCG alone administration group, the significance of the difference between the groups was determined by Dunnett's test or Dunn's test.

<Results>
(EGCG plasma concentration)
EGCG in plasma was measured after deconjugation with β-glucuronidase and sulfatase. The mean plasma concentration of EGCG in rats fed 0.3% EGCG diet (EGCG alone) was 0.21±0.05 μM (Figure 1). Addition of fructooligosaccharide had the effect of increasing the plasma concentration of EGCG in a dose-dependent manner. Addition of 5% (w/w) fructooligosaccharides to the diet significantly increased plasma EGCG concentration to 0.65±0.12 μM (p<0.0001).

(EGCG levels in cecal contents and feces)
The amount of EGCG in the cecal contents was significantly increased by dietary administration of fructooligosaccharides (FOS) in a dose-dependent manner (Table 1). The EGCG concentration in the caecal gastrointestinal tract of rats fed the EGCG + 5% fructooligosaccharide (FOS) diet was significantly increased compared to the EGCG alone diet group (p<0.0001).
The amount of EGCG in the cecal contents of each rat per rat was calculated by multiplying the EGCG concentration (the amount of EGCG per 1 g of cecal contents (μmol/g) by the wet weight of each individual cecal contents.As a result, the fecal content It was found that the amount of EGCG in feces increases dramatically depending on the amount of food.
The average dry weight of feces collected over two weeks in the EGCG + 5% fructooligosaccharide (FOS) group was significantly increased compared to the EGCG alone diet group (p<0.05) (Table 1).
In contrast to the increase in cecal digesta volume, no significant increase was observed when 1% and 3% fructooligosaccharides (FOS) were added to the diet, but EGCG + 5% fructooligosaccharides (FOS) diet was not significantly increased. The cumulative amount of EGCG excreted in rat feces was significantly increased compared to the EGCG alone diet (p<0.0001) (Table 1).
The fecal excretion rate of EGCG, which is calculated as the ratio of the amount of unchanged EGCG excreted in the feces to the amount of EGCG ingested from the diet, was estimated to be 3.86% on average when fed with EGCG alone; In the case of the EGCG + 5% fructooligosaccharide diet, it significantly increased to 21.83%.

(pH, lactic acid and SCFA levels of cecal contents)
The pH of the cecal contents in all fructooligosaccharide administration groups was significantly lower than that in the EGCG alone administration group, and the p-values were 0.0 and 1%, respectively, in the 1%, 3%, and 5% fructooligosaccharide (FOS) administration groups. 01, less than 0.0001, and less than 0.0001 (FIG. 2(a)). Conversely, when fructooligosaccharides were added to the diet, cecal lactic acid levels increased in a dose-dependent manner (Figure 2(b)).
The cecal contents of the EGCG + 5% fructooligosaccharide (FOS) group had an approximately 10-fold increase in the amount of lactic acid per rat compared to the EGCG alone group (p<0.0001).
On the other hand, dietary treatment with fructooligosaccharides had little relationship with cecal content levels of SCFA. There was no statistically significant difference in the fecal concentrations of acetic acid, propionic acid, and butyric acid between the groups (Figure 2(c)).

(Composition of fecal microflora)
The microbial composition of rat feces was determined by 16S rRNA gene amplicon sequencing using Illumina MiSeq. At the genus level, the differences in the taxonomic composition of the fecal microbiota between groups are displayed in Figure 3(a). Among the top 20 genera, Lactobacillus was included most frequently in the EGCG alone administration group.
Addition of fructooligosaccharides tended to increase the relative abundance of Lactobacillus. Collinsella increased dramatically upon administration of fructooligosaccharide, accounting for approximately 40% of the total abundance in the EGCG+5% fructooligosaccharide (FOS) group.
In the EGCG+5% fructooligosaccharide (FOS) group, only three genera, Lactobacillus, Collinsella, and Bacteroides, accounted for 90% of the total (Figure 3(b)).
When calculating the diversity of the fecal microbiome of each individual, it was significantly reduced with the addition of fructooligosaccharides compared to EGCG alone. Furthermore, a decrease in the Shannon index was observed in inverse proportion to the concentration of fructooligosaccharide added (FIG. 3(c)).

<Consideration>
It is known that catechins, especially EGCG, have low bioavailability. Factors contributing to the low bioavailability of catechins, such as EGCG, include EGCG undergoing autoxidation and polymerization, inactivation due to interaction with metal ions and milk proteins, methylation by liver enzymes and glucuronin. Examples include conjugation with acids and sulfuric acid, and decomposition into phenolic acids by intestinal bacteria. In fact, the intestinal absorption rate of theasinensin A, which has become a dimer of EGCG through autooxidation, is estimated to be one-tenth that of EGCG. In this sense, it can be assumed that preventing autoxidation of EGCG in the gastrointestinal tract is a factor in improving the bioavailability of EGCG. Considering that autoxidation occurs at high pH, it is thought that EGCG can be stabilized to some extent by lowering the pH within the lumen.

In this study, we investigated the combined effect of food factors that can lower intraluminal pH to increase the intestinal absorption rate of EGCG in rats.
Focusing on the fact that fermentable carbohydrates acidify the intraluminal pH, we selected fructooligosaccharides as a candidate for increasing the plasma concentration of EGCG. Fructooligosaccharides are well known as prebiotics that have beneficial effects on the host's intestinal environment. In rats, it has been revealed that ingestion of fructooligosaccharides promoted the production of lactic acid and SCFA by microorganisms, and lowered the intraluminal pH value. In this study, we revealed that in rats administered fructooligosaccharide, the pH value decreased in a dose-dependent manner, and the fermentative use of fructooligosaccharide by intestinal bacteria and the production of lactic acid lowered the pH value of the luminal lumen. This is considered to have made a significant contribution.

Furthermore, as a result of analyzing the microbial composition in feces, it was found that treatment using fructooligosaccharide and EGCG in combination increased the abundance of Lactobacillus and Collinsella genera. These two genera are thought to produce lactic acid. It can be seen that the increased production of lactic acid by bacteria of both genera is associated with a decrease in the pH value of the rat caecal gastrointestinal tract. Furthermore, it is known that fructooligosaccharide is fermented and converted not only to lactic acid but also to SCFA. However, in this test, no significant difference was observed in the total amount of SCFA (the sum of acetic acid, propionic acid, and butyric acid) regardless of the amount of fructooligosaccharide added. This is presumed to be because EGCG is involved in SCFA production by microorganisms.

Addition of fructooligosaccharides reduced the α-diversity of the fecal microbiota in a dose-dependent manner. This is thought to be because bacterial species with a high ability to utilize fructooligosaccharides preferentially proliferated. In particular, the explosive presence of Collinsella and Lactobacillus led to a decline in other microbial communities in the gut. Considering the past findings that the α-diversity index of rats did not decrease even after 12 weeks of ingestion of 15% (w/w) fructooligosaccharides, simultaneous intake of EGCG and fructooligosaccharides significantly reduced α-diversity. may have caused.

This study also revealed that adding fructooligosaccharides to the diet increased the amount of EGCG in the cecal contents. This is considered to mean that dietary fructooligosaccharides prevented autoxidation of EGCG in the gastrointestinal tract, and more EGCG was able to reach the cecum intact. It is possible that microorganisms produce lactic acid from fructooligosaccharides and acidify the lumen, which helps stabilize EGCG. Such a pH-lowering effect of fructooligosaccharides has been observed not only in the large intestine but also in the small intestine. In mice, the microbial community of the lower small intestine is dominated by the Lactobacillaceae family. From this, it is inferred that some of the fructooligosaccharides fermented and utilized by the bacteria also lived in the small intestine, which is the active site of intestinal absorption of EGCG. Since the amount of EGCG in the intestine is highly correlated with the plasma concentration of EGCG, it is highly conceivable that acidification stabilizes EGCG and increases intestinal absorption.

In this study, it was found that increasing the stability of EGCG in an acidified intestinal environment is a factor that improves bioavailability. In the above test, fructooligosaccharide was used as an example of a substance that has the effect of lowering the intraluminal pH value, but other fermentable carbohydrates can be considered to perform a similar function.
The uptake of EGCG into epithelial cells was thought to be through passive diffusion, but in recent years it has been discovered that a specific transporter responsible for intestinal absorption of EGCG is actively expressed on the surface of ileal epithelial cells. did. Delivery of EGCG to ileal epithelial cells is believed to be useful for increasing the understanding of the beneficial health effects expected from increasing plasma concentrations of EGCG.

The following is a summary of the above considerations.
Among catechins, (-)-epigallocatechin gallate (EGCG) undergoes autooxidation at physiological pH, and therefore may be hardly absorbed in the intestine. Fructooligosaccharides (fructooligosaccharides) are fermented by intestinal bacteria and mainly converted into lactic acid. This study was conducted to determine whether dietary fructooligosaccharides help increase plasma concentrations of EGCG in rats by preventing autooxidation. Rats were placed on their assigned diet for two weeks on either a 0.3% (w/w) EGCG diet or an EGCG diet supplemented with 1%, 3% or 5% (w/w) fructooligosaccharides. Ingested. As a result, the plasma concentration of EGCG was 0.21±0.05 μM in the EGCG alone group, and was significantly higher at 0.65±0.12 μM in the EGCG+5% fructooligosaccharide group. Fructooligosaccharide treatment dose-dependently increased lactic acid levels in the cecal contents, decreased the pH of the cecal contents, and altered the abundance of Lactobacillus and Collinsella. Since the EGCG concentration in the cecal contents of rats fed the fructooligosaccharide-containing diet remained at a relatively high level, it is likely that fructooligosaccharides contributed to protecting EGCG from autooxidation. In conclusion, it appears that fructooligosaccharides lowered the pH of the lumen of the intestinal tract, leaving EGCG intact to some extent, thereby allowing EGCG to be taken up from the intestine into the blood circulation.
Although the above test targeted EGCG, it is known that other catechins have the same pH stability as EGCG, so it is assumed that the same effects as EGCG can be obtained with other catechins as well. It is inferred.

Claims (14)

A composition for improving the bioavailability of catechins, which contains fermentable carbohydrates as an active ingredient. The composition for improving bioavailability of catechins according to claim 1, wherein the fermentable carbohydrate is a fructooligosaccharide. The composition for improving the bioavailability of catechins according to claim 1 or 2, wherein the catechins are epigallocatechin gallate. The composition for improving the bioavailability of catechins according to any one of claims 1 to 3, which also has the effect of suppressing autoxidation of catechins. The composition for improving the bioavailability of catechins according to any one of claims 1 to 4, which also has the effect of maintaining the lumen in a low pH state. The composition for improving bioavailability of catechins according to any one of claims 1 to 5, which also has the effect of increasing lactic acid production. The composition for improving the bioavailability of catechins according to any one of claims 1 to 6, which also has the effect of activating lactic acid-producing bacteria. The composition for improving bioavailability of catechins according to claim 7, wherein the lactic acid-producing bacterium belongs to the genus Lactobacillus or the genus Collinsella. A composition containing catechins and fermentable carbohydrates. The composition according to claim 9, wherein the content of fermentable carbohydrate is 333 to 1667 parts by mass per 100 parts by mass of catechins. The composition according to claim 9 or 10, wherein the fermentable carbohydrate is a fructooligosaccharide. The composition according to any one of claims 9 to 11, wherein the catechins are epigallocatechin gallate. A method for improving the bioavailability of catechins, which comprises orally ingesting fermentable carbohydrates. The method for improving the bioavailability of catechins according to claim 13, wherein the fermentable carbohydrate is a fructooligosaccharide.

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