JP7483958B2 - Intestinal environment improving agent and β-glucuronidase activity inhibitor - Google Patents

Description

The present invention relates to an intestinal environment improving agent and a β-glucuronidase activity inhibitor.

Flavonoids are substances found in large quantities in plants in general, especially in the young fruits and peels of citrus fruits. They are known to have physiological effects such as anti-inflammatory, anti-allergic, and antihypertensive properties, as well as UV absorbing and antioxidant properties. Flavonoids have poor solubility in water, which means they are difficult to absorb by the body when ingested. For this reason, attention has been focused on water-soluble flavonoids, which have been made more soluble in water by transferring α-glucose to flavonoids through a glycosyltransfer reaction, and are expected to be applied to foods, cosmetics, and pharmaceuticals. For example, quercetin, a type of flavonoid, is widely used as an ingredient in foods and pharmaceuticals.

Patent Document 1 describes a supplement for improving the intestinal environment and the intestinal barrier, which contains quercetin as a functional nutritional component that has an anti-inflammatory effect in the intestinal tract.
Furthermore, Patent Document 2 describes that a mixture of a plant extract and prebiotics promotes the growth of bifidobacteria and lactic acid bacteria, and describes a nutritional composition containing (iso)quercetin glycoside as one of the active ingredients contained in an extract of Ginkgo Bioloba (ginkgo tree), which is an example of a plant extract rich in polyphenols.

JP 2014-079208 A JP 2008-500032 A

As shown in Patent Document 1, supplements that improve the intestinal environment and intestinal barrier, which contain quercetin as a functional nutritional component, are known, but quercetin glycoside has not been studied. Also, as shown in Patent Document 2, nutritional compositions that contain (iso)quercetin glycoside are known, but there are many unknowns regarding their functionality in the intestine. In particular, there are many unknowns regarding the effect of quercetin glycoside on the intestinal flora.

The objective of the present invention is to investigate the effects of quercetin glycoside on the intestinal flora and other effects on the intestine, and to provide new uses for quercetin glycoside.

The inventors have investigated the functionality of quercetin glycoside in the intestine, particularly in the large intestine, and have found that quercetin glycoside has the effect of improving the intestinal environment and also has the ability to inhibit β-glucuronidase activity.

That is, the present invention is as follows.
[1] An intestinal environment improving agent containing quercetin glycoside.
[2] The intestinal environment improving agent according to claim 1, which is for oral intake.
[3] The intestinal environment improving agent according to [1] or [2], which contains α-glucosylrutin as a quercetin glycoside.
[4] An intestinal environment improving agent according to any one of [1] to [3] for relatively increasing the proportion of neutral bacteria in the large intestine.
[5] An intestinal environment improving agent described in any one of [1] to [4] for relatively increasing the proportion of beneficial bacteria in the large intestine.
[6] A food or medicine containing the intestinal environment improving agent according to any one of [1] to [5].
[7] A β-glucuronidase activity inhibitor, comprising a quercetin glycoside.
[8] The β-glucuronidase activity inhibitor according to [7], which contains at least one selected from isoquercitrin, rutin, and α-glucosylrutin as the quercetin glycoside.
[9] The β-glucuronidase activity inhibitor according to [7] or [8], which contains α-glucosylrutin as a quercetin glycoside.
[10] A food or pharmaceutical comprising the β-glucuronidase activity inhibitor according to any one of [7] to [9].

According to the present invention, it is possible to provide an intestinal environment improving agent containing quercetin glycoside, and a β-glucuronidase activity inhibitor containing quercetin glycoside. In particular, when quercetin glycoside is orally ingested, it is not absorbed in the small intestine and tends to reach the large intestine, where it is broken down and absorbed, thereby improving the proportion of neutral and/or beneficial bacteria in the large intestine and inhibiting β-glucuronidase activity.

FIG. 1 shows an experimental model for the small intestinal epithelial permeability test. FIG. 2 shows the results of a small intestinal epithelial permeation test. FIG. 3 shows the results of the lactic acid bacteria proliferation count in the lactic acid bacteria culture liquid test. FIG. 4 shows the relative DNA abundance of each enterobacteria group in mouse feces. FIG. 5 shows the results of a β-glucuronidase activity inhibition test.

The intestinal environment improving agent and β-glucuronidase activity inhibitor of the present invention will now be described.
[Intestinal environment improvement agent]
The intestinal environment improving agent of the present invention contains a quercetin glycoside and, if necessary, further contains other ingredients. The intestinal environment improving agent has an effect of improving the intestinal environment (intestinal environment improving effect) and is used for improving the intestinal environment.

Here, in this specification, "intestine" when used in the context of "intestine" means the small intestine and/or large intestine. The small intestine includes the duodenum, jejunum, and ileum. The large intestine includes the cecum, colon, and rectum. Furthermore, when improving the intestinal environment according to the present invention, it is more preferable that the "intestine" includes the large intestine. In other words, the intestinal environment improving agent of the present invention is preferably an agent for improving the large intestinal environment.

The intestinal environment improving effect of the present invention refers to an effect of relatively increasing beneficial or neutral bacteria in the intestinal flora, and refers to an effect of relatively increasing at least one selected from the genera Lactobacillus and Bifidobacterium, which are known as beneficial bacteria, and the phyla Bacteroides and Firmicutes, which are known as neutral bacteria. Note that although the genus names are generally written in italics, they are written in roman type in the text.

The intestinal environment improving effect of the present invention is preferably an effect of relatively increasing at least one of the phyla Bacteroides and Firmicutes, which are known as neutral bacteria, among these bacteria. The relative bacterial mass of the phylum Firmicutes as neutral bacteria is the value obtained by subtracting the relative bacterial mass of the genus Lactobacillus and the Clostridium cluster XIVa group from the entire phylum Firmicutes.

<Quercetin glycoside>
Quercetin glycosides are compounds in which various sugars are bound to quercetin, a type of flavonoid, and examples thereof include isoquercitrin, rutin, α-glucosylrutin, and α-glucosylisoquercitrin.

The quercetin glycoside used in the present invention is not particularly limited in terms of its origin or production method. As a preferred embodiment of the quercetin glycoside, a quercetin glycoside obtained by adding glucose to rutin or isoquercitrin by the action of a glycosyltransferase in the presence of a glycosyl donor can be used. Quercetin glycoside is also known to be contained in onions, Chinese sophora japonica, buckwheat, apples, tea, grapes, broccoli, petit vert, leafy vegetables, and citrus fruits, and extracts of these plants can be used.

As quercetin glycosides, rutin, α-glucosyl rutin, and enzyme-treated rutin (hereinafter also referred to as "enzyme-treated rutin") are preferred because they have a strong effect of relatively increasing the number of beneficial or neutral bacteria in the intestinal flora, with α-glucosyl rutin and enzyme-treated rutin being more preferred. Enzyme-treated rutin is rutin to which glucose has been added using a glycosyltransferase, contains α-glucosyl rutin, and has significantly higher water solubility than rutin.

As shown in the small intestinal epithelium permeability test described later, when the number of sugars bound to quercetin in quercetin glycoside increases, the small intestinal epithelium permeability decreases, and when quercetin glycoside is orally ingested, it is likely to reach the large intestine without being absorbed in the small intestine, making it useful as an agent for improving the large intestinal environment. Therefore, rutin, α-glucosylrutin, and enzyme-treated rutin containing α-glucosylrutin are preferred, and α-glucosylrutin and enzyme-treated rutin containing α-glucosylrutin are more preferred.
The quercetin glycosides may be used alone or in combination of two or more.

The intestinal environment improving agent of the present invention contains quercetin glycoside as an active ingredient. The content of quercetin glycoside is preferably 30 to 100% by mass, more preferably 60 to 100% by mass, and even more preferably 75 to 100% by mass, based on 100% by mass of the intestinal environment improving agent of the present invention.

<α-Glucosylrutin and enzyme-treated rutin>
α-Glucosylrutin is, for example, represented by the following formula, that is, a compound in which one or more (about 2 to 20) glucose residues are bound to a glucose residue in a rutinose residue of rutin via an α1→4 bond.

In the above formula, n is 1 or an integer of 2 to 20, preferably 1 or an integer of 2 to 10, and more preferably 1.
In this specification, among α-glucosylrutin, a compound with only one glucose bonded thereto is referred to as "α-monoglucosylrutin", and a compound with two or more glucose bonded thereto is referred to as "α-polyglucosylrutin". Since the molecular weight of α-monoglucosylrutin is smaller than that of α-polyglucosylrutin, the number of molecules per unit mass of α-monoglucosylrutin is greater, which is considered to be advantageous in terms of action and effect. Therefore, it is preferable that a part or all of α-glucosylrutin is α-monoglucosylrutin. The α-monoglucosylrutin is preferably 10 to 100% by mass, more preferably 50 to 100% by mass, and even more preferably 90 to 100% by mass in the α-glucosylrutin.

Among the enzyme-treated rutins, α-glucosylrutin has a particularly strong effect of increasing the neutral bacteria in the human intestinal flora, and also has particularly high water solubility.
Enzyme-treated rutin is a product (also referred to as "first enzyme-treated rutin" in this specification) obtained by reacting rutin with a glycosyltransferase in the presence of an α-glucosyl sugar compound. Examples of the α-glucosyl sugar compound include cyclodextrin and partial starch hydrolysates. The glycosyltransferase is an enzyme that has the function of adding glucose to rutin, such as cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19).

The first enzyme-treated rutin is a composition that contains an aggregate of various α-glucosylrutins with different numbers of bound glucose, i.e., α-monoglucosylrutin and other α-polyglucosylrutins, and unreacted rutin.

If necessary, the first enzyme-treated rutin can be purified using, for example, a porous synthetic adsorbent and an appropriate elution solution to remove α-glucosyl sugar compounds and other impurities, thereby reducing the rutin content and obtaining first enzyme-treated rutin (purified α-glucosylrutin) with an increased purity of α-glucosylrutin.

The first enzyme-treated rutin may also be treated with an enzyme having glucoamylase activity that cleaves α-1,4-glucosidic bonds into glucose units, such as glucoamylase (EC 3.2.1.3
), in α-polyglucosylrutin to which multiple glucose residues have been added, by cleaving off all glucose residues except for one glucose residue that has been directly added to the glucose residue (in the rutinose residue) of rutin itself, it is possible to obtain enzyme-treated rutin (also referred to as "second enzyme-treated rutin" in this specification) that contains a large amount of α-monoglucosylrutin. This enzyme treatment does not cleave the glucose residue in the rutinose residue that is directly bonded to the quercetin skeleton from the quercetin skeleton.

The amount of rutin in the enzyme-treated rutin is preferably 10 to 85% by mass, more preferably 40 to 85% by mass, and even more preferably 70 to 85% by mass. In this way, the enzyme-treated rutin obtained as described above may contain, for example, a small amount of unreacted rutin or quercetin, which is a decomposition product of rutin, together with α-glucosylrutin. It may also contain isoquercitrin, which has a structure in which a rhamnose residue has been removed from rutin.
The amount of rutin can be calculated in accordance with the description of the quantitative method for enzyme-treated rutin on page 202 of Voluntary Standards for Food Additives Other Than Chemically Synthesized Products, Second Edition (Published: October 1, 1993, Edited by Voluntary Standards Committee of the Japan Food Additives Association, Published by the Japan Food Additives Association). A rutin standard solution with a known concentration can be prepared using a commercially available rutin reagent (e.g., manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

Such enzyme-treated rutin is not particularly limited, but specific examples include αG Rutin PS (trade name, rutin equivalent amount 80-86% by mass, manufactured by Toyo Sugar Refining Co., Ltd.), αG Rutin P (trade name, rutin equivalent amount 40-46% by mass, manufactured by Toyo Sugar Refining Co., Ltd.), and αG Rutin H (trade name, rutin equivalent amount 22-26% by mass, manufactured by Toyo Sugar Refining Co., Ltd.). These commercially available products may be used as is, or may be purified.

Other Ingredients
The intestinal environment improving agent of the present invention may contain not only quercetin glycoside but also other components. Examples of the other components include binders for preparing the improving agent in a solid form, specifically cyclodextrin, dextrin, water-soluble dietary fiber, starch, etc.; and solvents for preparing the improving agent in a liquid form, specifically water, alcohol (e.g., ethyl alcohol, glycerin, propylene glycol). Examples of the other components include other pharmacy-acceptable additives.

[β-glucuronidase activity inhibitor]
The β-glucuronidase activity inhibitor of the present invention contains a quercetin glycoside. The quercetin glycoside is the same as the specific and preferred examples described in the column of [Intestinal environment improving agent], and is preferably at least one selected from isoquercitrin, rutin, and α-glucosylrutin, more preferably α-glucosylrutin. The α-glucosylrutin is preferably α-glucosylrutin contained in enzyme-treated rutin. In addition, it is preferable that the α-glucosylrutin is partly or entirely α-monoglucosylrutin.
The quercetin glycoside may be used alone or in combination of two or more kinds.

β-glucuronidase is an enzyme that hydrolyzes compounds (glucuronides) in which various alcohols, phenols, amines, etc. are conjugated with glucuronic acid, and is present in many organisms, including bacteria, fungi, plants, and animals.

Glucuronidation usually occurs when carcinogens are excreted from the body. Carcinogens are inactivated by glucuronidation in the liver, excreted in the bile, and then excreted from the body via the intestinal tract. However, it is known that conjugated carcinogens are hydrolyzed by β-glucuronidase produced by intestinal bacteria, becoming deconjugated carcinogens and being released, thereby increasing the risk of colon cancer.

Known β-glucuronidases are, for example, those derived from the genus Clostridium and from Escherichia coli.
In clinical practice, it is known that colon cancer patients have higher β-glucuronidase activity than healthy individuals, and that the intestinal bacteria Clostridium genus having β-glucuronidase activity are more prevalent in colon cancer patients than in healthy individuals. It is also known that β-glucuronidase activity is significantly increased when a typical Japanese dieter is made to consume a Western diet mainly consisting of meat, and that it is correlated with the fluctuation of the Clostridium genus. In the examples described below, β-glucuronidase derived from Escherichia coli Type VII-A was used.

The β-glucuronidase activity inhibitor of the present invention can inhibit the activity of β-glucuronidase derived from intestinal bacteria in the body, particularly Escherichia coli, and therefore can reduce the risk of colon cancer.

The β-glucuronidase activity inhibitor of the present invention contains quercetin glycoside as an active ingredient. The content of quercetin glycoside is preferably 30 to 100% by mass, more preferably 60 to 100% by mass, and even more preferably 75 to 100% by mass, based on 100% by mass of the β-glucuronidase activity inhibitor of the present invention.
Furthermore, the β-glucuronidase activity inhibitor of the present invention may contain other ingredients as described in the section [Intestinal environment improving agent].

[Specific uses]
The intestinal environment improving agent and β-glucuronidase activity inhibitor of the present invention can be added to various foods and used. Also, they can be used in various dosage forms (solution, tablet, powder, etc.) and methods of use (oral, suppository, etc.) for various purposes (as a component of oral preparations (e.g., antiflatulent agents)).

The intestinal environment improving agent and β-glucuronidase activity inhibitor of the present invention are preferably for oral intake.
Examples of foods include processed foods such as fermented foods, breads, pickles, dried foods, paste products, flours, canned foods, frozen foods, retort foods, instant foods (instant noodles, dry foods, powdered drinks, etc.), dairy products (processed milk, skim milk powder, etc.), processed fish products, and processed livestock products; luxury foods such as confectioneries; cooking and seasoning ingredients such as fats and oils, sweeteners, seasonings, and spices; health foods (functional foods) such as supplements; foods for special purposes (foods for the sick, foods for the elderly, foods for children); foods for specified health uses; foods with functional claims; processed materials such as gelling agents and leavening agents; preserved foods; emergency foods; space foods; and beverages such as water, soft drinks, alcoholic beverages, tea, and coffee.

The intestinal environment improving agent and β-glucuronidase activity inhibitor of the present invention can be used as a pharmaceutical product, or can be added to a known pharmaceutical product.
Examples of pharmaceuticals include tablets, powders (fine granules, granules, etc.), capsules, drinks, syrups, lozenges and other oral medications; topical medications; and injections.

The intestinal environment improving agent and β-glucuronidase activity inhibitor of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these.

[Test Example 1] Small intestinal epithelium permeation test Using human colon cancer-derived Caco-2 cells, which are used as a model cell for the small intestinal epithelium, the intestinal absorbability of quercetin and its glycosides was examined.

In this test, Caco-2 cells were cultured on a membrane filter for 2-3 weeks, and the Caco-2 cell monolayer membrane differentiated into a membrane similar to the small intestinal epithelium was used to conduct the small intestinal epithelium permeation test. Membrane permeability was evaluated by comparing the membrane permeability coefficient Papp. Papp is a value that depends on the rate of membrane permeation through the Caco-2 cell membrane, and is said to correlate with the actual amount of intestinal absorption in the living body.

Papp = (dQ/dt)/(A·Co) Formula (I)
In formula (I),
Papp: Apparent membrane permeability coefficient (cm/sec)
dQ/dt: slope of permeation amount (mol/sec)
A: Single layer membrane area ( ^cm2 )
Co: Addition concentration (mol/mL)
This shows that.

Formation of Caco-2 cell monolayers on membrane filters
The insert was placed in a culture plate, and several drops of 6 vol % collagen solution (dissolved in 0.02 N hydrochloric acid) were applied evenly to the cell culture surface of the insert, and the insert was left to stand for 15 to 30 minutes.

Next, the inside of the insert was washed three times with phosphate-buffered saline (PBS), the cell culture surface was coated with collagen, and 2 mL of medium was placed on the inside of the collagen-coated insert and 3 mL on the outside.

10 μL of the Caco-2 cell suspension was taken and mixed with 10 μL of trypan blue solution, which was then added to a hemocytometer, and the number of stained live cells was counted.
The cells were seeded in the insert at a ratio of 2.0 x ¹⁰⁵ cells/well and cultured in a 5% _CO2 incubator at 37°C for about 20 days to differentiate into small intestinal epithelium-like cells. The medium was changed every 2 to 3 days.

Preparation of spiked samples
Quercetin (Que), isoquercitrin (Iqr), rutin (Rut), and α-glucosylrutin (αG-Rut) were each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 5 mM, and sterilized by passing through a 0.2 μm membrane filter.
αG-Rut was prepared by removing impurities from αG Rutin PS (manufactured by Toyo Sugar Refining Co., Ltd.) through a column purification process, and the purity of α-monoglucosylrutin was approximately 100%.

Experimental Procedure
The small intestinal epithelium model Caco-2 cell monolayer cultured on the porous membrane filter was observed under an optical microscope to confirm that the membrane had no holes. Next, the medium on the basement membrane side, which is the membrane located on the blood vessel side of the small intestinal epithelium, was removed, washed three times with HBSS (Hanks' Balanced Salt Solutions), and then filled with 2 mL of HBSS. In addition, the medium on the luminal side, which is located on the intestinal tract side of the small intestinal epithelium, was removed, washed once with HBSS, and then filled with 1 mL of HBSS.

After preincubation for 1 hour in a 37°C, 5% _CO2 incubator, the membrane was checked using an optical microscope to confirm the absence of holes and whether the transepithelial electrical resistance (TEER) value was normal.

10 μL of each of the samples of Que, Iqr, Rut, and αG-Rut prepared above was added to the luminal side so as to give a final concentration of 50 μM, and the mixture was allowed to stand in a 37° C., 5% CO ₂ incubator.

After 30, 60, and 90 minutes, the cells were removed from the incubator, and 100 μL of the sample was taken from the basement membrane side to obtain a permeation sample. Before sampling, the membrane was checked for the absence of holes using an optical microscope, and the TEER value was checked for normality.
The configuration of the above-mentioned experimental model is shown in FIG.

Quantification of Permeate Sample Concentration Using HPLC
The permeation sample stored at -20°C was dissolved in a thermostatic bath at 37°C and centrifuged at 12000g for 5 minutes. The supernatant was subjected to HPLC for quantification. The analytical conditions were as follows:

Detector: UV-visible spectroscopic detector (SPD-10A VP) (manufactured by Shimadzu Corporation)
Detection wavelength: 285 nm
Column: Mightysil RP-18 GP (4.6 x 250 mm)
Column temperature: 40°C
Mobile phase: 0.2% acetic acid aqueous solution:methanol = 55:45 (v/v)
Flow rate: 0.7 mL/min Injection volume: 10 μL
The results are shown in Figure 2.

In FIG. 2, the vertical axis indicates membrane permeability coefficient (Papp; 10 ⁻⁶ cm/sec), and the horizontal axis indicates quercetin glycosides (Que: quercetin, Iqr: isoquercitrin, Rut: rutin, αG-Rut: α-glucosylrutin).
It has been shown that the permeability of the small intestinal epithelium decreases when the number of sugars bound to quercetin increases. It is presumed that α-glucosylrutin is not absorbed in the small intestine and easily reaches the large intestine.

[Test Example 2] Human intestinal bacterial community culture test (assimilatory test)
Sample Preparation
The medium used was prepared by mixing equal amounts of PYF medium (also referred to as "2xPYF medium" in this specification) adjusted to twice the final concentration and 2 w/v % quercetin glycoside (Rut or αG-Rut) solution in a 2 mL screw-cap tube (manufactured by Watson). The pH of all the media after mixing was in the range of 7.3 to 7.5, and no pH adjustment was performed.

The composition of 2xPYF medium per 100 mL was 2 g tryptone, 1 g yeast extract, 0.1 g L-cysteine hydrochloride monohydrate, 8 mL pepsin-digested horse blood, and 8 mL PYF salts.

The pepsin-digested horse blood contained in the 2xPYF medium was prepared from defibrinated horse blood (Nippon Bio-Test Laboratories). 1 g of pepsin derived from porcine gastric mucosa (Sigma-Aldrich) and 6 mL of hydrochloric acid were added to 50 mL of defibrinated horse blood, and the mixture was treated at 55°C for 24 hours, followed by the addition of 2 mL of chloroform.

The composition of PYF salts contained in 2×PYF medium was as follows per 100 mL: CaCl ₂ 0.02 g, MgSO ₄ .7H ₂ O 0.02 g, KH ₂ PO ₄ 0.10 g, NaHCO ₃ 1.00 g, and NaCl 0.20 g.

2xPYF medium was prepared immediately before use and autoclaved at 121°C for 20 minutes. After autoclaving, it was immediately cooled on ice and argon gas was blown in for about 5 minutes to prevent oxygen from dissolving.

A 2 w/v% Rut solution and a 2 w/v% αG-Rut solution were prepared using degassed ultrapure water. Rut was hardly soluble in ultrapure water, so it was suspended in ultrapure water and sterilized by autoclaving. It was confirmed by HPLC analysis before and after autoclaving that Rut was not decomposed by autoclaving. αG-Rut was completely dissolved in ultrapure water, so it was sterilized using a membrane filter (0.2 μm; Advantec).

(Culture test)
The fecal samples were inoculated into these media at 1 vol % and subjected to static culture at 37° C. under anaerobic conditions for 72 hours.

Fecal samples were prepared from five men in their twenties. It was confirmed that the stool donors were healthy at the time of collection, had no history of gastrointestinal disease, and had not been administered antibiotics within the past three months. The collected stool was used for culture testing within six hours. The weight of the stool was measured, and it was diluted two-fold (w/v) with 50% glycerol (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) and suspended. The 50% glycerol suspension was prepared by mixing equal amounts of five samples and diluting them ten-fold with sterilized and degassed 0.1 M PBS. The PBS-diluted suspension was filtered through sterilized gauze to remove fibrous material, and the fecal sample was prepared.

The composition of 0.1 M PBS was 0.45 g of Na ₂ HPO ₄ ·12H ₂ O, 0.03 g of KH ₂ PO ₄ , 1 g of NaCl, and 0.025 g of KCl per 100 mL.
The 0.1 M PBS was sterilized by autoclaving at 121° C. for 20 minutes, and then degassed by blowing in argon gas.

The anaerobic state of the culture medium during static culture was maintained by placing one Anaeropack Kenki in a 2.5 L Anaeropack square jar. The pH of the culture medium was measured to evaluate assimilation. These cultures were carried out in triplicate (n=3) under the same conditions.

As controls, experiments were also performed on the above 2xPYF medium to which sterilized ultrapure water was added instead of the quercetin glycoside solution (MilliQ: negative control: NC), and on which 2 w/v% glucose was added instead of the quercetin glycoside solution (1 w/v% Glu: positive control: PC).

The results are shown in Table 1. The pH of the culture medium with and without inoculation (non-inoculation) is shown for Rut, αG-Rut, 1% Glu, and MilliQ.

The pH of the culture medium containing Rut and αG-Rut decreased, suggesting the proliferation of organic acid-producing bacteria. This suggests that Rut and αG-Rut are digested in the large intestine, where most of them are broken down into the aglycone quercetin.

[Example 1]
<Human intestinal bacteria culture test>
(Preparation of culture medium)
400 μL of the culture medium prepared in Test Example 2 (culture test) was centrifuged at 14,000 rpm for 5 minutes, the supernatant was removed, and the mixture was suspended in 200 μL of ultrapure water.

(Extraction of total genomic DNA from culture medium)
Lysis of the enterobacteria in the culture medium and extraction of the total genomic DNA were performed using a Favorprep Stool DNA Isolation Mini kit (manufactured by Favorgen) according to the procedure attached to the kit. As a control, 200 μL of the bacterial sample (inoculums) before culture was similarly performed.
Total genomic DNA was eluted using 200 μL of the elution buffer provided in the kit and stored at -20°C.

(Quantification of relative DNA amount by real-time PCR)
The relative bacterial mass was quantified according to the procedures in J. Agric. Food. Chem, 59, 6511-6519, 2011 (Reference 1) and Appl. Environ. Microbiol, 5445-5451, 2002 (Reference 2). The relative bacterial mass of each target bacterial species was calculated by measuring the ratio of the DNA of the target bacterial species to the total DNA in the culture medium.

Using the total DNA extracted from the culture medium as a template, primers were designed targeting five groups: the genus Bifidobacterium, the phylum Firmicutes, the phylum Bacteroides, the genus Lactobacillus, and the Clostridium cluster XIVa group, and the amount of DNA in each group was measured by real-time PCR.

The primers were designed to specifically extend a portion of the 16S rRNA gene of each group. The primers for each group and references are shown in Table 2.

In addition, because the total bacterial mass and the amount of extracted DNA differed for each culture medium, a similar experiment was performed using primers targeting the 16S rRNA gene region common to all bacteria as a reference, and the total bacterial mass (Total bacteria) was measured.

The real-time PCR analysis equipment used was the Thermal cycler dice time system (manufactured by Takara Bio).
The PCR conditions are shown in Table 3.

The reaction solution was prepared in a total volume of 15 μL, containing 7.5 μL of SYBR premix Ex TaqII (Tli RNaseH Plus) (Takarabio), 0.6 μL of forward primer (10 μM), 0.6 μL of reverse primer (10 μM), 1.2 μL of template DNA, and 5.1 μL of water.

The Ct value of each reaction solution was calculated using a multi-wavelength detection real-time PCR device (Thermal cycler dice time system software) (Takara Bio). The amount of bacteria in each group in each culture solution was calculated as follows, with the total amount of bacteria in each culture solution being 100%.

The relative bacterial mass of the Firmicutes phylum was calculated by subtracting the relative bacterial mass of Lactobacillus and Clostridium cluster XIVa group from the total bacterial mass of the Firmicutes phylum.

The results of Example 1 are shown in Table 4. Compared to the fecal suspension, it can be seen that the quercetin glycoside αG-Rut has an increased amount of neutral bacteria of the phylum Firmicutes. In addition, when focusing on neutral bacteria of the phylum Bacteroidetes, αG-Rut is decreased compared to the fecal suspension, but shows a higher value compared to Glu and Rut.

[Example 2]
<Lactic acid bacteria culture test>
(Preparation of spiked samples)
Physiological saline adjusted to 0.9% and M.R.S. BROTH (diluted with ultrapure water) were sterilized in an autoclave. Next, αG-Rut and Rut were dissolved in dimethyl sulfoxide (DMSO) and then processed with a 0.45 μm membrane filter. Furthermore, lactic acid bacteria (powder) were diluted 1000-fold with sterilized 0.9% physiological saline.

(Preparation of culture medium)
Sterilized M.R.S. BROTH (hereinafter referred to as culture solution) was added to the plate wells to prepare a blank. For the control, culture solution and filtered DSMO were added to the plate wells so that the final concentration of the culture solution was 0.5%, and lactic acid bacteria dissolved in 0.9% physiological saline were added to the plate wells to give 1 x ¹⁰³ pieces. For the test sample, culture solution and the additive sample prepared above were added to the plate wells so that the final concentration of the culture solution sample was 100 ppm (and the final concentration of DMSO was 0.5%), and lactic acid bacteria dissolved in 0.9% physiological saline were added to the plate wells to give 1 x ¹⁰³ pieces.

(Culture test)
The culture medium prepared above was cultured in an incubator at 37° C. for 0 to 70 hours.
(Measurement and analysis)
In general, the number of lactic acid bacteria proliferation is proportional to the turbidity of the culture solution. In this test, the number of lactic acid bacteria proliferation was evaluated by measuring the turbidity of the culture solution by absorbance.
The absorbance at 600 nm of a blank sample containing no lactic acid bacteria was measured using a microplate reader (Molecular Devices Japan) and used as the blank value.

Similarly, for the control and test samples, after the above-mentioned culture test, the absorbance at 600 nm was measured using a microplate reader to obtain the control value and the test sample value.
The values obtained by subtracting the blank value from the control value and the test sample value were used as the proliferation number of lactic acid bacteria. The results are shown in Table 5 and FIG.

When comparing the control and test samples after incubation for 48, 68, and 70 hours, it can be seen that lactic acid bacteria proliferated more in the test samples. In particular, when considering the amount of rutin converted, it can be seen that αG-Rut proliferated lactic acid bacteria more effectively than Rut. Therefore, it was found that αG-Rut and Rut, which are quercetin glycosides, have the effect of promoting the proliferation of lactic acid bacteria.

[Example 3]
<Mouse intestinal bacterial group test>
(Acclimation of mice)
ICR mice (male, 7 weeks old) were purchased from CLEA Japan, Inc. The purchased mice were pre-bred for 4 days in a room at 22° C. The feed composition was AIN-93M as shown in Table 6 below, and the mice were given regular food ad libitum.

(Feeding test of Rut or αG-Rut)
The acclimatized mice were divided into three groups of six mice each (control, sample 1 Rut, sample 2 αG-Rut) and housed for 14 days.

For the control, AIN-93M was given ad libitum. For Sample 1 Rut or Sample 2 αG-Rut, the cellulose powder in AIN-93M was reduced by 1% and replaced with Rut or αG-Rut as shown in Table 3, and the mice were given ad libitum. The bedding for the mice was changed once a week.

The results of changes in food intake and body weight of mice reared under the above conditions for 14 days are shown below.
The amount of food consumed by the mice in each of the control, Sample 1 Rut, and Sample 2 αG-Rut groups was approximately 4 g per day, with no significant difference between groups.

There was no significant change in body weight for the mice in the control, sample 1 Rut, and sample 2 αG-Rut groups throughout the 14-day rearing period.

(Extraction of total genomic DNA from feces)
On the 14th day, mouse droppings were collected.

(Quantification of relative DNA amount by real-time PCR)
Feces collected from each of the control, sample 1 Rut, and sample 2 αG-Rut groups were freeze-dried and then ground using a mortar and pestle.
100 mg of each of the crushed feces was weighed out, and lysis and total genomic DNA were extracted in the same manner as in Example 1.
Using the same reaction solution and procedures as in Example 1, the relative DNA amount was quantified by real-time PCR.

Figure 4 shows the quantitative results of the relative DNA amount in the feces of Example 3. Compared to the control, Sample 2 αG-Rut showed a decrease in "others" and a slight increase in the Bifidobacterium genus.

[Example 4]
<β-glucuronidase inhibition test>
The enzyme β-glucuronidase was β-D-glucuronidase type VII-A (G7646) (Sigma-Aldrich) derived from Escherichia coli, and its inhibition test was carried out.

The reaction solution was prepared by mixing 25 μL of 0.04 VOL% 4-nitrophenyl-β-D-glucuronide (pNP-β-D-glucuronide), 25 μL of quercetin glycoside (dissolved in 3 VOL% DMSO solution), and 10 μL of β-glucuronidase (40 mU/μL). The solvent used in all cases was 20 mM phosphate buffer (pH 6.8) containing 3 VOL% DMSO.

It was confirmed that phosphate buffer containing 3 VOL % DMSO did not inhibit the activity of β-glucuronidase.
The preincubation was carried out by mixing the above-mentioned β-glucuronidase reaction solution with a quercetin glycoside reaction solution prepared to a final concentration of 25, 50, or 100 μM at 37° C. for 20 minutes, and then adding 0.04 VOL% 4-nitrophenyl-β-D-glucuronide to a final concentration of 0.017 VOL%, and reacting for 40 minutes at 37° C. The reaction was stopped by adding 60 μL of 1 M Na ₂ CO ₃ , and the absorbance at 405 nm was measured using a spectrophotometer (Multiskan GO: manufactured by Thermo Scientific).

The control was one that did not contain quercetin glycoside. In addition, for each sample, an experiment was also conducted in the same way without adding β-glucuronidase, which served as a blank. The inhibition rate was calculated according to the following formula (III).

Residual activity (%)={(absorbance sample−absorbance blank)/(absorbance control−absorbance blank)}×100
Formula (III)
The results of Example 4 are shown in FIG.

Quercetin and its glycosides showed residual activity lower than 100%, indicating that they have a β-glucuronidase activity inhibitory effect compared to the control. In particular, Iqr and αG-Rut showed a high effect at 100 μM.

As mentioned above, when quercetin glycoside is orally ingested, it is likely to reach the large intestine without being absorbed in the small intestine, and is therefore thought to be broken down in the large intestine to become quercetin. As shown in Figure 5, quercetin has a high inhibitory effect on β-glucuronidase activity even at relatively low concentrations. Furthermore, quercetin glycoside itself also has the ability to inhibit β-glucuronidase activity. Therefore, it can be seen that quercetin glycoside is useful as an inhibitor of β-glucuronidase derived from E. coli.

2: Sample insertion, 4: Luminal side, 6: Caco-2 cells, 8: Basement membrane side, 10: Porous membrane filter, 12: Insert

Claims (3)

Contains quercetin glycoside,
The quercetin glycoside contains α-glucosylrutin,
The α-glucosylrutin contains α-monoglucosylrutin, and the α-glucuronidase activity inhibitor in the large intestine is provided . The β-glucuronidase activity inhibitor according to claim 1 , wherein the α-glucosylrutin contains 50 to 100% by mass of α-monoglucosylrutin. A food or medicine for inhibiting β-glucuronidase activity in the large intestine, comprising the β-glucuronidase inhibitor according to claim 1 or 2 .