JP2019043952A - AGENTS FOR IMPROVING INTESTINAL ENVIRONMENT AND INHIBITORS OF β-GLUCURONIDASE - Google Patents

Abstract

To provide novel applications of quercetin glycosides.SOLUTION: The invention provides an intestinal environment-improving agent and β-glucuronidase inhibitor comprising a quercetin glycoside.SELECTED DRAWING: None

Description

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

  Flavonoids are substances that are abundant in plants in general, especially in fruits and skins of citrus fruits, and are known to have ultraviolet absorption and antioxidant effects in addition to anti-inflammatory, anti-allergic, antihypertensive and other physiological actions. There is. Flavonoids have a problem that they are difficult to be absorbed into the body when ingested because they have poor solubility in water. Therefore, attention is being paid to water-soluble flavonoids, which transfer α-glucose to flavonoids by a glycosyl transfer reaction and increase the solubility in water, and their application to foods, cosmetics, and pharmaceuticals is expected. For example, quercetin, which is a type of flavonoid, is widely used as a component of food and medicine.

Patent Document 1 describes an enteric environment and an intestinal barrier improvement supplement in which quercetin is mixed as one of functional nutrition components having an anti-inflammatory action in the intestinal tract.
Patent Document 2 describes that a mixture of plant extract and prebiotics promotes the growth of bifidobacteria and lactic acid bacteria, and as the plant extract, a plant extract rich in polyphenols is described. Among the examples, a nutritional composition is described which comprises (iso) quercetin glycoside as one of the active ingredients contained in the extract of Ginkgo Bioloba (Ginkgo biloba).

JP, 2014-079208, A Japanese Patent Publication No. 2008-500032

  As shown in Patent Document 1, an enteric environment and intestinal barrier improvement supplement containing quercetin as one of functional nutritional components is known, but quercetin glycosides have not been studied. Further, as shown in Patent Document 2, a nutritional composition containing an (iso) quercetin glycoside is known, but there are many unclear points in the functionality in the intestine. In particular, the effects of quercetin glycosides on the intestinal bacterial flora are unclear.

  The object of the present invention is to examine the influence of quercetin glycosides on intestinal flora and other influences on the intestine and to provide new uses of quercetin glycosides.

  As a result of examining the functionality of the quercetin glycoside in the intestine, particularly in the large intestine, the present inventors have the effect that the quercetin glycoside improves the intestinal environment and also has the ability to inhibit the β-glucuronidase activity. I found it to have.

That is, the present invention is as follows.
[1] An intestinal environment-improving agent containing quercetin glycoside.
[2] The agent for improving intestinal environment according to claim 1, which is for oral intake.
[3] The intestinal environment-improving agent according to [1] or [2], which comprises α-glucosylrutin as a quercetin glycoside.
[4] The agent for improving intestinal environment according to any one of [1] to [3] for relatively increasing the proportion of neutral bacteria in the large intestine.
[5] The agent for improving intestinal environment according to any one of [1] to [4], for relatively increasing the proportion of good bacteria in the large intestine.
[6] A food or medicine containing the agent for improving intestinal environment according to any one of [1] to [5].
[7] A β-glucuronidase activity inhibitor comprising quercetin glycoside.
[8] The β-glucuronidase activity inhibitor according to [7], which comprises at least one selected from isoquercitrin, rutin and α-glucosylrutin as a 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 medicine containing the inhibitor of β-glucuronidase activity 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, the quercetin glycoside is not absorbed in the small intestine, but is easily absorbed in the large intestine without being absorbed in the small intestine, so it is decomposed and absorbed in the large intestine, and neutral bacteria and / or good bacteria in the large intestine The ratio can be improved and β-glucuronidase activity can be inhibited.

FIG. 1 shows an experimental model of small intestine epithelial permeation test. FIG. 2 shows the results of small intestine epithelial permeation test. FIG. 3 shows the results of the number of growth of lactic acid bacteria in the lactic acid bacteria culture solution test. FIG. 4 shows the relative DNA content of each intestinal bacterial group in mouse dung. FIG. 5 shows the results of the β-glucuronidase activity inhibition test.

The intestinal environment improving agent and the β-glucuronidase activity inhibitor of the present invention will be described.
[Intestinal environment improving agent]
The intestinal environment-improving agent of the present invention contains quercetin glycoside, and further contains other components as needed. The enteric environment improving agent has an action to improve the intestinal environment (enteric environment improving action) and is used to improve the intestinal environment.

  Here, as used herein, "gut" when used in the context of "intestinal" means the small and / or large intestine. The small intestine includes the duodenum, jejunum and ileum. The large intestine includes the cecum, colon and rectum. Also, the "gut" in improving the intestinal environment according to the present invention more preferably includes the large intestine. That is, the intestinal environment-improving agent of the present invention is preferably a colonic environment-improving agent.

  The intestinal environment improving action of the present invention is an action to relatively increase good bacteria or neutral bacteria in the intestinal bacterial flora, and the Lactobacillus genus and Bifidobacterium known as good bacteria. It refers to the action of relatively increasing at least one selected from the genus Bifidobacterium and Bacteroides and Firmicutes known as neutral bacteria. The genus name is generally written in italics (italic letters), but in the text, it is written in roman.

  The intestinal environment-improving action of the present invention is a action to relatively increase at least one of Bacteroides and Firmicutes which are known as neutral bacteria among these bacteria. Is preferred. In addition, the relative bacterial mass of the Filmicutes phylum as a neutral bacterium is a value obtained by subtracting the relative bacterial mass of the Lactobacillus genus and the Clostridium cluster XIVa (Clostridium cluster XIVa) group from the entire Filmicutes. did.

«Quercetin glycoside»
The quercetin glycoside is a compound in which various kinds of sugars are linked to quercetin which is a kind of flavonoid, and examples thereof include isoquercitrin, rutin, α-glucosylrutin, and α-glucosyl isoquercitrin.

  The quercetin glycoside used in the present invention is not particularly limited as to its origin or production method. As a preferred embodiment of quercetin glycosides, quercetin glycosides in which glucose is added by reacting rutin or isoquercitrin with a glycosyltransferase in the presence of a glycosyl donor can be used. In addition, quercetin glycosides are known to be contained in onion, enju, buckwheat, apple, tea, grape, broccoli, petit ver, leaf vegetables and citrus fruits, and the use of extracts of these plants Can.

  As quercetin glycosides have a high effect of relatively increasing good bacteria or neutral bacteria in the intestinal bacterial flora, rutin, α-glucosylrutin, and an enzyme-treated product of rutin (hereinafter also referred to as “enzyme-treated rutin”) Is preferred, and α-glucosylrutin and enzyme-treated rutin are more preferred. The enzyme-treated rutin is one obtained by adding glucose to rutin using a glycosyltransferase, which contains α-glucosylrutin and is significantly enhanced in water solubility compared to rutin.

As shown in the small intestine epithelial permeation test described later, when the number of sugars binding to quercetin in the quercetin glycoside increases, the small intestinal epithelial permeability decreases, and when the quercetin glycoside is orally ingested, it is not absorbed in the small intestine. It easily reaches the large intestine and is useful as an agent for improving the environment in the large intestine. Therefore, enzyme-treated rutin containing rutin, α-glucosylrutin and α-glucosylrutin is preferred, and enzyme-treated rutin containing α-glucosylrutin and α-glucosylrutin is 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 still more preferably 75 to 100% by mass in 100% by mass of the intestinal environment-improving agent of the present invention.

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

In the above formula, n is an integer of 1 or 2 to 20, preferably an integer of 1 or 2 to 10, more preferably 1.
In the present specification, among α-glucosylrutin, a compound in which only one glucose is bound is referred to as “α-monoglucosylrutin”, and a compound in which two or more glucose are bound 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 is greater for α-monoglucosylrutin, which is considered to be advantageous in terms of action and effect. Therefore, it is preferable that a part or all of α-glucosylrutin be α-monoglucosylrutin. The amount of α-monoglucosylrutin in the α-glucosylrutin is preferably 10 to 100% by mass, more preferably 50 to 100% by mass, and still more preferably 90 to 100% by mass.

Among the enzyme-treated rutins, α-glucosylrutin has a strong action to increase, particularly, neutral bacteria of human intestinal bacteria, and in particular, the water solubility is enhanced.
The enzyme-treated rutin is a product (also referred to herein as “first enzyme-treated rutin”) obtained by reacting rutin with a glycosyltransferase in the coexistence of an α-glucosyl sugar compound. As an alpha-glucosyl sugar compound, a cyclodextrin, a starch partial decomposition product, etc. are mentioned. The glycosyltransferase is an enzyme having a function of adding glucose to rutin, such as cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19).

  The first enzyme-treated rutin consists of various α-glucosylrutins different in the number of bound glucose, ie, an aggregate consisting of α-monoglucosylrutin and other α-polyglucosylrutins, and rutin which is an unreacted product. It is a composition to contain.

  If necessary, the α-glucosyl sugar compound and other impurities are removed by purifying the first enzyme-treated rutin using, for example, a porous synthetic adsorbent and a suitable eluate, and the content of rutin is further reduced. The first enzyme-treated rutin (a purified product of α-glucosylrutin) is obtained which is reduced and the purity of α-glucosylrutin is increased.

In addition, an enzyme having glucoamylase activity that cleaves the first enzyme-treated rutin into glucose units of an alpha-1,4-glucosidic bond, such as glucoamylase (EC 3.2.1.3
In the α-polyglucosyl rutin treated with) and having a plurality of glucose added, the glucose other than the glucose residue which is directly added to the glucose residue (in the rutinose residue) of rutin itself remains. By cleaving the residue, an enzyme-treated rutin containing a large amount of α-monoglucosylrutin (also referred to herein as “a second enzyme-treated rutin”) can be obtained. This enzyme treatment does not cleave the glucose residue in the rutinose residue directly linked to the quercetin backbone from the quercetin backbone.

The amount of rutin equivalent in the enzyme-treated rutin is preferably 10 to 85% by mass, more preferably 40 to 85% by mass, and still more preferably 70 to 85% by mass. Thus, the enzyme-treated rutin obtained as described above may contain, for example, unreacted rutin or quercetin, which is a degradation product of rutin, in a small amount together with α-glucosylrutin. In addition, isoquercitrin having a structure in which a rhamnose residue is removed from rutin may be included.
The amount of rutin conversion is, for example, the second edition food additive voluntary standards other than chemically synthesized products (issue: October 1, 1993 Editor: Japan Food Additives Association Voluntary Standards Expert Committee Issuer: Japan Food Additives Association page 202 Enzyme-treated rutin It can be calculated in accordance with the description of the determination method. In addition, a rutin standard solution with a known concentration can be prepared using a commercially available rutin reagent (for example, Fujifilm Wako Pure Chemical Industries, Ltd.).

  Such enzyme-treated rutin is not particularly limited, but specifically, αG rutin PS (trade name, rutin equivalent amount 80 to 86 mass%, manufactured by Toyo Seisei Co., Ltd.), αG rutin P (trade name, rutin) The amount of conversion is 40 to 46% by mass, manufactured by Toyo Seisei Co., Ltd., αG rutin H (trade name, the amount of 22 to 26% by mass of conversion of rutin converted, manufactured by Toyosei Sugar Co., Ltd.) These commercially available products may be used as they are or purified products may be used.

<< Other ingredients >>
The intestinal environment-improving agent of the present invention may contain not only quercetin glycosides but also other components. Other components include, for example, binders for preparing the improver in solid form, specifically cyclodextrin, dextrin, water-soluble dietary fiber, starch, etc .; solvents for preparing the improver in liquid form Specifically, water and alcohols (eg, ethyl alcohol, glycerin, propylene glycol) can be mentioned. Other ingredients include other pharmaceutically acceptable additives.

[Β-glucuronidase activity inhibitor]
The β-glucuronidase activity inhibitors of the present invention include quercetin glycosides. The quercetin glycoside is the same as the specific example and preferred example described in the [Intestinal Environment Improving Agent] column, and at least one selected from isoquercitrin, rutin and α-glucosylrutin is preferable, and α-glucosyl is preferable. Rutin is more preferred. As α-glucosylrutin, α-glucosylrutin contained in enzyme-treated rutin is preferred. Moreover, it is preferable that one part or all is (alpha)-monoglucosyl rutin as (alpha)-glucosyl rutin.
The quercetin glycosides may be used alone or in combination of two or more.

  [beta] -glucuronidase is an enzyme that hydrolyzes a compound (glucuronide) in which various alcohols, phenols, amines and the like are glucuronidated, and is present in many organisms such as bacteria, fungi, plants and animals.

  Glucuronidation is usually performed when the carcinogen is excreted from the body in vivo. The carcinogen is inactivated by glucuronidation in the liver, excreted in bile, excreted in the intestinal tract and excreted outside the body. However, it is known that the risk of colon cancer is increased because the conjugated carcinogen is hydrolyzed by β-glucuronidase produced by enterobacteria to be released as a deconjugated carcinogen.

As β-glucuronidase, for example, those derived from Clostridium (E. coli) are known.
In the clinic, colon cancer patients are known to have higher β-glucuronidase activity than healthy people, and the genus Clostridium of the enteric bacteria having β-glucuronidase activity is more in comparison with healthy people. know. In addition, β-glucuronidase activity is known to be significantly increased when a general Japanese dieter is fed a carnivorous Western diet, and is found to be correlated with the disappearance of the genus Clostridium. ing. In Examples described later, β-glucuronidase derived from E. coli Type VII-A was used.

  The inhibitor of β-glucuronidase activity of the present invention can inhibit the activity of β-glucuronidase derived from enteric bacteria of an organism, particularly E. coli, and therefore can reduce the risk of colorectal cancer.

The β-glucuronidase activity inhibitor of the present invention contains quercetin glycoside as an active ingredient. The content of the quercetin glycoside is preferably 30 to 100% by mass, more preferably 60 to 100% by mass, still more preferably 75 to 100% by mass in 100% by mass of the β-glucuronidase activity inhibitor of the present invention .
Moreover, the (beta) -glucuronidase activity inhibitor of this invention may also contain the other component described in the column of the "enteric environment improvement agent."

[Specific use]
The intestinal environment improving agent and the β-glucuronidase activity inhibitor of the present invention can also be used by being added to various foods. In addition, it can be used for various uses (for example, components of orally administered preparations (for example, an enteric preparation) and the like) in various dosage forms (solutions, tablets, powders and the like) and methods (oral, suppositories and the like).

The intestinal environment-improving agent and the β-glucuronidase activity inhibitor of the present invention are preferably for oral ingestion.
Examples of foods include fermented foods, breads, pickles, dried products, paste products, flours, cans, frozen foods, retort foods, instant foods (immediate noodles, dry foods, powdered beverages, etc.), dairy products (processed milk, Processed foods such as skimmed milk powder, processed fish meat, processed livestock products; Favorite foods such as confectionery products; Ingredients for cooking and seasoning such as oils and fats, sweeteners, seasonings, spices, etc. Health foods such as supplements (functional Foods; Special-purpose food (food for the sick, food for the elderly, food for child care); food for specified health use; food for functional indication; processed materials such as gelling agents and expanders; preservative food; emergency food; Food; Water, soft drinks, alcoholic beverages, tea, beverages such as tea and coffee can be mentioned.

The intestinal environment improving agent and the β-glucuronidase activity inhibitor of the present invention can be used as a medicament, or can be used by adding to known medicaments.
Examples of the pharmaceutical include internal medicines such as tablets, powders (fine granules, granules etc.), capsules, drinks, syrups, troches and the like; external preparations; and injections.

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

Test Example 1 Small Intestinal Epithelial Permeation Test Using human colon cancer-derived cells Caco-2 cells used as small intestinal epithelial model cells, intestinal absorbability of quercetin and its glycoside was examined.

  In this test, Caco-2 cells were cultured on a membrane filter for 2 to 3 weeks, and a small intestinal epithelial permeation test was performed using a Caco-2 cell monolayer membrane differentiated into a small intestinal epithelial membrane. The evaluation of the membrane permeability was performed by comparison of the membrane permeability coefficient Papp. Papp is a value dependent on the rate of membrane permeation to Caco-2 cell membrane, and is considered to be correlated 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 (mol / sec)
A: monolayer film area (cm ² )
Co: Addition concentration (mol / mL)
Is shown.

Formation of Caco-2 cell monolayer on membrane filter
The insert was placed in a culture plate, and several drops of 6 VOL% collagen solution (dissolved in 0.02 N hydrochloric acid) were dropped on the cell culture surface of the insert, and the solution was evenly distributed and left 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 collagen-coated, and 2 mL of culture medium was placed inside the collagen-coated insert and 3 mL outside.

10 μL of the Caco-2 cell suspension was taken, mixed with 10 μL of trypan blue solution, added to a hemocytometer, and the number of stained viable cells was counted.
Cells were seeded at a rate of 2.0 × 10 ⁵ cells / well in the above insert, and cultured for about 20 days in a 5% CO ₂ incubator at 37 ° C. to differentiate into small intestinal epithelium-like. Medium change was performed at intervals of once every 2 to 3 days.

<Addition sample preparation>
Quercetin (Que), isoquercitrin (Iqr), rutin (Rut), α-glucosylrutin (αG-Rut) are each dissolved in dimethylsulfoxide (DMSO) to a concentration of 5 mM, and 0.2 μm membrane filter is used. Sterilized by passage. For αG-Rut, αG-rutin PS (manufactured by Toyo Seisei Co., Ltd.) was subjected to column purification to remove impurities, and α-monoglucosylrutin was used at a purity of about 100%.

<Experimental operation>
The small intestine epithelial model Caco-2 cell monolayers cultured on a porous membrane filter were observed with a light microscope to confirm that there were no holes in the membrane. Next, the medium on the basement membrane side, which is a membrane located on the blood vessel side in the small intestine epithelium, was removed, washed three times with HBSS (Hanks' Balanced Salt Solutions), and then filled with 2 mL HBSS. In addition, the luminal medium located on the intestinal side of the small intestine epithelium was removed, washed once with HBSS, and then filled with 1 mL of HBSS.

After pre-incubation for 1 hour at 37 ° C. in a 5% CO ₂ incubator, light microscopy was used to confirm that there were no holes in the membrane. In addition, it was confirmed whether the transepithelial electrical resistance value (TEER value) was normal.

10 μL of the above-prepared samples of Que, Iqr, Rut, and αG-Rut were added to the lumen side to a final concentration of 50 μM, respectively, and allowed to stand in a 37 ° C., 5% CO ₂ incubator.

After 30, 60 and 90 minutes, they were removed from the incubator, and 100 μL was sampled from the basement membrane side to obtain a permeation sample. Before sampling, after confirming that the film had no holes using an optical microscope, it was confirmed whether the TEER value was normal.
The configuration of the above experimental model is shown in FIG.

<Quantification of permeate sample concentration using HPLC>
The permeated sample stored at -20 ° C was dissolved in a thermostat at 37 ° C and centrifuged at 12000 g for 5 minutes. The supernatant was subjected to HPLC for quantification. The analysis conditions are as follows.

-Detector: Ultraviolet-visible spectral 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% aqueous acetic acid solution: methanol = 55: 45 (v / v)
Flow rate: 0.7 mL / min Injection volume: 10 μL
The results are shown in FIG.

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

[Test Example 2] Human intestinal bacteria group culture test (utilization test)
(Sample preparation)
PYF medium (also referred to as “2 × PYF medium” in this specification) prepared to twice the final concentration and a 2 w / v% quercetin glycoside (Rut or αG-Rut) solution in a 2 mL screw cap tube (Watson) Made in the same volume were used as a culture medium. The pH of the medium after mixing was all in the range of 7.3 to 7.5, and the pH was not adjusted.

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

  Pepsin-digested equine blood contained in 2 × PYF medium was prepared from equine defibrillated blood (Nippon bio-test laboratories). 1 g of pig gastric mucosa-derived pepsin (manufactured by Sigma-Aldrich) and 6 mL of hydrochloric acid were added to 50 mL of horse defibrillated blood, treated at 55 ° C. for 24 hours, and prepared by adding 2 mL of chloroform.

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

  The 2 × PYF medium was prepared immediately before use and autoclaved at 121 ° C. for 20 minutes. After autoclaving, ice cooling was immediately performed, and argon gas was blown for about 5 minutes to prevent oxygen from dissolving.

  The 2 w / v% Rut solution and the 2 w / v% αG-Rut solution were prepared using degassed ultrapure water. Since Rut was hardly dissolved in ultrapure water, it was sterilized by autoclaving the one suspended in ultrapure water. The fact that Rut was not decomposed by the autoclave was confirmed by HPLC analysis before and after the autoclave. Since αG-Rut was completely dissolved in ultrapure water, it was sterilized using a Membrene filter (0.2 μm; manufactured by Advantec).

(Culture test)
A stool sample was inoculated at 1 VOL% into these media, and static culture was performed for 72 hours in an anaerobic state at 37 ° C.

  Fecal samples prepared stools of five men in their twenties. The stool provider confirmed that she was healthy at the time of stool collection, that there was no gastrointestinal tract disease etc. in the past, and that no antibiotics were administered within 3 months. Collected stool was used for culture test within 6 hours. The weight of the stool was measured, diluted twice (w / v) with 50% glycerol (manufactured by Sakamoto Pharmaceutical Co., Ltd.) and suspended. The 50% glycerol suspension was mixed in equal aliquots of 5 samples and diluted 10-fold with sterile, degassed 0.1 M PBS. The PBS diluted suspension was filtered with sterile gauze to remove fibrous material and used as a stool sample.

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

  The anaerobic condition of the culture solution during static culture was maintained by placing one anelopack / kenki in a 2.5L volume anelopaque square jar. The pH of the culture solution was measured to evaluate assimilability. These cultures were performed in triplicate (n = 3) under the same conditions.

  As a control, 2x PYF medium to which sterile ultrapure water was added instead of quercetin glucoside solution (MilliQ: negative control: NC), 2 w / v% glucose was added instead of quercetin glucoside solution The experiment was carried out in the same manner as described above (1 w / v% Glu: positive control: PC).

  The results are shown in Table 1. For Rut, αG-Rut, 1% Glu, and MilliQ, the pHs of the culture solutions when inoculated and not inoculated (non-inoculated) are shown.

  The culture solution containing Rut and αG-Rut had a lowered pH, which suggested the growth of organic acid-producing bacteria. From these facts, it is considered that Rut and αG-Rut are digested in the large intestine and most of them are degraded in the large intestine to the aglycone quercetin.

Example 1
«Human Intestinal Bacterial Community Culture Test»
(Preparation of culture solution)
After 400 μL of the culture solution prepared in Test Example 2 (culture test) was centrifuged at 14000 rpm for 5 minutes to remove the supernatant, it was suspended in 200 μL of ultrapure water.

(Extraction of whole genome DNA in culture solution)
The lysis of the enterobacteria group in the culture solution and the extraction of the total genomic DNA were performed using Favorprep Stool DNA Isolation Mini kit (manufactured by Favorgen) according to the procedure attached to the kit. As a control, 200 μL of a cell sample (inoculum) before culture was also performed in the same manner.
Elution of total genomic DNA was performed using 200 μl of elution buffer of the kit and stored at -20 ° C.

(Quantification of relative DNA amount by real-time PCR)
For quantification of relative cell mass, see J. Agric. Food. Chem, 59, 6511-6519, 2011 (Reference 1), and Appl. Environ. It carried out according to operation of Microbiol, 5445-5451, 2002 (references 2). The relative amount of cells 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 solution.

  Using the total DNA extracted from the culture solution as a template, 5 groups of Bifidobacterium (Bifidobacterium), Firmicutes, Bacteroides, Lactobacillus, and Clostridium cluster XIVa groups. Primers targeted to were designed, and the amount of DNA in each group was measured by real-time PCR.

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

  In addition, since the total amount of bacterial cells and the amount of extracted DNA differ for each culture solution, the same experiment is performed using a primer targeting the 16S rRNA gene region common to all bacteria as a reference, and the total bacterial cell amount (Total bacteria) Was measured.

The analysis instrument of real time-PCR used Thermal cycler dice time system (made by Takara bio).
The conditions for PCR are shown in Table 3.

  The composition of the reaction solution is 7.5 μL of SYBR premix Ex Taq II (Tli RNase H Plus) (Takara bio), 0.6 μL of Forward primer solution (10 μM), Reverse primer solution (Reverse primer) 0.6 μL of (10 μM), 1.2 μL of template DNA solution (Template DNA) and 5.1 μL of water were prepared to a total volume of 15 μL.

  The Ct value of each reaction solution was calculated using a multi-wavelength detection real time PCR device (Thermal cycle time system software) (Takara bio). The amount of cells of each group in each culture solution was calculated as follows assuming that the total amount of cells (Total bacteria) in each culture solution was 100%.

  In addition, the relative amount of cells of the Filmictes was determined by subtracting the amount of relative cells of Lactobacillus and the Clostridium cluster XIVa group from the whole of the Field.

  The results of Example 1 are shown in Table 4. As compared with the fecal suspension, it can be seen that the quercetin glucoside αG-Rut is increased in the membrane fulmicum, which is a neutral bacterium. In addition, focusing on Bacteroides, which is a neutral bacterium, αG-Rut shows a larger value than that of Glu and Rut, although αG-Rut is smaller than that of fecal suspension.

Example 2
«Lactic acid bacteria culture fluid test»
(Preparation of additive sample)
Saline prepared to 0.9%; R. S. BROTH (diluted with ultrapure water) was sterilized in an autoclave. Next, αG-Rut and Rut were dissolved in dimethyl sulfoxide (DMSO) and then treated with a 0.45 μm membrane filter. Furthermore, the lactic acid bacteria (powder) were diluted 1000 times with sterilized 0.9% physiological saline.

(Preparation of culture solution)
The plate wells were sterilized in M. R. S. What added BROTH (henceforth a culture solution) was made into the blank. As a control, 1 × 10 ³ lactic acid bacteria dissolved in 0.9% physiological saline were added to the plate wells, and the culture solution and DSMO filtered so that the final concentration of the culture solution was 0.5% were added. Added to be The test sample is added to the plate well with the culture solution and the addition sample prepared above so that the final sample concentration of the culture solution is 100 ppm (and the final concentration of DMSO is 0.5%), and 0.9% Lactic acid bacteria dissolved in physiological saline was added to a concentration of 1 × 10 ³ .

(Culture test)
The culture solution prepared above was cultured in a 37 ° C. incubator for 0 to 70 hours.
(Measurement and analysis)
In general, the growth rate of lactic acid bacteria is proportional to the turbidity of the culture solution. In this test, the number of growth of lactic acid bacteria was evaluated by measuring the turbidity of the culture solution by absorbance.
The absorbance at 600 nm of a blank not receiving lactic acid bacteria was measured by a microplate reader (manufactured by Molecular Device Japan) and used as a blank value.

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

  When the culture time is 48, 68, 70 hours, comparison of the control and the test sample shows that the test sample has more lactic acid bacteria. In particular, in terms of rutin equivalent amount, it can be seen that αG-Rut propagates lactic acid bacteria more effectively than Rut. Thus, it was found that the quercetin glycosides αG-Rut and Rut have a growth effect of lactic acid bacteria.

[Example 3]
«Mouse intestinal bacteria group test»
(Mouse acclimation)
For mice, ICR mice (male, 7 weeks old) were purchased from CLEA Japan, Inc. The purchased mice were bred for four days in a room at 22 ° C. The composition of the feed was AIN-93M described in Table 6 below, and was given as a regular diet at free food and drink.

(Rut or αG-Rut feeding test)
The acclimated mice were divided into three groups of six animals each (control, sample 1 Rut, sample 2 αG-Rut) and kept for 14 days.

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

The results of changes in food consumption and body weight of mice reared for 14 days under the above conditions are shown below.
The amount of food intake was about 4 g per day for mice in each group of control, sample 1 Rut and sample 2 αG-Rut, and there was no significant difference due to the difference in groups.

  With respect to the change in body weight, there was no significant change in the control, sample 1 Rut, and sample 2 αG-Rut groups of mice throughout 14 days of the breeding period.

(Extraction of whole genome DNA of Hun)
Day 14 mice were collected.

(Quantification of relative DNA amount by real-time PCR)
The dung collected from each group of control, sample 1 Rut, sample 2 αG-Rut was ground using a mortar and pestle after lyophilization.
100 mg each of the ground hun was weighed, and lysis and total genomic DNA extraction were performed in the same manner as in Example 1.
In the same reaction solution and operation as in Example 1, quantification of the relative DNA amount was performed by real-time PCR.

  FIG. 4 shows the results of quantification of the relative amount of DNA of Example 3 according to the present invention. As compared with the control, sample 2 αG-Rut had a decrease in others and a slight increase in the genus Bifidobacterium.

Example 4
«Β-glucuronidase inhibition test»
The enzyme β-glucuronidase was subjected to the inhibition test using β-D-Glucuronidase type VII-A (G7646) (manufactured by Sigma-Aldrich) derived from E. coli.

  The reaction solution contained 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), 10 μL of β-glucuronidase (40 mU / μL) was mixed and prepared. The solvents used were all 3 VOL% DMSO-containing 20 mM phosphate buffer (pH 6.8).

In addition, it confirmed that 3VOL% DMSO containing phosphate buffer did not inhibit the activity of (beta) -glucuronidase.
Pre-incubation is 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 and performing at 37 ° C. for 20 minutes, and then 0.04 VOL% 4 -Nitrophenyl- (beta) -D- glucuronide was added so that it might become final concentration 0.017 VOL%, and it was made to react at 37 degreeC for 40 minutes. The reaction was stopped by adding 60 μL of 1 M Na ₂ CO ₃ , and the absorbance at 405 nm was measured with a spectrophotometer (Multiskan GO: manufactured by Thermo Scientific).

  Controls were free of quercetin glycosides. In addition, for each sample, one to which β-glucuronidase was not added was similarly tested and used as a blank. The inhibition rate was determined as in 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 glycoside show a residual activity value lower than 100%, and it is understood that they have a β-glucuronidase activity inhibitory action as compared with the control. In particular, Iqr and αG-Rut showed high effects at 100 μM.

  As described above, when taken orally, quercetin glycosides are considered to be degraded in the large intestine to be quercetin because they are likely to reach the large intestine without being absorbed in the small intestine. As shown in FIG. 5, quercetin has a large ability to inhibit β-glucuronidase activity even at relatively low concentrations. In addition, quercetin glycoside itself also has the ability to inhibit β-glucuronidase activity. Accordingly, quercetin glycoside is found to be useful as an activity inhibitor of β-glucuronidase derived from E. coli.

2: sample input, 4: lumen side, 6: Caco-2 cells, 8: basement membrane side, 10: porous membrane filter, 12: insert

Claims (10)

  An intestinal environment-improving agent containing quercetin glycoside.   The intestinal environment-improving agent according to claim 1, which is for oral intake.   The intestinal environment-improving agent according to claim 1 or 2, which contains α-glucosylrutin as a quercetin glycoside.   The intestinal environment-improving agent according to any one of claims 1 to 3, for relatively increasing the proportion of neutral bacteria in the large intestine.   The intestinal environment-improving agent according to any one of claims 1 to 4, which relatively increases the proportion of good bacteria in the large intestine.   A food or medicine containing the agent for improving intestinal environment according to any one of claims 1 to 5.   The beta-glucuronidase activity inhibitor containing quercetin glycoside.   The β-glucuronidase activity inhibitor according to claim 7, which comprises at least one selected from isoquercitrin, rutin and α-glucosylrutin as a quercetin glycoside.   The beta-glucuronidase activity inhibitor of Claim 7 or 8 which contains alpha-glucosyl rutin as a quercetin glycoside.   A food or medicament containing the inhibitor of β-glucuronidase activity according to any one of claims 7 to 9.