JP2001348382A - Dioxin toxicity inhibitor - Google Patents

Abstract

(57) [Problem] To provide an agent for suppressing or preventing the development of dioxin toxicity or for blocking a dioxin receptor. SOLUTION: The flavonoid contains, for example, a flavone, a flavanol, a flavonol, a chalcone, an isoflavone, a flavanone, a benzalkumaranone or an anthocyanin, and the agent for suppressing or inhibiting the development of toxicity of dioxin and a dioxin receptor. Blocking agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to uses and agents for using flavonoids to suppress or prevent the development of dioxin toxicity or to block dioxin receptors.

[0002]

2. Description of the Related Art Dioxins and their analogs are widely mixed in the living environment of Japanese people, causing serious anxiety in people. Dioxins mainly enter the body through the mouth together with food and the like, and their carcinogenic risk is estimated to be one. This is a risk value that if one million people eat, all one million people will develop cancer.
This is an extremely high risk when compared to 300. When dioxins enter the body, they activate (transform) their receptors present in somatic cells. The transformed receptor translocates to the cell nucleus, binds to a specific site of the gene called DRE, and strongly promotes gene expression of CYP enzymes located downstream thereof. CYP enzymes are enzymes that activate carcinogens to ultimate carcinogens, resulting in carcinogenesis and teratogenesis. Therefore, to suppress the toxicity of dioxins, it is sufficient to inhibit this transformation of the receptor. Currently, drugs such as naphthoflavones and nitroflavones are known to inhibit this transformation, but these drugs are toxic in their own right, and the fact that the drug is ineffective unless taken daily is important. Taken together, it doesn't seem very effective. Dioxins invade the body from everyday foods.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide an agent for suppressing or inhibiting the onset of toxicity of dioxin. Another object of the present invention is to provide a blocking agent that blocks a dioxin receptor to achieve the above object. Still another object of the present invention is to provide a novel use of a flavonoid to achieve the above two objects. Still other objects and advantages of the present invention will become apparent from the following description.

[0004]

According to the present invention, the above objects and advantages of the present invention are firstly achieved by an agent for suppressing or inhibiting the onset of dioxin toxicity characterized by containing a flavonoid. . Secondly, according to the present invention, the above objects and advantages of the present invention are achieved, secondly, by the use of flavonoids for suppressing or preventing the onset of dioxin toxicity.

Further, according to the present invention, the above objects and advantages of the present invention are achieved, thirdly, by a dioxin receptor blocking agent characterized by containing a flavonoid. Finally, according to the present invention, the above objects and advantages of the present invention are achieved fourthly by the use of flavonoids for blocking dioxin receptors. Hereinafter, the present invention will be described in detail.

As the flavonoids used in the present invention, natural flavonoids are preferred, and examples thereof include flavones, catechins (flavanols), flavonols, chalcones, isoflavones, flavanones, benzalkumaranones and anthocyanins. it can. These can be used alone or in combination of two or more. Of these, catechins are preferred. As catechins, for example, (+) catechin, (−) epicatechin,
(-) Gallocatechin, (-) epigallocatechin, (-)
Catechin gallate, (-) epicatechin gallate,
(-) Gallocatechin gallate, (-) epigallocatechin gallate and the like. In addition, specific examples of other natural flavonoids are described in `` Chemical Dictionary '' Kyoritsu Shuppan Co., Ltd.
It is listed in the table of flavonoids in 1989.

[0007] Flavonoids can also be used as natural product concentrates containing the above components, for example, green tea extract, oolong tea extract, black tea extract, tea catechin, tea polyphenol, tea flavonoid, and tea flavonol.

It is known that the mechanism by which dioxin develops toxicity is as follows. The dioxin receptor, or Ah receptor, is present in the cytoplasm and, before binding to the ligand, heat shock protein.
ein 90, HSP90) and form a heterotrimer with the dimer and exist as an inactive form. When this binds to a ligand and is activated, that is, transformed, it dissociates from HSP90 and translocates into the nucleus to ARN.
T forms a different heterodimer with a protein called T and a specific sequence of DNA (DRE: dioxin resp
once to initiate transcription of the CYP1A subfamily. This transformation of the Ah receptor is well known as the initial stage of the onset of dioxin toxicity. By the way, it is known that the transformed Ah receptor can bind to DRE in the cytoplasm without ARNT which is a nuclear protein. Therefore, in the present invention, it was examined whether or not flavonoids can suppress Ah receptor transformation by a method of measuring gel shift analysis, which is usually performed using a nuclear protein, with a cytoplasmic protein.

The agent of the present invention may contain a flavonoid in the form of a natural product containing the flavonoid, an extract of the natural product, or an isolated flavonoid. The agent of the present invention is desirably orally ingested or administered to humans or animals, for example, as beverages, foods, tablets, granules and the like. For example, when the natural product containing flavonoids is green tea or black tea, it can be infused with warm or hot water and consumed as so-called tea. As mentioned above, dioxin often enters the body from food,
The agent of the present invention is preferably taken during or after a meal.

Although the amount of the agent of the present invention varies depending on the type of flavonoid and the state of the flavonoid contained therein, when a person weighing 60 kg ingests three times a day, the amount of the flavonoid per dose is preferably about 10 mg to about 4000
mg, more preferably about 40 mg to about 1000 mg.

According to the study of the present inventors, flavonoids block the dioxin receptor and inhibit the transformation of the dioxin receptor, and as a result, can effectively suppress or inhibit the dioxin from developing toxicity. It became clear. This will be specifically understood from the description of the following examples.

[0012]

EXAMPLES (1) Catechin and tea extract: (+) catechin, (-) epicatechin, (-) gallocatechin,
(-) Epigallocatechin, (-) catechin gallate,
(-) Epicatechin gallate, (-) gallocatechin gallate, (-) epigallocatechin gallate was added to dimethyl sulfoxide (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 10 mM.
(Biochemical reagent) and stored at -20 ° C. Since theaflavin is a mixture, it was dissolved in dimethyl sulfoxide to a concentration of 10 mg / ml. Green tea, oolong tea,
Each of the three types of black tea extract was adjusted to 10 mg / ml in phosphate buffer saline (phosp).
hate buffered saline, PBS)
And used.

(2) Preparation of Ah receptor: Liver was excised from a male SD rat and perfused with 1.15% KCl and bled. 2 times the liver weight of HEDG buffer (25 mM
HEPES, pH 7.4, 1.5 mM EDTA, 1.0 m
M DTT, 10% glycerol) and homogenized, and the obtained homogenate was 105,000 × g,
Centrifuged at 70 ° C for 70 minutes. The supernatant was collected to obtain a cytoplasmic fraction containing the Ah receptor, and the amount of the protein was measured, aliquoted and stored frozen at -80 ° C.

(3) Transformation of Ah receptor: A cytoplasm equivalent to 400 μg as a protein is placed in a microtube, and HEDG buffer is added thereto for 9 hours.
The volume was 9 μl. 0.5 μl of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an agonist (a substance that transforms a receptor) and 0.5 μl of an antagonist (a substance inhibiting an agonist action) Catechin or tea extract was added and reacted at 20 ° C. for 2 hours to transform the Ah receptor.

(4) DRE probe adjustment: Ah receptor
Whether or not the gene has been transformed
It can be determined by whether or not it binds to the DRE site. So, the remains
Probe comparable to the DRE site of the gene, DRE3 probe
Created. 5'-GAT CTG GCTCTT C
TCACG CAA CTC CG-3 '(Cordin
G) 5'-GAT CCG GAG TTG CGT G
AG AAG AGC CA-3 '(non-coding)
Two types of single-stranded DNA oligonucleotides (underlined
Specific binding sequence), and mix them in equimolar amounts.
DRE3 (double-stranded DNA
Lignonucleotide probe). This DRE3
The probe was [γ- ³²P] ATP and T4 polynucleotide
The 5 'end of the protein using T4PK³²At P
Labeled. That is, 1 pmol of DRE3 probe
, 50 unit T4PK (Takara, # 2021
A), 50 μCi of [γ-³²P] ATP (Amasha
And AA0018) were mixed and reacted at 37 ° C. for 45 minutes.
I let you. To remove unreacted ATP, mix the reaction mixture with Quick
k Spin Column (Boehringer Manhai)
And Sephadex G25) for DNA.
Centrifuge at 1100xg for 5 minutes³²Labeled P
The DRE3 probe eluted. Measure the radioactivity of the probe
After the determination, the gel was used for the gel shift assay described below.

(5) Gel shift analysis: 2.5 μl of cytoplasm containing the transformed Ah receptor
(Equivalent to 10 μg of protein) and 9.5 μl of poly [dIdC] -KCl solution (final 200 ng poly [d
IdC], 150 mM KCl) and left at room temperature for 15 minutes to allow proteins and DNA (in this case poly [dId
C]). Then 0.5μ
m ³² P-labeled DRE3 probe (30 kcpm,
0.2-0.8 ng, 13-50 fmol), reacted at room temperature for 20 minutes, and transformed A
The h receptor and the DRE3 probe were specifically bound. A 4% lxTAE gel was prepared in advance, and lxTAE buffer (6.7 mM Tris-HCl, pH 7.5, 3.3) was prepared.
65 V in 1 mM sodium acetate, 1.0 mM EDTA).
For 30 minutes. Add the above DRE to this gel
The cytoplasmic proteins reacted with the three probes were supplied, and electrophoresed with a new buffer at 65 V for 100 minutes. After completion of the electrophoresis, the gel was dried at 80 ° C. for 40 minutes, applied with an X-ray film, and subjected to autoradiography. The transformed Ah receptor band detected on the film was analyzed with an imaging analyzer.

(6) Test Results First, the optimal concentration at which TCDD transforms the cytoplasmic Ah receptor of rat hepatocytes was determined. That is, FIG. 1 shows the results of detecting the transformed Ah receptor by the gel shift method after various concentrations of TCDD were allowed to act on the rat liver cytoplasmic fraction at 20 ° C. for 2 hours. The meanings of the numbers in FIG. 1 are as follows. 1: control; TCDD 0.05n
M, 3; 0.1 nM, 4; 0.2 nM, 5; 0.5 nM,
6; 1.0 nM, 7; 2.0 nM, 8; 5.0 nM, 9;
5.0 nM + competitor.

As shown in FIG. 1, the Ah receptor and the DRE
The concentration of the 3 bound bands increased in a TCDD concentration-dependent manner. The transformation was almost maximal at TCDD 0.5-1 nM. In subsequent experiments, TCDD
The Ah receptor was transformed using a concentration of 1 nM or 0.5 nM, and the inhibitory effect of catechins and tea extracts on it was examined. Next, as a preliminary study, it was examined whether or not catechin itself transforms the Ah receptor, that is, the effect of catechin as an agonist was examined. This experiment is necessary because if catechin has the same action as TCDD, there is no significance in examining the inhibitory effect on TCDD.

FIG. 2 shows the results obtained by reacting 100 μM of various catechins at 20 ° C. for 2 hours on the cytoplasmic fraction of rat liver and detecting the transformed Ah receptor by the gel shift method. The meanings of the numbers in FIG. 2 are as follows. 1; control, 2; TCDD
0.5 nM, 3; TCDD 0.5 nM + competitor, 4; (+) catechin, 5; (-) epicatechin,
6; (-) gallocatechin gallate, 7; (-) epigallocatechin, 8; (-) catechin gallate, 9; (-) epicatechin gallate, 10; (-) gallocatechin gallate, 1
1; (-) epigallocatechin gallate. FIG. 2 shows that none of the eight catechins used exhibited an agonistic action, and that they did not themselves transform the Ah receptor. Therefore, the inhibitory effect of catechin on TCDD-induced transformation (antagonistic effect) was measured.

FIG. 3 shows that 100 μM of various catechins and 1 nM of TCDD were added to rat liver cytoplasmic fraction at 20 ° C. for 2 hours.
The results of detecting the transformed Ah receptor by the gel shift method after allowing to act for a time are shown. FIG.
The meanings of the numbers in the table are as follows. 1: Control, 2: TCDD 1.0 nM, 3: TCDD 1.0 nM
+ Competitor, 4; TCDD + (+) catechin,
5; TCDD + (−) epicatechin; 6; TCDD +
(-) Gallocatechin, 7; TCDD + (-) epigallocatechin, 8; TCDD + (-) catechin gallate, 9;
TCDD + (−) epicatechin gallate, 10; TCD
D + (−) gallocatechin gallate, 11; TCDD +
(-) Epigallocatechin gallate.

That is, when 100 μM of catechin was added to the cytoplasm and then TCDD was allowed to act, four catechins having a gallate ester in the structure, ie, (−) catechin gallate, (−) epicatechin gallate, and (−) gallo Catechin gallate and (−) epigallocatechin gallate suppressed the transformation of the Ah receptor by TCDD. On the other hand, (+) catechin, (−) epicatechin, (−) gallocatechin, (−)
Four kinds of epigallocatechin had a weak inhibitory effect on TCDD.

Next, the concentration dependency was examined using (−) catechin gallate and (−) epicatechin gallate. FIG.
In the rat liver cytoplasmic fraction, various concentrations of catechin gallate or epicatechin gallate and TCDD1 were added.
The results of detecting the transformed Ah receptor by gel shift method after nM was allowed to act at 20 ° C. for 2 hours are shown. The meanings of the numbers in FIG. 4 are as follows.
1; control, 2; TCDD 1.0 nM, 3-7;
TCDD + (−) catechin gallate, 8-12; TCD
D + (−) epicatechin gallate, 3 and 8; 5 μM, 4
And 9; 10 μM, 5 and 10; 20 μM, 6 and 11; 50
μM, 7 and 12; 100 μM. From FIG. 4, it was found that (−) catechin gallate and (−) epicatechin gallate suppress the action of TCDD at 50 μM or more.

Finally, the inhibitory effect of a tea extract rich in catechin was measured. FIG. 5 shows that 100 μg / ml of various kinds of tea extracts and TCDD were added to the cytoplasmic fraction of rat liver.
The figure shows the results of detecting the transformed Ah receptor by the gel shift method after 1 nM was allowed to act at 20 ° C. for 2 hours. The meanings of the numbers in FIG. 5 are as follows. 1; control, 2; TCDD 1.0 nM, 3;
TCDD + one tea picking green tea, 4; TCDD + Bancha, 5; T
CDD + gyokuro, 6; TCDD + color, 7; TCDD + narcissus, 8; TCDD + iron Kannon, 9; TCDD + Uba, 1
0: TCDD + Dimbra, 11: TCDD + Nuwara Eliya.

FIG. 5 shows that when 100 μg / ml of the tea extract was added to the cytoplasm, the effect was observed in all nine extracts used. In particular, the extracts of all three types of black tea (Uva, Dimbra and Nuara) completely suppressed the action of TCDD. Two types of green tea (one tea picking green tea, gyokuro) and two types of oolong tea (color type, narcissus) also showed strong inhibitory effects. The inhibitory effect of the extract of bancha and iron Kannon was weaker than the other seven.

[Brief description of the drawings]

FIG. 1 is an autoradiograph showing the concentration dependence of TCDD on Ah receptor transformation.

FIG. 2 is an autoradiograph showing the agonist effect of catechin on Ah receptor transformation.

FIG. 3 is an autoradiograph showing the antagonistic effect of catechin on Ah receptor transformation.

FIG. 4 is an autoradiograph showing the inhibitory effect of catechin gallate and epicatechin gallate on the transformation of Ah receptor by TCDD.

FIG. 5 is an autoradiograph showing the inhibitory effect of various tea extracts on the transformation of Ah receptor by TCDD.

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[Procedure amendment]

[Submission date] March 15, 2001 (2001.3.1.1)
5)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 4

FIG. 3

FIG. 5

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 A61P 43/00 (72) Inventor Takami Tsunoda 21 Sagamimachi Goddess, Hara-gun, Shizuoka Prefecture ITO EN Co., Ltd. In Central Research Laboratory (72) Inventor Iwao Sakane 21 Goddess of Sagara-cho, Haibara-gun, Shizuoka Prefecture F-Term in Inouen Central Research Laboratory Co., Ltd. NA14 ZB26 ZC42

Claims (6)

[Claims] 1. An agent for suppressing or inhibiting the onset of dioxin toxicity, comprising a flavonoid. 2. The flavonoids are flavones, flavanols, flavonols, chalcones, isoflavones,
The inhibitor according to claim 1, which is at least one natural flavonoid selected from the group consisting of flavanones, benzalcumalanones and anthocyanins. 3. Use of a flavonoid for suppressing or preventing the onset of dioxin toxicity. 4. A dioxin receptor blocking agent comprising a flavonoid. 5. The flavonoids are flavones, flavanols, flavonols, chalcones, isoflavones,
The blocking agent according to claim 4, wherein the blocking agent is at least one natural flavonoid selected from the group consisting of flavanones, benzalkumaranones and anthocyanins. 6. Use of flavonoids for blocking dioxin receptors.