JP2008208038A - Nrf2 activator - Google Patents

Abstract

The present invention provides compounds and compositions for use in the treatment of diseases in which activation of Nrf2 is effective for treatment.
A composition for use in the treatment of a disease in which activation of Nrf2 is therapeutically effective, comprising a compound derived from Bunaharitake is provided. Specific examples thereof include the following compounds. 4- (1-hydroxy-ethyl) -3-methyl-dihydro-furan-2-one, 5-hydroxy-3,4,5-trimethyl-5-H-furan-2-one, acetic acid 1- (4- Methyl-5-oxo-tetrahydro-furan-3-yl) -ethyl ester, (5-ethyl-tetrahydro-furan-2-yl) -acetic acid.
[Selection figure] None

Description

  The present invention relates to an Nrf2 activator comprising a natural compound derived from Bunaharitake and a composition used for the treatment of a disease for which activation of Nrf2 is effective for treatment.

  The idea that oxidative stress promotes aging was proposed as The Free Radical Theory of Aging by Denham Harman in 1956 and has been reconsidered in recent years as an aging society has been reached (Harman D., J., Gerontol. 11, 298-300 (1956)). This theory is that tissue cell damage due to reactive oxygen species (ROS) generated in vivo during oxygen breathing causes aging. In recent years, research on the ROS generation system and its elimination system has progressed, and ROS is related not only to cell damage but also to the control of cell functions including intracellular information transmission, and the failure of the control function promotes aging Research is progressing.

ROS is oxygen having free radicals with unpaired electrons. Examples of ROS generated in vivo include superoxide (O ₂ ⁻ ), hydroxy radical (HO ⁻ ), hydrogen peroxide (H ₂ O ₂ ), and peroxynitrite (ONOO ⁻ ) (Shimosawa T. et al.). , Nippon Rinsho. 2005 Jun; 63 (6): 994-9.). ROS is physiologically produced in intracellular organelles such as mitochondria, cell membrane, cytoplasm and the like, and oxidizes and modifies proteins, lipids, nucleic acids and the like as oxidative stress. As a result, for example, proteins are oxidatively modified to cause denaturation and enzyme inactivation, and nucleic acid oxidation causes DNA translational failure and degradation. Accumulation of these oxidatively modified biological components causes damage in cellular tissues, which leads to the promotion of numerous diseases such as aging and carcinogenesis. Therefore, enhancing the protective ability against oxidative stress is effective in preventing these diseases and delaying their progression (Ferrari CK et al., Biomed Pharmacother. 2003 Jul-Aug; 57 (5-6): 251- 60 Toshikazu Yoshikawa et al. “All About Antioxidants” (1998), published by Advanced Medicine.

  In recent years, various compounds (bio-foreign substances) that are not originally present in the living body, such as environmental pollutants, have been increasing and affecting the human body. Normally, when these xenobiotics are absorbed into the body, they are excreted by being metabolized (detoxified) by xenobiotic-metabolizing enzymes. However, when its metabolic activity deteriorates, xenobiotics (especially those that affect the human body) accumulate in the human body, leading to metabolism to electrophilic substances and decreased liver function, leading to cancer, respiratory organs, etc. It leads to various diseases such as diseases and liver dysfunction. (Panuganti SD et al., J Pharmacol Exp Ther. 2006 Jul; 318 (1): 26-34. Epub 2006 Mar 28. Miller JA et al., Cancer Res. 1970 Mar; 30 (3): 559-76 Kohle C et al., Biochem Pharmacol. 2006 Sep 28; 72 (7): 795-805. Epub 2006 Apr 29). Therefore, increasing the detoxification ability of xenobiotics is effective in preventing these diseases and delaying their progression.

  In recent years, it has been clarified that a series of proteins called second-phase enzymes cooperate in defense against oxidative stress in the living body and detoxification of xenobiotics. The second phase enzymes include glutathione S-transferase (GST), NAD (P) H: quinone oxidoreductase 1 (NQO1), glutamylcysteine ligase (GCL), heme oxygenase 1 (HO1), thioredoxin reductase 1 (TXNRD1 ) Etc. are known. For example, GST catalyzes the formation of a complex of glutathione and a xenobiotic compound in a living body, and acts to protect against electrophilic chemical substances. NQO1 is involved in the two-electron reduction reaction of quinone, and can avoid the oxidative damage caused by the one-electron reduction of quinone. Furthermore, TXNRD1 is an enzyme responsible for the conversion of oxidized form of thioredoxin from reduced form, and reduced thioredoxin is involved in ROS elimination and intracellular redox control via a thiol group. (Toshikazu Yoshikawa et al., “All about Antioxidants” published by Advanced Medicine (1998), Masayasu Inoue et al., “Redox Control and Antioxidant Treatment Strategy” published by the Pharmaceutical Journal). There is a common sequence called an antioxidant responsive element (ARE) in the promoter of these second phase enzyme genes, and its expression is induced by the transcription factor NF-E2 related factor 2 (Nrf2). (Nguyen T et al., Annu Rev Pharmacol Toxicol. 43: 233-60 (2003), Itoh K et al., Free Radic Biol Med. 2004 May 15; 36 (10): 1208- 13).

  Nrf2 is localized in the cytoplasm as a Keap1-Nrf2 complex under non-stress conditions, and when activated substances such as electrophiles, active oxygen, and heavy metals act on it, it migrates into the nucleus, and heterozygous with small maf. Forms a mer and binds to ARE, inducing the expression of a second phase enzyme (Itoh K et al., Seikagaku. 2006 Feb; 78 (2): 79-92).

  In fact, it has been reported that mice and cells lacking Nrf2 have low resistance to oxidative stress and carcinogens (Ramos-Gomez M et al., Proc Natl Acad Sci US A. 2001 Mar 13; 98 ( 6): 3410-5, Ishii T et al., J Biol Chem. 2000 May 26; 275 (21): 16023-9).

  Thus, by activating the transcription factor Nrf2 and enhancing the activity of the second phase enzymes controlled by Nrf2, the defense function against oxidative stress can be enhanced to treat various diseases caused by oxidative stress. In addition, the xenobiotic detoxification system can be enhanced to reduce the risk of carcinogenesis caused by the xenobiotic. Accordingly, there is a strong demand for the development of pharmaceuticals or foods that can activate the transcription factor Nrf2 and that are highly safe for the living body.

  As an activator of Nrf2, isothiocyanate compounds (especially sulforaphane), which are abundant in cruciferous vegetables, are famous (Zhang Y et al., Proc Natl Acad Sci US A. 1992 Mar 15; 89 (6 ): 2399-403). Other Nrf2 activators include 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) (Itoh T et al., Mol Cell Biol. 2004 Jan; 24 (1): 36-45), hops Xanthohumol (Dietz BM et al., Chem Res Toxicol. 2005 Aug; 18 (8): 1296-305), ginger related ginger (Japanese Patent Laid-Open No. 2006-188444), tea Epigallocatechin gallate (Chen C et al., Arch Pharm Res. 2000 Dec; 23 (6): 605-12) and the like are known.

By the way, beech haritake (Mycoleptodonoides aitchisonii) is a white rot fungus that grows on fallen trees of beech and mizunara in early September-November. This mushroom has an umbrella diameter of 3-10cm, semi-circular, white, and later yellowish, the back is white and needle-like, as well as the surface, it is later yellowish, the meat is white, soft and water-absorbing, unique It is known for its aroma and taste and is valued as edible. It is mainly eaten in the Tohoku region and is also known as Kamiharitake. As an artificial cultivation method, it has been reported so far in JP-A-2006-6164, JP-A-2004-350589, and the like. So far, as a physiological action possessed by Bunaharitake, diabetes prevention / improvement action (patent document 1), antitumor action (patent document 2), brain function improving action (patent document 3), blood pressure lowering action (patent document 4), A thrombolytic action (Patent Document 5) has been reported. However, it has not been reported so far that Bunaharitake has an Nrf2 activating action and thus induces a second-phase enzyme group to enhance a defense system against oxidative stress and a detoxification system for xenobiotics. In addition, to date, knowledge of (E) -2-decene-diacid (compound 6) as a component of royal jelly (Japanese Patent Laid-Open No. 48-8723), 2-phenyl-5,6-dihydro-γ -Knowledge as mushroom flavor of pyrone (Compound 10) (Patent No. 2948870), Knowledge of tobacco component of 2- (2-formylpyrrol-1-yl) -4-methylvaleric acid (Compound 11) ( However, it has not been reported that these compounds have Nrf2 activating ability.
JP 2002-187851 A JP 2002-187850 A JP 2002-080390 A JP 2002-029995 A JP 2006-131550 A

Summary of the Invention

  The present inventor has found that a compound purified from the beech haritake ethyl acetate fraction has Nrf2 activating activity. The present invention is based on these findings.

  An object of the present invention is to provide a novel compound useful for activation of Nrf2 and a composition used for treatment of a disease for which activation of Nrf2 is effective for treatment.

［上記式中、
Ｒ^１は、酸素原子またはＣ_１−６アルキル基を表し、Ｒ^２は、水素原子またはＣ_１−６アルキル基を表し、Ｒ^３は、水素原子、水酸基で置換されていてもよいＣ_１−６アルキル基、またはＣ_１−４アルキル基、Ｃ_１−４アシル基、カルボキシル基、およびＣ_１−４アルコキシカルボニル基からなる群から選択される置換基で置換されていてもよいＣ_１−６アルキルカルボニルオキシＣ_１−６アルキル基を表し、Ｒ^４は、水素原子またはカルボキシルＣ_１−６アルキル基を表し、
- -は、単結合または二重結合を表し、
但し、Ｒ^１が酸素原子である場合は- - -は二重結合であり、Ｒ^１がＣ_１−６アルキル基である場合は- - -は単結合である。］。 According to the present invention, there is provided a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a solvate thereof (hereinafter referred to as “the novel compound according to the present invention”):

[In the above formula,
R ¹ represents an oxygen atom or a C _1-6 alkyl group, R ² represents a hydrogen atom or a C _1-6 alkyl group, and R ³ represents a C ₁ -C ₁ which may be substituted with a hydrogen atom or a hydroxyl group. _A C _1-6 optionally substituted with a ₆ alkyl group, or a substituent selected from the group consisting of a C _1-4 alkyl group, a C _1-4 acyl group, a carboxyl group, and a C _1-4 alkoxycarbonyl group Represents an alkylcarbonyloxy C _1-6 alkyl group, R ⁴ represents a hydrogen atom or a carboxyl C _1-6 alkyl group,
- - represents a single bond or a double bond,
However, when R ¹ is an oxygen atom, − − − is a double bond, and when R ¹ is a C _1-6 alkyl group, − − − is a single bond. ].

According to the present invention, in the above formula (I), or the following formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VIa), or formula (VII) Nrf2 activation comprising a compound represented by the formula or a pharmaceutically acceptable salt thereof or a solvate thereof (hereinafter referred to as “Nrf2 activating compound according to the present invention”) as an active ingredient is effective for treatment. Compositions for use in the treatment of disease are provided.

［上記式中、
Ｒ^１０１は、水素原子またはＣ_１−６アルキル基を表し、Ｒ^１０２は、水酸基で置換されていてもよいＣ_１−６アルキル基；Ｃ_１−６アルキルカルボニル基；Ｃ_１−４アルキル基、Ｃ_１−４アシル基、カルボキシル基、およびＣ_１−４アルコキシカルボニル基からなる群から選択される置換基で置換されていてもよいＣ_１−６アルキルカルボニルオキシＣ_１−６アルキル基；またはフェニル基を表し、Ｒ^１０３は、水素原子、水酸基、またはＣ_１−６アルキル基を表し、Ｒ^１０４は、水素原子、水酸基、またはＣ_１−６アルキル基を表し、
- - -は、単結合または二重結合を表す。］。 Compound of formula (II):

[In the above formula,
R ¹⁰¹ represents a hydrogen atom or a _{C 1-6} alkyl ^{group, R 102} is optionally substituted with a hydroxyl _{C 1-6} alkyl _{group; C 1-6} alkylcarbonyl _{group; C 1-4} alkyl group, A C _1-6 alkylcarbonyloxy C _1-6 alkyl group optionally substituted with a substituent selected from the group consisting of a C _1-4 acyl group, a carboxyl group, and a C _1-4 alkoxycarbonyl group; or phenyl R ¹⁰³ represents a hydrogen atom, a hydroxyl group, or a C _1-6 alkyl group, R ¹⁰⁴ represents a hydrogen atom, a hydroxyl group, or a C _1-6 alkyl group,
- - - represents a single bond or a double bond. ].

［上記式中、
Ｒ^１０５は、Ｃ_１−６アルキル基を表し、Ｒ^１０６は、カルボキシルＣ_１−６アルキル基を表す。］。 Compound of formula (III):

[In the above formula,
R ¹⁰⁵ represents a C _1-6 alkyl group, and R ¹⁰⁶ represents a carboxyl C _1-6 alkyl group. ].

［上記式中、
ｍは、３から９の整数を表す。］。 Compound of formula (IV):

[In the above formula,
m represents an integer of 3 to 9. ].

［上記式中、
Ｒ^１０７は、１以上の水酸基で置換されていてもよいＣ_１−６アルキル基を表す。］。 Compound of formula (V):

[In the above formula,
R ¹⁰⁷ represents a C _1-6 alkyl group which may be substituted with one or more hydroxyl groups. ].

［上記式中、
Ｈｅｔは、異種の環員原子として１つの酸素原子を有する５〜６員の飽和または不飽和複素環式基を表す。］。
式（ＶＩａ）の化合物：

［上記式中、
Ｗ、Ｘ、Ｙ、Ｚのうち、いずれか１つが酸素原子を表し、それ以外が−ＣＨ_２−、＞ＣＨ−、−ＣＨ＝、＞Ｃ＝、−ＣＨ_２−ＣＨ_２−、＞ＣＨ−ＣＨ_２−、−ＣＨ＝ＣＨ−、または＞Ｃ＝ＣＨ−を表す（但し、Ｗが酸素原子である場合は、Ｘ、Ｙ、およびＺが、−ＣＨ_２−または＞ＣＨ−であり、Ｚが酸素原子である場合は、Ｗ、Ｘ、およびＹが、−ＣＨ_２−または＞ＣＨ−であり、Ｘが酸素原子である場合は、Ｗが−ＣＨ_２−ＣＨ_２−、＞ＣＨ−ＣＨ_２−、−ＣＨ＝ＣＨ−、または＞Ｃ＝ＣＨ−であり、ＹおよびＺが−ＣＨ＝、＞Ｃ＝、−ＣＨ_２−、または＞ＣＨ−であり、Ｙが酸素原子である場合は、Ｚが−ＣＨ_２−ＣＨ_２−、＞ＣＨ−ＣＨ_２−、−ＣＨ＝ＣＨ−、または＞Ｃ＝ＣＨ−であり、ＷおよびＸが−ＣＨ＝、＞Ｃ＝、−ＣＨ_２−、または＞ＣＨ−である）。］。 Compound of formula (VI):

[In the above formula,
Het represents a 5- or 6-membered saturated or unsaturated heterocyclic group having one oxygen atom as a hetero ring member. ].
Compound of formula (VIa):

[In the above formula,
W, X, Y, of Z, any one of an oxygen atom, and the other is _{-CH 2 -,> CH -,} - CH =,> C =, - CH 2 -CH 2 -,> CH- CH ₂ —, —CH═CH—, or> C═CH— (in the case where W is an oxygen atom, X, Y, and Z are —CH ₂ — or> CH—, Z When X is an oxygen atom, W, X, and Y are —CH ₂ — or> CH—, and when X is an oxygen atom, W is —CH ₂ —CH ₂ —,> CH—CH. ₂ -, - CH = CH-, or> is C = CH-, = Y and Z is -CH,> C =, - CH 2 -, or> is CH-, Y is an oxygen atom , Z is _{-CH 2 -CH 2 -,> CH} -CH 2 -, - CH = CH-, or> is C = CH-, W and X are -CH = _{> C =, - CH 2 -} , or> CH- is it). ].

［上記式中、
Ｒ^１０８は、Ｃ_１−６アルキル基を表す。］ Compound of formula (VII):

[In the above formula,
R ¹⁰⁸ represents a C _1-6 alkyl group. ]

  According to the present invention, a composition used for the treatment of a disease in which activation of Nrf2 is effective for treatment, comprising an extract of the fruit beech fruit body (hereinafter referred to as “the extract according to the present invention”) as an active ingredient (Hereinafter, these compositions may be collectively referred to as “the composition according to the present invention”).

  According to the present invention, there is provided a method for producing an Nrf2 activating compound according to the present invention, comprising the step of purifying the extract according to the present invention and measuring the Nrf2 activating ability of the obtained fraction. Provided.

  The novel compounds, Nrf2 activating compounds, and extracts according to the present invention are obtained from Bunaharitake, which has been eaten since ancient times. Therefore, the novel compound, Nrf2 activating compound and extract according to the present invention are advantageous in that they can be used as a highly safe Nrf2 activating agent.

Detailed Description of the Invention

As used herein , the terms “alkyl”, “alkoxy”, and “acyl” as a group or part of a group refer to straight-chain or branched alkyl groups, alkoxy groups, and acyl groups. means.
C _1-6 alkyl is preferably C _1-4 alkyl. C _1-4 alkyl is preferably C _1-2 alkyl.
C _1-4 alkoxy is preferably C _1-2 alkoxy.
C _1-4 acyl is preferably C _1-2 acyl.
Examples of C _1-6 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl.
Examples of C _1-4 alkoxy include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy.
Examples of C _1-4 acyl include formyl, acetyl, propionyl, butyryl, isobutyryl.
As used herein, “optionally substituted alkyl” refers to an alkyl in which one or more hydrogen atoms on the alkyl are substituted by one or more substituents (which may be the same or different) and Means unsubstituted alkyl. It will be apparent to those skilled in the art that the maximum number of substituents can be determined depending on the number of substitutable hydrogen atoms on the alkyl. The same applies to groups having substituents other than alkyl.
The novel compound according to the present invention is represented by the formula (I).

In the formula (I), R ¹ is preferably an oxygen atom or an ethyl group.

In the formula (I), R ² is preferably a hydrogen atom or a methyl group.

In the formula (I), R ³ is preferably a hydrogen atom, an ethyl group which may be substituted with a hydroxyl group, or an acetyloxymethyl group which may be substituted with a methyl group.

In the formula (I), R ⁴ is preferably a hydrogen atom or a carboxymethyl group.

In a preferred embodiment of the novel compound according to the present invention, R ¹ is an oxygen atom or an ethyl group, R ² is a hydrogen atom or a methyl group, and R ³ may be substituted with a hydrogen atom or a hydroxyl group. Examples thereof include compounds represented by the formula (I) which are an ethyl group or an acetyloxymethyl group optionally substituted with a methyl group, and R ⁴ is a hydrogen atom or a carboxymethyl group.

  In the present invention, preferred examples of the compound represented by the formula (I) include 4- (1-hydroxy-ethyl) -3-methyl-dihydro-furan-2-one (compound 2), acetic acid 1- ( 4-methyl-5-oxo-tetrahydro-furan-3-yl) -ethyl ester (compound 4) and (5-ethyl-tetrahydro-furan-2-yl) -acetic acid (compound 5).

The novel compound according to the present invention can be produced by extracting and purifying from a fruit body of Mycoleptodonoides aitchisonii belonging to the genus Beechaharitake, according to a known method.

Nrf2-Activating Compound The Nrf2-activating compound according to the present invention can be represented by the formula (II).

In formula (II), R ¹⁰¹ is preferably a hydrogen atom or a methyl group. More preferably, R ¹⁰¹ is a hydrogen atom.

In the formula (II), R ¹⁰² is preferably a methyl group, an ethyl group optionally substituted with a hydroxyl group, a methylcarbonyl group, an acetyloxymethyl group optionally substituted with a methyl group, or a phenyl group. . More preferably, R ¹⁰² is an ethyl group, a methylcarbonyl group, or a phenyl group which may be substituted with a hydroxyl group. Even more preferably, R ¹⁰² is a phenyl group.

In the formula (II), R ¹⁰³ is preferably a hydrogen atom, a hydroxyl group, or a methyl group. More preferably, R ¹⁰³ is a hydrogen atom.

In the formula (II), R ¹⁰⁴ is preferably a hydrogen atom, a hydroxyl group, or a methyl group. More preferably, R ¹⁰⁴ is a hydrogen atom.

In a preferred embodiment of the Nrf2 activating compound according to the present invention, R ¹⁰¹ is a hydrogen atom or a methyl group, and R ¹⁰² is a methyl group, an ethyl group optionally substituted with a hydroxyl group, a methylcarbonyl group, or a methyl group. An optionally substituted acetyloxymethyl group or a phenyl group, R ¹⁰³ is a hydrogen atom, a hydroxyl group, or a methyl group, and R ¹⁰⁴ is a hydrogen atom, a hydroxyl group, or a methyl group (II) ).

In a further preferred embodiment of the Nrf2 activating compound according to the present invention, R ¹⁰¹ is a hydrogen atom or a methyl group, and R ¹⁰² is an ethyl group, a methylcarbonyl group, or a phenyl group optionally substituted with a hydroxyl group. , R ¹⁰³ is a hydrogen atom, and R ¹⁰⁴ is a hydrogen atom, a compound represented by the formula (II) is given.

In an even more preferred embodiment of the Nrf2 activating compound according to the present invention, R ¹⁰¹ is a hydrogen atom, R ¹⁰² is a phenyl group, R ¹⁰³ is a hydrogen atom, and R ¹⁰⁴ is a hydrogen atom. And a compound represented by formula (II) (compound 9).

  In the present invention, preferred examples of the compound represented by the formula (II) include 4-acetyl-3-methyl-dihydro-furan-2-one (compound 1), 4- (1-hydroxy-ethyl)- 3-methyl-dihydro-furan-2-one (compound 2), 5-hydroxy-3,4,5-trimethyl-5-H-furan-2-one (compound 3), acetic acid 1- (4-methyl- 5-oxo-tetrahydro-furan-3-yl) -ethyl ester (compound 4), dihydro-4-phenyl-2 (3H) -furanone (compound 9).

  The Nrf2 activating compound according to the present invention can be represented by the formula (III).

In formula (III), R ¹⁰⁵ is preferably an ethyl group.

In the formula (III), R ¹⁰⁶ is preferably a carboxymethyl group.

A preferred embodiment of the Nrf2-activated compound according to the present invention includes a compound (compound 5) represented by the formula (III), wherein R ¹⁰⁵ is an ethyl group and R ¹⁰⁶ is a carboxymethyl group.

  In the present invention, preferred examples of the compound represented by the formula (III) include (5-ethyl-tetrahydro-furan-2-yl) -acetic acid (Compound 5).

  The Nrf2 activating compound according to the present invention can be represented by the formula (IV).

  In the formula (IV), m is preferably an integer of 5 to 7. More preferably, m is 6.

  A preferred embodiment of the Nrf2 activating compound according to the present invention includes a compound represented by formula (IV) (compound 6) wherein m is 6.

  In the present invention, a preferred example of the compound represented by the formula (IV) is (E) -2-decene-diacid (compound 6).

  The Nrf2 activating compound according to the present invention can be represented by formula (V).

In the formula (V), R ¹⁰⁷ is preferably a pentyl group which may be substituted with one or more hydroxyl groups, and more preferably a pentyl group substituted with one or two hydroxyl groups.

A preferred embodiment of the Nrf2 activating compound according to the present invention includes a compound represented by the formula (V), wherein R ¹⁰⁷ is a pentyl group optionally substituted with one or more hydroxyl groups.

  In the present invention, a preferred example of the compound represented by the formula (V) is 1-phenyl-pentane-1,3-diol (compound 7).

  The Nrf2 activating compound according to the present invention can be represented by the formula (VI).

  The compound of the formula (VI) can be preferably represented by the formula (VIa).

In a preferred embodiment of the Nrf2 activated compound according to the present invention, W is an oxygen atom, X, Y, and Z are —CH ₂ — or> CH—, a compound represented by formula (VIa), and Z is The compound represented by the formula (VIa) which is an oxygen atom and W, X, and Y are —CH ₂ — or> CH— is exemplified.

  In a preferred embodiment of the Nrf2 activating compound according to the present invention, X is an oxygen atom, W is —CH═CH— or> C═CH—, and Y and Z are —CH═ or> C═. A compound represented by formula (VIa), and a formula (Y) in which Y is an oxygen atom, Z is -CH = CH- or> C = CH-, and W and X are -CH = or> C = And compounds represented by VIa).

In a preferred embodiment of the Nrf2 activating compound according to the present invention, X is an oxygen atom, W is —CH ₂ —CH ₂ — or> CH—CH ₂ —, and Y and Z are —CH═ or> A compound represented by formula (VIa) wherein C =, X is an oxygen atom, W is —CH═CH— or> C═CH—, and Y and Z are —CH ₂ — or> CH—. A compound represented by formula (VIa), Y is an oxygen atom, Z is —CH ₂ —CH ₂ — or> CH—CH ₂ —, and W and X are —CH═ or> C═. A compound represented by formula (VIa), and a compound in which Y is an oxygen atom, Z is —CH═CH— or> C═CH—, and W and X are —CH ₂ — or> CH—. And compounds represented by VIa).

  In the present invention, preferred examples of the compound represented by the formula (VI) include 2-phenyl-pyran-4-one (compound 8), dihydro-4-phenyl-2 (3H) -furanone (compound 9). And 2-phenyl-5,6-dihydro-γ-pyrone (compound 10).

  The Nrf2 activating compound according to the present invention can be represented by the formula (VII).

In the formula (VII), R ¹⁰⁸ is preferably a branched C _1-6 alkyl group, more preferably a branched butyl group.

A preferred embodiment of the Nrf2 activating compound according to the present invention includes a compound represented by the formula (VII), wherein R ¹⁰⁸ is a branched C _1-6 alkyl group.

  In the present invention, a suitable example of the compound represented by the formula (VII) is 2- (2-formylpyrrol-1-yl) -4-methylvaleric acid (Compound 11).

  In the present invention, the novel compound represented by the formula (I) can also be used for the treatment of diseases in which Nrf2 activation is effective for treatment.

  The compounds of formulas (I) to (VII) can be pharmaceutically acceptable salts, for example, acid addition salts. Acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, and sulfate; citrate, oxalate, malate, tartrate, fumarate, maleate, methanesulfonate And organic acid salts such as salicylate. Moreover, the compound which has a carboxyl group can also be made into salts with metals, such as sodium, potassium, calcium, magnesium, aluminum, and amino acids, such as a lysine.

  The compound represented by any one of formulas (I) to (VII) and a salt thereof can be a pharmaceutically acceptable solvate, such as a hydrate, an alcohol solvate (for example, a methanol solvate). , Ethanol solvate) and ether solvate.

  Commercially available Nrf2 activating compounds according to the present invention can be obtained.

  The Nrf2 activating compound according to the present invention can also be produced according to a known method.

  For example, compound 1 is disclosed in JP-A-51-041350, Forzato C et al., Tetrahedron Asymmetry (2005), 16 (18), 3011-3023, Piskov VB, Zhurnal Obshchei Khimii (1960), 30 1390-5. Can be manufactured according to.

  Compound 3 is prepared according to Fabian WM et al., Eur. J. Org. Chem. (2001), 2 303-309, Scheffold R at al., Helv. Chim. Acta (1967) 50 (3) 798-807. can do.

  Compound 6 can be prepared according to Noda N et al., Lipids (2005), 40 (8), 833-838, Fujisawa T et al., Chem. Lett. (1982), (2) 219-20. .

  Compound 7 is prepared according to Bartoli G et al., Eur. J. Org. Chem. (2001), 24 4679-4684, Emerson WS et al., J. Org. Chem. (1945), 10 464-9. be able to.

  Compound 8 was prepared by Groundwater PW et al., J. Chem. Soc., Perkin Trans. 1, (1997), (2), 163-169, Koreeda M et al., Tetrahedron Lett. (1980), 21 (13 ), 1197-200.

  Compound 9 is disclosed in JP 2004-269490 A, Bomb C et al., Synlett (2004), (9), 1619-1621, Pharm I et al., Archiv der Pharmazie (Weinheim, Germany) (1993), 326. (12), 941-5.

  Compound 10 can be produced according to Japanese Patent No. 2948870, Centro N C et al., Gazzetta Chimica Italiana (1966), 96 (8-9), 1073-83.

  Compound 11 can be produced according to Abell A D et al., Tetrahedron Letters (1992), 33 (39), 5831-2, Japanese Patent Application Laid-Open No. 51-123899.

  As the compound used as an active ingredient in the present invention, a compound purified from the fruit body of Bunaharitake can be used. When various mushrooms were evaluated for their ability to induce QR activity, Bunaharitake had a high ability to induce QR activity. In addition, beech haritake is an edible mushroom and can be artificially cultivated. Therefore, in the present invention, the beech haritake fruiting body is used.

  The compound purified from the beech fruit body may be any compound that is derived from the beech fruit body and has an Nfr2 activating action, preferably any one of formulas (II) to (VII) It is a compound represented by these. In the present invention, the beech haritake fruit body can be used as an active ingredient as it is, or the beech haritake fruit body extract can also be used.

  The fruit body of the beech tree used in the present invention is the fruit body of the beech tree (Mycoleptodonoides aitchisonii) belonging to the order of the Aphyllophorales genus Climacodontaceae.

  Examples of the fruit body of the beech tree include a natural fruit body that naturally grows in nature, and a fruit body that has been artificially cultivated. However, the quality of the ingredient is not affected by the habitat or climate, and the year-round stability. From the viewpoint of being able to be harvested, an artificially cultivated beech haritake fruit body is preferable.

  Examples of the artificial cultivation method include fungus bed cultivation, log cultivation, and the like, but fungus bed cultivation is preferable in that it can be harvested inexpensively and stably year-round for the quality of the component content and the like.

  Here, `` fungus bed cultivation '' means a method of inoculating a material consisting of a water retaining body and a nutrient source without using raw wood, and cultivating under an environment in which temperature, humidity, illuminance, etc. are controlled, Specific examples of the fungus bed cultivation method include the method described in JP-A-2003-024406, but are not limited to these methods.

  In the case where the beech haritake fruit body is used as it is as an active ingredient, the beech haritake fruit body may be used as it is, or a pulverized product obtained by pulverizing the beech haritake fruit body after air drying, hot air drying or freeze drying may be used. Further, the pulverized product may be granulated, encapsulated, tableted, paste or the like by a conventional method. Further, even a suspension or suspension of the pulverized product thus pulverized can be used as long as it has an Nrf2 activation effect.

In the present invention, the extract of a fruit beech fruit body used as an active ingredient can be extracted and prepared according to a known method.

  The extract of a beech haritake fruit body can be used for the extraction operation of the beech haritake fruit body as it is, but can also be used for the extraction operation after a drying process. For example, it can be prepared by pulverizing the freeze-dried beech haritake fruit body and then subjecting it to an extraction operation. The extraction operation can be carried out, for example, by suspending the fruit body of Bunaharitake in a solvent and crushing the cells with stirring or sonication, grinding, French press or the like. From the crude extract obtained by these extraction operations, solids can be removed by a single or a combination of solid-liquid separation operations such as centrifugation and filtration, if necessary. The obtained liquid can be further separated by separation based on a difference in solubility in ethanol or the like or liquid-liquid distribution using ethyl acetate or the like. The obtained beech haritake fruit body extract (liquid) may be further processed, for example, used as a liquid or solid form of a concentrate, a lyophilized product, a partially purified product, or the like.

  Solvents usable in extraction include water at 0 ° C. to 100 ° C., an appropriate buffer, an appropriate organic solvent (for example, alcohols such as methanol and ethanol), or a mixture of an appropriate organic solvent and water (for example, Ethanol water) and the like, preferably hot water, more preferably hot water at 100 ° C. The extraction time is not particularly limited, but is 5 minutes or more, preferably about 30 minutes. Subsequently, the extract of a fruit beech fruit body can be obtained by centrifugation or filtration. The amount of the extraction liquid is not particularly limited, but it is preferable to use about 10 times as much water as the dry mushroom weight in terms of cost, extraction efficiency, and the like. As the extraction solvent, a solvent such as ethanol can be added as appropriate, but it is desirable to extract only with water from the viewpoint of cost and ease of handling at the production site.

  The fruiting body may be extracted as it is, but it is preferable that the fruiting body is extracted from a fruiting body that can be stored after drying because an extract or extract with excellent quality can be obtained. Further, it is preferable to extract the dried fruit body after pulverization, since the extraction rate is improved.

  The drying method is not particularly limited as long as it is a known drying method such as a hot air drying method, an air drying method, a spray drying method, a vacuum (or reduced pressure) drying method, a freeze drying method, etc., but suppresses a decrease in Nrf2 activation action. In this respect, the freeze-drying method is preferable.

  According to a preferred embodiment of the present invention, the lyophilized beech garnish is suspended in hot water at 100 ° C., extracted with stirring, solid-liquid separated by filtration, and separated by the difference in solubility in ethanol solution. Thereafter, further extraction with ethyl acetate can yield a Bunaharitake fruiting body extract.

  A substance having the ability to activate Nrf2 present in the extract may be further purified, and in this case, known separation and purification methods can be combined appropriately.

  Methods that can be used for further purification of the active substance include, for example, methods that utilize solubility such as liquid-liquid partitioning and organic solvent precipitation, methods that utilize molecular weight differences such as gel filtration, and ion exchange chromatography. Such a method using a difference in charge, a method using a difference in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, a recrystallization treatment method, a reprecipitation treatment method, and a process for recovering precipitates generated by cooling. Examples thereof include a method, acid-base distribution, a separation method using a density gradient, and a separation method based on vapor pressure. Further, the concentration after extraction and purification may be any method as long as it is a publicly known method, and specific examples include heating under reduced pressure. Further, the drying after extraction and purification may be any known method as long as it is a known method, and specific examples include air drying, heat drying, spray drying, and freeze drying.

Production Method The production method according to the present invention is preferably a method for producing an Nrf2 activating compound comprising the following steps:
(A) a step of extracting a beech haritake fruit body using an extraction solvent;
(B) a step of fractionating the extract obtained in step (a);
(C) a method comprising measuring the Nrf2 activation ability of the fraction obtained in step (b); and (d) purifying the fraction determined to have Nrf2 activation ability. is there.

  In step (c), for example, by measuring QR activity, ARE promoter activity, expression of various second phase enzymes, etc., it can be determined whether or not the obtained fraction has Nrf2 activation ability ( See Examples 5-7 below). Here, for example, when an increase in QR activity, an increase in ARE promoter activity, and an increase in expression of various phase II enzymes are confirmed, the obtained fraction has Nrf2 activation ability, that is, the Nrf2 activity according to the present invention. It can be determined that it is a fraction containing a chemical compound.

  In the step (d), purification can be performed according to a known method. For example, the Nrf2 activating compound according to the present invention can be obtained by concentrating and crystallizing the obtained fraction.

Application Transcription factor Nrf2 is known to be deeply related to the ability to protect against oxidative stress in the body and detoxification of xenobiotics. Specifically, the transcription factor Nrf2 is known to induce the expression of the gene by binding to the ARE in the promoter with respect to the second phase enzyme group. It is known that increased expression of the two-phase enzymes enhances protection from oxidative stress and detoxification of xenobiotics.

  When Nrf2 enters an oxidative stress state, Nrf2 moves from the cytoplasm to the nucleus in response to the oxidative stress, binds to ARE, and induces the expression of a second phase enzyme that effectively works for oxidative stress defense and antioxidation. Increase the defense system. When the oxidative stress defense system is enhanced, the oxidative stress state is improved, and various diseases caused by oxidative stress are expected to be improved. For example, age-related macular degeneration, which is one of the chronic diseases associated with aging, is a typical disease that develops with aging and causes blindness in elderly people. The cause of this disease is suggested to be photooxidative stress on retinal pigment epithelial cells, and accumulation of oxidized products of lipids and proteins is known as a risk factor. When photooxidative stress is applied to retinal pigment epithelial cells, the amount of lipid peroxide in the cells increases and the cells die, but sulforaphane and bis (2-hydroxybenzylidene) acetone, which have the ability to induce phase II enzymes, do not. (Gao X et al., Proc Natl Acad Sci US A. 2004 Jul 13; 101 (28): 10446-51). In addition, a decrease in the ability of the living body to protect against oxidative stress caused by a decrease in the amount of glutathione, etc. may increase ROS in the blood and cause hypertension. Peroxynitrite, a kind of ROS, is also a strong inflammation-inducing substance, and it has been pointed out that its formation may lead to the formation of arteriosclerotic lesions. However, in stroke-prone spontaneously hypertensive rats (SHRsp), administration of broccoli sprout containing a large amount of glucoraphanin (a metabolic precursor of sulforaphane) can suppress an increase in blood pressure and improve vascular endothelial function. (Wu L et al., Proc Natl Acad Sci US A. 2004 May 4; 101 (18): 7094-9). This indicates that an increase in glutathione level and induction of phase 2 enzymes are effective in improving hypertension.

  Nrf2 also enhances the detoxification system in addition to the oxidative stress defense system. The xenobiotic metabolism system (detoxification system) in which substances that become foreign substances such as drugs and environmental chemicals are metabolized is a first phase enzyme group controlled by transcription factors AhR, CAR, etc., and a first phase controlled by Nrf2. It consists of two-phase enzymes. Xenobiotics are converted to oxo intermediates by the first phase enzymes, then converted to more water-soluble derivatives by the second phase enzymes and excreted from the body (Rushmore TH et al., Curr Drug Metab. 2002 Oct; 3 (5): 481-90). In this case, the oxo intermediate after the first phase enzyme reaction is often a very strong toxic substance. For example, benzopyrene known as a carcinogen is metabolized by a first phase enzyme group such as CYP1A1 and then converted to benzopyrene diol epoxide, and then conjugated with glutathione or glucuronic acid by a second phase enzyme group such as GST. Discharged. At this time, it is said that the intermediate benzopyrene diol epoxide binds to DNA and causes damage to the replication process of DNA, causing canceration (Motohashi H et al., Trends Mol Med. 2004 Nov; 10 ( 11): 549-57). From this, it is considered that carcinogenesis can be suppressed if the activity of the second-phase enzyme group can be increased and harmful intermediates can be metabolized faster and excreted from the body. DEP (diesel exhaust particles), a particulate contaminant exhausted by diesel engines and a kind of xenobiotic, is known to cause symptoms such as respiratory diseases, but GST induced by Nrf2 activation. , NQO1, and HO1 have been reported to protect against oxidative stress by DEP and the toxicity of quinone in DEP (Li N et al., J Immunol. 2004 Sep 1; 173 (5): 3467- 81). From this, it is considered that the Nrf2 activator is effective for the prevention and improvement of various diseases caused by environmental pollutants.

  In addition to the above, the Nrf2 activator is a disease caused by oxidative stress or xenobiotics such as inflammation (Proc Natl Acad Sci US A. 2004 May 4; 101 (18): 7094-9), carcinogenesis ( Proc Natl Acad Sci US A. 1992 Mar 15; 89 (6): 2399-403 Carcinogenesis.2000 Dec; 21 (12): 2287-91 Fahey Jwet al., Food Chem Toxicol. 1999 Sep-Oct; 37 (9- 10): 973-9), skin disorders (J Clin Invest. 2004 Jan; 113 (1): 65-73), Helicobacter pylori protection (Proc Natl Acad Sci US A. 2002 May 28; 99 (11): 7610- 5), neurodegenerative diseases (Curr Drug Targets CNS Neurol Disord. 2005 Jun; 4 (3): 267-81), environmental chemical detoxification (Toxicol Sci. 2006 Mar; 90 (1): 111-9. Epub 2005 Dec 13), liver dysfunction (Toxicol Sci. 2006 Mar; 90 (1): 111-9. Epub 2005 Dec 13), autoimmune disease (Am J Pathol. 2006 Jun; 168 (6): 1960-74), diabetes (Transplant Proc. 2006 Jan-Feb; 38 (1): 282-3) has been reported to improve symptoms.

  According to the examples, the ethyl beech halachacetate fraction used as an active ingredient in the present invention is the activity of quinone reductase (QR), which is known to increase due to increased expression of NQO1, which is a second phase enzyme (implementation) Example 5), ARE promoter activity (Example 6) and gene expression of various second phase enzymes (Example 7) were increased. In addition, the ethyl beech garlic acetate fraction according to the present invention suppressed cell death by active oxygen (Example 8). Furthermore, the expression of the phase II enzyme group (Example 9) and the QR activity in the liver (Example 10) were increased in the liver and small intestinal epithelium of mice that were orally administered with the ethyl beech extract of the present invention. From these results, it was shown that the ethyl beech haritake acetate has a function of activating the transcription factor Nrf2.

  According to the examples, the compounds 1 to 11 isolated and purified from the extract of Bunaharitake used as an active ingredient in the present invention increased the QR activity (Example 5). From these results, it was shown that the compounds 1 to 11 have a function of activating the transcription factor Nrf2.

  Therefore, the active ingredient according to the present invention, that is, the Nrf2 activating compound according to the present invention and the extract according to the present invention can be used for the treatment of diseases in which the activation of the transcription factor Nrf2 is effective for treatment.

  In the present specification, “diseases in which activation of Nrf2 is effective for treatment” include diseases caused by oxidative stress and diseases caused by xenobiotics.

  In the present specification, “oxidative stress” means a state in which the oxidative damage power of ROS or the like generated in the living body exceeds the antioxidant potential of the in-vivo antioxidant system. “Diseases caused by oxidative stress” include chronic diseases associated with aging (eg age-related macular degeneration), arteriosclerosis, hypertension, diabetes, carcinogenesis, liver dysfunction, ulcers, neurodegenerative diseases (eg , Parkinson's disease, Alzheimer's disease, etc.), autoimmune diseases, blemishes, wrinkles, eye diseases, respiratory diseases (eg, bronchitis), asthma, inflammation (eg, gastritis, hepatitis, dermatitis, etc.), cerebral infarction, myocardium Examples include infarctions.

  In the specification of the present application, the “xenobiotic” means a substance that does not originally exist in the living body, and particularly a compound that is harmful to the living body. Examples of “diseases caused by xenobiotics” include respiratory diseases (eg bronchitis), liver dysfunction, carcinogenesis, autoimmune diseases and the like.

  In the present specification, “treatment” is used in the meaning including prevention and improvement.

  In the present specification, “disease” is used in a meaning including a state.

  In the present specification, “treatment of disease” is used in the meaning including regulation, delay of progression, alleviation, prevention of onset, prevention of recurrence, suppression of disease or condition.

  According to the present invention there is provided an Nrf2 activator comprising an Nrf2 activating compound according to the present invention or an extract according to the present invention.

  According to the present invention there is provided an oxidative stress protective agent, an antioxidant and a xenobiotic antidote comprising the Nrf2 activating compound according to the present invention or the extract according to the present invention.

  According to the present invention, there is provided use of an Nrf2 activating compound according to the present invention or an extract according to the present invention for the manufacture of a therapeutic agent for a disease for which activation of Nrf2 is therapeutically effective.

  According to the present invention there is also provided the use of an Nrf2 activating compound according to the present invention or an extract according to the present invention for the manufacture of an Nrf2 activator.

  According to the present invention there is also provided the use of an Nrf2 activating compound according to the present invention or an extract according to the present invention for the manufacture of an oxidative stress protective agent, an antioxidant, or a xenobiotic antidote.

  According to the present invention, Nrf2 activation is therapeutically effective comprising administering to a mammal, including a human, a therapeutically effective amount of an Nrf2 activating compound according to the present invention or an extract according to the present invention. A method of treating a disease is provided.

  According to the present invention, there is also provided a method for activating Nrf2, comprising the step of administering an effective amount of an Nrf2 activating compound according to the present invention or an extract according to the present invention to a mammal including a human.

  According to the present invention, there is also provided a method for protecting against oxidative stress, an antioxidant method, and a living body comprising the step of administering an effective amount of an Nrf2 activating compound according to the present invention or an extract according to the present invention to mammals including humans. A method for detoxification of foreign matter is provided.

Pharmaceutical composition and food When the composition and preparation according to the present invention are used as a medicine, the Nrf2 activating compound according to the present invention or the extract according to the present invention is used as an active ingredient, and a pharmaceutically acceptable carrier, excipient It can be produced by mixing with an agent, binder, diluent and the like.

  The active ingredient may be, for example, a pulverized fruit body (for example, powder), a suspension, an extract, or a product obtained by further purifying the extract, or a mixture containing the active ingredient, or substantially The thing refined until it became single may be sufficient.

  The compositions and preparations according to the present invention can be administered orally or parenterally. Examples of oral preparations include granules, powders, tablets (including sugar-coated tablets), solutions, pills, soft or hard capsules, syrups, pastes, emulsions, suspensions and the like. Examples of parenteral preparations include injections (for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, and external preparations (for example, nasal preparations, transdermal preparations, ointments) ), Suppositories (for example, rectal suppositories, vaginal suppositories), eye drops and the like. These preparations can be formulated together with a pharmaceutically acceptable carrier (for example, an excipient and an additive) by a technique commonly used in the art. Pharmaceutically acceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, disintegrants. , PH adjusting agents, wetting agents, lubricants and the like. Examples of the pharmaceutically acceptable carrier include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like. It is done.

  The preparation can be produced, for example, as follows.

  An oral preparation contains, as an active ingredient, for example, excipients (for example, lactose, sucrose, starch, mannitol), disintegrants (for example, calcium carbonate, carboxymethylcellulose calcium), binders (for example, pregelatinized starch, gum arabic, carboxy Methylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose) or lubricant (eg talc, magnesium stearate, polyethylene glycol 6000) and compression molded, then, if necessary, taste masking, enteric or sustained purposes Therefore, it can be produced by coating by a method known per se. As the coating agent, for example, ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate and Eudragit (Rohm, Germany, methacrylic acid / acrylic acid copolymer) can be used.

  The injection comprises an active ingredient as a dispersant (for example, Tween 80 (manufactured by Atlas Powder, USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservative (for example, Methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol), isotonic agents (for example, sodium chloride, glycerin, sorbitol, glucose, invert sugar) and aqueous solvents (for example, distilled water, physiological saline, Ringer's solution, etc.) ) Or an oil-based solvent (for example, olive oil, sesame oil, cottonseed oil, vegetable oil such as corn oil, propylene glycol) or the like. At this time, if desired, additives such as a solubilizing agent (for example, sodium salicylate, sodium acetate), a stabilizer (for example, human serum albumin), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride) are added. Also good.

  In formulating, one or more active ingredients other than the active ingredient according to the present invention may be further blended. In the administration of the active ingredient according to the present invention, one or more medically effective active ingredients other than the active ingredient according to the present invention may be administered in combination.

  The compositions and preparations according to the present invention are intended not only for pharmaceutical applications but also for foods. Therefore, in the application of the composition and preparation according to the present invention to foods, reference can be made to the description relating to foods described below.

  The food according to the present invention is a food containing an effective amount of the Nrf2 activating compound according to the present invention or the extract according to the present invention. Here, “contained in an effective amount” refers to a content in which an active ingredient is ingested in a range as described later when an amount normally consumed in each food is ingested. In the food according to the present invention, the Nrf2 activating compound according to the present invention or the extract according to the present invention can be added to the food as it is or in the form of the composition as described above. More specifically, the food according to the present invention further comprises a Nrf2 activating compound according to the present invention or an extract according to the present invention as it is prepared as a food, and various proteins, sugars, fats, trace elements, vitamins and the like. , Liquid, semi-liquid or solid, paste, or added to general foods. The food according to the present invention comprises an Nrf2 activating compound according to the present invention or an extract according to the present invention and an anti-food such as cruciferous plants, hops, ginger, tea, etc. You may use in combination with food with high diuretic effects, such as food with reported oxidation activity, coffee, moss, and herbal tea. In the present invention, the “food product” is other than a medicine and is not particularly limited as long as it can be taken by a mammal.

  In the present invention, the term “food” is used to include health foods (for example, foods for specified health use, functional nutritional foods, nutritional supplements), functional foods, and foods for the sick.

  The health food may also be in the form of a normal food or in the form of a dietary supplement (eg supplement).

  The form of “food” is not particularly limited, and may be, for example, a beverage form.

  The food according to the present invention can be provided as a food suitable for consumers expecting an Nrf2 activation function, an improvement or alleviation of a state improved or alleviated with the activation of Nrf2, that is, a food for specified health use. it can. The “special health food” as used herein refers to health care when manufacturing or selling food for the purpose of Nrf2 activation, improvement or alleviation of conditions that are improved or alleviated with Nrf2 activation. Foods that may be subject to some legal restrictions from a viewpoint.

  According to the present invention, there is provided a food comprising an effective amount of the Nrf2 activating compound or Bunaharitake extract according to the present invention, wherein the Nrf2 activating function is improved or mitigated by the activation of Nrf2. Foods marked with are provided. Here, the various functions described above can be displayed on any of a food body, a container, a package, a manual, a package insert, or an advertisement.

  Examples of foods and drinks that are ingested daily, which are targets of addition / formulation of the Nrf2 activating compound according to the present invention or the extract according to the present invention, include yogurt, drink yogurt, juice, milk, soy milk, alcoholic beverages (alcoholic beverages), coffee , Tea, tea, oolong tea, sports beverages, baked confectionery such as cookies, bread, cakes, rice crackers, Japanese confectionery such as sheepskin, frozen confectionery such as pudding, jelly and ice cream, confectionery such as chewing gum and candy , Snacks such as crackers and chips, noodles such as udon and soba, fish paste products such as kamaboko, ham, and fish sausage Seasonings, tofu, konjac, other boiled sardines, dumplings, croquettes, salads, sushi Flop, it can be exemplified various delicatessen stew, etc., but not particularly limited thereto.

  According to a more preferred embodiment of the present invention, examples of the food to be added and blended include beverages mixed with vegetable juice and tea-based beverages.

    In the production of beverages (including health foods and functional foods in the form of beverages) provided in the present invention, sugars, fragrances, fruit juices, food additives, etc. used for normal beverage formulation design are added as appropriate. be able to. In the production of beverages, production techniques known in the art can also be referred to, for example, “Revised New Version Soft Drinks” (Kokai Co., Ltd.) can be referred to.

  The Nrf2 activating compound according to the present invention or the extract according to the present invention, which is an active ingredient of the composition according to the present invention, is contained in beech haritake that has been ingested by humans as a food for many years. It can be used safely for humans (eg, humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.). The dose or intake of the active ingredient according to the present invention can be appropriately determined according to the recipient, the age and weight of the recipient, symptoms, administration time, dosage form, administration method, drug combination, and the like. For example, when the active ingredient according to the present invention is orally administered as a medicine, it is administered in the range of about 100 mg to about 20 g / kg body weight / day, preferably 1 g to 5 g / kg body weight / day per adult. be able to. Drugs having other mechanisms of action used in combination with the active ingredient according to the present invention can also be appropriately determined based on the clinically used dose. Moreover, when ingesting as a foodstuff, the active ingredient by this invention can be mix | blended with a foodstuff so that it may become ingestion amount of about 0.1-100g per day, preferably about 1-10g per adult.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1: Preparation of Beech Haritake Fruit Body Dry Powder Using Fungus Bed Cultivation Method A total of 1000 g of 380 g of beech sawdust, 37.5 g of dried beer koji, 37.5 g of dried okara and 545 g of tap water was added to PP for 1.2 kg. A (polypropylene) bag was packed into a medium. In addition, a polypropylene cap with a filter sandwiched in this bag, autoclaved at 121 ° C for 50 minutes, and after sterilization of the culture medium, the inoculum of Bunaharitake deposited as FERM BP-6697 at the Patent Organism Depositary Was precultured in the dark at a temperature of 23 to 25 ° C. and a humidity of 60 to 80% for 40 days, and then shifted to the middle culture step. In the middle culture step, a beech haritake primordium was formed when cultured for 30 days under conditions of a temperature of 13 to 15 ° C., a humidity of 80 to 95%, and an illuminance of 200 lux. One portion of the bag portion where a primordium having a diameter of about 3 cm was recognized in the bag was cut out, and then the process was shifted to a post-culture step. In the post-culturing step, when cultured for 17 days under conditions of a temperature of 13 to 15 ° C., a humidity of 80 to 95%, and an illuminance of 200 lux, it grew into a single beech tree. The obtained beech haritake fruit body was 232.5 g, and the total number of days required for cultivation was 87 days.

  3 kg of beech seedlings obtained in the above-mentioned fungus bed cultivation are refrigerated at 4 ° C. for up to one week immediately after harvesting, and dried at 65 ° C. for 6 hours in a hot air dryer. 0.3 kg of dried product was obtained. The dried beech haritake fruit body was pulverized using a pulverizer to obtain a dried beech haritake fruit body powder.

Example 2: Crude Extraction Process Using Beech Haritake Fruit Body Dry Powder Hot Water Extract as a Starting Material 530 g of Beech Haritake fruit body dry powder prepared using the method of Example 1 for 30 minutes using 10 times the amount of purified water The hot water extracted and filtered with a filter paper was concentrated under reduced pressure in a rotary evaporator to obtain 208 g of a hot water extract of dried beech haritake fruit bodies. A 10-fold amount of 80% ethanol solution was added to the extract and stirred, followed by centrifugation at 5000 rpm for 10 minutes, and the supernatant was collected and concentrated under reduced pressure in an evaporator. This extract was redissolved in 5 volumes of purified water, followed by liquid-liquid partition with ethyl acetate three times, and then concentrated. Further, the extract was redissolved in an 80% methanol solution and subjected to liquid-liquid partition with hexane three times, and then the 80% methanol layer was concentrated under reduced pressure to obtain 6.0 g of a dried product.

Example 3: Isolation of active compound The dried product obtained in Example 2 was redissolved in chloroform: methanol = 50: 1 solution and applied to a column packed with silica gel (Wakogel: manufactured by Wako Pure Chemical Industries). The methanol content was eluted with chloroform: methanol = 50: 1, 20: 1, 1: 1 and methanol 100% in this order. Among these, fractions eluted with a mixed solution of chloroform: methanol = 50: 1 were collected, concentrated under reduced pressure, redissolved in a solution of chloroform: methanol = 40: 1, and reapplied to a column filled with silica gel. Chloroform: methanol = 40: 1 solution was added to a 5-fold column of silica gel, and the eluted fractions were collected 5 ml each. Each fraction was developed by thin layer chromatography and colored with phosphomolybthenic acid (manufactured by Sigma). The results were roughly divided into 6 fractions (F1 to F6).

The above 6 fractions were subjected to a QR assay, and the fraction F2, which showed the strongest activity among them, was further purified by high performance liquid chromatography (HPLC). First, crude purification was carried out under the following elution conditions (1), (2) and (3) for compounds 1 and 2, (4) for compound 3, (5) and (6) for compounds 4 and 5, (5) for compound 6, (7) and (8) for compound 7, (7) for compound 8, (9) for compounds 9 and 10, and (10) and (11) for compound 11 Purified.
(1) Column: YMC-Pack C-18 (10 × 250 mm, manufactured by YMC), flow rate: 1.5 ml / min, temperature: 40 ° C., detection: diode array detector, solvent: A) 0.1% formic acid B) Acetonitrile, elution conditions: B) 35-100%
(2) Column: CAPCELL PAK AQ C-18 (10 × 250 mm, manufactured by Shiseido), flow rate: 4.7 ml / min, temperature: 40 ° C., detection: diode array detector, differential refraction detector (both Shimadzu Corporation) Manufactured), solvent: A) 0.1% formic acid B) methanol, elution condition: B) constant 5% (3) column: TSK gel Amide-80 (4.6 × 250 mm, manufactured by Tosoh Corporation), flow rate: 1. 0 ml / min, temperature: 40 ° C., detection: diode array detector, differential refraction detector, solvent: A) acetonitrile, elution condition: A) 100% constant (4) column: CAPCELL PAK AQ C-18 (10 × 250 mm) ), Flow rate: 3.0 ml / min, temperature: 40 ° C., detection: diode array detector, solvent: A) 0.1% formic acid B) acetonitrile, elution conditions: B) constant 10% (5 ) Column: CAPCELL PAK AQ C-18 (10 × 250 mm), flow rate: 4.7 ml / min, temperature: 40 ° C., detection: diode array detector, differential refraction detector, solvent: A) 0.1% formic acid B ) Acetonitrile, elution conditions: B) 20% constant (6) Column: CAPCELL PAK AQ C-18 (4.6 × 250 mm), flow rate: 1.0 ml / min, temperature: 40 ° C., detection: diode array detector, Differential refraction detector, solvent: A) 0.1% formic acid B) acetonitrile, elution conditions: B) 10% constant (7) Column: Discovery HS PEG (10 × 250 mm, manufactured by SUPELCO), flow rate: 2.0 ml / Min, temperature: 40 ° C., detection: diode array detector, differential refraction detector, solvent: A) 0.1% formic acid B) acetonitrile, elution conditions: B) 10% (8) Column: Xterra phenyl (4.6 × 250 mm, manufactured by Waters), flow rate: 1.0 ml / min, temperature: 40 ° C., detection: diode array detector, solvent: A) 0.1% formic acid B ) Methanol, elution conditions: B) 18% constant (9) Column: CAPCELL PAK AQ C-18 (4.6 × 250 mm), flow rate: 1.5 ml / min, temperature: 40 ° C., detection: diode array detector, Solvent: A) 0.2% formic acid B) Acetonitrile, elution conditions: B) 18% constant (10) Column: CAPCELL PAK MG C-18 (4.6 × 250 mm), flow rate: 1.2 ml / min, temperature: 40 ° C., detection: diode array detector, solvent: A) 0.2% formic acid B) acetonitrile, elution conditions: B) 45% constant (11) column: CAPCELL PAK MG -18 (4.6 × 250 mm), flow rate: 1.2 ml / min, temperature: 40 ° C., detection: diode array detector, solvent: A) 0.2% formic acid B) acetonitrile, elution conditions: B) 30% Constant

Example 4: Determination of structure of isolated compound Various spectral data of the isolated compound are as follows.

¹H-NMR（CD₃OD，400 MHz）：δ4.52（1H, t, J=9.0），4.20（1H, t, J=9.0），3.27（1H, q, J=9.3），2.85（1H, dq），2.22（3H, s），1.29（3H, d, J=6.7）
¹³C-NMR（CD₃OD，100 MHz）：δ206.8，180.7，68.0，56.3，38.0，29.4，15.1
ESI-MS：m/z 142.9[M+H]⁺
[α]²⁰ _D = -28.7（c 0.035, CH₃OH） Chemical Formula 1: 4-acetyl-3-methyl-dihydro-furan-2-one

¹ H-NMR (CD ₃ OD, 400 MHz): δ4.52 (1H, t, J = 9.0), 4.20 (1H, t, J = 9.0), 3.27 (1H, q, J = 9.3), 2.85 ( 1H, dq), 2.22 (3H, s), 1.29 (3H, d, J = 6.7)
¹³ C-NMR (CD ₃ OD, 100 MHz): δ 206.8, 180.7, 68.0, 56.3, 38.0, 29.4, 15.1
ESI-MS: m / z 142.9 [M + H] ⁺
[α] ²⁰ _D = -28.7 (c 0.035, CH ₃ OH)

¹H-NMR（CD₃OD，400 MHz）：δ4.33（1H, t, J=8.6），3.96（1H, t, J=8.9），3.77（1H, m），2.59（1H, dq），2.26-2.17（1H, m），1.30（3H, d, J=7.6），1.17(1H, d, J=6.5)
¹³C-NMR（CD₃OD，100 MHz）：δ182.9，69.7，69.3，51.3，38.4，21.5，16.6
ESI-MS：m/z 145.0[M+H]⁺
[α]²⁰ _D = 15.9（c 0.013, CH₃OH） Compound 2: 4- (1-hydroxy-ethyl) -3-methyl-dihydro-furan-2-one

¹ H-NMR (CD ₃ OD, 400 MHz): δ 4.33 (1H, t, J = 8.6), 3.96 (1H, t, J = 8.9), 3.77 (1H, m), 2.59 (1H, dq) , 2.26-2.17 (1H, m), 1.30 (3H, d, J = 7.6), 1.17 (1H, d, J = 6.5)
¹³ C-NMR (CD ₃ OD, 100 MHz): δ182.9, 69.7, 69.3, 51.3, 38.4, 21.5, 16.6
ESI-MS: m / z 145.0 [M + H] ⁺
[α] ²⁰ _D = 15.9 (c 0.013, CH ₃ OH)

¹H-NMR（CDCl₃，400 MHz）：δ1.97（3H, s），1.79（3H, s），1.62（3H, s）
¹³C-NMR（CDCl₃，100 MHz）：δ172.3，158.8，124.3，105.6，23.4，10.5，8.4
ESI-MS：m/z 142.9[M+H]⁺ Compound 3: 5-hydroxy-3,4,5-trimethyl-5-H-furan-2-one

¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.97 (3H, s), 1.79 (3H, s), 1.62 (3H, s)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 172.3, 158.8, 124.3, 105.6, 23.4, 10.5, 8.4
ESI-MS: m / z 142.9 [M + H] ⁺

¹H-NMR（CD₃OD，400 MHz）：δ4.54-4.51（1H, m），4.28（1H, m），4.08（1H, m），2.99-2.97（1H, m），2.50-2.49（1H, m），2.02（3H, s），1.39（3H, d, J=5.9），1.18(3H, d, J=6.7)
¹³C-NMR（CD₃OD，100 MHz）：δ181.7，172.4，79.4，63.6，45.3，36.8，20.6，20.3，10.3
ESI-MS：m/z 186.9[M+H]⁺
[α]²⁰ _D = -3.7（c 0.013, CH₃OH） Compound 4: Acetic acid 1- (4-methyl-5-oxo-tetrahydro-furan-3-yl) -ethyl ester

¹ H-NMR (CD ₃ OD, 400 MHz): δ4.54-4.51 (1H, m), 4.28 (1H, m), 4.08 (1H, m), 2.99-2.97 (1H, m), 2.50-2.49 (1H, m), 2.02 (3H, s), 1.39 (3H, d, J = 5.9), 1.18 (3H, d, J = 6.7)
¹³ C-NMR (CD ₃ OD, 100 MHz): δ 181.7, 172.4, 79.4, 63.6, 45.3, 36.8, 20.6, 20.3, 10.3
ESI-MS: m / z 186.9 [M + H] ⁺
[α] ²⁰ _D = -3.7 (c 0.013, CH ₃ OH)

¹H-NMR（CD₃OD，400 MHz）：δ4.35-4.32（1H, m），3.91-3.88（1H, m），2.54-2.38（2H, m），2.14-2.02（2H, m），1.66-1.40（4H, m），0.90（3H, t, J=7.5）
¹³C-NMR（CD₃OD，100 MHz）：δ175.7，81.1，76.7，42.3，32.8，32.2，29.6，10.4
ESI-MS：m/z 156.8[M-H]^-
[α]²⁰ _D = 2.3（c 0.021, CH₃OH） Compound 5: (5-ethyl-tetrahydro-furan-2-yl) -acetic acid

¹ H-NMR (CD ₃ OD, 400 MHz): δ4.35-4.32 (1H, m), 3.91-3.88 (1H, m), 2.54-2.38 (2H, m), 2.14-2.02 (2H, m) , 1.66-1.40 (4H, m), 0.90 (3H, t, J = 7.5)
¹³ C-NMR (CD ₃ OD, 100 MHz): δ175.7, 81.1, 76.7, 42.3, 32.8, 32.2, 29.6, 10.4
ESI-MS: m / z 156.8 [MH] ^-
[α] ²⁰ _D = 2.3 (c 0.021, CH ₃ OH)

¹H-NMR（CD₃OD，400 MHz）：δ6.97-6.89（1H, m），5.79（1H, d, J=15.6），2.29-2.19（4H, m），1.62-1.58（2H, m），1.49-1.46（2H, m），1.38-1.34（4H, m）
¹³C-NMR（CD₃OD，100 MHz）：δ178.2，170.9，151.3，123.3，35.5，33.5，30.5，30.4，29.6，26.5
ESI-MS：m/z 198.9[M-H]^- Compound 6: (E) -2-decene-diacid

¹ H-NMR (CD ₃ OD, 400 MHz): δ 6.97-6.89 (1H, m), 5.79 (1H, d, J = 15.6), 2.29-2.19 (4H, m), 1.62-1.58 (2H, m), 1.49-1.46 (2H, m), 1.38-1.34 (4H, m)
¹³ C-NMR (CD ₃ OD, 100 MHz): δ 178.2, 170.9, 151.3, 123.3, 35.5, 33.5, 30.5, 30.4, 29.6, 26.5
ESI-MS: m / z 198.9 [MH] ^-

¹H-NMR（CDCl₃，400 MHz）：δ7.39-7.26（5H, m），4.95（1H, dd, J=8.8, 4.4），3.90-3.89（1H, m），1.84-1.80（2H, m），1.62-1.51（2H, m），0.94（3H, t, J=7.4）
¹³C-NMR（CDCl₃，100 MHz）：δ144.5，128.5, 125.7，127.6，75.5，74.1，44.9，30.9，9.6
ESI-MS：m/z 203.1[M+Na]⁺ Compound 7: 1-phenyl-pentane-1,3-diol

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.39-7.26 (5H, m), 4.95 (1H, dd, J = 8.8, 4.4), 3.90-3.89 (1H, m), 1.84-1.80 (2H , m), 1.62-1.51 (2H, m), 0.94 (3H, t, J = 7.4)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 144.5, 128.5, 125.7, 127.6, 75.5, 74.1, 44.9, 30.9, 9.6
ESI-MS: m / z 203.1 [M + Na] ⁺

¹H-NMR（CDCl₃，400 MHz）：δ7.82（1H, d, J=6.4），7.73（2H, dd），7.50-7.43（3H, m），6.77（1H, d, J=2.4），6.36（1H, dd, J=2.5, 6.1）
¹³C-NMR（CDCl₃，100 MHz）：δ179.2，164.0, 154.8，131.5，131.2，129.1，125.8，117.1，112.4
ESI-MS：m/z 173.0[M+H]⁺ Compound 8: 2-Phenyl-pyran-4-one

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.82 (1H, d, J = 6.4), 7.73 (2H, dd), 7.50-7.43 (3H, m), 6.77 (1H, d, J = 2.4 ), 6.36 (1H, dd, J = 2.5, 6.1)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 179.2, 164.0, 154.8, 131.5, 131.2, 129.1, 125.8, 117.1, 112.4
ESI-MS: m / z 173.0 [M + H] ⁺

¹H-NMR（CDCl₃，400 MHz）：δ7.39（2H, dd, J=7.0, 7.1），7.31（1H, dd, J=7.0, 7.0），7.24（2H, d, J=7.1），4.68（1H, dd, J=8.0, 9.2），4.28（1H, dd, J=8.0, 8.9），3.80（1H, m），2.94（1H, dd, J=8.3, 17.5），2.69（1H, dd, J=9.1, 17.5）
¹³C-NMR（CDCl₃，100 MHz）：δ176.4，139.4, 129.1，127.7，126.7，74.0，41.1，35.7
ESI-MS：m/z 163.0[M+H]⁺ Compound 9: Dihydro-4-phenyl-2 (3H) -furanone

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.39 (2H, dd, J = 7.0, 7.1), 7.31 (1H, dd, J = 7.0, 7.0), 7.24 (2H, d, J = 7.1) , 4.68 (1H, dd, J = 8.0, 9.2), 4.28 (1H, dd, J = 8.0, 8.9), 3.80 (1H, m), 2.94 (1H, dd, J = 8.3, 17.5), 2.69 (1H , dd, J = 9.1, 17.5)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 176.4, 139.4, 129.1, 127.7, 126.7, 74.0, 41.1, 35.7
ESI-MS: m / z 163.0 [M + H] ⁺

¹H-NMR（CDCl₃，400 MHz）：δ7.74（2H, d, J=7.8），7.48（1H, dd, J=7.7, 7.7），7.42（2H, dd, J=7.7, 7.8），6.03（1H, s），4.67（2H, t, J=6.6），2.67（2H, t, J=6.6）
¹³C-NMR（CDCl₃，100 MHz）：δ192.7，170.6, 132.7，131.7，128.7，126.5，102.5，68.2，36.0
ESI-MS：m/z 175.0[M+H]⁺ Compound 10: 2-phenyl-5,6-dihydro-γ-pyrone

¹ H-NMR (CDCl ₃ , 400 MHz): δ 7.74 (2H, d, J = 7.8), 7.48 (1H, dd, J = 7.7, 7.7), 7.42 (2H, dd, J = 7.7, 7.8) , 6.03 (1H, s), 4.67 (2H, t, J = 6.6), 2.67 (2H, t, J = 6.6)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 192.7, 170.6, 132.7, 131.7, 128.7, 126.5, 102.5, 68.2, 36.0
ESI-MS: m / z 175.0 [M + H] ⁺

¹H-NMR（CDCl₃，400 MHz）：δ9.48（1H, s），7.17（1H, m），6.99（1H, m），6.32（1H, m），5.98（1H, m），2.00（2H, m），1.41（1H, m），0.92（3H, d, J=7.2），0.90（3H, d, J=7.6）
¹³C-NMR（CDCl₃，100 MHz）：δ180.0，175.1, 131.7，130.0，126.0，110.7，57.8，40.8，24.8，22.9，21.4
EI-MS：m/z 209 [M]⁺ Compound 11: 2- (2-formylpyrrol-1-yl) -4-methylvaleric acid

¹ H-NMR (CDCl ₃ , 400 MHz): δ 9.48 (1H, s), 7.17 (1H, m), 6.99 (1H, m), 6.32 (1H, m), 5.98 (1H, m), 2.00 (2H, m), 1.41 (1H, m), 0.92 (3H, d, J = 7.2), 0.90 (3H, d, J = 7.6)
¹³ C-NMR (CDCl ₃ , 100 MHz): δ 180.0, 175.1, 131.7, 130.0, 126.0, 110.7, 57.8, 40.8, 24.8, 22.9, 21.4
EI-MS: m / z 209 [M] ⁺

Example 5: Quinone reductase activity test It is known that the quinone reductase (QR) activity is increased by the increase in the expression of NQO1 by Nrf2 activation (Proc Natl Acad Sci US A. 101 10446-51 (2004)). ). Therefore, whether or not the beech scallop ethyl acetate fraction prepared in Example 2 (hereinafter referred to as “beech sardines ethyl acetate fraction”) has Nrf2 activation ability was measured using QR activity as an index.

  The QR activity was measured according to the method described in Prochaska HJ et al., Anal Biochem. 1988 Mar; 169 (2): 328-36.

Mouse hepatoma cells Hepa1c1c7 cells (obtained from Dainippon Sumitomo Pharma Co., Ltd.) were seeded in 96-well microplates at 7 × 10 ³ cells / well, 10% fetal calf serum (Hyclone) and penicillin-streptomycin (100 units each) 50 ml of α-MEM medium (manufactured by Invitrogen) containing 50 μl / ml, 100 μg / ml, manufactured by Sigma) and cultured overnight at 37 ° C. and 5% CO ₂ . Next, the culture medium was replaced with a medium prepared such that the ethyl beech halachacetate fraction prepared in Example 2 and the compounds 1 to 11 isolated in Example 3 each had a concentration of 100 μg / ml, and Cultured for 48 hours. Thereafter, the cells in each well were disrupted with 0.08% digitonin and a 2 mM EDTA solution, and the absorbance at 610 nm by the menadion-formazan system was measured using a microplate reader.

  The QR-inducing activity was calculated as a relative activity where the activity of the additive-free cells was 1, (n = 3, average value ± standard deviation). As a positive control, sulforaphane (3 μM) (manufactured by Sigma), which is an Nrf2 activator, was used. Moreover, the significant difference was determined using the t test (* p <0.05, ** p <0.01).

  As a result, an increase in QR activity was observed due to the addition of the beech haritake ethyl acetate fraction and compounds 1 to 11 (FIG. 1). In particular, Compound 9 showed the highest QR activation ability.

Example 6: Evaluation of Nrf2 activation ability in ARE reporter assay system Nrf2 is known to bind to ARE and induce the expression of phase II enzymes (Itoh K et al., Seikagaku. 2006 Feb; 78 (2): 79-92). Therefore, whether or not the beech haritake ethyl acetate fraction has Nrf2 activation ability was measured using ARE promoter activity as an index.

  Measurement of ARE promoter activity was performed using an ARE reporter assay system.

  The reporter plasmid pGL3-mARE3 prepared a double-stranded oligo DNA in which a mouse NQO1-derived ARE (antioxidant response element), to which Nrf2 is bound, was repeated three times, and this was used as a firefly luciferase reporter vector pGL3-promoter vector (Promega). (By more available) was inserted into the MluI-NheI site.

Double-stranded oligo DNA containing ARE
5'-TCGACGCGTAGAGTCACAGTGAGTCGGCAAAATTAGAGTCACAGTGAGTCGGCAAAATTAGAGTCACAGTGAGTCGGCAAAATTGCTAGCTAG-3 '(SEQ ID NO: 1), and
5'-CTAGCTAGCAATTTTGCCGACTCACTGTGACTCTAATTTTGCCGACTCACTGTGACTCTAATTTTGCCGACTCACTGTGACTCTACGCGTCGA-3 '(SEQ ID NO: 2)
Were prepared by chemical synthesis and annealing. Moreover, it was confirmed by nucleotide sequence analysis that it was the objective plasmid.

Next, 4 × 10 ⁵ cells / well of mouse macrophage cell line RAW264.7 (obtained from American Type Culture Collection) was seeded in a 12-well microplate, and 10% fetal calf serum and penicillin-streptomycin ( 0.8 ml of RPMI 1640 medium (manufactured by Sigma) containing 100 units / ml and 100 μg / ml, respectively, was added and cultured overnight at 37 ° C. and 5% CO ₂ . The cells were transfected with 1.0 μg of pGL3-mARE3 and 0.01 μg of Renilla luciferase reporter vector phRL-TK (Promega) for correction using SuperFect Transfection reagent (Qiagen). After transfection for 3 hours, the medium was changed to a new one, and then the ethyl beech scallop acetate (100, 200 μg / ml) and sulforaphane (8 μM) were added as a positive control and cultured for 24 hours. It was collected. After cell disruption using a Dual-Luciferase reporter assay system (Promega), firefly luciferase activity and Renilla luciferase activity were measured using a luminometer (ARVO Light Luminescence Counter, PerkinElmer). ).

  The value obtained by dividing the firefly luciferase activity by the Renilla luciferase activity was defined as the relative luciferase activity (n = 3, mean ± standard deviation).

  As a result, it was confirmed that the ARE promoter activity was increased in a concentration-dependent manner by the addition of the beech haritake ethyl acetate fraction (FIG. 2). Therefore, it was suggested that the ability to induce QR activity of the ethyl beech sap was confirmed in Example 5 through Nrf2 activation.

Example 7: Expression level analysis of various genes controlled by Nrf2 Hepa1c1c7 cells were seeded in a 96-well microplate at 7 × 10 ³ cells / well, 10% fetal calf serum and penicillin-streptomycin (100 units / ml each) , 100 μg / ml) was added, and cultured overnight at 37 ° C. and 5% CO ₂ . Next, the culture medium was divided into a medium prepared so that the ethyl acetate fraction prepared in Example 2 had a concentration of 50, 100, and 200 μg / ml, and a medium containing sulforaphane (3 μM) as a positive control, respectively. They were replaced and cultured for another 12 hours.

Thereafter, total RNA was prepared using TRIzol (manufactured by Invitrogen) and purified using RNeasy Mini (manufactured by Qiagen) and RNase-Free DNase Set (manufactured by Qiagen). Furthermore, cDNA was synthesized from the purified 5 μg of total RNA using a Thermo Script RT-PCR system (Invitrogen), SYBR ^(R) Premix Ex Taq ^™ reagent (Takara Bio) and Light Cycler (Roche). The amount of mRNA expression of various second phase enzyme genes was quantified.

  The expression level of each gene was corrected by the expression level of GAPDH, and the relative expression level was calculated (n = 2, average value ± standard deviation).

The DNA primers used for gene expression analysis are
As sense and antisense primers for glutathione S-transferase mu1 (GSTM1)
5′-TGCCCGAAAGCACCACCTGGAT-3 ′ (SEQ ID NO: 3), and
5′-ACCATGGCCTCTTGCCCAGGAA-3 ′ (SEQ ID NO: 4),
NAD (P) H: Sense and antisense primer for quinone oxidoreductase (NAD (P) H: quinone oxidoreductase (NQO1))
5'-ACAGCATTGGCCACACTCCACCA-3 '(SEQ ID NO: 5), and
5′-TGATGGCCCACAGAGAGGCCAAA-3 ′ (SEQ ID NO: 6),
As sense and antisense primers for Glutamate cysteine ligase catalytic subunit (GCLC)
5'-TCTGCAAAGGCGGCAACGCTGT-3 '(SEQ ID NO: 7), and
5'-GCATCGGGTGTCCACATCAACTTCC-3 '(SEQ ID NO: 8),
As sense and antisense primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
5'-TCTGCCGATGCCCCCATGTTTG-3 '(SEQ ID NO: 13)
5'-TGGGTGGCAGTGATGGCATGGA-3 '(SEQ ID NO: 14)
All were synthesized by Invitrogen. GSTM1 and NQO1 are enzymes related to detoxification, and GCLC is an enzyme responsible for the synthesis of the antioxidant glutathione.

  As a result, when the variation in the expression level of various genes controlled by Nrf2 was examined for the ethyl beech cultivar that showed QR activity induction in Example 5, the target gene GSTM1, which is known to increase expression by Nrf2, The expression of NQO1, GCLC was increased similarly to the sulforaphane used as a positive control (FIG. 3). Therefore, it was revealed that the ethyl beech halotake acetate fraction activates Nrf2, thereby increasing the expression of a series of genes called second phase enzymes and enhancing the antioxidant and detoxification systems.

Example 8: Inhibition of cell death by hydrogen peroxide 2.5 × 10 ⁴ cells / well of RAW264.7 cells in 96-well microplates were added to each sample, 10% fetal calf serum, and penicillin-streptomycin (100 each). 100 μl of RPMI 1640 medium containing (unit / ml, 100 μg / ml) was added and cultured overnight at 37 ° C. and 5% CO ² . Here, the ethyl acetate fraction prepared in Example 2 was used for each sample, and the concentrations were 25, 50, and 100 μg / ml, and sulforaphane (3 μM) was used as a positive control. Hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto so as to have final concentrations of 0.2, 0.4, and 0.8 mM, and further cultured for 2 hours. Thereafter, the cell viability was measured by measuring the absorbance at 450 nm with a microplate reader using Cell Counting Kit-8 (manufactured by Dojindo). The cell viability was calculated for each sample with no hydrogen peroxide added as 100% (n = 3, average value ± standard deviation).

  As a result, it was confirmed that the ethyl beechaharitake acetate suppresses cell death by hydrogen peroxide, that is, active oxygen, like sulforaphane (FIG. 4).

Example 9: Change in gene expression level of mouse liver and small intestinal epithelium by oral gavage C57BL / 6NCr1Cr1j male male 7-week-old (obtained from Charles River, n = 7-8) AIN-76A (ethoxyquin free, research) Dieted) and acclimated for 1 week. Thereafter, the ethyl beech haritake acetate fraction was orally administered by gavage at 30 mg / animal / day or 15 mg / animal / day for 3 days. Similarly, purified water as a control group and sulforaphane (1 mg / animal / day) as a positive control group were administered to mice.

  The liver and small intestinal epithelium were collected under ether anesthesia 24 hours after the final administration, immediately frozen in liquid nitrogen, and stored at −80 ° C. until use. The RNA of each organ was purified by the same method as in Example 7, and after cDNA was synthesized, the mRNA expression level of each gene was quantified.

  The expression level of each gene was corrected by the expression level of GAPDH, and the relative expression level was calculated (n = 3, average value ± standard deviation). In the liver, GSTM1, NQO1, GCLC, Cyp1A1, and Cyp1A2 were measured, and in the small intestine epithelium, GSTM1, NQO1, and GCLC genes were measured. In addition to the same DNA primers used for the gene expression level analysis as in Example 7, the following were synthesized and used by Invitrogen.

As sense and antisense primers for cytochrome P450 1A1 (Cytochrome P450 1A1 (Cyp1A1))
5'-TGGGGCTTGCCCTTCATTGGTC-3 '(SEQ ID NO: 9), and
5'-TCTGGCCGGCCCTTGAAGTCAT-3 '(SEQ ID NO: 10)
It was used.

As sense and antisense primers for cytochrome P450 1A2 (Cytochrome P450 1A2 (Cyp1A2))
5'-TCGGCCATCGACAAGACCCAGA-3 '(SEQ ID NO: 11), and
5'-TGTTCACAGGTCCCGGGCTTCA-3 '(SEQ ID NO: 12)
It was used.

  Moreover, the significant difference was determined using the Steel test (* p <0.05, ** p <0.01).

The results are shown in Table 1.

  As a result, in the group administered with the beech haritake ethyl acetate fraction, significant increases in the expression of GSTM1, NQO1, and GCLC, which are representative of the second phase enzymes, were observed. GSTM1 and NQO1 are enzymes related to detoxification, and GCLC is an enzyme responsible for the synthesis of the antioxidant glutathione. For this reason, it is thought that the detoxification action and the antioxidant action were enhanced by the beech haritake ethyl acetate fraction. On the other hand, in the group to which the ethyl beech scallop ethyl acetate fraction was administered, there was no increase in the expression of Cyp1A1 or Cyp1A2 typical as a first phase enzyme involved in the activation of carcinogens. From this, it was also clarified that the ethyl beech halotake acetate fraction selectively increases the expression of the second phase enzyme group in the metabolism of the detoxification system. From this, it was shown that the beech haritake ethyl acetate fraction activates Nrf2 in the body even when ingested orally and increases the expression of a series of genes called second phase enzymes. In addition, no significant difference was observed in body weight change, food intake, and liver weight at the time of dissection during each sample administration period (data not shown).

Example 10: Evaluation of mouse liver QR activity by oral gavage The liver was collected from mice administered with each sample under the same conditions as in Example 9. A 10-fold amount of buffer (10 mM Tris-HCl, 0.5 mM EDTA, 0.25 M sucrose, pH 8.8) was added to about 100 mg of the liver, and the mixture was crushed with a homogenizer (manufactured by Hitachi, Ltd.). After centrifuging at 10,000 × g for 20 minutes at 4 ° C. and collecting the supernatant, the protein concentration was measured by the Bradford method. After dilution with the above buffer so that the protein concentration of each sample was 5 mg / ml, the absorbance at 610 nm by the menadion-formazan system was measured using a microplate reader to evaluate the QR activity.

  The QR activity was calculated as a relative activity with the control group being 1 (n = 3, mean ± standard deviation). The experimental method was carried out according to the method described in Hans J et al., J Biol Chem. 1986 Jan 25; 261 (3): 1372-8. Moreover, the significant difference was determined using the Steel test (* p <0.05, ** p <0.01).

  As a result, a dose-dependent increase in the enzyme activity of QR was significantly observed in the group administered with the beech haritake ethyl acetate fraction (FIG. 5). From this, it became clear that even when ingested orally, the beech haritake ethyl acetate fraction activates Nrf2 in the body, increases the expression level of the gene controlled by Nrf2, and increases the accompanying enzyme activity. It was.

In Example 5, it is the figure which showed the influence of the Nrf2 activation compound by this invention and the Bunaharitake ethyl acetate fraction with respect to QR activity. In Example 6, it is the figure which showed the influence of the Bunaharitake ethyl acetate fraction with respect to promoter activity. It is the figure which showed the influence in the Bunaharitake ethyl acetate fraction with respect to the expression level of the gene (GSTM1, NQO1, GCLC) controlled by Nrf2 in Example 7. In Example 8, it illustrates the effects of Bunaharitake ethyl acetate fraction on cell death induced by H ₂ O _2. In Example 10, it is the figure which showed QR activity in the liver of the mouse | mouth which administered the beech-haritake-ethyl acetate fraction.

Claims (30)

Formula (I):

[In the above formula,
R ¹ represents an oxygen atom or a C _1-6 alkyl group, R ² represents a hydrogen atom or a C _1-6 alkyl group, and R ³ represents a C ₁ -C ₁ which may be substituted with a hydrogen atom or a hydroxyl group. _A C _1-6 optionally substituted with a ₆ alkyl group, or a substituent selected from the group consisting of a C _1-4 alkyl group, a C _1-4 acyl group, a carboxyl group, and a C _1-4 alkoxycarbonyl group Represents an alkylcarbonyloxy C _1-6 alkyl group, R ⁴ represents a hydrogen atom or a carboxyl C _1-6 alkyl group,
- - - represents a single bond or a double bond,
However, when R ¹ is an oxygen atom, − − − is a double bond, and when R ¹ is a C _1-6 alkyl group, − − − is a single bond. ]
Or a pharmaceutically acceptable salt or solvate thereof. R ¹ is an oxygen atom or an ethyl group, R ² is a hydrogen atom or a methyl group, and R ³ is substituted with a hydrogen atom, an ethyl group which may be substituted with a hydroxyl group, or a methyl group. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein R ⁴ is a hydrogen atom or a carboxymethyl group. The compound of formula (I) is
4- (1-hydroxy-ethyl) -3-methyl-dihydro-furan-2-one,
The acetic acid 1- (4-methyl-5-oxo-tetrahydro-furan-3-yl) -ethyl ester and (5-ethyl-tetrahydro-furan-2-yl) -acetic acid are selected from the group consisting of: 3. The compound according to 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, which is derived from the fruit body of Mycoleptodonoides aitchisonii belonging to the genus Beech Haritake. Formula (II):

[In the above formula,
R ¹⁰¹ represents a hydrogen atom or a _{C 1-6} alkyl ^{group, R 102} is optionally substituted with a hydroxyl _{C 1-6} alkyl _{group; C 1-6} alkylcarbonyl _{group; C 1-4} alkyl group, A C _1-6 alkylcarbonyloxy C _1-6 alkyl group optionally substituted with a substituent selected from the group consisting of a C _1-4 acyl group, a carboxyl group, and a C _1-4 alkoxycarbonyl group; or phenyl R ¹⁰³ represents a hydrogen atom, a hydroxyl group, or a C _1-6 alkyl group, R ¹⁰⁴ represents a hydrogen atom, a hydroxyl group, or a C _1-6 alkyl group,
- - - represents a single bond or a double bond. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective. R ¹⁰¹ is a hydrogen atom or a methyl group, and R ¹⁰² is a methyl group, an ethyl group optionally substituted with a hydroxyl group, a methylcarbonyl group, an acetyloxymethyl group optionally substituted with a methyl group, or phenyl The composition according to claim 5, wherein R ¹⁰³ is a hydrogen atom, a hydroxyl group, or a methyl group, and R ¹⁰⁴ is a hydrogen atom, a hydroxyl group, or a methyl group. R ¹⁰¹ is a hydrogen atom or a methyl group, R ¹⁰² is an ethyl group, a methylcarbonyl group, or a phenyl group which may be substituted with a hydroxyl group, R ¹⁰³ is a hydrogen atom, R ¹⁰⁴ is The composition according to claim 6, which is a hydrogen atom. The composition according to claim 7, wherein R ¹⁰¹ is a hydrogen atom, R ¹⁰² is a phenyl group, R ¹⁰³ is a hydrogen atom, and R ¹⁰⁴ is a hydrogen atom. Formula (III):

[In the above formula,
R ¹⁰⁵ represents a C _1-6 alkyl group, and R ¹⁰⁶ represents a carboxyl C _1-6 alkyl group. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective. The composition according to claim 9, wherein R ¹⁰⁵ is an ethyl group and R ¹⁰⁶ is a carboxymethyl group. Formula (IV):

[In the above formula,
m represents an integer of 3 to 9. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective.   12. A composition according to claim 11, wherein m is 6. Formula (V):

[In the above formula,
R ¹⁰⁷ represents a C _1-6 alkyl group which may be substituted with one or more hydroxyl groups. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective. The composition according to claim 13, wherein R ¹⁰⁷ is a pentyl group optionally substituted with one or more hydroxyl groups. Formula (VI):

[In the above formula,
Het represents a 5- or 6-membered saturated or unsaturated heterocyclic group having one oxygen atom as a hetero ring member. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective. The compound of formula (VI) is of formula (VIa):

[In the above formula,
W, X, Y, of Z, any one of an oxygen atom, and the other is _{-CH 2 -,> CH -,} - CH =,> C =, - CH 2 -CH 2 -,> CH- CH ₂ —, —CH═CH—, or> C═CH— (in the case where W is an oxygen atom, X, Y, and Z are —CH ₂ — or> CH—, Z When X is an oxygen atom, W, X, and Y are —CH ₂ — or> CH—, and when X is an oxygen atom, W is —CH ₂ —CH ₂ —,> CH—CH. ₂ -, - CH = CH-, or> is C = CH-, = Y and Z is -CH,> C =, - CH 2 -, or> is CH-, Y is an oxygen atom , Z is _{-CH 2 -CH 2 -,> CH} -CH 2 -, - CH = CH-, or> is C = CH-, W and X are -CH = _{> C =, - CH 2 -} , or> CH- is it). The composition of Claim 15 represented by this. Formula (VII):

[In the above formula,
R ¹⁰⁸ represents a C _1-6 alkyl group. ]
Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, a composition used for the treatment of a disease for which Nrf2 activation is therapeutically effective. The composition of claim 17, wherein R ¹⁰⁸ is a branched C _1-6 alkyl group.   A compound comprising the compound of formula (I) according to claim 1 or 2 or a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient, for the treatment of a disease in which Nrf2 activation is therapeutically effective. The composition used. The active ingredient is
4-acetyl-3-methyl-dihydro-furan-2-one,
4- (1-hydroxy-ethyl) -3-methyl-dihydro-furan-2-one,
5-hydroxy-3,4,5-trimethyl-5-H-furan-2-one,
Acetic acid 1- (4-methyl-5-oxo-tetrahydro-furan-3-yl) -ethyl ester,
(5-ethyl-tetrahydro-furan-2-yl) -acetic acid,
(E) -2-decene-diacid,
1-phenyl-pentane-1,3-diol,
2-phenyl-pyran-4-one,
Dihydro-4-phenyl-2 (3H) -furanone,
21. Any one of claims 5 to 19 selected from the group consisting of 2-phenyl-5,6-dihydro- [gamma] -pyrone and 2- (2-formylpyrrol-1-yl) -4-methylvaleric acid. The composition according to item.   21. The composition according to any one of claims 5 to 20, wherein the active ingredient is derived from a beech haritake fruiting body.   A composition used for the treatment of a disease for which activation of Nrf2 is effective for treatment, comprising an extract of a beech haritake fruiting body.   The composition according to claim 22, wherein the fruit beech fruit body is a dry pulverized product.   24. The composition according to claim 22 or 23, wherein the extract of the fruit beech fruit body is an extract obtained by extracting the fruit beech fruit body with an aqueous solvent.   25. Composition according to claim 24, characterized in that it is extracted with an aqueous solvent and then partitioned with ethyl acetate.   26. The composition according to any one of claims 5 to 25, wherein the disease for which Nrf2 activation is effective for treatment is a disease caused by oxidative stress.   Diseases caused by oxidative stress include aging-related chronic diseases, arteriosclerosis, hypertension, diabetes, carcinogenesis, liver dysfunction, ulcers, neurodegenerative diseases, autoimmune diseases, spots, wrinkles, eye diseases, respiratory diseases, 27. The composition of claim 26, wherein the composition is selected from the group consisting of asthma, inflammation, cerebral infarction, and myocardial infarction.   The composition according to any one of claims 5 to 25, wherein the disease for which Nrf2 activation is effective for treatment is a disease caused by a xenobiotic.   29. The composition of claim 28, wherein the disease caused by the xenobiotic is selected from the group consisting of respiratory disease, liver dysfunction, carcinogenesis, and autoimmune disease.   It is a manufacturing method of the compound as described in any one of Claims 5-21, its pharmacologically acceptable salt, or those solvates, Comprising: Bunaharitakeko as described in any one of Claims 22-25 A method comprising the step of purifying an extract of an entity and measuring Nrf2 activation ability of the obtained fraction.

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