JP2008208054A - Inflammatory cytokine production inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new highly safe agent having sufficiently strong inhibitory actions on production of inflammatory cytokines such as TNF-α and useful as an anti-inflammatory agent etc. <P>SOLUTION: The inhibitor of inflammatory cytokine production in animal cells is characterized by comprising a compound represented by formula (1) (wherein, R<SB>1</SB>, R<SB>2</SB>and R<SB>3</SB>represent each a hydrogen atom or a hydroxy group) and a 16-22C unsaturated fatty acid or a glyceride thereof or a lower alkyl ester thereof as active ingredients. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug that suppresses the production of inflammatory cytokines TNF-α and IL-1β, which are useful in pharmaceuticals and health foods.

  It is known that various diseases such as rheumatoid arthritis, ulcerative colitis, Crohn's disease, type 2 diabetes, and allergic dermatitis are caused by abnormal production of endogenous cytokines. Accordingly, various cytokine production inhibitors have been developed for the prevention and treatment of such diseases. In addition to existing anti-inflammatory agents such as ibuprofen and indomethacin, thalidomide derivatives (see Non-Patent Document 1), pyrazolone derivatives ( Non-patent document 2), Synthetic chromene derivative (see non-patent document 3), Alberoids of siberian family (see patent document 1), Chromon derivative (see patent document 2), Hepatocyte growth factor (see patent document 3) However, since these diseases often follow a chronic course and treatment is prolonged, there is a particular need for a compound that can be taken orally and has no side effects. From this point of view, milk protein fragment peptides (see Patent Document 4), licorice and ginger extracts (Patent Document 5), highly unsaturated fatty acids such as docosahexaenoic acid (see Patent Documents 6 and 7), edible plants Food ingredients such as flavonoids such as quercetin and luteolin (Patent Documents 8 and 9, Non-Patent Documents 4, 5, 6, 7, and 8) contained in seed coats and pericarps are attracting attention. Further enhancement of activity is necessary.

Japanese Patent Laid-Open No. 2003-26586 JP 2005-247762 A JP 2000-239182 A JP 2004-196707 A WO2003 / 007974 Special table 2006-511514 gazette JP 2000-159667 A JP 2004-75619 A Japanese Patent Laid-Open No. 2001-114686 S. Niwayama et al., J. Med. Chem., 39, 3044-3045, 1996 M.P. Clark et al., J. Med. Chem., 47, 2724-2727, 2004 J-F. Cheng et al., Bioorg. Med. Chem. Lett., 13, 3647-3650, 2003 G.K.Rangan etal., Am. J. Physiol., 277 (5, Pt.2), F779-F789, 1999 A. Xagorari etal., J. Pharmacol. Exp. Ther., 296, 181-187, 2001 J. Wang et al., J. Agric. Food Chem., 50, 4183-4189, 2002 S. Hougee etal., Biochem. Pharmacol., 69, 241-248, 2005 M. Comalada etal., Biochem. Pharmacol., 72, 1010-1021, 2006

  An object of the present invention is to develop a novel drug useful as an anti-inflammatory agent, etc. in view of the above-mentioned present situation, which is highly safe and has a sufficiently strong inhibitory effect on the production of inflammatory cytokines such as TNF-α. .

In order to solve the above problems, the present inventors have conducted intensive studies, and among flavonoids distributed in a wide range of plants, formulas (1) such as chrysin and apigenin that have not been studied so far in terms of anti-inflammatory agents. (However, R ₁ = R ₂ = R ₃ = H, or R ₁ = R ₃ = H, R ₂ = OH) is a single macrophage-like differentiated compound, particularly in the presence of highly unsaturated fatty acids. It has been found that it strongly suppresses the production of inflammatory cytokines such as TNF-α and IL-1β when cell culture cells (acute monocytic leukemia cells, THP-1) are stimulated with lipopolysaccharide (LPS) of Escherichia coli. The present invention has been completed.

  Inflammatory response signal carriers contain active oxygen (L. Fialkow et al., Free Radic. Biol. Med., 42, 153-164, 2007). Therefore, anti-oxidant powerful flavonoids such as quercetin and luteolin have been taken up as promising anti-inflammatory agents and have been shown to suppress the production of inflammatory cytokines (T. Wadsworth et al., Biochem Pharmacol., 57, 941-949, 1999). However, as a result of investigations by the present inventors, chrysin and apigenin, which are flavonoids with little antioxidant property, have a production inhibitory action such as TNF-α, and in addition, a stronger inhibitory action in the presence of highly unsaturated fatty acids. Found to show. Furthermore, this combined effect of unsaturated fatty acids was also observed with quercetin and luteolin. It is already known that polyunsaturated fatty acids have an inhibitory effect on the production of inflammatory cytokines (Japanese translations 2006-511514, JP-A 2000-159667). Furthermore, it is enhanced by combined use with ferulic acid derivatives and chalcones. This has been recently clarified by the present inventors (Japanese Patent Application No. 2006-291726).

However, the biological effects of the combination of flavonoids in the narrow sense except for chalcones with highly unsaturated fatty acids have not been investigated. The present invention is the first to show that flavonoids can exert a strong anti-inflammatory action in combination with highly unsaturated fatty acids regardless of the presence or absence of antioxidant properties.

２）上記グリセリドが、炭素数１６〜２２の不飽和脂肪酸のモノ、ジまたはトリグリセリドであることを特徴とする、１）に記載の動物細胞における炎症性サイトカイン産生抑制剤。
３）上記低級アルキルエステルが、炭素数１６〜２２の不飽和脂肪酸のエチル、メチル、プロピル、もしくはブチルエステルであることを特徴とする、１）に記載の動物細胞における炎症性サイトカイン産生抑制剤。
４）炭素数１６〜２２の不飽和脂肪酸が4Z,7Z,10Z,13Z,16Z,19Z-ドコサヘキサエン酸、4Z,7Z,10Z,13Z,16Z-ドコサペンタエン酸、7Z,10Z,13Z,16Z,19Z-ドコサペンタエン酸、5Z,8Z,11Z,14Z,17Z-エイコサペンタエン酸、6Z,9Z,12Z,15Z-オクタデカテトラエン酸、α-リノレン酸、リノール酸、オレイン酸及びアラキドン酸からなる群から選ばれた１種以上
であることを特徴とする、１）〜３）のいずれかに記載の動物細胞における炎症性サイトカイン産生抑制剤。
５）式（１）で示される化合物がクリシン（式１においてR₁=R₂=R₃=H）、アピゲニン（式１においてR₁=R₃=H、R₂=OH）、ルテオリン（式１においてR₁=H, R₂=R₃=OH）、ケンフェロール（式１においてR₁=R₂=OH, R₃=H）、ケルセチン（式１においてR₁=R₂=R₃=OH）の中から選ばれる1つ以上の化合物であることを特徴とする、１）〜４）のいずれかに記載の動物細胞における炎症性サイトカイン産生抑制剤。 That is, the present invention is as follows.
1) An inflammatory cytokine production inhibitor in animal cells, comprising a compound represented by the following formula (1) and an unsaturated fatty acid having 16 to 22 carbon atoms or a glyceride or lower alkyl ester thereof as active ingredients.

2) The inflammatory cytokine production inhibitor in animal cells according to 1), wherein the glyceride is mono-, di- or triglyceride of an unsaturated fatty acid having 16 to 22 carbon atoms.
3) The inflammatory cytokine production inhibitor in animal cells according to 1), wherein the lower alkyl ester is an ethyl, methyl, propyl, or butyl ester of an unsaturated fatty acid having 16 to 22 carbon atoms.
4) Unsaturated fatty acids having 16 to 22 carbon atoms are 4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoic acid, 4Z, 7Z, 10Z, 13Z, 16Z-docosapentaenoic acid, 7Z, 10Z, 13Z, 16Z, 19Z-Docosapentaenoic acid, 5Z, 8Z, 11Z, 14Z, 17Z-Eicosapentaenoic acid, 6Z, 9Z, 12Z, 15Z-Octadecatetraenoic acid, α-linolenic acid, linoleic acid, oleic acid and arachidonic acid The inflammatory cytokine production inhibitor in animal cells according to any one of 1) to 3), which is one or more selected from the group.
5) The compound represented by formula (1) is chrysin (R ₁ = R ₂ = R ₃ = H in formula 1), apigenin (R ₁ = R ₃ = H, R ₂ = OH in formula 1), luteolin (formula 1 R ₁ = H, R ₂ = R ₃ = OH), kaempferol (R ₁ = R ₂ = OH, R ₃ = H in Formula 1), quercetin (R ₁ = R ₂ = R ₃ = in Formula 1) The inflammatory cytokine production inhibitor in animal cells according to any one of 1) to 4), which is one or more compounds selected from OH).

The mixed composition of a compound represented by formula (1) and a highly unsaturated fatty acid used in the present invention suppresses the production of inflammatory cytokines such as TNF-α and IL-1β in animal immune cells. As a result, it has the effect of alleviating symptoms of chronic inflammatory diseases such as rheumatoid arthritis and ulcerative colitis at the individual level.
On the other hand, conventional substances such as pyrazole derivatives known as substances that suppress the production of inflammatory cytokines have problems of safety and side effects, whereas the flavonoid compounds of the present invention have conventionally been used for vegetables, fruits and the like. It is a combination of these ingredients because it is eaten or taken as a component of health enhancers such as herbs, herbal medicines, and propolis, and highly unsaturated fatty acids such as docosahexaenoic acid have also been ingested as components of fish oil. It can be said that the drug of the present invention has high safety.
Inflammatory cytokines such as TNF-α are overproduced in various aspects such as obesity and aspiration, as well as rheumatism and colitis, and cause potential diseases. Therefore, such constant control of inflammatory cytokines such as TNF-α is extremely important for maintaining health. As described above, the drug of the present invention is composed of ingredients of food and related materials, and is highly safe. Therefore, it is considered that daily and long-term ingestion is possible. It is a drug useful as a food additive.

The inflammatory cytokine production inhibitor of the present invention can be obtained by blending a compound represented by the following formula (1) and an unsaturated fatty acid having 16 to 22 carbon atoms or a glyceride or lower alkyl ester thereof.

Among the compounds of formula (1) in the present invention, preferable compounds include, for example, chrysin (R ₁ = R ₂ = R ₃ = H in formula 1), apigenin (R ₁ = R ₃ = H, R ₂ in formula 1) = OH), luteolin (R ₁ = H, R ₂ = R ₃ = OH in Formula 1), kaempferol (R ₁ = R ₂ = OH, R ₃ = H in Formula 1), quercetin (R _{1 in} Formula 1) = R ₂ = R ₃ = OH). These O-glycoside type glycosides are also used.

  All of these flavonoid compounds inhibit TNF-α production in animal cells by 40-50% at a low concentration of 30 μM, but when combined with 10 μM docosahexaenoic acid, the inhibition rate reaches 80% or more. In addition, when these flavonoids are used in combination with 10 μM docosahexaenoic acid at 10 to 30 μM for IL-1β production, the inhibition rate of 65% is 80% or more with docosahexaenoic acid alone.

  Furthermore, the combination of the compound represented by the formula (1) of the present invention and a highly unsaturated fatty acid suppresses the production of inflammatory cytokines such as TNF-α and IL-1β, and further, nitric oxide (NO) in animal cells. Are more strongly suppressed than in the case of single use.

The unsaturated fatty acid having 16 to 22 carbon atoms used in the present invention is a naturally distributed fatty acid having 1 to 6 double bonds, specifically,
Docosahexaenoic acid (4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoic acid),
(n-6) docosapentaenoic acid (4Z, 7Z, 10Z, 13Z, 16Z-docosapentaenoic acid),
(n-3) docosapentaenoic acid (7Z, 10Z, 13Z, 16Z, 19Z-docosapentaenoic acid),
Eicosapentaenoic acid (5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoic acid),
Arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid),
Stearidonic acid (6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid),
Homo-γ-linolenic acid (8Z, 11Z, 14Z-eicosatrienoic acid),
α-linolenic acid (9Z, 12Z, 15Z-octadecatrienoic acid),
γ-linolenic acid (6Z, 9Z, 12Z-octadecatrienoic acid),
Linoleic acid (9Z, 12Z-octadecadienoic acid),
Oleic acid (9Z-octadecenoic acid),
Conjugated linolenic acid (10E, 12Z-octadecadienoic acid
Or 9Z, 11E-octadecadienoic acid).

Among these, highly unsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid contained in fish oil, among others, have a high inflammatory cytokine production inhibitory effect.
These unsaturated fatty acids may be glycerides, and may be monoglycerides, diglycerides, or triglycerides, and the binding positions of unsaturated fatty acids in glycerol may be any of 1, 2, and 3 positions. In the case of triglycerides, fatty acids other than the unsaturated fatty acid, such as palmitic acid or stearic acid, may be bound. In addition to ethyl ester, methyl, propyl, or butyl ester is also used as the lower alkyl ester.

  On the other hand, the compound represented by the formula (1) relating to the present invention is known per se and easy to synthesize organically, but is easily extracted and prepared from edible plants such as legume seed coats by a known method (special table) 2001-500546). In this case, if there are no inconvenient impurities, the crude extract can be used as it is. In particular, the glucosides of the flavonoids are often extracted from plants, but these glycosides are also enzymatically hydrolyzed in vivo to produce flavonoids, and thus are used in the same manner as free flavonoids. Unsaturated fatty acids, glycerides containing them, and lower alkyl esters are easily produced by known methods from fish oils, vegetable oils, microbial oils and fats, and the like.

When used in the present invention, the amount of the compound of formula (1) is adjusted within a physiologically safe range depending on the type of compound, the method and degree of purification, and the degree of effect required. For example, although it is set to several mg or less per 1 ml of drinking water or 1 g of food, it is appropriately increased or decreased within a physiologically safe range depending on the total intake amount or intake form. The amount of the unsaturated fatty acid or its glyceride or ester is appropriately increased or decreased within a physiologically safe range depending on the kind of compound, purity, degree of required effect, and the like.

Next, the present invention will be described in more detail based on examples.

[Example 1]
Human THP-1 acute monocytic leukemia cells (purchased from Dainippon Pharmaceutical Co., Ltd.) are pre-cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and then suspended at 1 x 10 ⁵ cells / ml After adding 0.1 μM phorbol 12-myristate 13-acetate (PMA), 0.1 ml was dispensed into a 96-well plate. After 3 days of culture, microscopically confirm that the cells differentiated into macrophages and stuck to the bottom, then add 10 μM docosahexaenoic acid (DHA) with or without 10% FBS to each well. Replaced with 0.2 ml of fresh medium. Next, 2 μl of an ethanol solution of a sample having a predetermined concentration (0.1 to 3 mM) or ethanol alone was added to the medium of each well, and further cultured for 3 hours.

  Subsequently, 6 μl of a phosphate buffer containing lipopolysaccharide (0127: B8, Sigma) of E. coli cell membrane at a concentration of 33 μg / ml was added to each well and cultured for 20 hours. The medium was collected and the TNF-α concentration was measured with an ELISA kit (Endogen). Cells collected in the well after collecting the medium are stained with 0.2% crystal violet-20% methanol solution, washed thoroughly with water, and 0.1 ml of 1% SDS aqueous solution is added to each well to elute the dye and plate reader (595 nm) The absorbance was measured by using as an index of cell number. Among the samples used, chrysin and kaempferol are from Alexis, luteolin is from Extrasynthese, apigenin and (+)-catechin are from Sigma, and quercetin is from Nacalai Tesque.

  The results are shown in FIG. The concentration (μM) of the sample in the culture solution is shown on the horizontal axis in the figure. From the results of FIG. 1A, it can be seen that the flavonoid of the present invention suppresses the production of TNF-α in a concentration-dependent manner (except for catechin), and further suppresses it more strongly when docosahexaenoic acid is simultaneously added to the medium. . As shown in Reference Example 1 below, chrysin and apigenin have little antioxidant property, while catechin shows strong antioxidant property. However, in this experiment, chrysin and apigenin show TNF-α production inhibitory activity. Since it was inactive, it is clear that there is no direct relationship between antioxidant properties and TNF-α inhibitory activity. As shown in FIG. 1B, the dye concentration, which is an indicator of the number of cells, usually had a difference of only 20% or less between the control and the sample after the experiment was completed. Therefore, a large decrease in TNF-α production is due to cell loss. It was confirmed that it was not.

[Example 2]
FIG. 2 shows the results of measuring the concentration of IL-1β in the culture medium obtained in the experiment of Example 1 by ELISA (manufactured by Endogen). Flavonoid concentration (μM) is shown on the horizontal axis. From this result, 3 μM apigenin, 10 μM luteolin and kaempferol (Kaempf. In the figure) alone have no inhibitory effect on IL-1β, but when used in combination with the highly unsaturated fatty acid DHA, the inhibitory action of DHA is remarkable. You can see that it strengthens. In addition, non-antioxidant chrysin and apigenin show stronger activity than anti-oxidant luteolin and quercetin in the suppression of IL-1β production shown here. This suppresses the inhibitory action.

Example 3
FIG. 3 shows the result of measuring the concentration of IL-6 in the culture medium obtained in the experiment of Example 1 by ELISA (manufactured by Endogen). The concentration of flavonoids is 10 μM in all cases. From these results, it can be seen that 10 μM apigenin and quercetin alone have the effect of suppressing the production of IL-6 and enhancing the inhibitory action of DHA.

Example 4
Nitric oxide (NO) also plays an important role as a signal molecule of the inflammatory response, and suppression of NO production also leads to suppression of inflammation. Mouse RAW264 macrophage cell line (purchased from RIKEN) was pre-cultured in DMEM medium containing 10% FBS, recovered by trypsinization to give a suspension of 2 x 10 ⁵ cells / ml, 96-well in 0.2 ml increments Implanted in a plate. After culturing for 2 days, the medium in each well was replaced with 0.2 ml of fresh medium containing 10% FBS with or without 10 μM docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA).

  Next, 2 μl of an ethanol solution of a sample having a predetermined concentration (0.1 to 3 mM) or ethanol alone was added to the medium of each well, and further cultured for 3 hours. Next, 6 μl of medium containing 100 μg / ml E. coli cell membrane lipopolysaccharide (0127: B8, manufactured by Sigma), 10 μg / ml arginine, and 1 μg / ml IFN-γ is added to each well and cultured for 18 hours. did. The medium was recovered, and nitrite ions converted from NO in the medium were quantified with a grease reagent (manufactured by Sigma). From the results shown in FIG. 4A, it can be seen that flavonoids suppress NO production in macrophage cells stimulated with LPS, and further, when DHA and EPA are simultaneously added, they are more strongly suppressed. FIG. 4B shows the result of staining the cells with crystal violet and measuring the absorbance to determine the number of cells.

[Reference Example 1]
The scavenging ability (antioxidant activity) of superoxide anion radical of flavonoids was measured by the method of literature (K. Hyland et al., Anal. Biochem., 135, 280-287, 1983). The results are shown in FIG. It can be seen that at concentrations (1 to 30 μM) used in the cell test, chrysin shows little antioxidant activity, and apigenin also shows very weak antioxidant properties compared to catechin and luteolin. In addition, since the antioxidant property of quercetin is difficult to measure by this method, it was compared with chrysin and apigenin at the DPPH radical scavenging rate described below, which serves as a guide for antioxidant property instead.
[Reference Example 2]
According to the method of the literature (L. Wang et al., J. Agric. Food Chem., 54, 9798-9804, 2006), the elimination rate of DPPH radical when flavonoid was added was measured from the change in absorbance. The results are shown in FIG. 6, and it can be seen that quercetin has a strong scavenging activity while chrysin and apigenin show little radical scavenging activity at a level of several tens of μM.

A is a graph showing that TNF-α production in THP-1 cells is suppressed by the combined use of flavonoids and DHA. The value on the vertical axis is the relative value (average and standard deviation of three wells) of the TNF-α concentration in the sample well when the TNF-α concentration in the control well is taken as 100. Concentration (μM). B is the absorbance (average and standard deviation of three wells) measured by staining cells in each well with crystal violet, washing, and extracting the dye. It is a graph which shows that IL-1 (beta) production in a THP-1 cell is suppressed by combined use of flavonoid (3-30micromol) and DHA. The value on the vertical axis is the relative value (average and standard deviation of three wells) of the IL-1β concentration in the sample well when the IL-1β concentration in the control well is 100. It is a graph which shows that IL-6 production in a THP-1 cell is suppressed by combined use of flavonoid (10 micromol) and DHA. The value on the vertical axis is the relative value (average and standard deviation of two wells) of the IL-6 concentration in the sample well when the IL-6 concentration in the control well is 100. A is a graph showing that NO production in RAW264 cells is suppressed by the combined use of flavonoids and PUFA (DHA or EPA). The value on the vertical axis is the relative value (average and standard deviation of the two wells) of the sample well when the absorbance of the color developed by Griess reagent in the control well is 100, and the number on the horizontal axis is the sample Concentration (μM). B is the relative value of absorbance measured by staining cells in each well with crystal violet, washing, extracting the dye, and measuring the value (control well value is 100 and expressed as the average and standard deviation of two wells). It is a graph which shows the active oxygen scavenging ability (antioxidant property) of a flavonoid. It is a graph which shows the DPPH radical scavenging ability of a flavonoid.

Claims (5)

An inhibitor of inflammatory cytokine production in animal cells, comprising as an active ingredient a compound represented by the following formula (1) and an unsaturated fatty acid having 16 to 22 carbon atoms or a glyceride or lower alkyl ester thereof.

  The inflammatory cytokine production inhibitor in animal cells according to claim 1, wherein the glyceride is mono-, di- or triglyceride of unsaturated fatty acid having 16 to 22 carbon atoms.   The inflammatory cytokine production inhibitor in animal cells according to claim 1, wherein the lower alkyl ester is an ethyl, methyl, propyl, or butyl ester of an unsaturated fatty acid having 16 to 22 carbon atoms.   Unsaturated fatty acids having 16 to 22 carbon atoms are 4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoic acid, 4Z, 7Z, 10Z, 13Z, 16Z-docosapentaenoic acid, 7Z, 10Z, 13Z, 16Z, 19Z- Docosapentaenoic acid, 5Z, 8Z, 11Z, 14Z, 17Z-eicosapentaenoic acid, 6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid, α-linolenic acid, linoleic acid, oleic acid and arachidonic acid The inflammatory cytokine production inhibitor in animal cells according to any one of claims 1 to 3, wherein the agent is one or more selected. Compounds represented by formula (1) are chrysin (R ₁ = R ₂ = R ₃ = H in formula 1), apigenin (R ₁ = R ₃ = H, R ₂ = OH in formula 1), luteolin (in formula 1) R ₁ = H, R ₂ = R ₃ = OH), kaempferol (R ₁ = R ₂ = OH, R ₃ = H in Formula 1), quercetin (R ₁ = R ₂ = R ₃ = OH in Formula 1) The inflammatory cytokine production inhibitor in animal cells according to any one of claims 1 to 4, wherein the compound is one or more compounds selected from the group consisting of.

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