JP2024041560A - Inhibitor of phenol generation by 4-hydroxybenzoic acid decarboxylase - Google Patents
Inhibitor of phenol generation by 4-hydroxybenzoic acid decarboxylase Download PDFInfo
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- JP2024041560A JP2024041560A JP2022146439A JP2022146439A JP2024041560A JP 2024041560 A JP2024041560 A JP 2024041560A JP 2022146439 A JP2022146439 A JP 2022146439A JP 2022146439 A JP2022146439 A JP 2022146439A JP 2024041560 A JP2024041560 A JP 2024041560A
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- Prior art keywords
- phenol
- hydroxybenzoic acid
- sesame lignan
- inhibitor
- compound
- Prior art date
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Abstract
Description
本発明は、ゴマ種子中に含まれる特定のリグナン化合物又はその誘導体を有効成分として含有する、4-ヒドロキシ安息香酸デカルボキシラーゼ(Hydroxyarylic acid decarboxylase)(以下、「Had」ということがある)によるフェノール生成の阻害剤に関する。 The present invention is directed to the production of phenol by 4-hydroxybenzoic acid decarboxylase (hereinafter sometimes referred to as "Had"), which contains a specific lignan compound or its derivative contained in sesame seeds as an active ingredient. Concerning inhibitors of.
糖尿病性腎症は、糖尿病の中でも罹患者数が多い2型糖尿病の進行に伴って発症する合併症の一つであり、腎機能が低下する疾患である。糖尿病性腎症の治療には、年間1.5兆円もの費用を要し、その最大の要因が透析治療にある。これまで、糖尿病性腎症の原因物質が多数同定されているが、未だその治療薬が開発されていないのが現状である。 Diabetic nephropathy is one of the complications that develop as type 2 diabetes progresses, which is the most prevalent type of diabetes, and is a disease in which kidney function declines. Treating diabetic nephropathy costs as much as 1.5 trillion yen per year, the largest factor being dialysis treatment. To date, many causative substances for diabetic nephropathy have been identified, but no therapeutic drugs have yet been developed.
最近、糖尿病性腎症の発症リスクや病期を判定できるバイオマーカーとして、フェニル硫酸(Phenyl Sulfate)が同定され(特許文献1)、かかるフェニル硫酸は、糖尿病性腎症による腎障害に関与していることも報告されている(非特許文献1)。 Recently, phenyl sulfate has been identified as a biomarker that can determine the risk of onset and stage of diabetic nephropathy (Patent Document 1), and phenyl sulfate has been implicated in renal damage caused by diabetic nephropathy. It has also been reported that there are some (non-patent document 1).
一方、フェノールは、フェニル硫酸の前駆体であり、腸に内在する腸内細菌が有するチロシンフェノールリアーゼ(TPL)により、L-チロシンから生成される。生成されたフェノールは、体内代謝を経てフェニル硫酸に変換されると考えられることから、TPLによるフェノール生成を阻害すれば、糖尿病性腎症による腎障害を改善することが予測される。実際、TPLの阻害剤である2-アザ-DL-チロシンを、腎不全モデルマウスに投与すると、血中のフェニル硫酸濃度が低下し、腎機能が改善することが報告されている(特許文献2、非特許文献1)。また、本発明者らは、ゴマ種子中に含まれる特定のリグナン化合物が、TPLによるフェノール生成を阻害する作用を有することを見いだしている(特願2022-26918)。 On the other hand, phenol is a precursor of phenyl sulfate, and is produced from L-tyrosine by tyrosine phenol lyase (TPL) possessed by intestinal bacteria resident in the intestines. Since the generated phenol is thought to be converted to phenyl sulfate through internal metabolism, it is predicted that inhibiting phenol production by TPL will improve renal damage caused by diabetic nephropathy. In fact, it has been reported that when 2-aza-DL-tyrosine, an inhibitor of TPL, is administered to renal failure model mice, blood phenyl sulfate concentration decreases and renal function improves (Patent Document 2). , Non-Patent Document 1). Furthermore, the present inventors have discovered that a specific lignan compound contained in sesame seeds has the effect of inhibiting phenol production by TPL (Japanese Patent Application No. 2022-26918).
また、TPL以外に、L-チロシンからフェノールが生成される経路として、L-チロシンから体内代謝により生成された4-ヒドロキシ安息香酸が、Hadにより生成される経路の存在が推定されている(非特許文献2)。しかしながら、ゴマ種子中に含まれる特定のリグナン化合物が、Hadによるフェノール生成を阻害する作用を有することは知られていなかった。 Furthermore, in addition to TPL, it is presumed that there is a pathway in which 4-hydroxybenzoic acid, which is generated from L-tyrosine through internal metabolism, is generated by Had (non-TPL). Patent Document 2). However, it was not known that specific lignan compounds contained in sesame seeds have the effect of inhibiting phenol production by Had.
本発明の課題は、摂取したときの安全性が高く、製造コストが比較的安価であり、かつ、Hadによるフェノール生成を阻害する作用を有する低分子化合物を提供することにある。 An object of the present invention is to provide a low-molecular compound that is highly safe when ingested, is relatively inexpensive to produce, and has the effect of inhibiting phenol production by Had.
ゴマ種子は、抗酸化成分を多く含むことから、飲食品や医薬品として幅広く応用されている。ゴマ種子由来の抗酸化成分の代表として、リグナンと呼ばれる代謝生成物(すなわち、ゴマリグナン化合物)が知られており、植物特化代謝産物と総称される。ゴマリグナン化合物は、2分子のフェニルプロパノイドが側鎖中央の炭素同士で結合した構造を有し、その中には、Hadの酵素反応の基質となるフェノールの基本骨格を有するものが多い。そこで、ゴマリグナン化合物に着目し、上記課題を解決すべく鋭意研究を続けた結果、特定のゴマリグナン化合物が、4-ヒドロキシ安息香酸を基質としたHad酵素によるフェノール生成を阻害する作用を有することを見いだした。さらに、上記特定のゴマリグナン化合物が、様々なHad産生菌におけるHadによるフェノール生成を阻害する作用を有することも確認した。本発明は、これらの知見に基づき、完成するに至ったものである。 Sesame seeds contain a large amount of antioxidant components, so they are widely used in foods, drinks, and medicines. Metabolic products called lignans (ie, sesame lignan compounds) are known as representative antioxidant components derived from sesame seeds, and are collectively referred to as plant-specific metabolites. Sesame lignan compounds have a structure in which two molecules of phenylpropanoid are bonded to each other through carbon atoms at the center of their side chains, and many of them have a basic skeleton of phenol, which serves as a substrate for the Had enzymatic reaction. Therefore, we focused on sesame lignan compounds and continued intensive research to solve the above problems, and as a result, we discovered that a specific sesame lignan compound has the effect of inhibiting phenol production by Had enzyme using 4-hydroxybenzoic acid as a substrate. Ta. Furthermore, it was confirmed that the above-mentioned specific sesame lignan compound has an effect of inhibiting Had-induced phenol production in various Had-producing bacteria. The present invention has been completed based on these findings.
すなわち、本発明は、以下のとおりである。
〔1〕以下の式(I)で表される化合物及びその生理的に許容される塩からなる群から選択される1種又は2種以上の化合物を含む、4-ヒドロキシ安息香酸デカルボキシラーゼによるフェノール生成の阻害剤。
That is, the present invention is as follows.
[1] Phenol produced by 4-hydroxybenzoic acid decarboxylase containing one or more compounds selected from the group consisting of the compound represented by the following formula (I) and its physiologically acceptable salts inhibitor of production.
式(I)中、R1は、OH、以下の式(II)又は(III)で示される基を表し、R2は、H、OH、 単糖類、(1→2)二糖類、又は三糖類を表す。 In formula (I), R 1 represents OH, a group represented by the following formula (II) or (III), and R 2 represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.
〔3〕式(I)で表される化合物が、セサモール、セサミノール、セサモリン、及びセサミノールモノグルコシドから選択される1種又は2種以上の化合物である、上記〔1〕又は〔2〕に記載の阻害剤。
〔4〕式(I)で表される化合物が、4-ヒドロキシ安息香酸デカルボキシラーゼによるフェノール生成を混合阻害する、上記〔1〕又は〔2〕に記載の阻害剤。
[3] In the above [1] or [2], the compound represented by formula (I) is one or more compounds selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside. Inhibitors as described.
[4] The inhibitor according to [1] or [2] above, wherein the compound represented by formula (I) mixedly inhibits phenol production by 4-hydroxybenzoic acid decarboxylase.
また本発明の実施の他の形態として、
以下の式(I)で表される化合物及びその生理的に許容される塩から選択される1種又は2種以上の化合物(以下、「本件ゴマリグナン化合物」ということがある)を、Hadによるフェノール生成の阻害を必要とする対象に摂取するステップを含む、当該生成を阻害する方法;や、
Hadによるフェノール生成の阻害における使用のための本件ゴマリグナン化合物;や、
Hadによるフェノール生成の阻害剤を製造するための、本件ゴマリグナン化合物の使用;
を挙げることができる。
Further, as another embodiment of the present invention,
One or more compounds selected from the compounds represented by the following formula (I) and physiologically acceptable salts thereof (hereinafter sometimes referred to as "the sesame lignan compound") are prepared using phenol by Had. A method of inhibiting the production, including the step of ingesting it into a subject in need of inhibition of the production;
the subject sesame lignan compounds for use in inhibiting phenol production by Had;
Use of the subject sesame lignan compounds to produce an inhibitor of phenol production by Had;
can be mentioned.
式(I)中、R1は、OH、以下の式(II)又は(III)で示される基を表し、R2は、H、OH、 単糖類、(1→2)二糖類、又は三糖類を表す。 In formula (I), R 1 represents OH, a group represented by the following formula (II) or (III), and R 2 represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.
また本発明の実施の他の形態として、
本件ゴマリグナン化合物を、Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質(例えば、フェニル硫酸;以下同じ)に起因する症状又は疾患の予防若しくは治療を必要とする対象に摂取するステップを含む、当該症状又は疾患を予防若しくは治療する方法;や、
Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患の予防若しくは治療における使用のための本件ゴマリグナン化合物;や、
Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患の予防剤若しくは治療剤を製造するための、本件ゴマリグナン化合物の使用;
を挙げることができる。
Further, as another embodiment of the present invention,
The present sesame lignan compound is ingested by a subject in need of prevention or treatment of symptoms or diseases caused by uremic substances (e.g., phenyl sulfate; the same shall apply hereinafter) whose precursor is phenol produced by Had in Had-producing bacteria. A method of preventing or treating the condition or disease, comprising:
The present sesame lignan compound for use in the prevention or treatment of symptoms or diseases caused by uremic substances having phenol precursors produced by Had in Had-producing bacteria;
Use of the present sesame lignan compound for producing a preventive or therapeutic agent for symptoms or diseases caused by uremic substances having phenol as a precursor produced by Had in Had-producing bacteria;
can be mentioned.
本件ゴマリグナン化合物は、Had産生菌由来のHadによるフェノール生成を阻害する作用を有する。また、本件ゴマリグナン化合物は、食品として使用されているゴマ由来の成分に関連する低分子化合物であることから、摂取したときの安全性が高く、かつ、製造コストが比較的安価である。このため、本件ゴマリグナン化合物を摂取すると、腸などの生体内のHad産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患の予防・治療効果が得られる。 The present sesame lignan compound has the effect of inhibiting phenol production by Had derived from Had-producing bacteria. Furthermore, the present sesame lignan compound is a low-molecular compound related to components derived from sesame seeds used as foods, so it is highly safe when ingested, and the manufacturing cost is relatively low. Therefore, when the sesame lignan compound of the present invention is ingested, it is possible to obtain preventive and therapeutic effects on symptoms and diseases caused by uremic substances whose precursors are phenols produced by Had in Had-producing bacteria in living organisms such as the intestines.
本発明の阻害剤は、本件ゴマリグナン化合物、すなわち、以下の式(I)の化合物及びその生理的に許容される塩から選択される1種又は2種以上の化合物を含み、かつ、「Hadによるフェノール生成を阻害するため」という用途に特定された剤(以下、「本件阻害剤」ということがある)である。本件阻害剤は、本件ゴマリグナン化合物を、単独で非ヒト哺乳動物用飼料や、飲食品又は医薬品(製剤)として使用してもよいし、さらに添加剤を混合し、組成物の形態(すなわち、非ヒト哺乳動物用飼料組成物や、飲食品組成物又は医薬組成物)として使用してもよい。上記飲食品としては、例えば、健康食品(機能性食品、栄養補助食品、健康補助食品、栄養強化食品、栄養調整食品、サプリメント等)、保健機能食品(特定保健用食品、栄養機能食品、機能性表示食品等)を挙げることができる。なお、本件阻害剤は、先願(特願2022-26918)におけるTPLによるフェノール生成の阻害剤と区別するために、「TPLによるフェノール生成を阻害するため」という用途に使用されない。 The inhibitor of the present invention contains the present sesame lignan compound, that is, one or more compounds selected from the following compounds of formula (I) and physiologically acceptable salts thereof, and It is an agent specified for the purpose of "inhibiting phenol production" (hereinafter sometimes referred to as "the subject inhibitor"). The sesame lignan compound of the present invention may be used alone as feed for non-human mammals, food/beverage products, or pharmaceuticals (preparations), or it may be mixed with additives to form a composition (i.e., a non-human mammal). It may also be used as a feed composition for human mammals, a food/beverage composition, or a pharmaceutical composition. Examples of the above-mentioned foods and drinks include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally adjusted foods, supplements, etc.), foods with health claims (foods for specified health uses, nutritionally functional foods, functional foods, etc.) (labeled foods, etc.). In order to distinguish the present inhibitor from the inhibitor of phenol production by TPL in the previous application (Japanese Patent Application No. 2022-26918), it is not used for the purpose of "inhibiting phenol production by TPL."
式(I)中、R1は、OH、以下の式(II)又は(III)で示される基を表し、R2は、H、OH、 単糖類、(1→2)二糖類、又は三糖類を表す。 In formula (I), R 1 represents OH, a group represented by the following formula (II) or (III), and R 2 represents H, OH, a monosaccharide, a (1→2) disaccharide, or a trisaccharide. Represents sugars.
式(I)中、R2における単糖類としては、例えば、グルコース、フルクトース、ガラクトース等を挙げることができ、R2における(1→2)二糖類としては、例えば、(1→2)ジグルコシド等を挙げることができ、R2における三糖類としては、例えば、トリグルコシド等を挙げることができる。 In formula (I), examples of the monosaccharide in R 2 include glucose, fructose, galactose, etc., and examples of the (1→2) disaccharide in R 2 include (1→2) diglucoside, etc. Examples of the trisaccharide for R2 include triglucoside.
後述する本実施例において、4種類のゴマリグナン化合物(セサモール、セサミノール、セサモリン、及びセサミノールモノグルコシド)が、Hadによるフェノール生成を特に効果的に阻害することが示されているため、式(I)中、R2としては、H、OH、又は単糖類が好ましく、式(I)の化合物としては、セサモール、セサミノール、セサモリン、及びセサミノールモノグルコシドから選択される1種又は2種以上の化合物がより好ましい。 In this example, which will be described later, four types of sesame lignan compounds (sesamol, sesaminol, sesamolin, and sesaminol monoglucoside) have been shown to particularly effectively inhibit phenol production by Had. ), R 2 is preferably H, OH, or a monosaccharide, and the compound of formula (I) is one or more selected from sesamol, sesaminol, sesamolin, and sesaminol monoglucoside. Compounds are more preferred.
本明細書において、「Had」とは、以下の反応式に示すとおり、反応1(4-ヒドロキシ安息香酸-二酸化炭素[CO2]→フェノール)と、かかる反応に逆行する反応2(フェノール+二酸化炭素[CO2]→4-ヒドロキシ安息香酸)とを触媒する酵素を意味する。 As used herein, "Had" refers to an enzyme that catalyzes reaction 1 (4-hydroxybenzoic acid - carbon dioxide [CO 2 ] → phenol) and the reverse reaction 2 (phenol + carbon dioxide [CO 2 ] → 4-hydroxybenzoic acid), as shown in the following reaction formula.
本件ゴマリグナン化合物は、上記反応式において、少なくとも反応1を阻害することにより、Hadによるフェノール生成を阻害する作用を有するものである。 The present sesame lignan compound has the effect of inhibiting phenol production by Had by inhibiting at least reaction 1 in the above reaction formula.
上記反応式における、本件ゴマリグナン化合物による阻害形式としては、特に制限されず、例えば、競合阻害(Competitive Inhibition)、不競合阻害(Uncompetitive Inhibition)、混合阻害(Mixed Inhibition)等を挙げることができる。 In the above reaction formula, the type of inhibition by the present sesame lignan compound is not particularly limited, and examples thereof include competitive inhibition, uncompetitive inhibition, mixed inhibition, and the like.
本明細書において、「競合阻害」とは、阻害剤(本件ゴマリグナン化合物)が、基質(4-ヒドロキシ安息香酸)に結合していない酵素(Had)における基質結合部位に結合する阻害形式を意味する。なお、競合阻害において、阻害剤は酵素・基質複合体には結合しない。 In this specification, "competitive inhibition" refers to an inhibition form in which an inhibitor (the sesame lignan compound of the present invention) binds to a substrate-binding site in an enzyme (Had) that is not bound to a substrate (4-hydroxybenzoic acid). In competitive inhibition, the inhibitor does not bind to the enzyme-substrate complex.
本明細書において、「不競合阻害」とは、阻害剤(本件ゴマリグナン化合物)が、基質(4-ヒドロキシ安息香酸)に結合していない酵素(Had)には結合せずに、酵素・基質複合体にのみ結合する阻害形式を意味する。 As used herein, "uncompetitive inhibition" means that the inhibitor (the present sesame lignan compound) does not bind to the enzyme (Had) that is not bound to the substrate (4-hydroxybenzoic acid) and inhibits the enzyme-substrate complex. means an inhibitory form that binds only to the body.
本明細書において、「混合阻害」とは、阻害剤(本件ゴマリグナン化合物)が、基質(4-ヒドロキシ安息香酸)に結合していない酵素(Had)と、酵素・基質複合体の両方に、異なる阻害定数(Ki)で結合する阻害形式を意味する。 As used herein, "mixed inhibition" means that the inhibitor (the present sesame lignan compound) has different effects on both the enzyme (Had) that is not bound to the substrate (4-hydroxybenzoic acid) and the enzyme-substrate complex. Refers to the type of inhibition that binds with the inhibition constant (Ki).
本件ゴマリグナン化合物としては、後述する本実施例でその効果が実証されているため、Hadによるフェノール生成を混合型阻害の形式で阻害(混合阻害)するものを好適に例示することができる。 As the present sesame lignan compound, since its effect has been demonstrated in the present example described below, a compound that inhibits phenol production by Had in the form of mixed inhibition (mixed inhibition) can be suitably exemplified.
上記Hadとしては、その由来は特に制限されないが、後述する本実施例でその効果が実証されているため、Had産生菌(より具体的には、哺乳動物生体内の各種臓器又は組織におけるHad産生菌)により産生されるHad(換言すると、Had産生菌由来のHad)を好適に例示することができる。したがって、本件阻害剤は、哺乳動物生体内のHad産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患の予防剤若しくは治療剤に有利に適用することができる。 The origin of the above-mentioned Had is not particularly limited, but since its effect has been demonstrated in the present embodiment described below, a suitable example is Had produced by Had-producing bacteria (more specifically, Had-producing bacteria in various organs or tissues in the living body of a mammal) (in other words, Had derived from Had-producing bacteria). Therefore, the present inhibitor can be advantageously used as a preventive or therapeutic agent for symptoms or diseases caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria in the living body of a mammal.
本明細書において、「Had産生菌」としては、Had産生腸内細菌であっても、Had産生非腸内細菌であってもよく、具体的には、例えば、シトロバクター(Citrobacter)属細菌(例えば、シトロバクター・コセリ[C. koseri]、シトロバクター・フロインディー[C. freundii])、クライベラ(Kluyvera)属細菌(例えば、クライベラ・インターメディア[K. intermedia])、エンテロバクター(Enterobacter)属細菌(例えば、エンテロバクター・クロアカ[E. cloacae]、エンテロバクター・アエロゲネス[E. aerogenes])、シゲラ(Shigella)属細菌(例えば、志賀赤痢菌[S. Shigella dysenteriae])、クレブシエラ(Klebsiella)属細菌(例えば、クレブシエラ・ニューモニエ[K. pneumoniae])、エシェリキア(Escherichia)属細菌(例えば、大腸菌[E. coli])、バシラス(Bacillus)属細菌(例えば、枯草菌[B. subtilis])等を挙げることができる。 In this specification, "Had-producing bacteria" may be Had-producing enterobacteria or Had-producing non-intestinal bacteria, and specifically, for example, bacteria of the genus Citrobacter ( For example, bacteria of the genus Kluyvera (e.g., C. koseri, C. freundii), bacteria of the genus Kluyvera (e.g., K. intermedia), spp. Bacteria (e.g., E. cloacae, E. aerogenes), Shigella (e.g., S. Shigella dysenteriae), Klebsiella Bacteria (e.g., Klebsiella pneumoniae), Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis), etc. can be mentioned.
本明細書において、「各種臓器又は組織」としては、例えば、大腸(結腸又は直腸)、胃、肝臓、心臓、脳、脊髄、肺、食道、十二指腸、小腸、皮膚、前立腺、膀胱、子宮、腎臓、膵臓、脾臓、気管、気管支、胆嚢、胆管等を挙げることができる。 As used herein, "various organs or tissues" include, for example, large intestine (colon or rectum), stomach, liver, heart, brain, spinal cord, lung, esophagus, duodenum, small intestine, skin, prostate, bladder, uterus, kidney. , pancreas, spleen, trachea, bronchi, gallbladder, bile duct, etc.
本明細書において、哺乳動物としては、ヒトや、非ヒト哺乳動物(例えば、サル、マウス、ラット、イヌ、ネコ、家畜[例えば、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカ])等を挙げることができる。 In this specification, mammals include humans, non-human mammals (e.g., monkeys, mice, rats, dogs, cats, livestock [e.g., rabbits, pigs, horses, cows, sheep, goats, deer]), etc. can be mentioned.
本明細書において、「Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患」としては、例えば、食欲不振、悪心、嘔吐、口臭、口内炎、腸炎等の消化器系異常;無欲、無関心、記銘力低下、うつ状態、傾眠、昏睡、多発神経炎、(発達性)協調運動障害、食思不振等の神経系異常;動脈硬化、貧血、赤血球造血障害、高血圧、虚血性心疾患、心膜炎、心筋炎、(血液)凝固異常、心不全、心血管障害等の循環器系異常;色素沈着、掻痒(感)、皮下出血、皮膚萎縮、感染、アトピー、脱毛等の皮膚異常(疾患);アルブミン尿、急性腎不全、急性尿細管壊死、腎性貧血、慢性腎不全、尿細管間質障害、急性腎炎、慢性腎炎、ネフローゼ、糖尿病性腎症、動脈硬化性腎症、腎性骨異常症(例えば、腎性骨異栄養症)、溢水等の腎機能障害;脳卒中;心筋梗塞;癌;自閉症;免疫不全;骨異常症;副甲状腺亢進症;インスリン抵抗性;栄養不良;炎症;震戦;などを挙げることができる。また、上記症状又は疾患には、IgA腎症、嚢胞腎、肝機能障害(例えば、劇症肝炎、脂肪肝、NASH、NAFLD)、癌(例えば、胆汁発がん)等のLPS(Lipopolysaccharide)が関与する症状又は疾患;潰瘍性大腸炎、クローン病等の炎症性腸疾患;動脈硬化関連疾患;ループス、強皮症、リウマチ等の自己免疫疾患;肥満;メタボリックシンドローム;糖尿病;自閉症;パーキンソン病;アルツハイマー病;サルコペニアも含まれる。なお、上記症状又は疾患には、Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因せずに、これら尿毒症物質以外の要因に起因する症状又は疾患は含まれない。 In this specification, "symptoms or diseases caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria" include, for example, anorexia, nausea, vomiting, bad breath, stomatitis, enteritis, etc. Gastrointestinal system abnormalities; nervous system abnormalities such as avolition, apathy, impaired memory, depression, somnolence, coma, polyneuritis, (developmental) coordination disorder, anorexia; arteriosclerosis, anemia, erythropoietic disorders , hypertension, ischemic heart disease, pericarditis, myocarditis, (blood) coagulation abnormalities, heart failure, cardiovascular disorders such as cardiovascular disorders; pigmentation, itching (feeling), subcutaneous bleeding, skin atrophy, infection, atopy. , skin abnormalities (diseases) such as hair loss; albuminuria, acute renal failure, acute tubular necrosis, renal anemia, chronic renal failure, tubulointerstitial disorder, acute nephritis, chronic nephritis, nephrosis, diabetic nephropathy, arteries Renal dysfunction such as sclerosing nephropathy, renal bone abnormalities (e.g., renal osteodystrophy), and effusion; stroke; myocardial infarction; cancer; autism; immunodeficiency; bone abnormalities; hyperparathyroidism ; insulin resistance; malnutrition; inflammation; tremor; etc. In addition, the above-mentioned symptoms or diseases include IgA nephropathy, cystic kidney disease, liver dysfunction (e.g., fulminant hepatitis, fatty liver, NASH, NAFLD), cancer (e.g., biliary carcinogenesis), etc., which involve LPS (Lipopolysaccharide). Symptoms or diseases; inflammatory bowel diseases such as ulcerative colitis and Crohn's disease; arteriosclerosis-related diseases; autoimmune diseases such as lupus, scleroderma, and rheumatism; obesity; metabolic syndrome; diabetes; autism; Parkinson's disease; Alzheimer's disease; also includes sarcopenia. Note that the above symptoms or diseases do not include symptoms or diseases that are not caused by uremic substances whose precursor is phenol produced by Had in Had-producing bacteria, but are caused by factors other than these uremic substances. .
本件ゴマリグナン化合物は、市販品を購入するか、あるいは公知の反応を適宜組み合わせることによって合成することができる。原料は市販購入品や市販購入品に一般的な保護基等を付与して合成することができる。また、いずれの工程も有機化学で汎用される保護・脱保護による合成、精製等を適応することができる。 The sesame lignan compound of the present invention can be synthesized by purchasing a commercially available product or by appropriately combining known reactions. The raw material can be synthesized by adding a general protective group to a commercially purchased product or a commercially purchased product. In addition, synthesis, purification, etc. by protection and deprotection, which are commonly used in organic chemistry, can be applied to any of the steps.
本明細書において、「生理的に許容される塩」とは、妥当な医学的、薬学的、又は生物学的判断の範囲内で、哺乳動物の組織と接触して用いるのに、過度の毒性、刺激性、アレルギー応答、及びその他の問題や合併症を伴うことなく、適度な受益性/危険性比率に相応して適している塩を意味する。生理的に許容される塩としては、例えば、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩等の酸付加塩;アンモニウム塩;ナトリウム塩、リチウム塩、カリウム塩等のアルカリ金属塩;アルミニウム塩;カルシウム塩、マグネシウム塩等のアルカリ土類金属塩;ジシクロヘキシルアミン塩、N-メチル-D-グルカミン等の有機塩基との塩;アルギニン、リシン、オルニチン等のアミノ酸との塩;塩基性窒素含有基により生じる塩;などを挙げることができる。 As used herein, "physiologically acceptable salts" are salts that, within the scope of reasonable medical, pharmaceutical, or biological judgment, have excessive toxicity when used in contact with mammalian tissue. , salts that are suitable according to a moderate benefit/risk ratio, without irritation, allergic reactions, and other problems and complications. Physiologically acceptable salts include, for example, hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, and succinate. Acid addition salts such as salts, oxalates, lactates, tartrates, glycolates, methanesulfonates, butyrates, valerates, citrates, fumarates, maleates, malates; ammonium Salts; alkali metal salts such as sodium salts, lithium salts, potassium salts; aluminum salts; alkaline earth metal salts such as calcium salts and magnesium salts; salts with organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine. ; Salts with amino acids such as arginine, lysine, ornithine; Salts formed by basic nitrogen-containing groups; and the like.
本件ゴマリグナン化合物には、種々の異性体(例えば、光学異性体、位置異性体、互変異性体等)、水和物等の溶媒和物、結晶多形、及びエステル体等のプロドラッグも包含される。また、式(I)の化合物を構成する1個若しくは2個以上の原子が同位体である化合物も本件ゴマリグナン化合物に含まれる。本件ゴマリグナン化合物に含まれる同位体の例としては、水素、炭素、窒素、酸素、リン、臭素、及び塩素の同位体、例えば、2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、75Br、76Br、77Br、82Br、及び37Clなどを挙げることができる。 The present sesame lignan compound also includes various isomers (e.g., optical isomers, positional isomers, tautomers, etc.), solvates such as hydrates, crystal polymorphs, and prodrugs such as esters. be done. Further, compounds in which one or more atoms constituting the compound of formula (I) are isotopes are also included in the present sesame lignan compound. Examples of isotopes contained in the sesame lignan compound include hydrogen, carbon, nitrogen, oxygen, phosphorus, bromine, and chlorine isotopes, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N , 15 N, 15 O, 17 O, 18 O, 75 Br, 76 Br, 77 Br, 82 Br, and 37 Cl.
本件阻害剤の摂取(投与)対象としては、特に制限されず、通常、Hadによるフェノール生成の阻害を必要とする哺乳動物や、Had産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質に起因する症状又は疾患の予防若しくは治療を必要とする哺乳動物である。 The targets for ingestion (administration) of this inhibitor are not particularly limited, but usually include mammals that require inhibition of phenol production by Had, and uremic diseases in which phenol produced by Had in Had-producing bacteria is a precursor. A mammal in need of prevention or treatment of symptoms or diseases caused by the substance.
本件阻害剤を、対象へ摂取(投与)する方法としては、例えば、粉末剤、顆粒剤、錠剤(タブレット)、カプセル剤、シロップ剤、懸濁液など形態で経口的に摂取する方法(経口摂取);溶液、乳剤、懸濁液などの形態(剤型)で注射(例えば、皮下注射、静脈内注射、筋肉内注射、局所投与)する方法(非経口投与);スプレー剤の形態で鼻孔内投与する方法(非経口投与);等を挙げることができる。 Examples of methods for ingesting (administering) the subject inhibitor include oral ingestion in the form of powder, granules, tablets, capsules, syrups, suspensions, etc. ); method of injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, local administration) in the form (dosage form) of a solution, emulsion, suspension, etc. (parenteral administration); intranasal administration in the form of a spray. Methods of administration (parenteral administration); and the like.
経口摂取用の本件阻害剤は、徐放性剤や腸溶性剤とすることができる。かかる腸溶性剤は、例えば、有効成分である本件ゴマリグナン化合物を含む顆粒を、腸溶性被膜(すなわち、胃液に対しては抵抗性があり、小腸内で溶解する基剤[腸溶性成分]を主成分とする被膜)で充填するか、あるいは、有効成分である本件ゴマリグナン化合物を含む顆粒に、滑沢剤を加えて打錠して得た錠剤を、腸溶性被膜でコーティングすることにより得ることができる。 The subject inhibitor for oral ingestion can be in the form of sustained release or enteric-coated formulations. Such enteric-coated agents, for example, coat granules containing the sesame lignan compound as an active ingredient with an enteric coating (i.e., a base [enteric component] that is resistant to gastric juice and dissolves in the small intestine). Alternatively, tablets obtained by adding a lubricant to granules containing the sesame lignan compound as an active ingredient and compressing the same may be coated with an enteric coating. can.
剤型がタブレットである本件阻害剤を得る方法としては、例えば、有効成分である本件ゴマリグナン化合物と、必要に応じて配合され得る任意の成分(添加剤)とを混合した後、この混合物を圧縮成形してタブレットを得る方法や、さらに、圧縮成形後に得られるタブレットを腸溶性成分によりコーティングする方法(腸溶剤とする方法)等を挙げることができ、後者の方法が好ましい。 As a method for obtaining the present inhibitor whose dosage form is a tablet, for example, after mixing the present sesame lignan compound as an active ingredient and optional components (additives) that may be added as necessary, this mixture is compressed. Examples include a method of obtaining a tablet by molding, and a method of coating the tablet obtained after compression molding with an enteric component (a method of forming an enteric agent), with the latter method being preferred.
上記腸溶性成分としては、例えば、シェラック、ツェイン、ヒドロキシメチルセルロースフタレート、カルボキシメチルセルロース、カルボキシメチルエチルセルロース、酢酸フタル酸セルロース、メタクリル酸コポリマー、水に不溶のエチルセルロース、アミノアルキルメタアクリレートコポリマー、ビール酵母細胞壁(例えば、商品名イーストラップなど)、タピオカデンプン、ゼラチン、ペクチン等を挙げることができ、中でもシェラックが好ましい。なお、腸溶性剤であるか否かは、第14改正日本薬局方 崩壊試験法により確認可能である。 Examples of the enteric components include shellac, zein, hydroxymethyl cellulose phthalate, carboxymethyl cellulose, carboxymethyl ethyl cellulose, cellulose acetate phthalate, methacrylic acid copolymers, water-insoluble ethyl cellulose, aminoalkyl methacrylate copolymers, beer yeast cell walls (e.g. , trade name Yeast Wrap, etc.), tapioca starch, gelatin, pectin, etc., among which shellac is preferred. In addition, whether or not it is an enteric-coated drug can be confirmed by the disintegration test method of the 14th edition of the Japanese Pharmacopoeia.
本件阻害剤に含まれる本件ゴマリグナン化合物の摂取量は、摂取対象(哺乳動物)の年齢、体重、性別、症状、薬剤への感受性、生物種等に応じて適宜決定され、例えば、本件ゴマリグナン化合物に換算したときの濃度が0.1μg~200mg/kg(体重)/日の投与量の範囲である。なお、本件阻害剤は、一日あたり単回又は複数回(例えば、2~4回)に分けて摂取してもよい。 The intake amount of the Sesame Lignan Compound contained in the Inhibitor is appropriately determined depending on the age, weight, sex, symptoms, drug sensitivity, species, etc. of the subject (mammal). The calculated concentration ranges from 0.1 μg to 200 mg/kg (body weight)/day. The inhibitor may be taken once or in multiple doses (for example, 2 to 4 times) per day.
本明細書において、「添加剤」としては、生理的に許容される通常の担体、結合剤、安定化剤、賦形剤、希釈剤、pH緩衝剤、崩壊剤、等張剤、添加剤、被覆剤、可溶化剤、潤滑剤、滑走剤、溶解補助剤、滑沢剤、風味剤、甘味剤、溶剤、ゲル化剤、栄養剤等の配合成分を例示することができる。かかる配合成分としては、具体的に、水、生理食塩水、動物性脂肪及び油、植物油、乳糖、デンプン、ゼラチン、結晶性セルロース、ガム、タルク、ステアリン酸マグネシウム、ヒドロキシプロピルセルロース、ポリアルキレングリコール、ポリビニルアルコール、グリセリンを例示することができる。 As used herein, "additives" include conventional physiologically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives, Examples of ingredients include coating agents, solubilizers, lubricants, sliding agents, solubilizing agents, lubricants, flavoring agents, sweeteners, solvents, gelling agents, and nutrients. Specifically, such ingredients include water, physiological saline, animal fat and oil, vegetable oil, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropylcellulose, polyalkylene glycol, Examples include polyvinyl alcohol and glycerin.
本件阻害剤としては、Hadによるフェノール生成の阻害成分として、本件ゴマリグナン化合物以外の成分を含むものであってもよいが、本件ゴマリグナン化合物単独でも優れた阻害効果を発揮するため、本件ゴマリグナン化合物以外の上記阻害成分(例えば、タンパク質、アミノ酸、DNA、RNA、ポリマー等の化合物;植物由来の抽出物[例えば、ゴマリグナン化合物])を含まないものが好ましい。 The present inhibitor may contain components other than the present sesame lignan compound as an inhibitory component of phenol production by Had, but since the present sesame lignan compound alone exhibits an excellent inhibitory effect, Preferably, it does not contain the above-mentioned inhibitory components (eg, compounds such as proteins, amino acids, DNA, RNA, polymers, etc.; plant-derived extracts [eg, sesame lignan compounds]).
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
1.本件ゴマリグナン化合物が、Hadによるフェノール生成を阻害することの確認(1)
ゴマ種子中に含まれるリグナン化合物(すなわち、ゴマリグナン化合物)について、Hadによるフェノール生成を阻害する作用を有するかどうかを、枯草菌JCM1465株由来Hadを用いて解析した。
1. Confirmation that the sesame lignan compound inhibits phenol production by Had (1)
Lignan compounds contained in sesame seeds (ie, sesame lignan compounds) were analyzed using Had derived from Bacillus subtilis strain JCM1465 to determine whether they have the effect of inhibiting phenol production by Had.
1-1 材料及び方法
[Hadの粗酵素液の調製]
Hadの粗酵素液は、以下の手順〔1〕~〔7〕に従って調製し、その後の[Had阻害活性試験]に用いた。
〔1〕Had産生大腸菌BL21株を、0.1%のグリセロール、1%の4-ヒドロキシ安息香酸、及び100μg/mLのアンピシリンを含むGAM糖分解用半流動液体培地(ニッスイ社製)50mL中で、37℃、120rpmの条件下で、OD600=0.5になるまで振盪培養した。
〔2〕氷上で急冷した後、15℃で30分間静置した。
〔3〕1mMのIPTGを添加し、15℃で24時間、アネロパック入りジャー内にて静置培養(嫌気培養)した。
〔4〕培養したHad産生大腸菌BL21株を含む培養液を、遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(液体培地)を除去後、反応用緩衝液(50mMのKPB[pH7.0]及び2mMの2-メルカプトエタノールを含む液)中に懸濁した。
〔5〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(上記反応用緩衝液)を除去後、上記反応用緩衝液中に懸濁した。
〔6〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(上記反応用緩衝液)を除去後、上記反応用緩衝液中に懸濁した。
〔7〕SONIFIER(登録商標) SFX250(EMERSON社製)を用いて超音波破砕処理を行った後、遠心処理し、上清をHadの粗酵素液として回収した。
1-1 Materials and methods [Preparation of Had crude enzyme solution]
A crude Had enzyme solution was prepared according to the following procedures [1] to [7] and used in the subsequent [Had inhibitory activity test].
[1] Had-producing E. coli strain BL21 was grown in 50 mL of a semi-solid liquid medium for GAM glycolysis (manufactured by Nissui) containing 0.1% glycerol, 1% 4-hydroxybenzoic acid, and 100 μg/mL ampicillin. The cells were cultured with shaking at 37° C. and 120 rpm until OD 600 =0.5.
[2] After rapidly cooling on ice, the mixture was allowed to stand at 15°C for 30 minutes.
[3] 1mM IPTG was added and cultured statically (anaerobic culture) at 15°C for 24 hours in a jar containing Aneropack.
[4] The culture solution containing the cultivated Had-producing E. coli strain BL21 was collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (liquid medium), the reaction buffer was (a solution containing 50mM KPB [pH 7.0] and 2mM 2-mercaptoethanol).
[5] Bacteria were collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (the above reaction buffer), they were suspended in the above reaction buffer.
[6] Bacteria were collected by centrifugation (15,000 x g, 10 minutes, 4°C), and after removing the supernatant (the above reaction buffer), they were suspended in the above reaction buffer.
[7] After performing ultrasonic disruption using SONIFIER (registered trademark) SFX250 (manufactured by EMERSON), centrifugation was performed, and the supernatant was collected as a Had crude enzyme solution.
[Had阻害活性試験]
Had阻害活性試験は、以下の手順〔1〕~〔4〕に従って行った。
〔1〕1mMの4-ヒドロキシ安息香酸及び100μMの各種ゴマリグナン化合物(表1に示す8種類のゴマリグナン化合物)を含む反応用緩衝液(200mMのリン酸カリウム緩衝液[KPB][pH7.0]、及び4mMの2-メルカプトエタノールを含む液)95μLを、37℃で10分間インキュベートした。なお、比較対照として、4-ヒドロキシ安息香酸を含み、かつゴマリグナン化合物を含まない反応用緩衝液も同様にインキュベートした。
〔2〕Hadの粗酵素液5μLを添加し、37℃で3時間インキュベートした。
〔3〕反応停止液(40%のアセトニトリル及び4%のトリフルオロ酢酸[TFA]を含む液)100μLを添加後、遠心処理(15,000×g、10分間、4℃)した。
〔4〕上清を回収し、液体クロマトグラフ(HPLC)分析を表2に示す条件に従って行い、フェノール量を定量した。フェノール量は、ゴマリグナン化合物を含まない反応用緩衝液中のフェノール濃度を100とし、各種ゴマリグナン化合物を含む反応用緩衝液中のフェノール濃度の相対値として算出した(図1の縦軸参照)。
[Had inhibitory activity test]
The Had inhibitory activity test was conducted according to the following procedures [1] to [4].
[1] Reaction buffer (200 mM potassium phosphate buffer [KPB] [pH 7.0], and 95 μL of a solution containing 4 mM 2-mercaptoethanol) was incubated at 37° C. for 10 minutes. As a comparison, a reaction buffer containing 4-hydroxybenzoic acid and no sesame lignan compound was similarly incubated.
[2] 5 μL of Had's crude enzyme solution was added and incubated at 37° C. for 3 hours.
[3] After adding 100 μL of a reaction stop solution (a solution containing 40% acetonitrile and 4% trifluoroacetic acid [TFA]), the mixture was centrifuged (15,000×g, 10 minutes, 4° C.).
[4] The supernatant was collected and subjected to liquid chromatography (HPLC) analysis according to the conditions shown in Table 2 to quantify the amount of phenol. The amount of phenol was calculated as the relative value of the phenol concentration in the reaction buffer containing various sesame lignan compounds, with the phenol concentration in the reaction buffer containing no sesame lignan compound being 100 (see the vertical axis in FIG. 1).
1-2 結果
組換え大腸菌由来Hadと、その基質である4-ヒドロキシ安息香酸とを、表1に示す8種類のゴマリグナン化合物の存在下でインキュベートすると、そのうち、7種類のゴマリグナン化合物(セサモール、セサミノール[SML]、セサモリン、セサミノールモノグルコシド[SMG]、セサミン、ジグルコシド[1-2 SDG]、及びセサミノールモノグルコシド[SMG])を用いた場合、これらゴマリグナン化合物非存在下でインキュベートした場合と比べ、生成されるフェノール量が低下し、特に4種類のゴマリグナン化合物(セサモール、SML、セサモリン、及びSMG)については、有意にフェノール量が低下することが示された(図2参照)。
この結果は、本件ゴマリグナン化合物に包含される上記7種類のゴマリグナン化合物が、Hadによるフェノール生成を阻害する作用を有することを示している。
1-2 Results When recombinant E. coli-derived Had and its substrate, 4-hydroxybenzoic acid, were incubated in the presence of the eight sesame lignan compounds shown in Table 1, the amount of phenol produced was reduced when seven of the sesame lignan compounds (sesamol, sesaminol [SML], sesamolin, sesaminol monoglucoside [SMG], sesamin, diglucoside [1-2 SDG], and sesaminol monoglucoside [SMG]) were used compared to incubation in the absence of these sesame lignan compounds. In particular, the amount of phenol produced was significantly reduced for four of the sesame lignan compounds (sesamol, SML, sesamolin, and SMG) (see Figure 2).
This result indicates that the above seven types of sesame lignan compounds included in the present sesame lignan compounds have an inhibitory effect on phenol production by Had.
2.本件ゴマリグナン化合物によるHadの阻害形式及び阻害定数
次に、本件ゴマリグナン化合物によるHadの阻害形式の決定と、本件ゴマリグナン化合物によるHadの阻害定数、具体的には、Hadによるフェノール生成の50%阻害が認められる本件ゴマリグナン化合物濃度の算出を行った。
2. Inhibition type and inhibition constant of Had by the sesame lignan compound Next, we determined the type of inhibition of Had by the sesame lignan compound, and the inhibition constant of Had by the sesame lignan compound. Specifically, 50% inhibition of phenol production by Had was observed. The concentration of the sesame lignan compound was calculated.
2-1 方法
100μMの2種類のゴマリグナン化合物(セサモール及びSML)を用いて、実施例1のHad阻害活性試験を行い、フェノール生成速度(μM/分)(以下、「v」という)を算出した。次いで、縦軸にvを、横軸に4-ヒドロキシ安息香酸濃度(mM)(以下、[S]という)をそれぞれプロットし、SigmaPlot(ヒューリンクス社製)を用いて近似曲線にフィッティングを行い、速度論パラメータを算出した。その後、縦軸に1/v、横軸に1/[S]をプロットし(Lineweaver Burk plot)、近似直線の形状より、各種ゴマリグナン化合物によるHadの阻害形式の決定と、Hadの阻害定数の算出を行った。
2-1 Method The Had inhibitory activity test of Example 1 was conducted using 100 μM of two types of sesame lignan compounds (sesamol and SML), and the phenol production rate (μM/min) (hereinafter referred to as "v") was calculated. . Next, v is plotted on the vertical axis and 4-hydroxybenzoic acid concentration (mM) (hereinafter referred to as [S]) is plotted on the horizontal axis, and an approximate curve is fitted using SigmaPlot (manufactured by Hulinx) to determine the speed. The theoretical parameters were calculated. Then, plot 1/v on the vertical axis and 1/[S] on the horizontal axis (Lineweaver Burk plot), and from the shape of the approximate straight line, determine the form of Had inhibition by various sesame lignan compounds and calculate the Had inhibition constant. I did it.
2-2 結果
その結果、2種類のゴマリグナン化合物(セサモール及びSML)によるHadの阻害形式及び阻害定数(Ki)は、表3に示すとおりであることが明らかとなった。
2-2 Results As a result, it was revealed that the inhibition type and inhibition constant (Ki) of Had by two types of sesame lignan compounds (sesamol and SML) are as shown in Table 3.
3.本件ゴマリグナン化合物が、Hadによるフェノール生成を阻害することの確認(2)
ゴマリグナン化合物について、Hadによるフェノール生成を阻害する作用を有するかどうかを、枯草菌JCM1465株由来Had遺伝子を導入した大腸菌BL21株(本明細書において、「Had産生大腸菌BL21株」ということがある)由来Hadを用いて解析した。
3. Confirmation that the sesame lignan compound inhibits phenol production by Had (2)
Regarding sesame lignan compounds, whether or not they have the effect of inhibiting phenol production by Had was determined using the E. coli BL21 strain (herein sometimes referred to as "Had-producing E. coli BL21 strain") into which the Had gene derived from Bacillus subtilis strain JCM1465 was introduced. Analyzed using Had.
3-1 材料及び方法
[Hadの粗酵素液の調製]
Hadの粗酵素液は、以下の手順〔1〕~〔7〕に従って調製し、その後の[Had阻害活性試験]に用いた。
〔1〕Had産生大腸菌BL21株を、0.1%のグリセロール、1%の4-ヒドロキシ安息香酸、及び100μg/mLのアンピシリンを含むGAM糖分解用半流動液体培地(ニッスイ社製)50mL中で、37℃、120rpmの条件下で、OD600=0.5になるまで振盪培養した。
〔2〕氷上で急冷した後、15℃で30分間静置した。
〔3〕1mMのIPTGを添加し、15℃で24時間、アネロパック入りジャー内にて静置培養(嫌気培養)した。
〔4〕培養したHad産生大腸菌BL21株を含む培養液を、遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(液体培地)を除去後、反応用緩衝液(50mMのKPB[pH7.0]及び2mMの2-メルカプトエタノールを含む液)中に懸濁した。
〔5〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(上記反応用緩衝液)を除去後、上記反応用緩衝液中に懸濁した。
〔6〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(上記反応用緩衝液)を除去後、上記反応用緩衝液中に懸濁した。
〔7〕SONIFIER(登録商標) SFX250(EMERSON社製)を用いて超音波破砕処理を行った後、遠心処理し、上清をHadの粗酵素液として回収した。
3-1 Materials and Methods [Preparation of crude enzyme solution of Had]
A crude enzyme solution of Had was prepared according to the following steps (1) to (7) and used in the subsequent [Had inhibitory activity test].
[1] Had-producing Escherichia coli BL21 strain was cultured with shaking at 37°C and 120 rpm in 50 mL of semi-solid liquid medium for GAM glycolysis (manufactured by Nissui Co., Ltd.) containing 0.1% glycerol, 1% 4-hydroxybenzoic acid, and 100 μg/mL ampicillin until OD 600 reached 0.5.
[2] After rapid cooling on ice, the mixture was allowed to stand at 15°C for 30 minutes.
[3] 1 mM IPTG was added, and the mixture was subjected to static culture (anaerobic culture) in an Anaeropack-containing jar at 15° C. for 24 hours.
[4] The culture solution containing the cultured Had-producing E. coli BL21 strain was centrifuged (15,000 × g, 10 minutes, 4°C) to harvest the bacteria. After removing the supernatant (liquid medium), the bacteria were suspended in a reaction buffer (a solution containing 50 mM KPB [pH 7.0] and 2 mM 2-mercaptoethanol).
[5] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[6] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[7] After ultrasonic disruption using SONIFIER (registered trademark) SFX250 (manufactured by EMERSON), the cells were centrifuged and the supernatant was collected as a crude enzyme solution of Had.
[Had阻害活性試験]
Had阻害活性試験は、以下の手順〔1〕~〔2〕に従って行った。
〔1〕Hadの粗酵素液に、4-ヒドロキシ安息香酸及びセサモールをそれぞれ終濃度が5mMとなるように添加し、37℃で各種時間(30分間、1時間、3時間、又は24時間)、アネロパック入りジャー内にてインキュベートした。なお、比較対照として、4-ヒドロキシ安息香酸及びセサモールのいずれか一方を添加したHadの粗酵素液と、4-ヒドロキシ安息香酸及びセサモールの両方を添加しなかったHadの粗酵素液も同様にインキュベートした。
〔2〕遠心処理(15,000×g、10分間、4℃)し、上清を0.45μm親水性フィルターに通した後、HPLC分析を表2に示す条件に従って行い、フェノール量を定量した(図2参照)。
[Had inhibitory activity test]
The Had inhibitory activity test was conducted according to the following procedures [1] to [2].
[1] 4-hydroxybenzoic acid and sesamol were added to Had's crude enzyme solution to a final concentration of 5 mM, and incubated at 37°C for various times (30 minutes, 1 hour, 3 hours, or 24 hours). It was incubated in a jar containing an anero pack. As a comparison, Had's crude enzyme solution to which either 4-hydroxybenzoic acid or sesamol was added and Had's crude enzyme solution to which neither 4-hydroxybenzoic acid nor sesamol were added were incubated in the same way. did.
[2] After centrifugation (15,000 x g, 10 minutes, 4°C) and passing the supernatant through a 0.45 μm hydrophilic filter, HPLC analysis was performed according to the conditions shown in Table 2 to quantify the amount of phenol. (See Figure 2).
3-2 結果
Had産生大腸菌BL21株由来Hadを、その基質である4-ヒドロキシ安息香酸と、本件ゴマリグナン化合物(セサモール)の存在下で少なくとも30分間インキュベートすると、本件ゴマリグナン化合物非存在下でインキュベートした場合と比べ、生成されるフェノール量が有意に低下することが示された(図2参照)。
この結果は、本件ゴマリグナン化合物が、Hadによるフェノール生成を短期間で阻害することを示している。
3-2 Results When Had-producing E. coli strain BL21-derived Had was incubated with its substrate 4-hydroxybenzoic acid in the presence of the present sesame lignan compound (sesamol) for at least 30 minutes, when it was incubated in the absence of the present sesame lignan compound It was shown that the amount of phenol produced was significantly reduced compared to the above (see Figure 2).
This result shows that the present sesame lignan compound inhibits phenol production by Had in a short period of time.
4.本件ゴマリグナン化合物が、Had産生菌によるフェノール生成を阻害することの確認
Hadによるフェノール生成の阻害作用を有する本件ゴマリグナン化合物が、Had産生菌体中でも阻害作用を発揮するかどうかを解析した。
4. Confirmation that the present sesame lignan compound inhibits phenol production by Had-producing bacteria It was analyzed whether the present sesame lignan compound, which has an inhibitory effect on phenol production by Had, also exerts an inhibitory effect on Had-producing bacteria.
4-1 材料及び方法
[Had産生菌]
Had産生菌として、Hadを産生する4種類の腸内細菌、具体的には、シトロバクター・コセリ(Citrobacter koseri)JCM1658株由来Had産生菌、及びクライベラ・インターメディア(Kluyvera intermedia)JCM1238株由来Had産生菌(すべて、理化学研究所 バイオリソース研究センターより入手)と、エンテロバクター属細菌(Enterobacter sp.)ALC0001株由来Had産生菌、及びシゲラ属細菌(Shigella sp.)ALC0002株由来Had産生菌(いずれも研究室保有株)を、以下の[Had阻害活性試験]に使用した。
4-1 Materials and methods [Had-producing bacteria]
As Had-producing bacteria, there are four types of intestinal bacteria that produce Had, specifically, Had-producing bacteria derived from Citrobacter koseri JCM1658 strain, and Had-producing bacteria derived from Kluyvera intermedia JCM1238 strain. bacteria (all obtained from the RIKEN BioResource Research Center), Had-producing bacteria derived from Enterobacter sp. ALC0001 strain, and Had-producing bacteria derived from Shigella sp. ALC0002 strain (both were research strain) was used in the following [Had inhibitory activity test].
[Had阻害活性試験]
Had阻害活性試験は、以下の手順〔1〕~〔5〕に従って行った。
〔1〕Had産生菌を、0.1%のグリセロール及び1%の4-ヒドロキシ安息香酸を含むGAM糖分解用半流動液体培地(ニッスイ社製)中で、37℃で24時間、アネロパック入りジャー内にて静置培養(嫌気培養)した。
〔2〕培養した菌体を含む培養液を、遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(液体培地)を除去後、反応用緩衝液(50mMのKPB[pH7.0]及び2mMの2-メルカプトエタノールを含む液)中に懸濁した。
〔3〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(上記反応用緩衝液)を除去後、上記反応用緩衝液中に懸濁した。
〔4〕遠心処理(15,000×g、10分間、4℃)にて集菌し、上清(反応用緩衝液)を除去後、OD600=2.0となるように、Had産生菌を5mMの4-ヒドロキシ安息香酸及び5mMのセサモールを含む上記反応用緩衝液中に懸濁し、37℃で各種時間(30分間、1時間、3時間、又は24時間)、アネロパック入りジャー内にてインキュベートした。なお、比較対照として、Had産生菌を、4-ヒドロキシ安息香酸及びセサモールのいずれか一方を含む上記反応用緩衝液、並びに4-ヒドロキシ安息香酸及びセサモールの両方を含まない上記反応用緩衝液にそれぞれ懸濁し、同様にインキュベートした。
〔5〕遠心処理(15,000×g、10分間、4℃)し、上清を0.45μm親水性フィルターに通した後、HPLC分析を表2に示す条件に従って行い、フェノール量を定量した(図3~6参照)。
[Had inhibitory activity test]
The Had inhibitory activity test was carried out according to the following procedures [1] to [5].
[1] Had-producing bacteria were statically cultured (anaerobic culture) in a semi-solid liquid medium for GAM sugar decomposition (manufactured by Nissui Co., Ltd.) containing 0.1% glycerol and 1% 4-hydroxybenzoic acid at 37°C for 24 hours in a jar containing an Anaeropack.
[2] The culture solution containing the cultured cells was centrifuged (15,000 × g, 10 minutes, 4 ° C.) to collect the cells, and after removing the supernatant (liquid medium), the cells were suspended in a reaction buffer (a solution containing 50 mM KPB [pH 7.0] and 2 mM 2-mercaptoethanol).
[3] The cells were harvested by centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant (the above-mentioned reaction buffer) was removed, and the cells were suspended in the above-mentioned reaction buffer.
[4] The bacteria were harvested by centrifugation (15,000×g, 10 min, 4° C.), and the supernatant (reaction buffer) was removed. The Had-producing bacteria were then suspended in the reaction buffer containing 5 mM 4-hydroxybenzoic acid and 5 mM sesamol to give an OD 600 of 2.0, and incubated in a jar containing an Anaeropack for various times (30 min, 1 h, 3 h, or 24 h) at 37° C. As comparative controls, Had-producing bacteria were suspended in the reaction buffer containing either 4-hydroxybenzoic acid or sesamol, and in the reaction buffer containing neither 4-hydroxybenzoic acid nor sesamol, and incubated in the same manner.
[5] After centrifugation (15,000×g, 10 minutes, 4° C.), the supernatant was passed through a 0.45 μm hydrophilic filter, and then HPLC analysis was carried out according to the conditions shown in Table 2 to quantify the amount of phenol (see FIGS. 3 to 6).
4-2 結果
4種類のHad産生菌(シトロバクター・コセリJCM1658株由来Had産生菌、クライベラ・インターメディアJCM1238株由来Had産生菌、エンテロバクター属細菌ALC0001株由来Had産生菌、及びシゲラ属細菌ALC0002株由来Had産生菌)を、その基質である4-ヒドロキシ安息香酸と、本件ゴマリグナン化合物(セサモール)の存在下で少なくとも30分間インキュベートすると、本件ゴマリグナン化合物非存在下でインキュベートした場合と比べ、生成されるフェノール量が有意に低下することが示された(図3~6参照)。
この結果は、セサモール等の本件ゴマリグナン化合物が、Hadを産生する腸内細菌の菌体内に取り込まれ、Had産生菌が産生するHadによるフェノール生成を阻害したことを示している。
4-2 Results When four types of Had-producing bacteria (Citrobacter koseri JCM1658 strain-derived Had-producing bacteria, Kluyvera intermedia JCM1238 strain-derived Had-producing bacteria, Enterobacter sp. ALC0001 strain-derived Had-producing bacteria, and Shigella sp. ALC0002 strain-derived Had-producing bacteria) were incubated for at least 30 minutes in the presence of their substrate, 4-hydroxybenzoic acid, and the present sesame lignan compound (sesamol), the amount of phenol produced was significantly reduced compared to when incubated in the absence of the present sesame lignan compound (see Figures 3 to 6).
This result indicates that the present sesame lignan compounds such as sesamol are taken up into the cells of enterobacteria that produce Had, and inhibit the production of phenol by Had produced by the Had-producing bacteria.
本発明は、生体内のHad産生菌におけるHadによって生成されるフェノールを前駆体とする尿毒症物質(例えば、フェニル硫酸)に起因する症状又は疾患の予防・治療に資するものである。 The present invention contributes to the prevention and treatment of symptoms and diseases caused by uremic substances (eg, phenyl sulfate) whose precursor is phenol produced by Had in Had-producing bacteria in vivo.
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