JP2017132762A - MRNA maturation inhibitor - Google Patents
MRNA maturation inhibitor Download PDFInfo
- Publication number
- JP2017132762A JP2017132762A JP2017011067A JP2017011067A JP2017132762A JP 2017132762 A JP2017132762 A JP 2017132762A JP 2017011067 A JP2017011067 A JP 2017011067A JP 2017011067 A JP2017011067 A JP 2017011067A JP 2017132762 A JP2017132762 A JP 2017132762A
- Authority
- JP
- Japan
- Prior art keywords
- group
- chlorogenic acid
- maturation inhibitor
- mrna
- hydrogen atom
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Abstract
Description
本発明は、安全で活性が高く、抗腫瘍剤の有効成分としても好適なｍＲＮＡ成熟阻害剤に関する。 The present invention relates to an mRNA maturation inhibitor that is safe and highly active, and is also suitable as an active ingredient of an antitumor agent.
日本では、がんによる死亡が３人に１人となっている。特に高齢者は、染色体中にこれまでに蓄積された変異を多く含むために、細胞ががん化しやすい傾向がある。がんは、細胞の増殖異常によるものであるため、食品から細胞増殖を抑制する機能性化合物を積極的に摂取することによって、効果的にがんの発症を抑制できると期待できる。現代社会の多様化した生活環境から、このような機能性化合物は、食品だけでなくサプリメント等として摂取する視点も重要となっている。すなわち、抗腫瘍作用を有する生理活性化合物を、食品やサプリメント等から継続的に摂取する方法論を確立することは、超高齢化社会を迎えた日本において健康な長寿社会を築いていく上で極めて重要な社会課題となる。 In Japan, cancer deaths account for 1 in 3 people. In particular, elderly people tend to cancer cells because they contain many mutations accumulated so far in their chromosomes. Since cancer is caused by abnormal cell growth, it can be expected that the onset of cancer can be effectively suppressed by actively ingesting a functional compound that suppresses cell growth from food. From the diversified living environment of modern society, the viewpoint of taking such functional compounds as supplements as well as foods is important. In other words, it is extremely important to establish a methodology for continually ingesting physiologically active compounds with antitumor activity from foods and supplements in order to build a healthy and long-lived society in Japan, which has reached a super-aging society. Social issues.
従来、抗腫瘍剤としては、化学的合成品が主体であるが、これらは使用濃度の制限や副作用等の課題があり、天然に存在する安全性が高くかつ活性の強い抗腫瘍剤への要望が高まっている。今までに、食品や天然物から抗腫瘍成分の探索が数多くなされている。例えば特許文献１には、茶由来のエピガロカテキンガレートを有効成分とする抗腫瘍剤が開示されている。また、特許文献２には、シノブヒバ中のジテルペンに抗腫瘍活性があることが記載されている。特許文献３には、リンゴ由来のプロアントシアニジンの抗腫瘍活性が提案されている。特許文献４には、広く野菜や果物に存在しているフラボノイドの一種モリンを有効成分とする抗腫瘍剤であって、口腔ガンを抑制する抗腫瘍剤が開示されている。特許文献５には、桂皮酸誘導体を含有するヒアルロニダーゼ阻害剤が記載されている。特許文献６には、アンニンコウから無極性溶媒で抽出される成分を含有する抗腫瘍剤が提案されている。さらに、非特許文献１には、コーヒーのクロロゲン酸に肝細胞がん増殖抑制効果があることが記載されている。 Conventionally, as anti-tumor agents, chemical synthetic products have mainly been used, but these have problems such as restrictions on the concentration used and side effects, and there is a demand for naturally occurring highly safe and highly active anti-tumor agents. Is growing. To date, many searches for antitumor components from foods and natural products have been made. For example, Patent Document 1 discloses an antitumor agent containing epigallocatechin gallate derived from tea as an active ingredient. Patent Document 2 describes that diterpenes in Shinobuhiba have antitumor activity. Patent Document 3 proposes an antitumor activity of proanthocyanidins derived from apples. Patent Document 4 discloses an antitumor agent containing morin, which is a kind of flavonoid widely present in vegetables and fruits, as an active ingredient and suppressing oral cancer. Patent Document 5 describes a hyaluronidase inhibitor containing a cinnamic acid derivative. Patent Document 6 proposes an antitumor agent containing a component extracted from anninko with a nonpolar solvent. Furthermore, Non-Patent Document 1 describes that chlorogenic acid of coffee has a hepatocellular carcinoma growth inhibitory effect.
一方で、ｍＲＮＡの成熟は、核内において、５’−キャッピング、スプライシング、３’ −エンドプロセッシング等の多段階の反応によりなされ、成熟ｍＲＮＡは、核外に運ばれる。成熟段階のいずれかが適切に行われず、ｍＲＮＡの成熟が阻害された場合には、核内に未成熟のｍＲＮＡが蓄積される。最近、ｍＲＮＡ成熟過程の阻害因子が、抗がん剤としての機能を期待されている。本発明者らは、抗腫瘍活性を持つ化合物の新たなスクリーニング系として、蛍光ラベルされたオリゴｄＴプローブを用いたＲＮＡ−ＦＩＳＨを利用し、ｍＲＮＡ成熟阻害活性を有する物質をスクリーニングするアッセイ系を開発した（非特許文献２参照。）。 On the other hand, mRNA maturation is carried out in the nucleus by a multi-step reaction such as 5'-capping, splicing, 3'-end processing, and the mature mRNA is carried out of the nucleus. If any of the maturation stages is not performed properly and mRNA maturation is inhibited, immature mRNA accumulates in the nucleus. Recently, inhibitors of mRNA maturation processes are expected to function as anticancer agents. As a new screening system for compounds having antitumor activity, the present inventors have developed an assay system for screening substances having mRNA maturation inhibitory activity using RNA-FISH using fluorescently labeled oligo dT probes. (See Non-Patent Document 2).
特許文献１〜６に記載の抗腫瘍剤は、天然物由来であり、比較的安全性が高いものの、抗腫瘍効果が不充分であったりする。このため、飲食品に本来含まれていて安全であることに加えて、より充分な抗腫瘍活性を有する化合物が求められている。そこで、本発明は、安全で活性が高く、より高い抗腫瘍活性を有する成分を提供することを目的とする。 Although the antitumor agents described in Patent Documents 1 to 6 are derived from natural products and have relatively high safety, the antitumor effect may be insufficient. For this reason, in addition to being originally contained in food and drink and being safe, there is a need for a compound having more sufficient antitumor activity. Therefore, an object of the present invention is to provide a safe and highly active ingredient having higher antitumor activity.
本発明者らは、上記課題を解決すべく鋭意研究した結果、新たに開発したｍＲＮＡ成熟阻害活性を有する物質をスクリーニングするアッセイ系を用いて各種食品素材のスクリーニングを実施したところ、焙煎コーヒー豆に含まれているクロロゲン酸ラクトンが、高いｍＲＮＡ成熟阻害作用を有していることを見出し、本発明を完成させた。 As a result of earnest research to solve the above problems, the present inventors conducted screening of various food materials using a newly developed assay system for screening a substance having an activity of inhibiting maturation of mRNA. Was found to have a high mRNA maturation inhibitory effect, and the present invention was completed.
［１］ 本発明の第一の態様に係るｍＲＮＡ成熟阻害剤は、下記一般式（１）〜（３） [1] The mRNA maturation inhibitor according to the first aspect of the present invention includes the following general formulas (1) to (3):
［式（１）〜（３）中、Ｒ１、Ｒ２、及びＲ３はそれぞれ独立して、水素原子、カフェオイル基、フェルロイル基、又はクマロイル基を表す。ただし、Ｒ１〜Ｒ３が全て水素原子である場合を除く。］
のいずれかで表されるクロロゲン酸ラクトンを有効成分とすることを特徴とする。
［２］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つが水素原子であり、残る１つがカフェオイル基、フェルロイル基、又はクマロイル基であることが好ましい。
［３］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つがそれぞれ独立して、カフェオイル基、フェルロイル基、又はクマロイル基であり、残る１つが水素原子であることが好ましい。
［４］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つが水素原子であり、残る１つがカフェオイル基、フェルロイル基、又はクマロイル基であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［５］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つがそれぞれ独立して、カフェオイル基、フェルロイル基、又はクマロイル基であり、残る１つが水素原子であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［６］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ３が水素原子であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［７］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ３が水素原子であり、前記Ｒ１及びＲ２がそれぞれ独立して、カフェオイル基、フェルロイル基、又はクマロイル基であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［８］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ３が水素原子であり、前記Ｒ１及びＲ２がそれぞれ独立して、水素原子又はカフェオイル基であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［９］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記一般式（１）で表され、前記Ｒ３が水素原子であり、前記Ｒ１及びＲ２がカフェオイル基であるクロロゲン酸ラクトンを有効成分とすることが好ましい。
［１０］ 前記［１］〜［９］のいずれかのｍＲＮＡ成熟阻害剤としては、腫瘍の治療又は再発予防に用いられることが好ましい。
［１１］ 本発明の第二の態様に係る医薬品は、前記［１］〜［１０］のいずれかのｍＲＮＡ成熟阻害剤を含有することを特徴とする。
[In the formulas (1) to (3), R 1 , R 2 , and R 3 each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where all of R 1 to R 3 are hydrogen atoms is excluded. ]
The chlorogenic acid lactone represented by any of the above is used as an active ingredient.
[2] As the mRNA maturation inhibitor of [1], any two of R 1 , R 2 , and R 3 are hydrogen atoms, and the remaining one is a caffeoyl group, a feruloyl group, or a coumaroyl group. It is preferable that
[3] As the mRNA maturation inhibitor of [1], any two of R 1 , R 2 , and R 3 are each independently a caffeoyl group, a feruloyl group, or a coumaroyl group, The remaining one is preferably a hydrogen atom.
[4] The mRNA maturation inhibitor of [1] is represented by the general formula (1), and any two of R 1 , R 2 , and R 3 are hydrogen atoms, and the remaining one is A chlorogenic acid lactone which is a caffeoyl group, a feruloyl group or a coumaroyl group is preferably used as an active ingredient.
[5] The mRNA maturation inhibitor of [1] is represented by the general formula (1), and any two of R 1 , R 2 , and R 3 are each independently a caffeoyl group. Chlorogenic acid lactone, which is a feruloyl group or coumaroyl group and the remaining one is a hydrogen atom, is preferably used as an active ingredient.
[6] The mRNA maturation inhibitor of [1] preferably contains a chlorogenic acid lactone represented by the general formula (1), wherein R 3 is a hydrogen atom, as an active ingredient.
[7] The mRNA maturation inhibitor of the above [1] is represented by the general formula (1), the R 3 is a hydrogen atom, the R 1 and R 2 are each independently a caffeoyl group, It is preferable to use a chlorogenic acid lactone which is a feruloyl group or a coumaroyl group as an active ingredient.
[8] The mRNA maturation inhibitor of [1] is represented by the general formula (1), wherein R 3 is a hydrogen atom, and R 1 and R 2 are each independently a hydrogen atom or a café. It is preferable to use chlorogenic lactone which is an oil group as an active ingredient.
[9] The mRNA maturation inhibitor of [1] is a chlorogenic acid lactone represented by the general formula (1), wherein R 3 is a hydrogen atom, and R 1 and R 2 are caffeoyl groups. The active ingredient is preferred.
[10] The mRNA maturation inhibitor of any one of [1] to [9] is preferably used for tumor treatment or recurrence prevention.
[11] The pharmaceutical product according to the second aspect of the present invention is characterized by containing the mRNA maturation inhibitor of any one of [1] to [10].
クロロゲン酸ラクトンは、高いｍＲＮＡ成熟阻害活性を備えることに加えて、天然にはコーヒーに比較的多く含まれている成分であり、比較的安全に投与可能である。このため、クロロゲン酸ラクトンを有効成分とする本発明に係るｍＲＮＡ成熟阻害剤は、サプリメントや医薬品等の有効成分として好適であり、特に、腫瘍等のように細胞の過増殖が原因となる疾患に対する治療又は再発防止のための医薬品の有効成分として好適である。 In addition to having a high mRNA maturation inhibitory activity, chlorogenic acid lactone is a component that is naturally contained in coffee in a relatively large amount and can be administered relatively safely. For this reason, the mRNA maturation inhibitor according to the present invention containing chlorogenic acid lactone as an active ingredient is suitable as an active ingredient for supplements, pharmaceuticals, and the like, and particularly for diseases caused by cell overgrowth such as tumors. It is suitable as an active ingredient of pharmaceuticals for treatment or prevention of recurrence.
本発明に係るｍＲＮＡ成熟阻害剤は、クロロゲン酸ラクトンを有効成分とする。クロロゲン酸ラクトンは、細胞内に取り込まれた後、ｍＲＮＡの成熟工程のいずれかの段階を阻害し、タンパク質合成を阻害する。このｍＲＮＡ成熟阻害の結果、細胞増殖が抑制される。このため、本発明に係るｍＲＮＡ成熟阻害剤は、細胞の過増殖により引き起こされる疾患の治療や予防のための医薬品、特に、腫瘍の治療や再発防止のための医薬品の有効成分として好適である。 The mRNA maturation inhibitor according to the present invention contains chlorogenic acid lactone as an active ingredient. Chlorogenic acid lactone, after being taken up into the cell, inhibits any stage of the maturation process of mRNA and inhibits protein synthesis. As a result of this mRNA maturation inhibition, cell proliferation is suppressed. For this reason, the mRNA maturation inhibitor according to the present invention is suitable as an active ingredient of a drug for the treatment or prevention of a disease caused by cell overgrowth, particularly a drug for the treatment of tumor or prevention of recurrence.
本発明に係るｍＲＮＡ成熟阻害剤は、具体的には、下記一般式（１）〜（３）のいずれかで表されるクロロゲン酸ラクトンを有効成分とすることを特徴とする。下記一般式（１）〜（３）中、Ｒ１、Ｒ２、及びＲ３はそれぞれ独立して、水素原子、カフェオイル基、フェルロイル基、又はクマロイル基を表す。ただし、Ｒ１〜Ｒ３が全て水素原子である場合を除く。 Specifically, the mRNA maturation inhibitor according to the present invention comprises chlorogenic acid lactone represented by any one of the following general formulas (1) to (3) as an active ingredient. In the following general formulas (1) to (3), R 1 , R 2 , and R 3 each independently represent a hydrogen atom, a caffeoyl group, a feruloyl group, or a coumaroyl group. However, the case where all of R 1 to R 3 are hydrogen atoms is excluded.
カフェオイル基を下記式（ａ）に、フェルロイル基を下記式（ｂ）に、クマロイル基を下記式（ｃ）に、それぞれ示す。式（ａ）〜（ｃ）中、「＊」が付された結合手は、一般式（１）〜（３）中のシクロヘキサン環に結合した酸素原子との結合手を示す。 The caffeoyl group is represented by the following formula (a), the feruloyl group is represented by the following formula (b), and the coumaroyl group is represented by the following formula (c). In formulas (a) to (c), a bond marked with “*” represents a bond with an oxygen atom bonded to the cyclohexane ring in general formulas (1) to (3).
一般式（１）〜（３）で表されるクロロゲン酸ラクトンとしては、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つが水素原子であり、残る１つがカフェオイル基、フェルロイル基、又はクマロイル基である化合物であってもよく、前記Ｒ１、Ｒ２、及びＲ３のうち、いずれか２つがそれぞれ独立して、カフェオイル基、フェルロイル基、又はクマロイル基であり、残る１つが水素原子であってもよく、前記Ｒ１、Ｒ２、及びＲ３の全てがそれぞれ独立して、カフェオイル基、フェルロイル基、又はクマロイル基であってもよい。 As chlorogenic acid lactones represented by the general formulas (1) to (3), any two of the R 1 , R 2 and R 3 are hydrogen atoms, and the remaining one is a caffeoyl group and a feruloyl group. Or a compound that is a coumaroyl group, and any two of R 1 , R 2 , and R 3 are each independently a caffeoyl group, a feruloyl group, or a coumaroyl group, and the remaining 1 One of them may be a hydrogen atom, and all of R 1 , R 2 , and R 3 may be each independently a caffeoyl group, a feruloyl group, or a coumaroyl group.
Ｒ１、Ｒ２、及びＲ３のうち、いずれか１つが水素原子である場合、残る２つの基は、同種の基であってもよく、異種の基であってもよい。同様に、Ｒ１、Ｒ２、及びＲ３の全てがカフェオイル基、フェルロイル基、又はクマロイル基の場合、これら３つ基は全て同種の基であってもよく、２種以上の基が組み合わせられていてもよい。 When any one of R 1 , R 2 , and R 3 is a hydrogen atom, the remaining two groups may be the same group or different groups. Similarly, when all of R 1 , R 2 , and R 3 are a caffeoyl group, a feruloyl group, or a coumaroyl group, these three groups may all be the same group, or two or more groups may be combined. It may be done.
一般式（１）〜（３）で表されるクロロゲン酸ラクトンのＲ１、Ｒ２、及びＲ３の基の組み合わせを表１に示す。表１中、「Ｈ」は水素原子、「（ａ）」はカフェオイル基、「（ｂ）」はフェルロイル基、「（ｃ）」はクマロイル基を示す。 Table 1 shows combinations of R 1 , R 2 , and R 3 groups of the chlorogenic acid lactones represented by the general formulas (1) to (3). In Table 1, “H” represents a hydrogen atom, “(a)” represents a caffeoyl group, “(b)” represents a feruloyl group, and “(c)” represents a coumaroyl group.
本発明に係るｍＲＮＡ成熟阻害剤の有効成分としては、一般式（１）で表されるクロロゲン酸ラクトンが好ましく、一般式（１）で表され、かつＲ１、Ｒ２、及びＲ３のうち、いずれか２つがそれぞれ独立して、カフェオイル基、フェルロイル基、若しくはクマロイル基であり、残る１つが水素原子であるクロロゲン酸ラクトン、又は一般式（１）で表され、かつＲ１、Ｒ２、及びＲ３のうち、いずれか２つがそれぞれ独立して、カフェオイル基、フェルロイル基、若しくはクマロイル基であり、残る１つが水素原子であるクロロゲン酸ラクトンがより好ましく、一般式（１）で表され、かつＲ３が水素原子であるクロロゲン酸ラクトン（表１の組み合わせ１〜６、１０〜１２のいずれかの一般式（１）で表されるクロロゲン酸ラクトン）がさらに好ましく、一般式（１）で表され、かつＲ３が水素原子であり、Ｒ１及びＲ２がそれぞれ独立して水素原子又はカフェオイル基であるクロロゲン酸ラクトン（表１の組み合わせ１、４、又は１０のいずれかの一般式（１）で表されるクロロゲン酸ラクトン）がよりさらに好ましく、一般式（１）で表され、Ｒ３が水素原子であり、Ｒ１及びＲ２がカフェオイル基であるクロロゲン酸ラクトン（表１の組み合わせ１０の一般式（１）で表されるクロロゲン酸ラクトン）が特に好ましい。 The active ingredient of the mRNA maturation inhibitor according to the present invention is preferably a chlorogenic acid lactone represented by the general formula (1), represented by the general formula (1), and among R 1 , R 2 , and R 3 Any two of them are independently a caffeoyl group, a feruloyl group or a coumaroyl group, and the remaining one is a hydrogen atom, or a chlorogenic acid lactone, or the general formula (1), and R 1 , R 2 And R 3 are more preferably chlorogenic acid lactones, each independently being a caffeoyl group, a feruloyl group, or a coumaroyl group, and the remaining one being a hydrogen atom, represented by the general formula (1) is, and chlorogenic acid lactate represented by the chlorogenic acid lactones R 3 is a hydrogen atom (any of formulas combinations of Table 1 1～6,10～12 (1) Down) is more preferably represented by the general formula (1), and R 3 is a hydrogen atom, a combination of chlorogenic acid lactones (Table 1 R 1 and R 2 are each independently a hydrogen atom or a caffeoyl group 1, 4 or 10 chlorogenic acid lactone represented by general formula (1) is more preferred, represented by general formula (1), R 3 is a hydrogen atom, R 1 and R 2 Is particularly preferably a chlorogenic acid lactone (a chlorogenic acid lactone represented by the general formula (1) of the combination 10 in Table 1), which is a caffeoyl group.
一般式（１）〜（３）で表されるクロロゲン酸ラクトンは、例えば、焙煎コーヒー豆から精製することができる。具体的には、焙煎コーヒー豆の熱水抽出物又は熱水抽出物残渣から、ＯＤＳカラム（Ｃ１８カラム）等の逆相系カラムを用いた高速液体カラムクロマトグラフィー（ＨＰＬＣ）法により単離精製することができる。 The chlorogenic acid lactone represented by the general formulas (1) to (3) can be purified from roasted coffee beans, for example. Specifically, it is isolated and purified from a hot water extract or hot water extract residue of roasted coffee beans by a high performance liquid column chromatography (HPLC) method using a reverse phase system column such as an ODS column (C18 column). can do.
一般式（１）〜（３）で表されるクロロゲン酸ラクトンは、例えば、キナ酸ラクトン（一般式（１）〜（３）であって、Ｒ１、Ｒ２、及びＲ３の全てが水素原子である化合物）の水酸基と、カフェ酸、フェルラ酸、又はｐ−クマル酸のカルボキシル基とを、脱水縮合させることによって合成することができる。脱水縮合反応は、常法により行うことができる。 The chlorogenic acid lactone represented by the general formulas (1) to (3) is, for example, a quinic acid lactone (general formulas (1) to (3), in which all of R 1 , R 2 , and R 3 are hydrogen The compound can be synthesized by dehydration condensation of the hydroxyl group of the compound (atom) and the carboxyl group of caffeic acid, ferulic acid, or p-coumaric acid. The dehydration condensation reaction can be performed by a conventional method.
また、一般式（１）で表されるクロロゲン酸ラクトンは、例えば、１−カフェオイルキナ酸（CAS ID：1241-87-8）、クロロゲン酸（３−カフェオイルキナ酸）（CAS ID：327-97-9）、シクロヘキサンカルボン酸（CAS ID：327161-03-5）、クリプトクロロゲン酸（CAS ID：905-99-7）、ネオクロロゲン酸（CAS ID：906-33-2）、１，３−ジカフェオイルキナ酸（CAS ID：19870-46-3）、１，３−ジカフェオイルキナ酸（CAS ID：14534-61-3）、４，５−ジカフェオイルキナ酸（CAS ID：57378-72-0）、１，３−ジカフェオイルキナ酸（CAS ID：30964-13-7）、３，５−ジカフェオイルキナ酸（CAS ID：2450-53-5）、３−フェルロイルキナ酸（CAS ID：1899-29-2）、４−フェルロイルキナ酸（CAS ID：2613-86-7）、５−フェルロイルキナ酸、４，５−ジフェルロイルキナ酸、３，５−ジフェルロイルキナ酸、３，４−ジフェルロイルキナ酸、３，５−ジフェルロイルキナ酸、３−ｐ−クマロイルキナ酸（CAS ID：1899-30-5）、４−ｐ−クマロイルキナ酸（CAS ID：93451-44-6）、５−ｐ−クマロイルキナ酸（CAS ID：32451-86-8）、１−ｐ−クマロイルキナ酸、３−カフェオイル−４−フェルロイルキナ酸、３−カフェオイル−５−フェルロイルキナ酸、４−カフェオイル−５−フェルロイルキナ酸、１−カフェオイル−５−フェルロイルキナ酸、３−フェルロイル−４−カフェオイルキナ酸、３−フェルロイル−５−カフェオイルキナ酸、４−フェルロイル−５−カフェオイルキナ酸等のクロロゲン酸を加熱処理し、シクロヘキサン環に結合したカルボキシル基と水酸基の脱水反応により合成することができる。クロロゲン酸の加熱処理によるラクトン化は、常法により行うことができる。 The chlorogenic acid lactone represented by the general formula (1) is, for example, 1-caffeoylquinic acid (CAS ID: 1241-87-8), chlorogenic acid (3-caffeoylquinic acid) (CAS ID: 327). -97-9), cyclohexanecarboxylic acid (CAS ID: 327161-03-5), cryptochlorogenic acid (CAS ID: 905-99-7), neochlorogenic acid (CAS ID: 906-33-2), 1, 3-dicaffeoylquinic acid (CAS ID: 19870-46-3), 1,3-dicaffeoylquinic acid (CAS ID: 14534-61-3), 4,5-dicaffeoylquinic acid (CAS ID) : 57378-72-0), 1,3-dicaffeoylquinic acid (CAS ID: 30964-13-7), 3,5-dicaffeoylquinic acid (CAS ID: 2450-53-5), 3- Ferroyl quinic acid (CAS ID: 1899-29-2), 4-feruloyl quinic acid (CAS ID: 2613-86-7), 5-feruloyl quinic acid, 4,5-diferroyl quinic acid, 3 , 5-Diferloylquinic acid, , 4-Diferloylquinic acid, 3,5-Diferloylquinic acid, 3-p-coumaroylquinic acid (CAS ID: 1899-30-5), 4-p-coumaroylquinic acid (CAS ID: 93451-44- 6), 5-p-coumaroylquinic acid (CAS ID: 32451-86-8), 1-p-coumaroylquinic acid, 3-caffeoyl-4-feruloylquinic acid, 3-caffeoyl-5-feruloylquinic acid 4-caffeoyl-5-feruloylquinic acid, 1-caffeoyl-5-feruloylquinic acid, 3-feruloyl-4-caffeoylquinic acid, 3-feruloyl-5-caffeoylquinic acid, 4-feruloyl It can be synthesized by subjecting chlorogenic acid such as -5-caffeoylquinic acid to heat treatment and dehydration reaction of a carboxyl group and a hydroxyl group bonded to a cyclohexane ring. Lactonization by heat treatment of chlorogenic acid can be performed by a conventional method.
本発明に係るｍＲＮＡ成熟阻害剤は、一般式（１）〜（３）のいずれかで表されるクロロゲン酸ラクトンを、１種類のみ含有していてもよく、２種類以上を組み合わせて含有していてもよい。例えば、本発明に係るｍＲＮＡ成熟阻害剤は、有効成分として、表１の組み合わせ１０の一般式（１）で表されるクロロゲン酸ラクトンのみを含有するものであってもよく、表１の組み合わせ１０の一般式（１）で表されるクロロゲン酸ラクトンと表１の組み合わせ１の一般式（１）で表されるクロロゲン酸ラクトンと表１の組み合わせ４の一般式（１）で表されるクロロゲン酸ラクトンを含有するものであってもよい。 The mRNA maturation inhibitor according to the present invention may contain only one type of chlorogenic acid lactone represented by any one of the general formulas (1) to (3), or a combination of two or more types. May be. For example, the mRNA maturation inhibitor according to the present invention may contain only the chlorogenic acid lactone represented by the general formula (1) of the combination 10 in Table 1 as an active ingredient. The chlorogenic acid lactone represented by the general formula (1) and the chlorogenic acid lactone represented by the general formula (1) in the combination 1 in Table 1 and the chlorogenic acid represented by the general formula (1) in the combination 4 in Table 1 It may contain a lactone.
本発明に係るｍＲＮＡ成熟阻害剤は、有効成分である一般式（１）〜（３）のいずれかで表されるクロロゲン酸ラクトンのみからなるものであってもよく、他の成分を含有するものであってもよい。当該他の成分としては、一般式（１）〜（３）のいずれかで表されるクロロゲン酸ラクトンによるｍＲＮＡ成熟阻害作用を損なわないものであればよく、例えば、賦形剤、結合剤、流動性改良剤（固結防止剤）、安定剤、保存剤、ｐＨ調整剤、溶解補助剤、懸濁化剤、乳化剤、粘稠剤、矯味剤、甘味料、酸味料、香料、着色料等として用いられている各種物質を、所望の製品品質に応じて適宜含有させてもよい。 The mRNA maturation inhibitor according to the present invention may be composed only of chlorogenic acid lactone represented by any one of the general formulas (1) to (3) which are active ingredients, and contains other ingredients. It may be. The other component may be any component as long as it does not impair the maturation inhibition effect of chlorogenic acid lactone represented by any one of the general formulas (1) to (3). As a property improver (anti-caking agent), stabilizer, preservative, pH adjuster, solubilizer, suspending agent, emulsifier, thickener, corrigent, sweetener, acidulant, flavor, colorant, etc. Various substances used may be appropriately contained according to desired product quality.
本発明に係るｍＲＮＡ成熟阻害剤の剤型は、特に限定されるものではなく、各種の剤型を適用できる。当該剤型としては、例えば、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、スプレー剤、注射剤、坐剤、点眼剤、点鼻剤等が挙げられる。服用が容易であることから、本発明に係るｍＲＮＡ成熟阻害剤の剤型としては、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤等の経口投与に適したものが好ましい。 The dosage form of the mRNA maturation inhibitor according to the present invention is not particularly limited, and various dosage forms can be applied. Examples of the dosage form include tablets, capsules, granules, powders, syrups, sprays, injections, suppositories, eye drops, and nasal drops. Since the dosage is easy, the dosage form of the mRNA maturation inhibitor according to the present invention is preferably a tablet, capsule, granule, powder, syrup or the like suitable for oral administration.
本発明に係るｍＲＮＡ成熟阻害剤の有効成分であるクロロゲン酸ラクトンは、焙煎コーヒー豆に比較的多く含まれている物質であり、比較的安全に服用できる。そこで、これらは、飲食品、飼料、化粧料、医薬品等の原料として好適であり、特に腫瘍の治療又は再発予防に用いられる医薬品やサプリメントの有効成分として有用である。例えば、本発明に係るｍＲＮＡ成熟阻害剤を配合させたサプリメントを継続的に摂取することにより、細胞増殖が抑制され、細胞の過増殖によって引き起こされる各種疾患（例えば、腫瘍など）の発症や再発リスクを低減できることが期待できる。 Chlorogenic acid lactone, which is an active ingredient of the mRNA maturation inhibitor according to the present invention, is a substance that is contained in a relatively large amount in roasted coffee beans and can be taken relatively safely. Therefore, these are suitable as raw materials for foods and drinks, feeds, cosmetics, pharmaceuticals and the like, and are particularly useful as active ingredients for pharmaceuticals and supplements used for tumor treatment or recurrence prevention. For example, by continuously ingesting a supplement containing the mRNA maturation inhibitor according to the present invention, cell proliferation is suppressed, and the onset and recurrence risk of various diseases (for example, tumors) caused by cell overgrowth Can be expected to be reduced.
その他、本発明に係るｍＲＮＡ成熟阻害剤は、細胞のタンパク質発現のメカニズム解明のためのツールとしても好適である。 In addition, the mRNA maturation inhibitor according to the present invention is also suitable as a tool for elucidating the mechanism of cellular protein expression.
次に、実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例等に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example etc.
［実施例１］
クロロゲン酸、前記一般式（１）で表され、Ｒ２及びＲ３が水素原子であり、Ｒ１がカフェオイル基であるクロロゲン酸ラクトン（３−ＣＱＬ：表１の組み合せ１）、前記一般式（１）で表され、Ｒ３が水素原子であり、Ｒ１及びＲ２がカフェオイル基であるジカフェオイルキナ酸ラクトン（３，４−ｄｉＣＱＬ：表１の組み合せ１０）、カフェ酸、及びキナ酸について、ｍＲＮＡ成熟阻害活性を調べた。ポジティブコントロールとして、ｍＲＮＡスプライシングに対する阻害活性を有するＧＥＸ１Ａを用いた。
[Example 1]
Chlorogenic acid, a chlorogenic acid lactone (3-CQL: combination 1 in Table 1) represented by the general formula (1), wherein R 2 and R 3 are hydrogen atoms, and R 1 is a caffeoyl group, the general formula Dicaffeoylquinic acid lactone (3,4-diCQL: combination 10 of Table 1) represented by (1), wherein R 3 is a hydrogen atom, and R 1 and R 2 are caffeoyl groups, caffeic acid, and For quinic acid, mRNA maturation inhibitory activity was examined. As a positive control, GEX1A having an inhibitory activity against mRNA splicing was used.
ｍＲＮＡ成熟阻害活性は、非特許文献２に記載されているＲＮＡ−ＦＩＳＨ（Ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ Ｓｉｔｕ Ｈｙｂｒｉｄｉｚａｔｉｏｎ）を利用した方法に準じて行った。ＲＮＡ−ＦＩＳＨには、ヒト骨肉腫細胞から樹立された培養細胞株Ｕ２ＯＳ細胞を用いた。Ｕ２ＯＳ細胞は、１０％ＦＢＳ（ウシ胎児血清）含有ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ）中で、３７℃、５％二酸化炭素環境下で培養した。 The mRNA maturation inhibitory activity was performed according to a method using RNA-FISH (Fluorescence in Situ Hybridization) described in Non-Patent Document 2. For RNA-FISH, cultured cell line U2OS cells established from human osteosarcoma cells were used. U2OS cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) at 37 ° C. in a 5% carbon dioxide environment.
具体的には、まず、５×１０４／ｍＬとなるように細胞を１２ウェルプレートのカバーグラス上に接種し、２４時間培養した。次いで、各ウェルに、ＧＥＸ１Ａ溶液（ＧＥＸ１Ａを３０ｎｇ／ｍＬとなるようにＤＭＳＯに溶解させた溶液）、クロロゲン酸溶液（クロロゲン酸を１５０μＭとなるようにＤＭＳＯに溶解させた溶液）、カフェ酸溶液（カフェ酸を１５０μＭとなるようにＤＭＳＯに溶解させた溶液）、３−ＣＱＬ溶液（３−ＣＱＬを１００μＭとなるようにＤＭＳＯに溶解させた溶液）、又は３，４−ｄｉＣＱＬ溶液（３，４−ｄｉＣＱＬを１００μＭとなるようにＤＭＳＯに溶解させた溶液）を添加し、さらに２４時間培養した。各ウェルに添加されるＤＭＳＯ量は等しくなるように調整し、ネガティブコントロールとして等量のＤＭＳＯを添加したウェルも、同様に２４時間培養した。 Specifically, first, cells were inoculated on a cover glass of a 12-well plate at 5 × 10 4 / mL and cultured for 24 hours. Next, in each well, a GEX1A solution (a solution in which GEX1A was dissolved in DMSO so as to be 30 ng / mL), a chlorogenic acid solution (a solution in which chlorogenic acid was dissolved in DMSO so as to be 150 μM), a caffeic acid solution ( A solution in which caffeic acid is dissolved in DMSO so as to be 150 μM), a 3-CQL solution (a solution in which 3-CQL is dissolved in DMSO so as to be 100 μM), or a 3,4-diCQL solution (3,4- A solution in which diCQL was dissolved in DMSO to a concentration of 100 μM) was added, and the cells were further cultured for 24 hours. The amount of DMSO added to each well was adjusted to be equal, and wells to which an equal amount of DMSO was added as a negative control were similarly cultured for 24 hours.
培養後、カバーグラス上の培養細胞を１０％ホルムアルデヒドを含むＰＢＳ（リン酸生理食塩水）にて２０分間固定処理した後、０．１％ ＴｒｉｔｏｎＸ−１００を含むＰＢＳにて１０分間透過処理（透過性付与処理）を行った。続いて、当該細胞を、ＰＢＳにて１回当たり１０分間インキュベートする洗浄処理を３回行い、次いで２×ＳＣＣバッファー（２×クエン酸ナトリウム標準）にて５分間洗浄処理した。 After culturing, the cultured cells on the cover glass were fixed with PBS containing 10% formaldehyde (phosphate physiological saline) for 20 minutes, and then permeabilized with PBS containing 0.1% Triton X-100 (permeation). Sex imparting treatment). Subsequently, the cells were washed 3 times with PBS for 10 minutes, and then washed with 2 × SCC buffer (2 × sodium citrate standard) for 5 minutes.
洗浄後の細胞に対して、ＲＮＡ−ＦＩＳＨを行った。すなわち、加湿チャンバー内にて、細胞をoligo hybridization buffer (Ambion社製)により４２℃、１時間予備ハイブリダイズした後、２０ｐＭのＣｙ３ラベルされたオリゴｄＴ４５プローブを含むoligo hybridization buffer (Ambion社製)により４２℃、２４時間ハイブリダイズした。その後、２×ＳＣＣバッファーにて４２℃、２０分間洗浄した後、０．５×ＳＣＣバッファーにて１回、０．１×ＳＣＣバッファーにて１回、順次洗浄した。 RNA-FISH was performed on the washed cells. That is, cells were prehybridized with oligo hybridization buffer (Ambion) at 42 ° C. for 1 hour in a humidified chamber, and then oligo hybridization buffer containing 20 pM Cy3-labeled oligo dT 45 probe (Ambion) For 24 hours at 42 ° C. Thereafter, the plate was washed with 2 × SCC buffer at 42 ° C. for 20 minutes, and then washed with 0.5 × SCC buffer once and 0.1 × SCC buffer once.
さらに、洗浄後の細胞の核を、ＤＡＰＩ（4',6-diamidino-2-phenylindole）染色して可視化した後、核内と細胞全体のＣｙ３蛍光強度を画像解析ソフトウェアImageＪ（imagej.nih.gov/ij/）を用いて測定し、細胞当たりのポリ（Ａ）＋ＲＮＡの核内の局在比（［核内のＣｙ３蛍光量］／［細胞全体のＣｙ３蛍光量］）を求めた。結果を図１〜５に示す。 Further, the nucleus of the washed cell is visualized by staining with DAPI (4 ′, 6-diamidino-2-phenylindole), and then the Cy3 fluorescence intensity in the nucleus and the whole cell is image analysis software ImageJ (imagej.nih.gov / ij /) to determine the localization ratio of poly (A) + RNA per cell ([Cy3 fluorescence in the nucleus] / [Cy3 fluorescence in the whole cell]) per cell. The results are shown in FIGS.
t検定を行ったところ、ポリ（Ａ）＋ＲＮＡの核内の局在比は、ネガティブコントロールであるＤＭＳＯを添加した細胞に比べて、クロロゲン酸、カフェ酸、３−ＣＱＬ、及び３，４−ｄｉＣＱＬを添加した細胞では有意差が見られ、核内のポリ（Ａ）＋ＲＮＡ量が有意に多くなっていた。なお、３，４−ｄｉＣＱＬを５０μＭ又は２００μＭとなるようにＤＭＳＯに溶解させた溶液を添加した細胞におけるｍＲＮＡの核内蓄積量は、３，４−ｄｉＣＱＬを１００μＭとなるようにＤＭＳＯに溶解させた溶液を添加した細胞（図５）と同程度であった（図示せず。）。つまり、クロロゲン酸、カフェ酸、３−ＣＱＬ、及び３，４−ｄｉＣＱＬは、ｍＲＮＡ成熟阻害活性を有していた。一方で、キナ酸を添加した細胞では、ポリ（Ａ）＋ＲＮＡの核内の局在比はＤＭＳＯを添加した細胞と同程度であり、キナ酸にはｍＲＮＡ成熟阻害活性がなかった。 When the t-test was performed, the localization ratio of poly (A) + RNA in the nucleus was higher than that of cells to which DMSO as a negative control was added. Chlorogenic acid, caffeic acid, 3-CQL, and 3,4- A significant difference was observed in cells to which diCQL was added, and the amount of poly (A) + RNA in the nucleus was significantly increased. It should be noted that the amount of mRNA accumulated in cells to which a solution in which 3,4-diCQL was dissolved in DMSO to 50 μM or 200 μM was added was dissolved in DMSO so that 3,4-diCQL was 100 μM. It was the same level as the cells to which the solution was added (FIG. 5) (not shown). That is, chlorogenic acid, caffeic acid, 3-CQL, and 3,4-diCQL had mRNA maturation inhibitory activity. On the other hand, in cells to which quinic acid was added, the localization ratio of poly (A) + RNA in the nucleus was comparable to that in cells to which DMSO was added, and quinic acid had no mRNA maturation inhibitory activity.
クロロゲン酸はカフェ酸のカルボキシル基がキナ酸の３位のヒドロキシ基と脱水縮合した構造をもつ化合物であり、３−ＣＱＬはクロロゲン酸のキナ酸残基の一部に環状構造ができた形をした化合物であり、３，４−ｄｉＣＱＬは３−ＣＱＬにカフェオイル基が導入された化合物である。このため、これらの３種の化合物に共通しているカフェ酸の構造が、ｍＲＮＡ成熟阻害活性に関係しているものと考えられた。クロロゲン酸ラクトンに強いｍＲＮＡ成熟阻害活性があったのに対して、クロロゲン酸やカフェ酸では当該活性が低かったことから、より高いｍＲＮＡ成熟阻害活性を達成するためには、ラクトン構造が重要であることが分かった。クロロゲン酸ラクトンが、クロロゲン酸よりもｍＲＮＡ成熟阻害活性が高かったのは、ラクトン構造により、培地中から細胞の核内へより効率よく移行したためと推察された。 Chlorogenic acid is a compound having a structure in which the carboxyl group of caffeic acid is dehydrated and condensed with the hydroxy group at the 3-position of quinic acid, and 3-CQL has a form in which a cyclic structure is formed in part of the quinic acid residue of chlorogenic acid. 3,4-diCQL is a compound in which a caffeoyl group is introduced into 3-CQL. For this reason, it was considered that the structure of caffeic acid common to these three compounds is related to the mRNA maturation inhibitory activity. Chlorogenic acid lactone had strong mRNA maturation inhibitory activity, whereas chlorogenic acid and caffeic acid had low activity, so lactone structure is important to achieve higher mRNA maturation inhibitory activity. I understood that. The reason why chlorogenic acid lactone had higher mRNA maturation inhibitory activity than chlorogenic acid was presumed to be due to more efficient transfer from the medium into the cell nucleus due to the lactone structure.
Claims (11)
のいずれかで表されるクロロゲン酸ラクトンを有効成分とすることを特徴とする、ｍＲＮＡ成熟阻害剤。 The following general formulas (1) to (3)
A maturation inhibitor for mRNA, comprising a chlorogenic acid lactone represented by any of the above as an active ingredient.
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