JP2020158465A - mRNA MATURING INHIBITOR - Google Patents
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本発明は、天然物由来で活性が高く、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 derived from a natural product, has high activity, 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, one in three people die from cancer. In particular, elderly people tend to have cancerous 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. Due to the diversified living environment of modern society, it is important to take such functional compounds not only as foods but also as supplements. In other words, establishing a method for continuously ingesting bioactive compounds with antitumor activity from foods and supplements is extremely important for building a healthy longevity society in Japan, which has entered a super-aging society. It becomes a social issue.
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、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。また、カテコール（非特許文献２）とカフェ酸メチル（非特許文献３）には抗腫瘍活性があることが報告されている。 Conventionally, chemically synthesized products have been mainly used as antitumor agents, but these have problems such as restrictions on the concentration used and side effects, and there is a demand for naturally occurring antitumor agents with high safety and strong activity. Is increasing. So far, many searches for antitumor components have been made from foods and natural products. For example, Non-Patent Document 1 describes that chlorogenic acid in coffee has an effect of suppressing the growth of hepatocellular carcinoma. Further, it has been reported that catechol (Non-Patent Document 2) and methyl caffeate (Non-Patent Document 3) have antitumor activity.
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４参照。）。発明者らはまた、コーヒーの持つ寿命延長効果に着目し、コーヒー成分について、当該アッセイ系を用いたスクリーニングの結果、クロロゲン酸ラクトン類に強いｍＲＮＡ成熟阻害活性があることを明らかにした（特許文献１）。 On the other hand, mRNA maturation takes place in the nucleus by a multi-step reaction such as 5'-capping, splicing, 3'-end processing, etc., and the mature mRNA is carried out of the nucleus. If any of the maturation stages are not properly performed and mRNA maturation is inhibited, immature mRNA accumulates in the nucleus. Recently, inhibitors of mRNA maturation process 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 a fluorescently labeled oligo dT probe. (See Non-Patent Document 4). The inventors also focused on the life-prolonging effect of coffee, and as a result of screening the coffee components using the assay system, it was clarified that chlorogenic acid lactones have a strong mRNA maturation inhibitory activity (Patent Documents). 1).
特許文献１に記載されているクロロゲン酸ラクトン類は、高いｍＲＮＡ成熟阻害活性を有するものの、より幅広く適用可能な成分が求められている。また、カテコール、カフェ酸メチル、カフェ酸エチル、及びフェルラ酸エチルは、抗腫瘍作用は見られるものの、そのメカニズムは未解明である。 Although the chlorogenic acid lactones described in Patent Document 1 have high mRNA maturation inhibitory activity, a component that can be widely applied is required. In addition, although catechol, methyl caffeate, ethyl caffeate, and ethyl ferurate have antitumor effects, their mechanisms have not been elucidated.
本発明は、天然物由来であり、比較的安全性が高いことが期待でき、かつ充分な抗腫瘍活性を有する成分を提供することを目的とする。 An object of the present invention is to provide a component which is derived from a natural product, can be expected to have relatively high safety, and has sufficient 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、焙煎コーヒー豆の可溶性成分に対するスクリーニングを実施したところ、１−(５−Ｈｙｄｒｏｘｙ ｐｙｒｉｄｉｎ−２−ｙｌ)ｅｔｈａｎｏｎｅ（アセチルピリジノール）、カテコール、及びカフェ酸メチルが強いｍＲＮＡ成熟阻害活性を有することを新たに見出した。更に、カテコール類縁体であるレソルシノールと、カフェ酸メチル関連物のカフェ酸エチル及びフェルラ酸エチルにｍＲＮＡ成熟阻害活性があることも見出し、本発明を完成させた。 As a result of diligent research to solve the above problems, the present inventors have conducted screening for soluble components of roasted coffee beans using a newly developed assay system for screening substances having mRNA maturation inhibitory activity. , 1- (5-Hydroxypyridin-2-yl) assay (acetylpyridinol), catechol, and methyl caffeate have been newly found to have strong mRNA maturation inhibitory activity. Furthermore, they have also found that resorcinol, which is a catechol analog, and ethyl caffeate and ethyl ferulate, which are related to methyl caffeate, have mRNA maturation inhibitory activity, and completed the present invention.
［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）〜（６）のいずれかで表される化合物を有効成分とすることを特徴とする。 [1] The mRNA maturation inhibitor according to the first aspect of the present invention is characterized by containing a compound represented by any of the following general formulas (1) to (6) as an active ingredient.
［２］ 前記［１］のｍＲＮＡ成熟阻害剤としては、前記化合物が、焙煎コーヒー豆に由来する化合物であることが好ましい。
［３］ 前記［１］又は［２］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。
［４］ 本発明の第二の態様に係るｍＲＮＡ成熟阻害剤の製造方法は、コーヒーの可溶性成分をメタノール抽出し、得られたメタノール抽出物から、液体クロマトグラフィーにより下記式（１）、（２）、又は（４）で表される化合物を含む画分を分取する。
［５］ 前記［４］のｍＲＮＡ成熟阻害剤の製造方法としては、前記コーヒーの可溶性成分が、焙煎コーヒー豆の可溶性成分であることが好ましい。
［６］ 本発明の第三の態様に係る医薬品は、前記［１］又は［２］のｍＲＮＡ成熟阻害剤を含有することを特徴とする。
[2] As the mRNA maturation inhibitor of the above [1], it is preferable that the compound is a compound derived from roasted coffee beans.
[3] The mRNA maturation inhibitor according to [1] or [2] is preferably used for treating tumors or preventing recurrence.
[4] In the method for producing an mRNA maturation inhibitor according to the second aspect of the present invention, the soluble component of coffee is extracted with methanol, and the obtained methanol extract is subjected to the following formulas (1) and (2) by liquid chromatography. ) Or the fraction containing the compound represented by (4).
[5] As a method for producing the mRNA maturation inhibitor of the above [4], it is preferable that the soluble component of the coffee is a soluble component of roasted coffee beans.
[6] The pharmaceutical product according to the third aspect of the present invention is characterized by containing the mRNA maturation inhibitor of the above [1] or [2].
アセチルピリジノール、カテコール、及びカフェ酸メチルは、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含まれている成分であり、比較的安全に投与可能である。また、レソルシノール、カフェ酸エチル、及びフェルラ酸エチルは、植物に存在している、ｍＲＮＡ成熟阻害活性を備える化合物である。このため、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。 Acetylpyridinol, catechol, and methyl caffeate are components naturally contained in coffee in addition to having high mRNA maturation inhibitory activity, and can be administered relatively safely. In addition, resorcinol, ethyl caffeate, and ethyl ferurate are compounds present in plants and having mRNA maturation inhibitory activity. Therefore, the mRNA maturation inhibitor according to the present invention is suitable as an active ingredient of supplements, pharmaceuticals, etc., and in particular, a pharmaceutical for treating or preventing recurrence of a disease caused by cell hyperproliferation such as a tumor. It is suitable as an active ingredient of.
本発明に係るｍＲＮＡ成熟阻害剤は、アセチルピリジノール（下記式（１）で表される化合物）、カテコール（下記式（２）で表される化合物）、レソルシノール（下記式（３）で表される化合物）、カフェ酸メチル（下記式（４）で表される化合物）、カフェ酸エチル（下記式（５）で表される化合物）、及びフェルラ酸エチル（下記式（６）で表される化合物）のいずれかを有効成分とする。本発明に係るｍＲＮＡ成熟阻害剤は、これらの６種の化合物のうち、１種類のみを有効成分としてもよく、２種類以上を有効成分としてもよい。これらの６種の化合物は、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 is represented by acetylpyridinol (compound represented by the following formula (1)), catechol (compound represented by the following formula (2)), and resorcinol (represented by the following formula (3)). Compound), methyl caffeate (compound represented by the following formula (4)), ethyl caffeate (compound represented by the following formula (5)), and ethyl ferurate (represented by the following formula (6)). Compound) is used as an active ingredient. The mRNA maturation inhibitor according to the present invention may contain only one of these six compounds as an active ingredient, or two or more of them as an active ingredient. After being taken up into cells, these six compounds inhibit any stage of the mRNA maturation process and inhibit protein synthesis. As a result of this inhibition of mRNA maturation, cell proliferation is suppressed. Therefore, the mRNA maturation inhibitor according to the present invention is suitable as an active ingredient of a drug for treating or preventing a disease caused by cell hyperproliferation, particularly a drug for treating a tumor or preventing recurrence.
本発明に係るｍＲＮＡ成熟阻害剤は、有効成分である前記６種の化合物のうちの１種又は２種以上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、前記６種の化合物による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 consist of only one or more of the above six compounds as active ingredients, or may contain other components. .. The other components may be any as long as they do not impair the mRNA maturation inhibitory action of the above 6 compounds, for example, excipients, binders, fluidity improvers (anti-caking agents), stabilizers, and storage. Various substances used as agents, pH adjusters, solubilizing agents, suspending agents, emulsifiers, thickeners, flavoring agents, sweeteners, acidulants, flavors, coloring agents, etc., depending on the desired product quality. May be appropriately contained.
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, nasal drops and the like. Since it is easy to take, the dosage form of the mRNA maturation inhibitor according to the present invention is preferably one suitable for oral administration such as tablets, capsules, granules, powders and syrups.
本発明に係るｍＲＮＡ成熟阻害剤の有効成分のうち、アセチルピリジノール、カテコール、及びカフェ酸メチルは、コーヒーに由来する成分、より詳細には、焙煎コーヒー豆に比較的多く含まれている可溶性成分（焙煎コーヒー豆の熱水抽出物に含まれる成分）であり、比較的安全に服用できる。また、レソルシノール、カフェ酸エチル、及びフェルラ酸エチルは、植物に存在している物質である。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。 Among the active ingredients of the mRNA maturation inhibitor according to the present invention, acetylpyridinol, catechol, and methyl caffeate are relatively abundant in coffee-derived ingredients, more specifically, roasted coffee beans. It is a soluble component (a component contained in the hot water extract of roasted coffee beans) and can be taken relatively safely. In addition, resorcinol, ethyl caffeate, and ethyl ferurate are substances present in plants. Therefore, these are suitable as raw materials for foods and drinks, feeds, cosmetics, pharmaceuticals, etc., and are particularly useful as active ingredients for pharmaceuticals and supplements used for treating tumors or preventing recurrence. For example, continuous ingestion of a supplement containing the mRNA maturation inhibitor according to the present invention suppresses cell proliferation and risks the onset and recurrence of various diseases (for example, tumors) caused by cell hyperproliferation. 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 protein expression in cells.
本発明に係るｍＲＮＡ成熟阻害剤の有効成分とする前記６種の化合物は、いずれも、天然物から抽出され粗精製されたものであってもよく、天然物から単一成分にまで精製されたものであってもよく、化学合成されたものであってもよい。アセチルピリジノール、カテコール、及びカフェ酸メチルは、例えば、コーヒーの可溶性成分をメタノール抽出し、得られたメタノール抽出物から、液体クロマトグラフィーによってこれらの化合物を含む画分（フラクション）を分取することによって精製して製造することができる。液体クロマトグラフィーは、ＨＰＬＣ等の常法により行うことができる。原料とするコーヒーの可溶性成分としては、焙煎コーヒー豆の可溶性成分であることが好ましく、インスタントコーヒー粉末の原料となるような焙煎コーヒー豆の熱水抽出物がより好ましい。 All of the above six compounds as active ingredients of the mRNA maturation inhibitor according to the present invention may be those extracted from natural products and crudely purified, or purified from natural products to single components. It may be a product or a chemically synthesized product. For acetylpyridinol, catechol, and methyl caffeate, for example, soluble components of coffee are extracted with methanol, and fractions containing these compounds are fractionated from the obtained methanol extract by liquid chromatography. It can be purified and produced. Liquid chromatography can be performed by a conventional method such as HPLC. The soluble component of coffee as a raw material is preferably a soluble component of roasted coffee beans, and a hot water extract of roasted coffee beans that can be used as a raw material for instant coffee powder is more preferable.
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。 Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples and the like.
＜ｍＲＮＡ阻害活性の測定＞
以降の実験において、各化合物のｍＲＮＡ成熟阻害活性は、非特許文献４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。
<Measurement of mRNA inhibitory activity>
In the subsequent experiments, the mRNA maturation inhibitory activity of each compound was carried out according to the method using RNA-FISH (Fluorescence in Situ Fluorescence) described in Non-Patent Document 4. For RNA-FISH, a cultured cell line U2OS cell established from human osteosarcoma cells was used. U2OS cells were cultured in DMEM (Dulvecco'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、活性を測定する対象の化合物のＤＭＳＯ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。また、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、ＧＥＸ１Ａ溶液（ＧＥＸ１Ａを３０ｎｇ／ｍＬとなるようにＤＭＳＯに溶解させた溶液）を各ウェルに添加して、同様に２４時間培養した。 Specifically, first, cells were inoculated on a cover glass of a 12-well plate so as to have a concentration of 5 × 10 4 / mL, and cultured for 24 hours. Then, to each well, a DMSO solution of the compound whose activity was to be measured 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 the wells to which an equal amount of DMSO was added as a negative control were similarly cultured for 24 hours. In addition, as a positive control, GEX1A having an inhibitory activity against mRNA splicing was used, a GEX1A solution (a solution in which GEX1A was dissolved in DMSO to 30 ng / mL) was added to each well, and the cells 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 in PBS (phosphate saline) containing 10% formaldehyde for 20 minutes, and then permeated (permeation) in PBS containing 0.1% Triton X-100 for 10 minutes. Gender-imparting processing) was performed. Subsequently, the cells were washed 3 times by incubating the cells with PBS for 10 minutes each time, 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）で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 are prehybridized in a humidification chamber at 42 ° C. for 1 hour with an oligo hybridization buffer (Ambion), and then an oligo hybridization buffer (Ambion) containing a 20 pM Cy3-labeled oligo dT 45 probe. Hybridized at 42 ° C. for 24 hours. Then, after washing with 2 × SCC buffer at 42 ° C. for 20 minutes, it was washed once with 0.5 × SCC buffer and once with 0.1 × SCC buffer.
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。 Furthermore, the nuclei of the washed cells are stained with DAPI (4', 6-diamidino-2-phenylindole) to visualize them, and then the Cy3 fluorescence intensity in the nuclei and the entire cells is measured by image analysis software ImageJ (imagej.nih.gov). Measurement was performed using / ij /), and the localization ratio of poly (A) + RNA per cell in the nucleus ([Cy3 fluorescence amount in the nucleus] / [Cy3 fluorescence amount in the whole cell]) was determined.
［実施例１］
焙煎コーヒー豆の可溶性成分から、ｍＲＮＡ成熟阻害活性を有する化合物を単離し、同定した。焙煎コーヒー豆の可溶性成分としては、インスタントコーヒー（商品名：〈〈ブレンディ〉インスタントコーヒー〉、味の素ＡＧＦ社製）を用い、中圧カラムクロマトグラフィーを繰り返すことによる分画精製を行い、ｍＲＮＡ成熟阻害活性のある画分からｍＲＮＡ成熟阻害活性を備える化合物を同定した。中圧カラムクロマトグラフィーには、シリカゲル（Wakogel C-200、粒子サイズ７５〜１５０μｍ）を充填したカラムを用いた。インスタントコーヒーの分画精製のフローと各フラクションの回収量を図１に示す。最終的に６段階の精製で、３種類の単一の活性化合物を単離した。
[Example 1]
Compounds having mRNA maturation inhibitory activity were isolated and identified from the soluble components of roasted coffee beans. Instant coffee (trade name: <<Blendy> instant coffee, manufactured by Ajinomoto AGF Co., Ltd.) is used as a soluble component of roasted coffee beans, and fractional purification is performed by repeating medium-pressure column chromatography to inhibit mRNA maturation. Compounds with mRNA maturation inhibitory activity were identified from the active fraction. For medium pressure column chromatography, a column packed with silica gel (Wakogel C-200, particle size 75 to 150 μm) was used. The flow of fractional purification of instant coffee and the amount of each fraction recovered are shown in FIG. Finally, 6-step purification isolated 3 single active compounds.
具体的には、まず、インスタントコーヒー５００ｇをメタノール２．５Ｌと混合した後、濾過して固形分を除去することにより、インスタントコーヒーメタノール抽出物を得た。このインスタントコーヒーメタノール抽出物にｍＲＮＡ成熟阻害活性が確認されたため、酢酸エチルと水を混合した後、酢酸エチル層を回収した。この酢酸エチル層を蒸発乾固したところ、固形分が３５．６ｇであった。この酢酸エチル層固形分に対して、中圧カラムクロマトグラフィーにより分画し、各フラクションについてｍＲＮＡ成熟阻害活性を測定し、活性が確認されたフラクションを回収するという精製工程を６回繰り返した。最初の４回の精製工程における中圧カラムクロマトグラフィーはクロロホルムとメタノールのグラジエントを移動相とし、５回目はヘキサンと酢酸エチルのグラジエントを移動相とし、６回目は０．１質量％の酢酸水溶液とメタノールのグラジエントを移動相とした。 Specifically, first, 500 g of instant coffee was mixed with 2.5 L of methanol and then filtered to remove solids to obtain an instant coffee methanol extract. Since mRNA maturation inhibitory activity was confirmed in this instant coffee methanol extract, the ethyl acetate layer was recovered after mixing ethyl acetate and water. When this ethyl acetate layer was evaporated to dryness, the solid content was 35.6 g. The solid content of the ethyl acetate layer was fractionated by medium pressure column chromatography, the mRNA maturation inhibitory activity was measured for each fraction, and the purification step of recovering the fraction whose activity was confirmed was repeated 6 times. Medium-pressure column chromatography in the first four purification steps used a gradient of chloroform and methanol as the mobile phase, a fifth with a gradient of hexane and ethyl acetate as the mobile phase, and a sixth with a 0.1 mass% aqueous acetic acid solution. The gradient of methanol was used as the mobile phase.
１回目の精製工程では、Ｆｒ．２（固形分１８．３１ｇ）に活性が観察された。そこで、このＦｒ.２に対して２回目の分画を行ったところ、得られたＦｒ．２−ａ〜Ｆｒ．２−ｄのうち、Ｆｒ.２−ａ（固形分０．２２６５ｇ）、Ｆｒ.２−ｃ３（固形分２．４４ｇ）、及びＦｒ.２−ｃ４（固形分１．４１ｇ）に顕著なｍＲＮＡ成熟阻害活性が見られた。Ｆｒ.２−ａの回収量が極めて少なかったことから、Ｆｒ．２−ｃ３とＦｒ．２−ｃ４を混合したものを、３回目の精製に供した。３回目の精製において、Ｆｒ.Ａ−１ａ〜Ｆｒ.Ａ−６のうち、Ｆｒ.Ａ−１ａ（固形分４．８ｍｇ）とＦｒ.Ａ−３（固形分２５２０．９ｍｇ）に顕著なｍＲＮＡ成熟阻害活性が見られた。Ｆｒ.Ａ−３では活性に濃度依存性が見られ、かつ回収量も多かったことから、Ｆｒ.Ａ−３を４回目の精製に供した。４回目の精製において、Ｆｒ．Ａ３−ａ〜Ｆｒ.Ａ３−ｄのうち、Ｆｒ．Ａ３−ｂ４（固形分１９９．２ｍｇ）、Ｆｒ．Ａ３−ｂ５（固形分６７６．０ｍｇ）、Ｆｒ．Ａ３−ｃ（固形分６８１．６ｍｇ）に顕著なｍＲＮＡ成熟阻害活性が見られた。Ｆｒ．Ａ３−ｂ５は低濃度で最もｍＲＮＡ成熟阻害活性が見られたフラクションであったため、Ｆｒ．Ａ３−ｂ５を５回目の精製に供した。 In the first purification step, Fr. Activity was observed in 2 (solid content 18.31 g). Therefore, when the second fractionation was performed on this Fr.2, the obtained Fr. 2-a to Fr. Among 2-d, remarkable mRNA maturation in Fr.2-a (solid content 0.2265 g), Fr.2-c3 (solid content 2.44 g), and Fr.2-c4 (solid content 1.41 g). Inhibitory activity was observed. Since the amount of Fr.2-a recovered was extremely small, Fr.2-a. 2-c3 and Fr. The mixture of 2-c4 was subjected to the third purification. In the third purification, among Fr.A-1a to Fr.A-6, Fr.A-1a (solid content 4.8 mg) and Fr.A-3 (solid content 2520.9 mg) had remarkable mRNA maturation. Inhibitory activity was observed. Since the activity of Fr.A-3 was concentration-dependent and the amount recovered was large, Fr.A-3 was subjected to the fourth purification. In the fourth purification, Fr. Of A3-a to Fr. A3-d, Fr. A3-b4 (solid content 199.2 mg), Fr. A3-b5 (solid content 676.0 mg), Fr. Significant mRNA maturation inhibitory activity was observed in A3-c (solid content 681.6 mg). Fr. Since A3-b5 was the fraction in which the mRNA maturation inhibitory activity was most observed at a low concentration, Fr. A3-b5 was subjected to the fifth purification.
５回目の精製において、Ｆｒ．Ａ３−ｂ５−ａ〜Ｆｒ．Ａ３−ｂ５−ｆのうち、Ｆｒ．Ａ３−ｂ５−ｃ２（固形分１２．６ｍｇ）、Ｆｒ．Ａ３−ｂ５−ｄ１（固形分７７．１ｍｇ）、Ｆｒ．Ａ３−ｂ５−ｄ２（固形分５．２ｍｇ）、Ｆｒ．Ａ３−ｂ５−ｄ３（固形分２２．７ｍｇ）、Ｆｒ．Ａ３−ｂ５−ｅ（固形分１９３．９ｍｇ）に顕著なｍＲＮＡ成熟阻害活性が見られた。ただし、Ｆｒ．Ａ３−ｂ５−ｃ２〜Ｆｒ．Ａ３−ｂ５−ｄ３では、これまでと同じ濃度（１５０μｇ/ｍＬ）で添加すると、細胞が死んでしまったため観察ができなかった。そこで、これらに関しては、低濃度（２５μｇ/ｍＬ）でｍＲＮＡ成熟阻害活性を測定した。また、ＵＶ２５４ｎｍの吸収ピークから、Ｆｒ．Ａ３−ｂ５−ｃ２とＦｒ．Ａ３−ｂ５−ｄ１を合わせたピークは１つであった。しかし、Ｆｒ．Ａ３−ｂ５−ｄ１の方が、５μｇ／ｍＬ又は１０μｇ／ｍＬ添加時のｍＲＮＡ成熟阻害活性が強かったために 、より純度が高いと予想されたため、Ｆｒ．Ａ３−ｂ５−ｄ１を、１Ｈ−ＮＭＲと１３Ｃ−ＮＭＲとＬＣ−ＭＳによって解析した。標準品のＮＭＲの結果との比較により、Ｆｒ．Ａ３−ｂ５−ｄ１に含まれているｍＲＮＡ成熟阻害活性を備える物質は、カテコール（式（２））と同定された。 In the fifth purification, Fr. A3-b5-a to Fr. Of A3-b5-f, Fr. A3-b5-c2 (solid content 12.6 mg), Fr. A3-b5-d1 (solid content 77.1 mg), Fr. A3-b5-d2 (solid content 5.2 mg), Fr. A3-b5-d3 (solid content 22.7 mg), Fr. A remarkable mRNA maturation inhibitory activity was observed in A3-b5-e (solid content 193.9 mg). However, Fr. A3-b5-c2-Fr. In A3-b5-d3, when it was added at the same concentration (150 μg / mL) as before, the cells died and could not be observed. Therefore, regarding these, mRNA maturation inhibitory activity was measured at a low concentration (25 μg / mL). Further, from the absorption peak of UV 254 nm, Fr. A3-b5-c2 and Fr. The combined peak of A3-b5-d1 was one. However, Fr. Since A3-b5-d1 was expected to have higher purity because it had stronger mRNA maturation inhibitory activity when 5 μg / mL or 10 μg / mL was added, Fr. A3-b5-d1 was analyzed by 1 1 H-NMR, 13 C-NMR and LC-MS. By comparison with the NMR results of the standard product, Fr. The substance having mRNA maturation inhibitory activity contained in A3-b5-d1 was identified as catechol (formula (2)).
一方、Ｆｒ．Ａ３−ｂ５−ｄ３は、ＵＶ２５４ｎｍの吸収ピークから、まだ複数の化合物が混在していると考えられたので、６回目の精製に供して更に精製を続けることとした。６回目の精製において、Ｆｒ．Ａ３−ｂ５−ｄ３−１〜Ｆｒ．Ａ３−ｂ５−ｄ３−６のうち、Ｆｒ．Ａ３−ｂ５−ｄ３−２（固形分０．９ｍｇ）とＦｒ．Ａ３−ｂ５−ｄ３−４（固形分０．８ｍｇ）に顕著なｍＲＮＡ成熟阻害活性が見られた。Ｆｒ．Ａ３−ｂ５−ｄ３−４の方が、ｍＲＮＡ成熟阻害活性性が顕著であったものの、ＵＶ２５４ｎｍの小さいピークが多数存在しており多くの化合物が混在していたこと、回収量も０．８ｍｇしかなかったことから、これ以上精製を進めも化合物の特定は困難であると考えた。一方で、Ｆｒ．Ａ３−ｂ５−ｄ３−２は、低濃度（２０μｇ／ｍＬ）ではｍＲＮＡ成熟阻害活性はなかったものの、高濃度ではｍＲＮＡの核内蓄積を確認することができた。そこで、Ｆｒ．Ａ３−ｂ５−ｄ３−２とＦｒ．Ａ３−ｂ５−ｄ３−４を１Ｈ−ＮＭＲとＬＣ−ＭＳを用いて解析した。その結果、Ｆｒ．Ａ３−ｂ５−ｄ３−４に含まれているｍＲＮＡ成熟阻害活性を備える物質は、カフェ酸メチル（式（４））であると同定された。また、Ｆｒ．Ａ３−ｂ５−ｄ３−４のＮＭＲスペクトルが、カフェ酸メチルの標品のスペクトルと一致していることも確認した。また、Ｆｒ．Ａ３−ｂ５−ｄ３−２に含まれているｍＲＮＡ成熟阻害活性を備える物質は、１−(５−Ｈｙｄｒｏｘｙ ｐｙｒｉｄｉｎ−２−ｙｌ)ｅｔｈａｎｏｎｅ（アセチルピリジノール：式（１））と同定された。 On the other hand, Fr. Since it was considered that a plurality of compounds were still mixed in A3-b5-d3 from the absorption peak at UV254 nm, it was decided to continue the purification for the sixth purification. In the sixth purification, Fr. A3-b5-d3-1 to Fr. Of A3-b5-d3-6, Fr. A3-b5-d3-2 (solid content 0.9 mg) and Fr. Remarkable mRNA maturation inhibitory activity was observed in A3-b5-d3-4 (solid content 0.8 mg). Fr. Although A3-b5-d3-4 had more remarkable mRNA maturation inhibitory activity, many small peaks at UV254 nm were present and many compounds were mixed, and the amount recovered was only 0.8 mg. Since there was no such compound, it was considered difficult to identify the compound even if further purification was carried out. On the other hand, Fr. Although A3-b5-d3-2 did not have mRNA maturation inhibitory activity at a low concentration (20 μg / mL), nuclear accumulation of mRNA could be confirmed at a high concentration. Therefore, Fr. A3-b5-d3-2 and Fr. A3-b5-d3-4 was analyzed using 1 1 H-NMR and LC-MS. As a result, Fr. The substance having mRNA maturation inhibitory activity contained in A3-b5-d3-4 was identified as methyl caffeate (formula (4)). In addition, Fr. It was also confirmed that the NMR spectrum of A3-b5-d3-4 was consistent with the spectrum of the standard of methyl caffeate. In addition, Fr. The substance having mRNA maturation inhibitory activity contained in A3-b5-d3-2 was identified as 1- (5-Hydroxypyridin-2-yl) ethanone (acetylpyridinol: formula (1)).
ついで、同定されたアセチルピリジノール、カテコール、及びカフェ酸メチルの標品について、ｍＲＮＡ成熟阻害活性を測定した。アセチルピリジノール処理（１００μＭ、３００μＭ、６００μＭ）後のポリ（Ａ）＋ＲＮＡの核内の局在比を調べた結果（ｎ＝２０）を図２に、カテコール処理（５０μＭ、１００μＭ、２５０μＭ）後のポリ（Ａ）＋ＲＮＡの核内の局在比を調べた結果（ｎ＝２０）を図３に、カフェ酸メチル処理（１０μＭ、２５μＭ、５０μＭ）後のポリ（Ａ）＋ＲＮＡの核内の局在比を調べた結果（ｎ＝２０）を図４に、それぞれ示す。 The mRNA maturation inhibitory activity of the identified acetylpyridinol, catechol, and methyl caffeate preparations was then measured. The results (n = 20) of examining the localization ratio of poly (A) + RNA in the nucleus after acetylpyridinol treatment (100 μM, 300 μM, 600 μM) are shown in FIG. 2 and catechol treatment (50 μM, 100 μM, 250 μM). The results of examining the localization ratio of poly (A) + RNA in the nucleus afterwards (n = 20) are shown in FIG. 3, which shows the nucleus of poly (A) + RNA after methyl caffeate treatment (10 μM, 25 μM, 50 μM). The results (n = 20) of examining the localization ratio in the inside are shown in FIG. 4, respectively.
ＡＮＯＶＡ及びＤｕｎｎｅｔｔ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、アセチルピリジノール、カテコール、及びカフェ酸メチル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。つまり、アセチルピリジノール、カテコール、及びカフェ酸メチルは、ｍＲＮＡ成熟阻害活性を有していた。特に、カフェ酸メチル１０μｇ／ｍＬ（５０μＭ）添加時のｍＲＮＡ成熟阻害活性は、Ｆｒ．Ａ３−ｂ５−ｄ３−４を２０μｇ／ｍＬ添加時よりも顕著であった。 When ANOVA and Dunnett's test were performed, the localization ratio of poly (A) + RNA in the nucleus was higher than that of cells supplemented with DMSO, which is a negative control, and acetylpyridinol, catechol, and methyl caffeate were added. A significant difference was observed in the cells, and the amount of poly (A) + RNA in the nucleus was significantly increased. That is, acetylpyridinol, catechol, and methyl caffeate had mRNA maturation inhibitory activity. In particular, the mRNA maturation inhibitory activity when methyl caffeate 10 μg / mL (50 μM) was added was determined by Fr. It was more remarkable than when A3-b5-d3-4 was added at 20 μg / mL.
［実施例２］
カテコール関連物質及びカフェ酸関連物質について、実施例１と同様にしてｍＲＮＡ成熟阻害活性を測定した。被験化合物は、レソルシノール（５０μＭ、１００μＭ、２５０μＭ）、カフェ酸エチル（２００μＭ）、フェルラ酸メチル（２００μＭ）、フェルラ酸エチル（２００μＭ）、桂皮酸メチル（２００μＭ）、桂皮酸エチル（２００μＭ）、及びクマル酸メチル（２００μＭ）とした。
[Example 2]
The mRNA maturation inhibitory activity of the catechol-related substance and the caffeic acid-related substance was measured in the same manner as in Example 1. The test compounds were resorcinol (50 μM, 100 μM, 250 μM), ethyl caffeate (200 μM), methyl ferurate (200 μM), ethyl ferurate (200 μM), methyl cinnamate (200 μM), ethyl cinnamate (200 μM), and cumal. It was methyl acid (200 μM).
レソルシノール処理後のポリ（Ａ）＋ＲＮＡの核内の局在比を調べた結果（ｎ＝２０）を図５に、カフェ酸関連物質処理後のポリ（Ａ）＋ＲＮＡの核内の局在比を調べた結果（ｎ＝２０）を図６に、それぞれ示す。２５０μＭ（２５μｇ／ｍＬ）のレソルシノール処理した細胞と、２００μＭのカフェ酸エチル処理した細胞と、２００μＭのフェルラ酸エチル処理した細胞に、有意なｍＲＮＡ成熟阻害活性が認められた。 The results of examining the nuclear localization ratio of poly (A) + RNA after resorcinol treatment (n = 20) are shown in FIG. 5, and the localization of poly (A) + RNA after caffeic acid-related substance treatment in the nucleus is shown in FIG. The results of examining the ratio (n = 20) are shown in FIG. 6, respectively. Significant mRNA maturation inhibitory activity was observed in 250 μM (25 μg / mL) resorcinol-treated cells, 200 μM ethyl caffeate-treated cells, and 200 μM ethyl ferurate-treated cells.
Claims (6)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019061856A JP7197793B2 (en) | 2019-03-27 | 2019-03-27 | mRNA maturation inhibitor |
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| JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 62, JPN6022034486, 2014, pages 5054 - 5060, ISSN: 0004853667 * |
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