JP2019178070A - Mitol production promoter - Google Patents
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Abstract
Description
本発明は、MITOL減少抑制又はMITOL産生促進剤に関する。 The present invention relates to a MITOL reduction inhibitor or a MITOL production promoter.
ミトコンドリアは細胞内に存在する細胞内小器官の一つであり、主な機能としてエネルギーであるATPを産生することが挙げられる。従って、ミトコンドリア機能を維持することは、細胞ひいては体の機能を維持することに繋がる。ミトコンドリアの機能は、ミトコンドリアダイナミクスとも呼ばれるミトコンドリアの融合および***や、他の細胞内小器官との相互作用などにより制御されていることがわかってきている。ミトコンドリア外膜に局在するユビキチンリガーゼであるMITOLは、ミトコンドリアダイナミクスやミトコンドリアと小胞体との接着制御に関与していることが報告されている（非特許文献１）。心臓特異的にMITOLを欠損させたマウスは、心機能の低下が認められ、さらに、心臓老化を示す所見であるリポフスチンの沈着とSA-β-galの発現亢進が観察されている（非特許文献２）。また、皮膚表皮特異的にMITOLを欠損させたマウスは、白髪・脱毛といった皮膚老化様所見が（非特許文献３）、神経特異的にMITOLを欠損させたアルツハイマー病モデルマウスではアルツハイマー病の病態悪化が観察されている（非特許文献２）。これらのことから、MITOL低下が心臓老化、皮膚老化、脳の老化など広く老化症状の促進に関与していると考えられる。また、MITOLをノックダウンしたHELA細胞では老化マーカーであるSA-β-galの発現上昇がみられ、MITOLノックインによりレスキューされることが報告されている（非特許文献４）。このことから、MITOL低下により老化が促進され、低下したMITOLを元に戻すことで老化が回復することが示唆される。以上のことから、MITOL発現を上昇させるまたは活性化させるようなアプローチは抗老化に有用であると考えられる。しかしながら、MITOL発現を上昇させる化合物は見出されていない。 Mitochondria are one of the organelles in the cell, and the main function is to produce ATP, which is energy. Therefore, maintaining the mitochondrial function leads to maintaining the function of cells and the body. It has been found that mitochondrial functions are controlled by mitochondrial fusion and division, also called mitochondrial dynamics, and interactions with other organelles. It has been reported that MITOL, a ubiquitin ligase localized in the mitochondrial outer membrane, is involved in mitochondrial dynamics and regulation of adhesion between mitochondria and the endoplasmic reticulum (Non-patent Document 1). In mice specifically deficient in MITOL, decreased cardiac function was observed, and in addition, lipofuscin deposition and SA-β-gal expression, which are signs of cardiac aging, were observed (Non-patent Document) 2). In addition, mice with deficient MITOL specifically for skin epidermis have skin aging-like findings such as white hair and hair loss (Non-patent Document 3), but Alzheimer's disease model mice with deficient MITOL specifically for nerves worsen the pathology of Alzheimer's disease. Has been observed (Non-Patent Document 2). From these facts, it is considered that MITO decrease is widely involved in promoting aging symptoms such as heart aging, skin aging, and brain aging. In addition, the expression of SA-β-gal, which is an aging marker, is increased in HELA cells in which MITOL is knocked down, and it has been reported that it is rescued by MITOL knock-in (Non-patent Document 4). This suggests that aging is promoted by a decrease in MITOL, and that aging is restored by restoring the reduced MITOL. Based on the above, an approach that increases or activates MITOL expression is considered useful for anti-aging. However, no compound has been found that increases MITOL expression.
ベルベリンは抗菌（非特許文献５）、抗炎症（非特許文献６）などの様々な生物活性を有するアルカロイドの一種であり、その誘導体においても生物活性が維持されていることが報告されている（非特許文献７）。 Berberine is a kind of alkaloid having various biological activities such as antibacterial (Non-patent Document 5), anti-inflammatory (Non-patent Document 6), and it has been reported that its biological activity is also maintained in its derivatives ( Non-patent document 7).
本発明の目的は、MITOL減少抑制又はMITOL産生促進剤を提供することである。 An object of the present invention is to provide a MITOL reduction inhibitor or a MITOL production promoter.
そこで発明者らは鋭意検討した結果、表皮角化細胞を用いた試験により、塩化ベルベリン及びその誘導体である塩化ベルベルビンがMITOL遺伝子発現促進作用を有することを見出した。更にマウスを用いた試験により、塩酸ベルベリンが複数臓器においてMITOLタンパク発現促進作用を有することを見出し、本発明を完成するに至った。 As a result of intensive studies, the inventors have found that berberine chloride and berbervin chloride, which is a derivative thereof, have a MITOL gene expression promoting effect by a test using epidermal keratinocytes. Furthermore, through a test using mice, it was found that berberine hydrochloride has a MITOL protein expression promoting action in a plurality of organs, and the present invention has been completed.
すなわち本発明は、
（１）ベルベリン、その誘導体、及びそれらの塩からなる群から選択される少なくとも１種を含有することを特徴とする、MITOL減少抑制又は産生促進剤、
（２）請求項1に記載のMITOL減少抑制又は産生促進剤を含むことを特徴とする医薬品、医薬部外品、化粧品又は飲食品
である。
That is, the present invention
(1) A MITOL decrease inhibitor or production promoter, comprising at least one selected from the group consisting of berberine, derivatives thereof, and salts thereof,
(2) A pharmaceutical, a quasi-drug, a cosmetic or a food or drink characterized by including the MITLol reduction-suppressing or production-promoting agent according to claim 1.
本発明のベルベリン、その誘導体、及びそれらの塩は、MITOL減少を抑制又はMITOL産生を促進することより例えば心機能低下などの心臓老化症状、白髪や脱毛といった皮膚老化症状、アルツハイマー病などの脳の老化症状の予防又は改善効果が期待できる。また、素材スクリーニング等に際してポジティブコントロールとして用いることができる。 The berberine of the present invention, derivatives thereof, and salts thereof suppress the decrease in MITOR or promote the production of MITO, for example, cardiac aging symptoms such as decreased cardiac function, skin aging symptoms such as gray hair and hair loss, brain such as Alzheimer's disease Expected to prevent or improve aging symptoms. It can also be used as a positive control for material screening and the like.
本発明におけるベルベリンとは、2,3-メチレンジオキシ-9,10-ジメトキシ-5,6-ジヒドロジベンゾ[a,g]キノリジニウムで表される化合物である。
本発明における誘導体とは、もとの化合物の分子内の小部分を変化させて得られる化合物のことである。特に限定されるものではないが例えば、ベルベリンのメトキシ基の一つがヒドロキシ基に変化したベルベルビンが挙げられる。
本発明における塩とは、例えば塩化物、塩酸、硫酸やタンニン酸との塩が挙げられるが、特に限定されるものではない。
Berberine in the present invention is a compound represented by 2,3-methylenedioxy-9,10-dimethoxy-5,6-dihydrodibenzo [a, g] quinolizinium.
The derivative in the present invention is a compound obtained by changing a small part in the molecule of the original compound. Although not particularly limited, for example, berbervin in which one of the methoxy groups of berberine is changed to a hydroxy group can be mentioned.
Examples of the salt in the present invention include salts with chloride, hydrochloric acid, sulfuric acid and tannic acid, but are not particularly limited.
本発明におけるMITOL産生促進とは、MITOL mRNA又はMITOLタンパク質の発現促進作用のことである。MITOL減少抑制とはMITOL mRNA又はMITOLタンパク質の減少抑制作用のことである。MITOL産生促進又は減少抑制作用は、細胞や臓器におけるMITOL発現量を評価することで確認できるが、その方法は特に限定されるものではない。 In the present invention, “mitol production promotion” refers to the expression promoting action of MITOL mRNA or MITOL protein. The suppression of MITOL decrease is the effect of suppressing the decrease of MITOL mRNA or MITOL protein. The MITOL production promotion or reduction-suppressing action can be confirmed by evaluating the MITOL expression level in cells or organs, but the method is not particularly limited.
本発明に用いるベルベリン、その誘導体、及びそれらの塩の市販品としては塩化ベルベリン（Sigma-Aldrich製）、塩化ベルベルビン（長良サイエンス株式会社製）、ベルベリン塩酸塩（ナカライテスク製）等が挙げられる。 Examples of commercially available products of berberine, derivatives thereof, and salts thereof used in the present invention include berberine chloride (manufactured by Sigma-Aldrich), berberine chloride (manufactured by Nagara Science Co., Ltd.), berberine hydrochloride (manufactured by Nacalai Tesque), and the like.
投与形態としては、特に限定されるものではないが、外用や内服が挙げられる。本発明を外用で適用する場合の剤形としては、例えば、ローション剤、液剤、クリーム剤、軟膏剤、ゲル剤、スプレー剤、シャンプー、コンディショナー、石鹸等が挙げられ、内服で適用する場合の剤形としては、錠剤、粉末剤、散剤、顆粒剤、液剤、カプセル剤、ドライシロップ剤、ゼリー剤、液状食品、半固形食品、固形食品等が挙げられる。 Although it does not specifically limit as a dosage form, External use and internal use are mentioned. Examples of the dosage form when the present invention is applied externally include, for example, lotions, liquids, creams, ointments, gels, sprays, shampoos, conditioners, soaps, and the like. Examples of the form include tablets, powders, powders, granules, liquids, capsules, dry syrups, jellies, liquid foods, semi-solid foods, solid foods and the like.
これらは、公知の方法で製造することができる。製造に際しては、本発明の効果を損なわない範囲で、化粧品、医薬部外品、医薬品、飲食品又は試薬に含有可能な種々の添加物を配合することができる。 These can be produced by known methods. In the production, various additives that can be contained in cosmetics, quasi-drugs, pharmaceuticals, foods and drinks, or reagents can be blended within a range that does not impair the effects of the present invention.
本発明のMITOL減少抑制又は産生促進剤は、特に限定されるものではないが例えば心機能低下などの心臓老化症状、白髪や脱毛といった皮膚老化症状、アルツハイマー病などの脳の老化症状を予防又は改善するため、あるいは、MITOL産生を促進するための試薬として用いることも可能であり、好適には素材スクリーニング等を行なうに際し陽性対照薬として利用可能である。また、本発明のMITOL減少抑制又はMITOL産生促進剤は、これを含む製品（医薬品、医薬部外品、化粧品、飲食品、又は試薬）又はその説明書に、MITOL減少抑制又はMITOL産生促進剤を促進するために用いられる旨の表示を付することができる。ここで、「製品またはその説明書に表示を付した」とは、製品の本体、容器、包装などに表示を付したこと、あるいは製品の情報を開示する説明書、添付文書、宣伝物、申請資料、その他の印刷物又は広告などに表示を付したことを意味する。また、これら表示においては、MITOL減少抑制又はMITOL産生促進に起因する疾患や症状の予防又は治療のために用いられることに関する情報を含むことができる。 The MITOL decrease inhibitor or production promoter of the present invention is not particularly limited, but prevents or improves cardiac aging symptoms such as decreased cardiac function, skin aging symptoms such as gray hair and hair loss, and brain aging symptoms such as Alzheimer's disease. Therefore, it can also be used as a reagent for accelerating MITO production, and is preferably used as a positive control drug when performing material screening and the like. In addition, the MITOL reduction inhibitor or MITOL production promoter of the present invention includes a MITOL reduction inhibitor or MITOL production promoter in a product (pharmaceutical, quasi-drug, cosmetics, food or drink, or reagent) containing the same or an instruction thereof. An indication that it is used for promotion can be attached. Here, “labeled product or its instructions” means that the product body, container, packaging, etc. are labeled, or that the product information is disclosed, attached document, promotional material, application It means that a label is attached to a document, other printed matter or an advertisement. Moreover, in these indications, the information regarding being used for the prevention or treatment of the disease and symptom resulting from MITO reduction suppression or MITO production promotion can be included.
本発明のベルベリン、その誘導体、及びそれらの塩の配合量は、化粧品、医薬部外品、医薬品、飲食品又は試薬で提供する場合、それぞれ組成物全体に対して０．０００００１〜１０質量％、好ましくは０．０００１〜５質量％、より好ましくは０．００１〜１質量％である。 The amount of berberine of the present invention, derivatives thereof, and salts thereof is 0.000001 to 10% by mass with respect to the entire composition when provided as cosmetics, quasi drugs, pharmaceuticals, foods and drinks, or reagents. Preferably it is 0.0001-5 mass%, More preferably, it is 0.001-1 mass%.
以下に実施例および試験例を挙げ、本発明をさらに具体的に説明するが、本発明は実施例に限定されない。 EXAMPLES The present invention will be described more specifically with reference to examples and test examples. However, the present invention is not limited to the examples.
（試験例１）塩化ベルベリン及び塩化ベルベルビンのMITOL遺伝子発現促進作用の評価
表皮角化細胞（倉敷紡績製）を培地Humedia-KG2（倉敷紡績，添付サプリメントを添加）にて、2500cells/cm2になるように24穴プレートに播種し、37℃、CO2 5%にセットしたインキュベーター内で72時間培養した。培地を吸引除去し、被験物質を含有する培地に交換し，更に24時間培養した．培養終了後、培地を除去し、ライセートバッファーを添加して細胞を溶解し、細胞溶解液を回収した。細胞溶解液より、RNeasy Mini Kit（キアゲン）を用いて添付のプロトコールに従いRNAを回収し、これを鋳型として、Prime Script RT Master mix（タカラバイオ）を用いた逆転写反応によりcDNAを合成した。合成したcDNAから、リアルタイムPCRシステム（Step One Plus、サーモフィッシャーサイエンティフィック）により、GAPDH、MITOLそれぞれのmRNAの発現量を測定し（SYBR Green法）、MITOLの発現量をGAPDH発現量により補正した。プライマーは、次の型番のものを用いた。GAPDH：HA067812、MITOL：HA192576（いずれもタカラバイオ）。測定したmRNA発現量をコントロール群（被験物質を含有していない培地）と比較し、被験物質によるMITOL遺伝子発現促進作用を評価した。
(Test Example 1) Evaluation of MITOL gene expression promoting action of berberine chloride and berberbin chloride to 2500 cells / cm 2 in the culture medium Humedia-KG2 (Kurashiki Spinning, supplemented with supplement) Thus, the cells were seeded in 24-well plates and cultured for 72 hours in an incubator set at 37 ° C. and 5% CO 2. The medium was removed by aspiration, replaced with a medium containing the test substance, and further cultured for 24 hours. After completion of the culture, the medium was removed, lysate buffer was added to lyse cells, and the cell lysate was collected. RNA was collected from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio) using this as a template. From the synthesized cDNA, mRNA expression levels of GAPDH and MITOL were measured by the real-time PCR system (Step One Plus, Thermo Fisher Scientific) (SYBR Green method), and the expression level of MITOL was corrected by GAPDH expression level . Primers with the following model numbers were used. GAPDH: HA067812, MITOL: HA192576 (both Takara Bio). The measured mRNA expression level was compared with the control group (medium not containing the test substance), and the MITOR gene expression promoting action by the test substance was evaluated.
＜試験結果＞
結果を図１に示す。塩化ベルベリン及び塩化ベルベルビンによりMITOLのmRNA発現量が上昇することが確認された。よって、塩化ベルベリン、塩化ベルベルビンは白髪や脱毛といった皮膚老化症状に対して効果があることが示唆された。
<Test results>
The results are shown in FIG. It was confirmed that mRNA expression level of MITOL was increased by berberine chloride and berberbin chloride. Therefore, it was suggested that berberine chloride and berbervin chloride are effective for skin aging symptoms such as gray hair and hair loss.
（試験例２）ベルベリン塩酸塩によるMITOLタンパク発現促進作用の評価
（１）動物及びその飼育
C57BL/6J（SLC、♂、8週齢）を購入し、食餌 は標準固形飼料CE-2（オリエンタル酵母工業）を自由摂食させ、純水を自由摂水させた。紫外線照射実験においては、Hos:HR-1（SLC、♂、8週齢）を用いた。TP-102（東洋理工株式会社）マウスケージにて飼育し、床敷にはクリーンチップCL-4161（SLC）を使用した。飼育環境は、室温を25±2°Cとし、明期を7〜19時とした。
（２）ベルベリン投与条件
ベルベリン塩酸塩（ナカライテスク）を、純水を用いて0.2%（w/v）に可溶し、９日間または８週間自由摂水させた。
（３）表皮ウェスタンブロッティング
マウス耳を採取し、Dispase II（Thermo Fisher Scientific）により表皮を分取した後、SDS Sample Bufferを加え超音波処理（BRANSON SONIFIR 150）を行った。95℃ 5minのヒートショックを行い、12000rpm、4°Cで10分間遠心した上清をタンパク質溶液として得た。
（４）心臓・脳・骨格筋のウェスタンブロッティング
マウスをイソフルラン吸引麻酔科で開胸し、臓器を摘出した。臓器の重さを電子天秤により測定し、1mg/30uLになるようにSDS Sample Bufferを加え超音波処理（BRANSON SONIFIR 150）を行った。95℃ 5minのヒートショックを行い、12000rpm、4°Cで10分間遠心した上清をタンパク質溶液として得た。
SDS-PAGEは、Immobilon-P Transfer Membrane（Merck Millipore）に転写し、以下の手順でブロッキング、抗体反応を行い、Immobilon Western Chemiluminescent HRP Substrate（Millipore）用いて感光、LuminoGraph（ATTO）を用いてバンドを検出した。
5% スキムミルク（雪印メグミルク）/TBS-T、1時間（室温）
↓ TBS-Tにて洗浄
一次抗体/Can Get Signal TM solution1 （TOYOBO）、１時間（室温）
↓ TBS-Tにて洗浄
二次抗体/Can Get Signal TM solution2 （TOYOBO）、３０分（室温）
↓ TBS-Tにて洗浄
検出
（材料）
TBS-T：0.1% Tween20/Tris Buffered Saline（TBS）
SDS Sample Buffer：溶媒を超純水として、トリス0.0625M、２−メルカプトエタノール5%（v/v）、SDS 2%（w/v）、スクロース30%（w/v）、BPB 0.005%（w/v）
一次抗体: anti-MITOL3-2抗体（EMBO J. 2006 Aug 9;25(15):3618-26. Epub 2006 Jul 27. （PMID:16874301）に記載の方法に従って作成）、1000倍希釈
anti-alpha-Tubulin（DM1A）（Sigma T-9026）、5000倍希釈
二次抗体: anti-rabbit IgG, HRP linked（Jackson 111-035-003）、2000倍希釈
anti-mouse IgG, HRP linked（Jackson 115-035-003）、2000倍希釈
（５）実験動物麻酔方法
実験動物麻酔装置SN-487-0T（SHINANO）を用いてイソフルランによる吸引麻酔を、導入麻酔を0.5%、維持麻酔を0.2%で行った。
（６）紫外線照射方法
UV Bench Lamp、15W、xx-15M、302nm、115V（UVP）を使用し、UVX Radiometer（Digital）を使用し線量を確認した。単匹飼育を行い、UVBをUVB（1週目：70mJ、2週目：80mJ、3週目：90mJ、４週目以降：100mJ）、3回/週の照射を行った。８週の照射が完了後に表皮のサンプルを回収した。
(Test Example 2) Evaluation of MITOL protein expression promoting action by berberine hydrochloride (1) Animals and their breeding
C57BL / 6J (SLC, salmon, 8 weeks old) was purchased, and the standard chow CE-2 (Oriental Yeast Industry) was freely fed as a diet, and pure water was freely fed. In the ultraviolet irradiation experiment, Hos: HR-1 (SLC, silkworm, 8 weeks old) was used. TP-102 (Toyo Riko Co., Ltd.) was bred in a mouse cage, and clean chip CL-4161 (SLC) was used for the flooring. The breeding environment was a room temperature of 25 ± 2 ° C and a light period of 7-19 o'clock.
(2) Berberine administration conditions Berberine hydrochloride (Nacalai Tesque) was dissolved in 0.2% (w / v) using pure water and allowed to freely drink water for 9 days or 8 weeks.
(3) Epidermal Western Blotting Mouse ears were collected and the epidermis was collected by Dispase II (Thermo Fisher Scientific), and then SDS Sample Buffer was added and sonication (BRANSON SONIFIR 150) was performed. A heat shock at 95 ° C. for 5 minutes was performed, and the supernatant obtained by centrifugation at 12000 rpm, 4 ° C. for 10 minutes was obtained as a protein solution.
(4) Western blotting of heart, brain, and skeletal muscle Mice were thoracotomized by isoflurane aspiration anesthesiology to remove organs. The weight of the organ was measured with an electronic balance, and SDS Sample Buffer was added to 1 mg / 30 uL, and sonication (BRANSON SONIFIR 150) was performed. A heat shock at 95 ° C. for 5 minutes was performed, and the supernatant obtained by centrifugation at 12000 rpm, 4 ° C. for 10 minutes was obtained as a protein solution.
SDS-PAGE is transferred to Immobilon-P Transfer Membrane (Merck Millipore), blocked and antibody-reacted by the following procedures, exposed using Immobilon Western Chemiluminescent HRP Substrate (Millipore), and banded using LuminoGraph (ATTO). Detected.
5% skim milk (Snow Brand Megmilk) / TBS-T, 1 hour (room temperature)
↓ Washed with TBS-T Primary antibody / Can Get Signal ™ solution1 (TOYOBO), 1 hour (room temperature)
↓ Washed with TBS-T Secondary antibody / Can Get Signal ™ solution2 (TOYOBO), 30 minutes (room temperature)
↓ Wash with TBS-T. Detection (material)
TBS-T: 0.1% Tween20 / Tris Buffered Saline (TBS)
SDS Sample Buffer: Tris 0.0625M, 2-mercaptoethanol 5% (v / v), SDS 2% (w / v), sucrose 30% (w / v), BPB 0.005% (w / v)
Primary antibody: anti-MITOL3-2 antibody (EMBO J. 2006 Aug 9; 25 (15): 3618-26. Epub 2006 Jul 27. (PMID: 16874301))
anti-alpha-Tubulin (DM1A) (Sigma T-9026), diluted 5000 times Secondary antibody: anti-rabbit IgG, HRP linked (Jackson 111-035-003), diluted 2000 times
anti-mouse IgG, HRP linked (Jackson 115-035-003), diluted 2000 times (5) Experimental animal anesthesia method
Using an experimental animal anesthesia device SN-487-0T (SHINANO), aspiration anesthesia with isoflurane was performed with induction anesthesia at 0.5% and maintenance anesthesia at 0.2%.
(6) UV irradiation method
A UV Bench Lamp, 15W, xx-15M, 302nm, 115V (UVP) was used, and the dose was confirmed using a UVX Radiometer (Digital). A single animal was raised and UVB was irradiated 3 times / week with UVB (1 week: 70 mJ, 2 weeks: 80 mJ, 3 weeks: 90 mJ, 4 weeks and beyond: 100 mJ). Epidermal samples were collected after 8 weeks of irradiation.
＜試験結果＞
図２にウェスタンブロット及びバンドを定量化した結果を示す。ベルベリン塩酸塩によりMITOLのタンパク発現量が上昇することが確認された。よって、ベルベリン塩酸塩は心臓、皮膚、脳などの老化症状に対して効果があることが示唆された。図１及び図２の結果より、ベルベリン、その誘導体、及びそれらの塩がMITOL発現促進作用を有することが示唆された。
<Test results>
FIG. 2 shows the results of quantification of Western blot and bands. It was confirmed that berberine hydrochloride increased the protein expression level of MITOL. Therefore, it was suggested that berberine hydrochloride is effective for aging symptoms such as heart, skin, and brain. From the results of FIG. 1 and FIG. 2, it was suggested that berberine, its derivatives, and their salts have a MITOL expression promoting action.
本発明のMITOL減少抑制又は産生促進剤は、例えば心機能低下などの心臓老化症状、白髪や脱毛といった皮膚老化症状、アルツハイマー病などの脳の老化症状 老化症状を予防又は改善するための化粧品、医薬部外品、医薬品又は飲食品の分野に利用可能である。また、MITOL産生を促進する成分のスクリーニングに際し、ポジティブコントロールとして用いることができる。 Mitol reduction inhibitor or production promoter of the present invention is, for example, cardiac aging symptoms such as reduced cardiac function, skin aging symptoms such as white hair and hair loss, brain aging symptoms such as Alzheimer's disease cosmetics and medicines for preventing or improving aging symptoms It can be used in the field of quasi-drugs, pharmaceuticals or foods and drinks. In addition, it can be used as a positive control in screening for components that promote MITOL production.
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