Development of a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method for screening mitochondria-targeted bioactive constituents from complex matrixes: Herbal medicines as a case study
Development of a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method for screening mitochondria-targeted bioactive constituents from complex matrixes: Herbal medicines as a case study
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开发基于线粒体的离心超滤/液相色谱/质谱方法，用于从复杂基质中筛选线粒体靶向生物活性成分：以草药为案例研究
DOI：
10.1016/j.chroma.2015.08.014
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发表时间：
2015-09-25
影响因子：
4.1
通讯作者：
Cai, Shao-Qing
中科院分区：
文献类型：
--
作者：
Yang, Xing-Xin;Xu, Feng;Cai, Shao-Qing
线粒体是重要的细胞内药理学靶标，因为对该细胞器的损伤导致多种人类病症，并且因为线粒体参与复杂过程，如能量产生、细胞凋亡和脂质代谢。为了加快天然生物活性化合物对线粒体的搜索，我们最初开发了一种高效的基于芦荟的筛选方法，该方法将离心超滤（CU）与液相色谱/质谱（LC/MS）相结合，称为芦荟靶向生物活性成分的筛选方法（SM-MBC），并且与从复杂基质（如草药提取物）中搜索芦荟靶向化合物兼容。功能活性，结构完整和纯的线粒体从大鼠心肌线粒体分离，包括细胞器释放，然后通过差速和Nycodenz密度梯度离心的优化协议。以噻苯咪唑（TZ）、鱼藤酮（RN）、胺碘酮（AR）和曲美他嗪（TD）为阳性对照，对该方法的可靠性进行了评价，并将其成功应用于虎杖（PCRR）和丹参（SR）提取物中活性成分的筛选。通过LC/MS检测和鉴定了19个活性化合物，其中17个是新的靶向化合物。19个目标化合物中有9个的活性通过体外药理试验得到证实。这些结果表明，SM-MBC可用于有效筛选的药物靶向成分的复杂制剂用于治疗线粒体疾病，如PCRR和SR。该结果可能是有意义的深入了解药物的作用机制和药物发现从草药。(C)2015 Elsevier B. V.版权所有。
Mitochondria are an important intracellular pharmacological target because damage to this organelle results in a variety of human disorders and because mitochondria are involved in complex processes such as energy generation, apoptosis and lipid metabolism. To expedite the search for natural bioactive compounds targeting mitochondria, we initially developed an efficient mitochondria-based screening method by combining centrifugal ultrafiltration (CU) with liquid chromatography/mass spectrometry (LC/MS), which is called screening method for mitochondria-targeted bioactive constituents (SM-MBC) and is compatible with the search of mitochondria-targeted compounds from complex matrixes such as herbal medicines extracts. Functionally active, structurally intact and pure mitochondria were obtained from rat myocardium using an optimized protocol for mitochondrial isolation comprising organelle release followed by differential and Nycodenz density gradient centrifugation. After evaluating the reliability of the method using thiabendazole (TZ), rotenone (RN), amiodarone (AR) and trimetazidine (TD) as positive controls, this method was successfully applied to screen bioactive constituents from extracts of Polygoni Cuspidati Rhizoma et Radix (PCRR) and Scutellariae Radix (SR). Nineteen active compounds were detected and identified by LC/MS, of which 17 were new mitochondria-targeted compounds. The activity of 9 of the 19 hit compounds was confirmed by in vitro pharmacological trials. These results demonstrate that SM-MBC can be used for the efficient screening of mitochondria-targeted constituents in complex preparations used to treat mitochondrial disorders, such as PCRR and SR. The results may be meaningful for an in-depth understanding of drug mechanism of action and drug discovery from medicinal herbs. (C) 2015 Elsevier B.V. All rights reserved.