title: 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.
abstract: 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.
authors: State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 38 Xueyuan Road, Beijing 100191, PR China; Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Xing-Xin
authors: XX
authors: Yang
authors: State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Feng
authors: F
authors: Xu
authors: State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 38 Xueyuan Road, Beijing 100191, PR China; Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Dan
authors: D
authors: Wang
authors: Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Zhi-Wei
authors: ZW
authors: Yang
authors: Department of Pharmacology, School of Basic Medical Science, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Huan-Ran
authors: HR
authors: Tan
authors: State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 38 Xueyuan Road, Beijing 100191, PR China.
authors: Ming-Ying
authors: MY
authors: Shang
authors: Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, PR China. Electronic address: xuanwang6818@bjmu.edu.cn.
authors: Xuan
authors: X
authors: Wang
authors: State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 38 Xueyuan Road, Beijing 100191, PR China. Electronic address: sqcai@bjmu.edu.cn.
authors: Shao-Qing
authors: SQ
authors: Cai
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014462:Ultrafiltration
chemical_list: D004365:Drugs, Chinese Herbal; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore
keywords: Bioactive compounds; Centrifugal ultrafiltration; Liquid chromatography/mass spectrometry; Mitochondria; Screening method