Multiple perspectives of qingkailing injection-fraction-single compound in revealing the hepatotoxicity of baicalin and hyodeoxycholic acid

Publication date: 6 April 2018
Source:Journal of Ethnopharmacology, Volume 215
Author(s): Jiayu Zhang, Shifeng Wang, Lulu Xu, Qiao Zhang, Zhanpeng Shang, Yanling Zhang, Qinghua Wu, Shiyou Li, Yanjiang Qiao
Ethnopharmacological relevanceThe complexity of ingredients in traditional Chinese medical formulas and the limited consideration of toxicological responses are fundamental issues that hamper prognostic information of drug quality control.Materials and methodsA multidisciplinary approach for quality control of Qingkailing injection (QKL) regarding drug induced liver toxicity was described for the first time. High content image analysis (HCA) was combined with reverse-phase chromatographic separation and high-resolution MS detection technologies to provide the dynamic responses of drug induced HepG2 cell injury. Firstly, a simple and rapid method for simultaneous qualification and quantification of 21 major constituents in QKL was established and validated using ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap), which were operated in full MS/dd-MS² mode and thus simultaneously acquired quantitative high resolution (HR) full scan data and confirmatory HR MS² data. Secondly, repeated semi-preparation HPLC was applied to obtain four fractions (F1-F4) for HCS analysis. Finally, potential hepatotoxicity was determined by five hepatotoxicity biomarkers, including cell loss, DNA condense, glutathione (GSH) depletion, reactive oxygen species (ROS) formation, and mitochondria membrane potential (MMP) depolarization.ResultsThe detection in polarity switching mode empowered the coverage of comprehensive constituents with different chemical properties. Satisfactory linearity precisions, repeatability, stability, and recovery were achieved. QKL injection significantly induced HepG2 cell injury above the concentration of 1.25% (v/v). Meanwhile, flavone glycosides (F3) and stinasterols (F4) fractions exhibited hepatotoxicity above 75μg/mL and 50μg/mL, respectively. Still further, baicalin originated from F3 significantly caused cell loss and glutathione (GSH) depletion. In parallel, hyodeoxycholic acid from F4 induced cell loss, nucleus condense, and GSH reduction as well.ConclusionsOur work provides multiple perspectives based on injection-fractions-single compound format to improve QKL pharmacovigilance through revealing the potential hepatotoxic material basis. Additionally, our study provides an integrating paradigm for the comprehensive and systematic quality control of traditional Chinese medical formulas.

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