An integrated strategy for rapid discovery and identification of quality markers in Guanxin Kangtai preparation using UHPLC-TOF/MS and multivariate statistical analysis

Background: Guanxin Kangtai preparation (GXKT), consisting of Panax ginseng, Panax notoginseng and Rex pubescens, is a new proprietary Chinese medicines under development for treating coronary heart disease. Like other Chinese medicines, the components of GXKT were complex and the bioactive compounds remained unclear. Purpose: To discover bioactive compounds as quality markers (Q-markers) for better quality control of GXKT. Study design: Chinese medicines was separated into fractions. The correlation between chemical information and bioactivity of these fractions were analyzed with multivariate statistical methods to discover bioactive compounds responsible for the actions of Chinese medicine. Method: GXKT was separated into fractions by using high-performance liquid chromatography (HPLC). Ultra HPLC coupled with time-of-flight mass spectrometer (UHPLC-TOF/MS) was applied to detect compound information from these fractions to form a chemical database. The bioactivity of these fractions in protecting cardiomyocytes from ischemia/reperfusion injury was examined in H9c2 cells that were exposed to hypoxia followed by reoxygenation (H/R). Then, partial least square model and orthogonal projections to latent structures discriminant analysis were employed to discover bioactive compounds from the chemical database that were positively correlated with the bioactivity of GXKT fractions. Finally, the bioactivity of these compounds was confirmed by bioassay in H9c2 cells. Results: The chemical information of 120 fractions separated from GXKT was detected and extracted by UHPLCTOF/MS, and a chemical database including 61 high abundance compounds were formed from all fractions. These fractions produced different extent of protective effect to H9c2 cell underwent H/R treatment with cell viability ranging from 33.43% to 74.91%, demonstrating the separation of bioactive compounds among different fractions. The multivariate analysis discovered 16 compounds from GXKT positively correlated with the bioactivity of GXKT. Of these compounds, 6 compounds, i.e.: ginsenoside Rc, ilexsaponin A(1), and chikusetsusaponin IVa were chemical identified and also confirmed for their responsibility to the action of GXKT by bioassay. Conclusion: Ginsenoside Rg(1), Rb-1, Rh-1, Rc, ilexsaponin A(1), and chikusetsusaponin IVa were bioactive compounds and qualified as Q-markers for quality control of GXKT. This research provided a useful reference for the quality research of Chinese medicines.