An Integrated Strategy to Discover the Quality Control Markers of Herbal Formulae of Danning Tablet with Anti-cholestasis Applications

Quality makers (Q-markers) are pivotal for the standardization of quality control for traditional Chinese medicine (TCM) drugs. However, it’s a great challenge to identify the Q-markers of TCM drugs especially for TCM formulas. This study was designed to provide a method to discover Q-markers of TCM formulas and applied to identify the Q-markers responsible for the anti-cholestatic effect of Danning tablet (DNT), a TCM formulae consisting with seven herbal ingredients. In this study, the anti-cholestatic effect of DNT was evaluated in mice with cholestasis induced by α-naphthyl isothiocyanate (ANIT). An untargeted metabolomic profiling were performed using serum and liver samples of mice. Xenobiotics derived from DNT and endogenous compounds responsible for the anti-cholestatic effect of DNT were screened by statistical analysis and the Q-markers of DNT were illustrated, followed by predicting the potential targets and mechanisms via network pharmacology study. DNT exhibited a protective effect against liver injury in mice with ANIT-induced cholestasis. Three endogenous compounds, namely taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and glycocholic acid (GCA), were identified as the endogenous markers responsible for the anti-cholestatic effect of DNT. By setting a cutoff value of the correlation co-efficient at 0.8, 6 promising Q-markers, including luteolin, kaempferol, apigenin, emodin, luteolin-7-glucoside, and 5,4'-dihydroxy-3,6,7,8,3'-pentamethoxyflavone, were screened out. These Q-markers were stably existed in DNT and predicted to be highly active in treating cholestasis. This study established a pipeline to screen out the potential Q-markers of TCM formulas and 6 key Q-markers responsible for the anti-cholestatic effect of DNT were identified.