Background: Tanshinone IIA (TIIA), protocatechuic aldehyde (PA), danshensu (DSS), salvianolic acid B (SAB) and hydroxysafflor yellow A (HSYA) are the major components of Salvia miltiorrhiza Bge. (Danshen) and Carthamus tinctorius L. (Honghua) herbal pair. These active components may contribute to the potential synergistic effects of the herbal pair.

Objective: This study aimed to investigate the metabolites of TIIA, PA, DSS, SAB and HSYA in zebrafish, and to explore the influence of HSYA on the metabolism of TIIA, PA, DSS, and SAB.

Method: 48 h post-fertilization zebrafish embryos were exposed either to each compound alone, TIIA (0.89 μg/mL), PA (0.41 μg/mL), DSS (0.59 μg/mL), SAB (2.15 μg/mL), and HSYA (1.83 μg/mL) and in combination with HSAY (1.83 μg/mL). The metabolites of TIIA, PA, DSS, SAB, and HSYA in zebrafish were characterized using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) and quantitatively determined by HPLC-MS with single and combined exposure.

Results: Among the 26 metabolites detected and characterized from these five compounds, methylation, hydroxylation, dehydrogenation, hydrolysis, sulfation and glucuronidation were the main phase I and phase II metabolic reactions of these compounds, respectively. Furthermore, the results showed that HSYA could either enhance or reduce the amount of TIIA, PA, DSS, SAB, and their corresponding metabolites.

Conclusion: The results provided a reference for the study on drug interactions in vivo. In addition, the zebrafish model which required much fewer amounts of test samples, compared to regular mammal models, had higher efficiency in predicting in vivo metabolism of compounds.