Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish

Rubiae Radix et Rhizoma (called “Qiancao”, QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called “Qiancaotan”, QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC–MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile–water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.