Discovery of processing-associated Q-marker of carbonized traditional Chinese medicine: An integrated strategy of metabolomics, systems pharmacology and in vivo high-throughput screening model

Background: Carbonized traditional Chinese medicine (TCM) is a kind of distinctive traditional medicine, which has been widely used to cure various bleeding syndromes in clinic for over 2000 years. However, there are no effective quality control methods developed on carbonized TCM so far. Purpose: This study aimed at developing a processing-associated quality marker (Q-marker) discovery strategy, which would enable to promote the quality control study of carbonized TCM. Methods: Carbonized Typhae Pollen (CTP), a typical carbonized TCM with fantastic efficacy of stanching bleeding and removing blood stasis, was used as an example. First, a ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) method was established to characterize four types of CTP in different processing degrees. Second, chemometric method was applied to screen candidate Q markers. Third, peak area changes and Aratio changes of each candidate markers in 57 batches samples were described (Traceability and Transitivity). Fourth, systems pharmacology and two high-throughput zebrafish models: cerebral hemorrhage model and thrombus model were used to furtherly screen Q-markers (Effectiveness). Finally, a ultraperformance liquid chromatographic coupled with triple quadrupole tandem mass spectrometry (UPLC-TQ-MS) method was established and applied to quantify Q-markers in additional 10 batches of CTP samples (Measurability). Results: The chemical profiles of Typhae Pollen during the carbonized process were investigated. Then, 12 candidate compounds were screened in chemometric part. Six Q-markers (isorhamnetin-3-O-neohesperidoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-neohesperidoside, naringenin, quercetin and isorhamnetin) were subsequently screened out using three principles of Q-markers combined with content changes and two in vivo zebrafish models. Their average contents in additional 10 batches of CTP were 316.8 mu g/g, 13.7 mu g/g, 6.1 mu g/g, 197.8 mu g/g, 12.9 mu g/g and 199.3 mu g/g, respectively. Their content proportion was about 25: 1: 0.5: 15: 1: 15. Conclusion: A processing-associated Q-marker discovery strategy was developed for carbonized TCM. It might provide a novel insight to solve the problem of 'Chao Tan Cun Xing' in carbonized process.