Systematically characterize the absorbed effective substances of Wutou Decoction and their metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network pharmacological analysis
Introduction
Wu-tou Decoction (WTD), is a classical formula in traditional Chinese medicine (TCM), and was originally recorded in “Jin Kui Yao Lve” written by traditional Chinese medical sage Zhang Zhongjing. The formula has been used to treat arthritis with excellent clinical effects. Its recipe contains five herbal medicines including Aconiti Radix Preparata (10 g), Ephedrae Herba (15 g), Paeoniae Radix Alba (15 g), Astragali Radix (15 g) and Glycyrrhiza Radix Preparata (15 g). In which, Aconiti Radix Preparata and Ephedrae Herba act as the monarch herbs due to the internal alkaloids revealing anti-inflammatory and analgesic effects [1], [2]. The main constituents in Paeoniae Radix Alba are monoterpene glycosides which possess neuroprotective [3] and anti-inflammatory effects [4], and the major bioactive constituents in Astragali Radix and Glycyrrhiza Radix Preparata are flavonoids and saponins [5], as which show anti-oxidant and attenuate toxicity effects [6]. Recently, many researchers poured attention into the pharmacological mechanisms of WTD acting on arthritis and inflammation. In our laboratory and co-laboratories, we have investigated the major constituents in WTD [5], [7], the comprehensive metabolomic evaluation of the therapeutic effects of WTD in treating rheumatic arthritis (RA) [6], [8], [9], the intestinal absorption of the alkaloids in WTD [10], the possible ion channels that WTD participated in inhibiting the chronic inflammatory [11], as well as the systems biology based approaches that integrated with network analysis to reveal the pharmacological mechanisms of WTD [12], [13]. However, few reports were concerning on the absorbed effective substances of WTD, whereas the possible metabolic pathways of WTD in vivo remains largely unclear. To interpret the pharmacological effects and the material basis, it is of great significance and warrant to analyze the absorbed substances and their affecting targets systematically.
As we know, herbal formula, characterized in TCM, is achieving its therapeutic effectiveness by regulating the systems of the body with the effective substances. For the numerous compounds act on multiple targets, it has great challenge to investigate the pharmacological effects of herbal formula, and hard to address this bottleneck by analyze the compounds solely with traditional methods. What’s more, the complex and dynamic interaction between the compounds and the targets are underlying the therapeutic effects and biological process of the formula. Network pharmacology, a novel method which combined the system network analysis and the pharmacology, could clarify the synergistic effects and underlying mechanisms among the networks of compound-compound, compound-target and target-disease in the molecular level, which let us know the interactions among the compounds, genes, proteins and diseases [14], [15]. However, previous studies gathered compounds from various databases to generate compound-target maps, some substances with plenty of targets but possess low bioavailability are also taken into account, which herein produce false-positive results, this method could define as nontarget-network pharmacology. In this context, we focused on the absorbable bioactive constituents as selected target compounds, and these exposed compounds were thought as playing a therapeutic role, then we integrated network pharmacology maps of the target compounds and corresponding target proteins, the prospered method could name as target-network pharmacology (T-NP).
More recently, hybrid instruments combined with mass spectrometry are increasingly employed in bio-sample analysis with favorable advantages in sensitivity and high sample throughput. In this work, we used ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) for the characterization of absorbed prototype constituents and the metabolites of WTD. Besides, T-NP method was used to integrate the absorbed constituents with corresponding targets and generated network pharmacological maps. As far as our concerned, this is the first time to reveal the comprehensive results of the absorbed effective substances, the metabolic pathways and target pharmacological network analysis of WTD in vivo.
Section snippets
Materials
Aconiti Radix Preparata, Ephedrae Herba, Paeoniae Radix Alba, Astragali Radix and Glycyrrhiza Radix Preparata were purchased from Ji Lin Pharmacy. All the botanical characters of the herbs were identified by Prof. Shumin Wang (Changchun University of Chinese Medicine). Acetonitrile, isopropanol, methanol, acetone and formic acid (HPLC-grade) were obtained from Fisher Scientific (Loughborough, UK). The distilled water was prepared by a Milli-Q plus (Milford, MA, USA). Leucine enkephalin and
Organic solvent selection
The endogenous proteins in plasma could cause great interferences with the absorbed substances and even result in ion suppression. Therefore, selecting a suitable organic solvent in plasma protein precipitation is vital to remove the interfered proteins. Here, we selected five different organic solvent systems to optimize an efficient solvent in protein precipitation and peak separation improvement. Fig. S1 exhibited the base peak chromatograms (BPCs) of dosed plasma with different organic
Conclusion
The present study has developed a sensitive and rapid method for the separation and identification of the absorbed constituents and metabolites of WTD. In the 20 min analysis, a total of 22 prototype constituents and 49 metabolites were identified or tentatively characterized. The major metabolic reactions of the absorbed constituents were sulfation, glucuronidation, demethylation and hydroxylation, and the possible metabolic pathways of these constituents were visualized in the photographs.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 81274046) and the National Basic Research Progrom of China (973 Program) (Nos. 2011CB505300, 2011CB505305)
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