Metabolites Identification and Pharmacokinetic Studies of Chinese Herbal Medicines

Traditional Chinese Medicines (TCMs) have been used in clinical practice for a long history with credible therapeutic effects. However, due to the complex chemical composition, their metabolic study has been a great challenge. When TCMs are used in the traditional manner, i.e. clinical oral dose of water decoction, a number of compounds may get into circulation and then be converted into even more metabolites. The knowledge of TCM metabolism is usually hindered by fairly low concentrations of these metabolites and poor understanding in metabolic pathways. Current studies to overcome these difficulties have mostly focused on a few major constituents, or used very high dosage to ensure the detection of metabolites. These studies could hardly represent the metabolism of herbal medicine in clinical therapy. In order to systematically characterize the metabolites of TCMs at an oral clinical dosage, a new strategy is needed.

In the present work, a novel strategy was proposed to systematically characterize in vivo metabolites of TCMs at a normal clinical dosage. Licorice was selected as the model herb, for its frequent appearance in about 60% of all TCM prescriptions. The strategy consisted of three steps. First, ten selected single compounds of licorice were administered to rats and studied separately. These compounds included three flavanones, three chalcones, two isoflavones, one flavone, and one saponin. They represent different structural types, and are also the major constituents of licorice. A total of 68 metabolites were characterized by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MSⁿ) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC/qTOF-MS) analyses, assisted with enzyme hydrolysis. Among them, 13 compounds were confirmed by comparison with reference standards. Metabolic pathways for each type of compounds were proposed. Secondly, in order to characterize metabolites other than those derived from the above ten compounds, a high dosage (equivalent to 20-fold clinical dosage) of licorice water extract was administered. Based on the proposed pathways, compounds of the same structural type had their metabolic patterns deduced. As a result, 22 more metabolites were characterized. Structures and MS/MS spectral data of all metabolites were used to set the ion pairs for selected reaction monitoring (SRM) detection. Finally, a clinical dosage of licorice water decoction (0.9g crude drug per kilogram) was orally administered to rats. The characterized metabolites (including constituents of licorice) were determined by a highly sensitive and selective LC/SRM-MS method. A total of 42 metabolites in plasma and 62 metabolites in urine were detected.

This is the first attempt to fully profile the metabolites of licorice at a normal clinical dosage, which led to the characterization of 90 phytochemicals in vivo, and elucidated their derivation from only 19 licorice compounds. A number of licorice metabolites in trace amount were successfully detected at a clinical dosage. The study was a preliminary groundwork for future pharmacokinetic studies. Moreover, the new strategy could be generally used for the characterization of TCM metabolites.