Characterization Of UDP-glucuronosyltransferases Responsible For The Metabolism Of Strychnine And Brucine In Vitro

Object iceSemen strychni is the dried seeds of Strychnos nux-vomica L. (Loganiaceae), which has been used for centuries as herbal medicine in China. It has a variety of pharmacological activity, however overdose of Semen strychni or improper processing can cause toxic reaction, and it is one of the toxic drug varieties prescribed by Ministry of Health. As two typical kinds of alkaloids, strychnine and brucine have been used as important ingredients in various remedies of traditional herbal medicines. In recent years, the responsibility of CYP450s for metabolism of strychnine and brucine was investigated, but few reports focus on the metabolic enzymes in phase II such as UGTs, SULTs, and so on. The polarity of most toxic drugs becomes stronger after phaseâ…¡ metabolic reactions, which makes it easier to be removed from body so as to reduce the deleterious effects caused by the toxic ingredients. Therefore, the study of phase II metabolism is of vital importance to reduce the adverse reaction of Semen Strychni clinically.In the present study, both strychnine and brucine glucuronidation were examined in great detail from the treatment by incubating with HLM and human recombinant UGTs.MethodsA analytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for determination of strychnine, brucine and glucuronidation in vitro. The difference of strychnine and brucine metabolism among different species and organs were compared by incubating with HLM. The kinetic parameters for the glucuronidation of strychnine and brucine were calculated and HLM, UGTs chemical inhibitors and recombinant UGT isoforms were employed to the determination of the main UGT isoforms that involved in the metabolism of strychnine and brucine to expound the detoxification pathway of Semen Strychni.ResultsN+-glucuronidation was found as the major metabolites of strychnine and brucine in vitro. The glucuronide metabolites were detected not in RLM or HIM but in HLM, showing that both brucine and strychnine were mainly metabolized in liver. The kinetics of strychnine (or brucine) glucuronidation in HLMs was monophasic, which suggesting that a single UGT isoform was responsible for the glucuronidation. Furthermore, the Km values of both strychnine and brucine glucuronidation by HLMs was similar to that by recombinant UGT1A4. Moreover, the findings were also supported by the results of the subsequent inhibition studies.ConclusionIn conclusion, the present study provides a comprehensive analysis of strychnine and brucine glucuronidation by human liver tissues and UGTs in vitro, and identifies UGT1A4 as a major contributor for strychnine and brucine glucuronidation in human liver microsomes. Species difference exist and both brucine and strychnine were mainly metabolized in liver. This research sets a basis for the prevention of ADRs and illustrating its possible mechanism for detoxification.