The ESI-HR-MS/MS fragmentation behaviors of limonin and obacunone were investigated for the first time. A qualitative and semi-quantitative method was developed for the in vivo and in vitro metabolic analysis of limonin and obacunone. The results demonstrated that the metabolic processes of limonin and obacunone were different between LMs and zebrafish. However, both of these two parent compounds presented similar metabolic processes in five species of LMs. This was caused by the metabolic difference between mammals and fish or because limonin probably cannot be absorbed in zebrafish.
1. Zebrafish has been used in metabolic study of drugs as a powerful tool in recent years. In this study, we make a feasible metabolism investigation of five protoberberine alkaloids (PBAs) applied in zebrafish model for the first time, including berberine (BBR), palmatine (PAL), jatrorrhizine (JAT), coptisine (COP) and epiberberine (EBBR). 2. After exposure for 24 hours, 19 metabolites were identified by LTQ Orbitrap mass spectrometer, including 9 phase I metabolites and 10 phase II metabolites. Demethylation, hydroxylation, sulfation and glucuronidation were the major metabolic transformation of PBAs in zebrafish, which were similar to mammals. Compared with reported literatures, BBR and JAT showed high consistency between human and zebrafish in metabolic pathways. 3. To our knowledge, this is the first time to study in vivo metabolism of COP, which provides useful information to other researchers. 4. This study indicated that zebrafish model is feasible and reasonable to predict the metabolism of PBAs. It showed great potential for developing a novel and rapid method for predicting the metabolism of trace compounds of botanical drugs, with the advantages of lower cost, higher performance and easier set up.
1. Huang-Lian-Jie-Du Decoction (HLJDD) is widely used for the treatment of hypertension, diabetes, inflammation and neural system diseases in clinic. In the present study, the comprehensive metabolic profile of HLJDD was demonstrated reliably and rapidly followed by the metabolic pathway analysis of six typical pure compounds (four alkaloids, one flavonoid and one iridoid) in HLJDD using LC-IT-MS combined with high resolution LC-FT-ICR-MS. 2. Totally, 85 compounds, including 32 prototype components and 53 biotransformed metabolites were detected and characterized in the urine and feces after oral administration of HLJDD and six pure compounds to rats, respectively. Among them, 17 prototypes were identified definitely with standard references. 3. Hydroxylation, demethylation and glucuronidation reactions of alkaloids, as well as glucuronidation and sulfonation reactions of iridoids and flavonoids, were observed as the major metabolic pathways of HLJDD. Flavonoids, iridoids and their metabolites were mainly excreted from urine. However, amount of alkaloids were detected in feces. 4. In general, the distinctive metabolic process of three kinds of representative components in HLJDD was clarified. The in vivo metabolic network of HLJDD was demonstrated. Meanwhile, the investigation of representative pure compounds in metabolic study provided a valuable strategy to elucidate the full-scale metabolic fate of HLJDD. This might be helpful to understand the in vivo mechanism of Traditional Chinese medicine (TCM).
The results demonstrated that reduction at C-7 and C-16, hydroxylation and reaction of glycine with reduction limonoids were the major metabolic pathways of limonoids in HLMs. Among them, glycination with reduction was the unique metabolic process of limonoids observed for the first time. CYP2D6 and CYP3A4 played an important role in the isomerization and glycination of limonoids in HLMs, whereas other CYP isoforms were considerably less active. The results might help to understand the metabolic process of limonoids in vitro such as the unidentified metabolites of limonin glucoside observed in the medium of microbes and the biotransformation of limonin in juices. Moreover, it would be beneficial for us to further study the pharmacokinetic behavior of limonoids in vivo systematically.
Huang-Lian-Jie-Du Decoction (HLJDD) is a classical Traditional Chinese Medicine (TCM) formula with heat-dissipating and detoxifying effects. It is used to treat inflammation-associated diseases. However, no systematic pharmacokinetic (PK) and pharmacodynamic (PD) data concerning the activity of HLJDD under inflammatory conditions is available to date. In the present study, the concentration-time profiles and the hepatic clearance rates (HCR) of 41 major components in rat plasma in response to the oral administration of a clinical dose of HLJDD were investigated by LC-QqQ-MS using a dynamic multiple reaction monitoring (DMRM) method. Additionally, the levels of 7 cytokines (CKs) in the plasma and the body temperature of rats were analyzed. Furthermore, a PK-PD model was established to describe the time course of the hemodynamic and anti-inflammatory effects of HLJDD. As one of the three major active constituents in HLJDD, iridoids were absorbed and eliminated more easily and quickly than alkaloids and flavonoids. Compared with the normal controls, the flavonoids, alkaloids and iridoids in inflamed rats exhibited consistently changing trends of PK behaviors, such as higher bioavailability, slower elimination, delays in reaching the maximum concentration (Tmax) and longer substantivity. The HCR of iridoids was different from that of alkaloids and flavonoids in inflamed rats. Furthermore, excellent pharmacodynamic effects of HLJDD were observed in inflamed rats. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, IL-10, and macrophage inflammatory protein-2 (MIP-2) and body temperature significantly decreased after the administration of HLJDD. Based on PK-PD modeling with the three-phase synchronous characterization of time-concentration-effect, flavonoids exhibited one mechanism of action in the anti-inflammatory process, while iridoids and alkaloids showed another mechanism of action. Taken together, the results demonstrated that HLJDD may restrain inflammation synergistically via its major constituents (alkaloids, flavonoids and iridoids). A correlation between the exposure concentration of different types of compounds and their anti-inflammatory effects in the body was shown. This study provides a comprehensive understanding of the anti-inflammatory activity of HLJDD.
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