Toad skin and toad venom, as two kinds of Chinese medicine, are prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. However, they display distinct properties in traditional application, and the hydrophilic ingredients are the possible distinguishing chemicals between them. In this work, 36 and 22 hydrophilic components were characterized from toad skin and venom, respectively, by UHPLC-HR-MS/MS, including amino acids, nucleosides, polypeptides, and indolealkylamines (IAAs). Among them, 15 compounds were unambiguously confirmed by comparison with standards. The CID-MS/MS fragmentation behaviors of seven indolealkylamine references were investigated to ascertain three types of structures. Subsequently, 11 high abundance contents of hydrophilic ingredients were determined from 11 batches of toad skin and 4 batches of toad venom by UPLC-QqQ-MS/MS. The quantitative results showed that the content of main IAAs in toad venom was much higher than in skin. In addition, the N-methyl serotonin (free IAA), bufothionine (combined IAA), and total IAAs sample were selected for anti-inflammatory evaluation in lipopolysaccharide (LPS) stimulated zebrafish embryo models. The obvious anti-inflammatory activities of IAAs were observed, especially for the free IAAs. This study illustrated IAAs were the main distinct hydrophilic components that probably lead to the difference between toad skin and toad venom in traditional applications.

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).

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 (T_max) 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.

NASH is a highly prevalent metabolic syndrome that has no specific approved agents up to now. BBBP, which mainly contains bile acids, possess various pharmacological properties and some bile acids are available for NASH treatment. Herein, the therapeutic effects and underlying mechanisms of BBBP against NASH were systemically evaluated. In this study, mice received an HFHS diet over a 20-week period to induce NASH with or without BBBP intervention were used to evaluate the effect and underlying mechanisms of BBBP against NASH. Our results demonstrated that BBBP attenuated hepatic steatosis, reduced body weight gain and lipid concentrations, and improved sensitivity to insulin and tolerance to glucose in mice fed an HFHS diet. Metabolomics and transcriptomic analysis revealed that BBBP suppressed the arginine biosynthesis by up-regulating NOS3 expression and the PI3K-Akt signaling pathway was also regulated by BBBP, as indicated by 55 DEGs. Bioinformatic analysis predicted the regulatory effect of the FXR/PXR-PI3K-AKT-NOS3 axis on arginine biosynthesis-related metabolites. These results were further confirmed by the significantly increased mRNA and protein levels of NOS3, PI3K (Pik3r2), and AKT1. And the increased levels of arginine biosynthesis related-metabolites, such as urea, aspartic acid, glutamic acid, citrulline, arginine, and ornithine, were confirmed accurately based on targeted metabolomics analysis. Together, our study uncoded the complicated mechanisms of anti-NASH activities of BBBP, and provided critical evidence inspiring the discovery of innovative therapies based on BBBP in the treatment of NASH.