Brucine (BRU) and brucine N-oxide (BNO) are prominent, bioactive, and toxic alkaloids in crude and processed Semen Strychni. Studies have demonstrated that BRU and BNO possess comprehensive pharmacological activities, such as anti-inflammatory and analgesic. In this context, a comparative study of BRU and BNO was performed by combination analysis of in silico ADMET prediction, in vivo toxicity evaluation, and potential action mechanism exploration. ADMET prediction showed that BRU and BNO might induce liver injury, and BRU may have a stronger hepatoxic effect. The prediction was experimentally verified using the zebrafish model. The BRU-induced hepatotoxicity of zebrafish larvae had a dose-response relationship. The mechanism of BRU-induced hepatotoxicity might relate to phosphorylation, kinase activity, and signal transduction. By comparison, signal transduction and gap junctions might involve BNO-induced hepatotoxicity. Our results provided a better understanding of BRU- and BNO-induced hepatotoxicity. We also built a foundation to elucidate the material base of the hepatotoxicity of traditional Chinese medicine Semen Strychni.

Estimating the extent to which drugs inhibit uridine 5'-diphosphate-glucuronosyltransferases1A1 (UGT1A1) enzyme activity is important for predicting hepatotoxicity and neurotoxicity. UGT1A1 enzyme activity is commonly evaluated by detecting the elimination of bilirubin substrate or the generation of bilirubin glucuronides. However, the present methods are inadequate for accurately assessing bilirubin metabolism, selecting incubation conditions, and comparing different systems. Therefore, in our study, we first established a ultra-performance liquid chromatography (UPLC)-MS/MS method to identify bilirubin and bilirubin glucuronides. To ensure the reaction was linear, we performed assays to optimize the protein concentration and incubation time. Finally, we measured UGT1A1 activity using three different systems. Data revealed the optimum incubation conditions were 10 min with 0.5 mg/mL human liver microsomes (HLM), recombinant human UGT1A1 (rUGT1A1), and rat liver microsomes (RLM). Bilirubin glucuronidation obeyed Michaelis-Menten kinetics in all three systems. The diversity in bilirubin metabolism among species was revealed. rUGT1A1 had the strongest binding affinity for bilirubin, but the lowest metabolism velocity. Compared with the other systems, RLM exhibited a significant difference. It has the lowest CLint and the highest Km. The difference in parameters between three systems may be attributable to the species differences. In conclusion, these in vitro studies provide useful information regarding drug interactions and the prediction of toxicity for future studies.

Houttuyniae herba, the Saururaceae plant Houttuynia cordata Thunb., has multiple therapeutic effects, including clearing heat, eliminating toxins, reducing swelling, discharging pus, and relieving stagnation. It has a long history as an edible and medicinal plant in China. Phytochemical studies show that the main constituents include volatile oil, flavonoids, and alkaloids. Aristolactam is a major alkaloid with a structure similar to toxic aristolochic acids. However, there has been no systematic study on aristolochic acids and alkaloids in Houttuyniae herba. Therefore, in this study, an LC–MS/MS method was developed to simultaneously detect seven alkaloids and five aristolochic acids in Houttuyniae herba from different origins. Six alkaloids (O-demethyl nornucifrine, N-nornucifrine, aristololactam AII, aristololactam FI, aristololactam BII, cepharadione B) were found and quantitatively determined in 75 batches of samples. Meanwhile, no aristolochic acids or aristololactams were found in Houttuyniae herba at a limit of detection (LOD) of ≤4 ng/mL. The method developed was fully validated in terms of LOD, limit of quantification (LOQ), linearity, precision, accuracy, and stability. These data clarify the content of the above safety-related components in Houttuyniae herba and provide a reference for further research into its safety.

Tripterygium glycosides tablets (TGTs) are widely used in clinical practice to treat rheumatoid arthritis and other autoimmune diseases, with significant beneficial effects but also high toxicity, necessitating rigorous quality evaluation and control. In current study, a rapid resolution liquid chromatography tandem electrospray ionization triple quadrupole mass spectrometry (RRLC–ESI–MS/MS) method was developed and validated for the quantitative analysis of 14 components of ten batches of TGTs produced by different manufacturers, including four diterpenoids, three triterpenoids, and seven sesquiterpene alkaloids. Meanwhile, the NO inhibition effects of these TGTs were evaluated in LPS-induced RAW264.7 cells for their downstream anti-inflammatory activities, as well as their cytotoxicity. The results indicate that the TGTs from different manufacturers showed poor quality consistency, as evidenced by large variations in chemical profiles and biological effects, which may increase the risks associated with clinical use. To improve the quality status of TGTs, it is crucial to identify indicator components whose characterization can accurately reflect the efficacy and toxicity of TGTs from which they were derived. Our study reveals that triptolide, triptoquinone B, celastrol, and demethylzelaysteral considerably contributed to the anti-inflammatory activity and/or cytotoxicity of TGTs, implying that they should be further investigated as candidate indicator components for TGT quality control.

Long dan Xie gan pill is a traditional complex compound preparation with a long history for treatment of diseases, including hepatocolic hygropyrexia, dizziness, tinnitus, and deafness. Quality of products from different manufacturers may be varied. Since the current standard could not control the quality of products in a comprehensive and effective way, this study aimed at establishing a practical and convenient approach for holistic quality control of the preparation. This study included both qualitative and quantitative works to get information on the overall composition and main components, respectively. As a result, HPLC fingerprint (UV 240 nm) similarities of all fifty samples were in the range of 0.65∼0.99. Results indicated that there was a difference among products from different manufacturers. Additionally, ten characteristic peaks of the fingerprint were tentatively identified by LC-MS. Further chemometrics analysis was utilized to evaluate the products from different manufacturers. At the same time, the HPLC (UV 285 nm) multicomponent quantification result showed that contents of gentiopicrin, baicalin, baicalein, and wogonin were in the range of 0.61–5.40, 1.96–5.33, 0.10–3.40, and 0.046–1.16 mg·g⁻¹, respectively. Data analysis verified the main different component of baicalein from the fingerprint statistical analysis. It is worth mentioning that the qualitative fingerprint and quantitative multicomponent determination were simultaneously accomplished by HPLC-DAD with dual channels. The study provided sound basis for improving quality control standards. This study also provided practical strategy for overall quality control of traditional Chinese medicines.