Integrated UHPLC-Q-TOF/MS and Liver-on-a-Chip Evaluation of Chemical Composition Changes and Hepatotoxicity Differences in Yaomu Before and After Fermentation

Background: Huafeng Dan (HFD) is a traditional famous medicine from Guizhou Province, commonly used for the treatment of stroke-induced hemiplegia and epilepsy. Yaomu is a key component and serves as the sovereign herb in the formula. Most of the components of Yaomu are toxic Chinese herbal medicines. Traditional fermentation processing methods are required to reduce its toxicity. Purpose: Current studies have not yet systematically analyzed the chemical constituents before and after fermentation. Meanwhile, there is a lack of safety evaluation before and after the fermentation of Yaomu, which can provide a basis for safe clinical medication. Method: Chemical constituents of Yaomu before and after processing were analyzed using UHPLC-Q/TOF-MS to compare compositional changes induced by fermentation. To further screen potential toxic components, representative compounds were selected from these differential compounds based on statistical indicators (such as VIP value), low cost and easy availability, as well as criteria from the literature, and the content changes before and after fermentation were investigated. In vitro toxicity was evaluated using a microfluidic liver organ-on-a-chip model to assess the toxic effects of Yaomu extracts before and after fermentation. Results: Studies have shown that in both positive and negative ionization modes, a total of 361 compounds were annotated in unfermented Yaomu. After fermentation, a total of 350 compounds were annotated. Multivariate statistical analysis revealed significant differences in the chemical composition of Yaomu before and after fermentation. Quantitative analysis demonstrated that the levels of diester-type diterpenoid alkaloids were significantly reduced after fermentation, accompanied by concurrent decreases in lysophosphatidylcholine (LPC) species, compared with unfermented Yaomu. In contrast, the concentrations of amino alcohol-type diterpenoid alkaloids were significantly increased. The microfluidic liver organ-on-a-chip results demonstrated that the post-fermentation extract caused significantly attenuated impairment of hepatocellular function and viability. The in vitro toxicity findings showed good concordance.