Pterocephalus hookeri(C. B. Clarke) Höeck, recorded in the Chinese Pharmacopoeia (2015 version) as a Tibetan medicine for the treatment of various diseases, especially rheumatoid arthritis, was believed to possess a slight toxicity. However, hardly any research has been carried out about it. The present study aimed to evaluate the toxicity in vivo and in vitro. Toxicity was observed by the evaluation of mice weight loss and histopathological changes in the liver. Then, the comparison research between ethyl acetate extract (EAE) and n-butanol extract (BUE) suggested that liver toxicity was mainly induced by BUE. The mechanical study suggested that BUE-induced liver toxicity was closely associated with necrosis detected by MTT and propidium iodide (PI) staining, via releasing lactate dehydrogenase (LDH), reducing the fluidity, and increasing the permeability of the cell membrane. Western blot analysis confirmed that the necrosis occurred molecularly by the up-regulation of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), as well as the activation of the nuclear factor-kappa-gene binding (NF-κB) signaling pathway in vivo and in vitro. This finding indicated that the liver toxicity induced by BUE fromP. hookeriwas mainly caused by necrosis, which provides an important theoretical support for further evaluation of the safety of this folk medicine.Keywords:Pterocephalus hookeri;liver toxicity;n-butanol extract;necrosis;inflammationKey Contribution:(1)Pterocephalus hookericould induce liver toxicity in vivo and in vitro. (2) The liver toxicity was induced by n-butanol extract ofP. hookeri. (3) The liver toxicity was mainly caused by necrosis associated with inflammation. (4) This finding provides an important theoretical support for further evaluation of the safety of this Tibetan medicine.
Background Pterocephalus hookeri (C. B. Clarke) Höeck , a Tibetan medicine widely used for treatment of rheumatoid arthritis , was recorded in Chinese Pharmacopoeia (2020 version) with slight toxicity. The liver injury was observed in mice with administration of n-butanol extract (BUE) in our previously study. However, the toxic components and the mechanism were still unrevealed. Purpose The present study was aimed to isolate and structural elucidate of the toxic compound pterocephin A (PA), as well as evaluate its liver toxicity and investigate its mechanism. Methods PA was isolated from the BUE of P. hookeri . Its structure was determined by analysis of HRMS, NMR and ECD data. L-02 cellular viability , LDH , ALT , AST , ROS, intracellular Ca 2+ and the fluidity of cell membrane were assessed by multifunctional microplate reader. The PI staining, cell membrane permeability assessment, and mitochondrial fluorescence staining analysis were determined through the fluorescence microscope . Liver samples for mice were assessed by pathological and immunohistochemistry analysis. Expression levels of indicated proteins were measured by western blotting assays. Results PA was determined as a previously undescribed oleanolane-type triterpenoid saponin . In vitro study revealed PA significantly induced hepatotoxicity by inhibition of L-02 cell growth, abnormally elevation of ALT and AST. Mechanically, PA induced the damage of cell membrane, fragmentation of mitochondria, and subsequently increase of intracellular Ca 2+ and ROS levels, which trigged by necroptosis with the activation of RIP1 and NF-κB signaling pathways . In vivo study confirmed PA could induce liver injury in mice with observation of the body weight loss , increasing of serum ALT and AST, and the histopathological changes in liver tissues. Conclusion Our present study indicated that PA was an undescribed toxic constituent in P. hookeri to induce liver injury in mice by activation of necroptosis and inflammation. And the findings are of great significance for the clinical use safely of this herb.
危险品化学品经营许可证（带存储）