ABSTRACTBackgroundCelastrol is known as one of the most medicinally valuable compounds. However, the pharmaceutical application of celastrol is significantly limited due to high toxicity, while there are few reports on the mechanism of toxicity.MethodsThis study searched for possible toxic metabolites through phase I invitrometabolism and glutathione capture experiments. Then invivometabolism experiments in mice and rats were conducted to look for metabolites invivo. Finally, mice invivotoxicity experiment was conducted to verify the toxicity of different doses of celastrol to mice.ResultsIn the invivoand in vitrometabolism experiments, we found 7 phase I metabolites invitro, 9 glutathione conjugation metabolites invitro, and 20 metabolites invivo. The metabolic soft points of celastrol could be the quinone methyl structure at C3-OH and C6. In vivotoxicity experiments show that celastrol causes weight loss, diarrhea, gastrointestinal tract and liver inflammation in mice.ConclusionsThis study analyzed the metabolites and possible metabolic soft spots of celastrol, and its hepatotoxicity and gastrointestinal toxicity were demonstrated through invivostudies for the first time. The results might provide an important basis for potential structural modification to increase the druggability of celastrol.