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Alkaloids derived from Rhizoma Coptis (RC) has been widely applied to clinical treatments in China. However, the toxicity of RC and the alkaloids from RC remained controversial. The research is designed to clarify the cardiotoxic compounds found in RC.In this study, the real-time cellular analysis cardio system and the high-content analysis were applied to monitor the function of cardiomyocytes (CMs) in the treatment of nine alkaloids in RC. Luciferase-coupled adenosine triphosphate (ATP) assay was used to detect cell viability.The results showed that berberine, palmatine, berbamine, and oxyberberine were cardiotoxic, which resulted in arrhythmia and cardiac arrest on CMs in a time- and dose-dependent manner. Meanwhile, berbamine and oxyberberine caused shrinkage and detachment on CMs at 10 μM. Cytotoxicity was induced by these two compounds with decline in cell index and ATP depletion. Cardiotoxicity or cytotoxicity was not observed in the other five alkaloids within 10 μM.For the first time, the cardiotoxicity of the nine alkaloids was evaluated to clarify the cardiotoxic components in RC. Furthermore, the experimental evidences were provided to support the safety of drug application.