Untargeted metabolomics using UHPLC‐Q‐Orbitrap HRMS for identifying cytotoxic compounds on MCF‐7 breast cancer cells from Annona muricata Linn leaf extracts as potential anticancer agents

Abstract Introduction The leaves of Annona muricata L., known as “soursop” or “ sirsak ” in Indonesia, are used traditionally for cancer treatment. However, the bioactive components remain largely unidentified. Objective This study used untargeted liquid chromatography–tandem mass spectrometry (LC‐MS/MS)‐based metabolomics to identify potential cytotoxic compounds in A. muricata leaf extracts on MCF‐7 breast cancer cells in vitro. Methods A. muricata leaves were macerated with water, 99% ethanol, and aqueous mixtures containing 30%, 50%, and 80% ethanol. Cytotoxic activity of the extracts against MCF‐7 breast cancer cells was determined using the MTT assay. Ultra‐high‐performance liquid chromatography–Q‐Orbitrap high‐resolution mass spectroscopy (UHPLC‐Q‐Orbitrap‐HRMS) was used to characterize the metabolite composition of each extract. The correlations between metabolite profile and cytotoxic activities were evaluated using orthogonal partial least square discriminant analysis (OPLS‐DA). The binding of these bioactive compounds to the tumorigenic alpha‐estrogen receptor (3ERT) was then evaluated by in silico docking simulations. Results Ninety‐nine percent ethanol extracts demonstrated the greatest potency for reducing MCF‐7 cell viability (IC 50 = 22 μg/ml). We detected 35 metabolites in ethanol extracts, including alkaloids, flavonoids, and acetogenins. OPLS‐DA predicted that annoreticuin, squadiolin C, and xylopine, and six unknown acetogenin metabolites, might reduce MCF‐7 cell viability. In silico analysis predicted that annoreticuin, squadiolin C, and xylopine bind to 3ERT with an affinity comparable to doxorubicin. Conclusion Untargeted metabolomics and in silico modeling identified cytotoxic compounds on MCF‐7 cells and binding affinity to 3ERT in A. muricata leaf extracts. The findings need to be further verified to prove the screening results.