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

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: = 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.