Safranal, crocin, crocetin, and picrocrocin are major known compounds in the stigma extract of Crocus sativus with various medicinal properties. Crocus cancellatus is another Crocus species that grows extensively in Iran’s various regions, such as the Kurdistan province. The predominant metabolites and biological properties of C. cancellatus have not yet been investigated. The ingredients of the stigma ethanol extract of C. cancellatus were investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS). The ROIMCR approach was performed to analyze the LC-MS full scan data sets. This method searches the MS regions of interest (ROI) data in the m/z domain and analyses the results using the multivariate curve-resolution alternating least squares (MCR-ALS) chemometrics technique for simultaneous resolution of two extracts. Also, the antiproliferative properties of C. cancellatus against MDA-MB-231 and MCF-7 cancer cells were examined by MTT, dual acridine orange/ethidium bromide test, Annexin V-FITC/PI, and zymography. The GC-MS and LC-MS untargeted metabolomics data analysis of the extract indicated the presence of cytotoxic agents including safranal, crocin, picrocrocin, and crocetin in the stigma ethanol extract of C. cancellatus. Biological tests showed that the viability of MDA-MB-231 and MCF-7 cancer cells is decreased following C. cancellatus treatment in a time- and dose-dependent way in both monolayer and 3D cell cultures. The MCF-7 cell spheroids had greater resistance to the cytotoxic activity of the extract in 3D cell culture than the MDA-MB-231 cell spheroids. The morphological changes of the cells treated with C. cancellatus stigmas extract were indicative of apoptosis. Zymography analysis revealed a similar trend of matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) activity in the treated cells with C. cancellatus extract in comparison with doxorubicin treatment as a positive control. The findings of this research indicate that the ethanolic extract of C. cancellatus stigmas was a good source of bioactive metabolites with anticancer activity.
