HPLC-Based Phytochemical Profiling and Untargeted Proteomics with Cytotoxicity Assessment of Ethanolic Extracts of Punica granatum on MCF-7 and C6 Cancer Cell Lines

Background and Aim: To perform HPLC-based phytochemical profiling and untargeted proteomic analysis of ethanolic extracts from Punica granatum, and evaluate their cytotoxic activity against MCF-7 and C6 cancer cell lines. The study aims to identify key bioactive compounds in the extract, analyze their molecular mechanisms through proteomic changes, and assess their potential anticancer properties. Materials and Methods: Pomegranate peels extracts were prepared using ethanol analyzed for phytochemical content, including alkaloids, flavonoids, and saponins. Cell viability was evaluated through the MTT assay. Advanced techniques like HPLC and LC-HRMS were used to analyze chemical compositions, while proteomics assays identified and quantified proteins through processes such as chromatography, and mass spectrometry. Results: RP-HPLC analysis revealed a greater diversity of compounds in inner Punica granatum peels across different wavelengths compared to outer peels. Comparative analysis of retention times and phytocomponents demonstrated both unique and shared compounds between the two peel types. LC-HRMS analysis of the outer peel identified 36 secondary metabolites, including important compounds like gallic acid, citric acid, and syringic acid. Finally, proteomic data highlighted significant changes in protein expression profiles in both MCF-7 (cancer) and HEK (normal) cells, suggesting potential biological effects of the pomegranate peel extracts on cellular processes. Conclusion: Anticancer activity was confirmed on MCF-7 (breast cancer) and C6 (glioblastoma) cell lines, with no significant toxicity on HEK 293 (non-cancerous) cells. These findings suggest potential for further cancer therapy development, with additional proteomic analysis planned.