Screening plant extracts for anticancer potential Characterization of active fractions from Melia dubia Cav and elucidation of their effects on cancer cell lines

Abstract

Cancer is a disease which still needs better therapeutic intervention in spite of the advances in the treatment modalities currently available, as some of the cancers are hard to cure and may cancers develop resistance against the currently used drugs. Plants are a repository of molecules with anticancer activities. To explore the anticancer activity of molecules in the plant extracts, we screened plant extracts and identified extracts with potential anticancer compounds and purified enriched fractions with anticancer molecules. In this study 426 extracts from 65 plants were screened for their cytotoxic effects on cancer cell lines. Among the 10 cancer cell lines tested many extracts showed high toxicity against many of the cell lines tested with minimum toxicity to normal cell line HaCaT. Notably, 28 extracts showed over 50% mortality in HT29 cells with minimal toxicity to HaCaT cells, demonstrating differential cytotoxicity. Similar results were obtained for other cancer lines, with 18 extracts selective for HCT116, 24 for A431, 21 for A498. This is the first report of anticancer activity in plants, such as Pancratium triflorum and Anamirta cocculus, Chassalia curviflora, Antigonon leptopus, Otacanthus caeruleus, Hewittia malabarica and Melia dubia. The chloroform extract of Hewittia malabarica aerial parts exhibited notable cytotoxicity against the human colorectal carcinoma cell line HT29, with an IC50 of 31.97±3.07 μg/mL. GC-MS analysis revealed 17 compounds, with tritetracontane and neodene being the most abundant. Pancratium triflorum extracts also displayed selective cytotoxicity with chloroform extract showing 92.36±0.81% mortality in HeLa cells. Conyza canadensis petroleum ether extract exhibited nearly complete mortality against PANC1 and HT29 cancer cell lines. Anamirta cocculus extract was most potent on the PA1 ovarian cancer cell line, achieving 98.64±0.40% mortality. Melia dubia extracts also demonstrated selective cytotoxicity with petroleum ether extract showed 97.48±1.77% mortality in PANC1 cancer cell lines. GC-MS analysis identified a variety of bioactive compounds, including tetratetracontane, (E)-Phytol, and 3-eicosene, which could contribute to their anticancer properties. The ethyl acetate extract of Anamirta cocculus exhibited the potent anticancer activity, showing high cytotoxicity against ovarian cancer (PA1) and colorectal adenocarcinoma (HT29) cell lines, with IC50 values of 8.30±0.38 μg/mL and 17.97±0.63 μg/mL, respectively. The ethyl acetate extract further inhibited colony formation in HT29 and PA1 cells, induced apoptosis, which was confirmed by characteristic markers such as membrane blebbing, chromatin condensation, and DNA fragmentation. The chloroform extract of Melia dubia leaves was subjected to fractionation through silica gel column chromatography to enrich fractions with anticancer activity. The crude extract showed an IC50 of 61.26±2.55 μg/mL, while the more purified fractions SC1-F8, SC2-F8, SC3-F8, and TLC-B3 exhibited IC50 values of 31.07±4.46, 17.86±1.29, 10.27±0.79, and 7.87±1.32 μg/mL, respectively. The fraction, TLC-B3, was subjected to GC-MS analysis, revealing ten compounds that are likely responsible for its potent anticancer effects. The cytotoxicity analysis of Melia dubia purified extracts revealed significant mortality in HT29, MCF7, PA1, and UM-SCC-83B cancer cell lines. The purified fraction, SC3-F8, effectively inhibited colony formation and cell migration and apoptosis in these lines. The purified fraction SC3-F8 demonstrated reduction in mitochondrial membrane potential (ΔΨM) after 48 hours. Annexin-V/propidium iodide co-staining showed an increase in apoptotic cells in a dose-dependent manner. DNA damage was evident as longer comet tail lengths were observed in all treated cancer cell lines. Additionally, SC3-F8 induced G1 phase cell cycle arrest and triggered the up regulation of pro-apoptotic genes while down regulating anti-apoptotic genes. The fraction also inhibited cancer cell migration and suppressed components of the epithelial-mesenchymal transition (EMT) pathway, potentially disrupting angiogenesis. SC1-F8 when incorporated into liposome resulted in decrease in IC50 from 31.07±4.46 μg/mL to 16.73±0.77 μg/mL indicating its usefulness for the delivery of anticancer molecules enriched from M. dubia. In silico molecular docking studies on the ligands identified by GC- MS from the TLC-B3 fraction of M.dubia on key apoptotic proteins, including caspase-3, caspase-9, and P53,resulted in identification of Petroselinic acid and 9-Octadecenoic acid as probable modulators of apoptosis in M.dubia. It is worth investigating the potential of these compounds as anticancer agents.