The phytochemical analysis of Carissa macrocarpa, cytotoxic potential of fractions, and the molecular modulation on p53 expression in HT-29 and LS174T cell lines

Natural products remain an invaluable source of anticancer agents, with flavonoids and phenolic compounds being particularly recognized for their ability to modulate tumour suppressor pathways. Carissa macrocarpa (C. macrocarpa) is an edible plant rich in secondary metabolites. This study sought to identify the secondary metabolites of C. macrocarpa leaves using LC–MS/MS, evaluate their cytotoxic and pro-apoptotic effects in colorectal cancer cell lines (HT-29 and LS174T), and elucidate potential molecular interactions with p53 through in silico docking, thereby uncovering mechanistic insights into its anticancer activity. LC–MS/MS revealed abundant flavonoid glycosides, particularly derivatives of kaempferol, quercetin, and isorhamnetin. Biological assays demonstrated potent cytotoxicity of the extracts, with the total extract showing the strongest effect against LS174T cells (IC₅₀ = 0.5 µg/mL). Extracts induced apoptosis, caused G1/S/G2 cell cycle arrest, and upregulated p53 expression. Docking confirmed strong binding affinities (−7.87 to −9.28 kcal/mol) for glycosylated flavonoids such as kaempferol-3-O-robinoside-7-O-rhamnoside, hesperidin, and isorhamnetin-3-O-rutinoside. Also, 100 ns molecular dynamics studies confirmed the stable binding of both kaempferol-3-O-robinoside-7-O-rhamnoside and hesperidin against the p53 pocket. In conclusion, this integrative study demonstrates that C. macrocarpa exerts anticancer effects in colorectal cancer cells by modulating p53 and provides a mechanistic rationale for its therapeutic potential.