Comparative Metabolomics Identifies Diterpene-Rich Marine Macroalgae with Potent Antiproliferative Activity Against...

Marine macroalgae constitutes an important source of structurally diverse secondary metabolites with significant pharmaceutical potential. In the present study, an untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomic approach was integrated with biological screening to investigate the relationship between metabolite composition and antiproliferative activity in eight Mediterranean macroalgal species. Crude organic extracts were evaluated against the human colorectal cancer cell lines HT-29 and HCT-116, while the most active extracts were further investigated by flow cytometry to explore their cellular effects. Comparative metabolomic analysis revealed marked interspecific differences, with the brown algae Dictyota dichotoma and Rugulopteryx okamurae exhibiting metabolomes enriched in lipophilic diterpenoids and showing the highest cytotoxic activity. Although the bioactive compounds were not isolated, the antiproliferative effects were associated with diterpene-rich metabolite profiles containing tentatively annotated compounds such as dictyodial- and dilkamural-related diterpenoids tentatively. D. dichotoma displayed potent activity against both HT-29 and HCT-116 cells (GI₅₀ ≈ 4.5-5 μg mL^-1), whereas R. okamurae was markedly more active against HT-29 than HCT-116 cells, demonstrating that tumour genetic background substantially influences cellular susceptibility to macroalgal extracts and highlighting the importance of evaluating multiple cancer cell models during preliminary screening. Flow cytometry demonstrated increased intracellular reactive oxygen species (ROS), reduced metabolic viability, and decreased cell concentration following treatment with the most active extracts, whereas Annexin V/propidium iodide staining showed no detectable increase in apoptotic cell populations. These findings suggest that oxidative stress-associated metabolic impairment, rather than classical apoptosis, contributes to the observed antiproliferative activity. Overall, this study demonstrates the value of integrating untargeted metabolomics with comparative biological screening to identify promising marine macroalgae and metabolite classes for future bioassay-guided isolation and development of marine-derived anticancer agents.