**New Affordable Methods for Large-Scale Isolation of Major Olive Secoiridoids and Systematic Comparative Study of Their Antiproliferative/Cytotoxic Effect on Multiple Cancer Cell Lines of Different Cancer Origins**

**Aikaterini Papakonstantinou 1,2, Petrina Koumarianou 1,3,†, Aimilia Rigakou 2,†, Panagiotis Diamantakos 2, Efseveia Frakolaki 4,‡, Niki Vassilaki 4, Evangelia Chavdoula 5, Eleni Melliou 2,6, Prokopios Magiatis 2,\* and Haralabia Boleti 1,3,\***


**Abstract:** Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for largescale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with halfmaximal effective concentrations (EC50) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC50 ≤ 50 μM). Oleocanthal (**1**) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC50: 9–20 μM). The relative effectiveness of the six OOPs was: oleocanthal (**1**) > oleuropein aglycone (**3a,b**) > ligstroside aglycone (**4a,b**) > oleacein (**2**) > oleomissional (**6a,b,c**) > oleocanthalic acid (**7**). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers.

**Keywords:** olive oil phenols; olive secoiridoids; oleocanthal; oleacein; antiproliferative bioactivity; cytotoxic bioactivity; pro-apoptotic effect; human cancer cells; health protection
