**1. Introduction**

Epidemiological data support the hypothesis that the Mediterranean diet (MD) may have an important role in preventing several types of cancer [1–4]. Furthermore, a clinical trial in which the diet was modified toward an improved adherence to MD showed a reduced total mortality and cancer risk after a 4-year follow-up [2]. A particular characteristic of the MD is that olive oil is the primary source of dietary lipids. The importance of olive oil in cancer prevention began to be highlighted by several epidemiological studies initiated in the mid-nineties which showed a decreased risk of cancer in different sites associated

**Citation:** Papakonstantinou, A.; Koumarianou, P.; Rigakou, A.; Diamantakos, P.; Frakolaki, E.; Vassilaki, N.; Chavdoula, E.; Melliou, E.; Magiatis, P.; Boleti, H. 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. *Int. J. Mol. Sci.* **2023**, *24*, 3. https://doi.org/ 10.3390/ijms24010003

Academic Editors: Barbara De Filippis, Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 25 October 2022 Revised: 12 December 2022 Accepted: 14 December 2022 Published: 20 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

with the uptake of olive oil [5]. Although these results have to be confirmed by extensive clinical trials, a recent pilot clinical trial with patients suffering from chronic lymphocytic leukemia demonstrated, for the first time, the anti-cancer therapeutic properties of olive oil containing high amounts of the polyphenols oleocanthal (**1**) and oleacein (**2**) [6]. Although the term polyphenols is not chemically accurate when used for olive oil phenols, it is widely used in the formal legislation regarding the health claims of olive oil in the EU and for this reason it is also used herein.

The cancer-preventive capacity of olive oil could be mediated at least in part by the presence of minor components which include more than 230 chemical compounds present in a small amount (about 2% of oil weight). Among these components, of particular interest are the different classes of phenolic compounds represented by phenolic acids, phenolic alcohols, flavonoids, secoiridoids and lignans. In particular, the phenolic alcohols hydroxytyrosol and tyrosol are abundantly present in olives, olive leaves and olive oil as both free compounds and linked to either elenolic acid (EA) or its dialdehydic form (EDA) giving rise to the secoiridoid derivatives oleuropein aglycone (3,4, DHPEA-EA) (**3a,b**), ligstroside aglycone (*p*-HPEA-EA) (**4a,b**), oleocanthal (*p*-HPEA-EDA) (**1**) and oleacein (3,4, DHPEA-EDA) (**2**) [7]. These compounds are not generally present in other types of oil and in other foods of vegetable origin. Their concentration in olive oil varies and depends upon several factors such as the variety of the olive tree, the agronomic conditions during cultivation and the maturity of the fruit during harvesting as well as the technological aspects of olive oil production, especially the time and temperature of malaxation [8]. Moreover, although compounds (**1**–**4**) are also found in other plants, olive oil is the only edible source providing them. The polyphenols found in olive oil have well-established beneficial effects on human health and metabolism [9–12].

A cancer chemo-preventive activity of olive oil has been attributed to hydroxytyrosol and tyrosol and their secoiridoid derivatives oleocanthal (**1**), oleacein (**2**), oleuropein aglycone (**3a,b**) and ligstroside aglycone (**4a,b**) [7,9,13–19]. Several studies have demonstrated that certain of these compounds can inhibit proliferation and induce apoptosis in different tumor cell lines and most animal studies have confirmed the ability of certain olive oil polyphenols (OOPs) to inhibit carcinogenesis both in the initiation and in the promotion/progression phases [19–33]. However, further investigations are necessary to clarify the real chemo-preventive potential of OOPs in humans, such as performing intervention studies on populations at high cancer risk [7]. This shows the urgency of performing in-depth investigations on the mechanism(s) of action of OOPs, in cell- and animal-based cancer models. Additionally, it is important to investigate the safety/toxicity issues of all the main phenolic ingredients of olive oil.

Recent advances in the development of a simple and rapid methodology for the direct identification and concentration measurement of each phenol in olive oil using quantitative 1H-nuclear magnetic resonance spectroscopy (qNMR) have offered a new perspective on the quality control of the health-protecting properties of olive oil [34,35]. During the last few years, several chromatographic techniques have been established for the isolation of each OOP, enlarging the spectrum of these natural products identified in olive oil [30,34–41]. In addition, several methods for their chemical synthesis have been published [42,43]. However, at present, there are no available methods based on selective extraction that could override the need for chromatographic purification. All existing methods are quite complicated or expensive for large-scale application, thus limiting the availability of OOPs as material for research or commercial purposes. In the present work, a variety of new methods for OOPs' isolation are presented which are easily applicable on a large scale, permitting the compounds' acquisition at appropriate amounts for further investigation of their bioactivities or for the production of commercial products.

Most studies concerning the cytotoxic or antitumor properties of OOPs have been performed so far with olive oil extracts, [16,25,44] hydroxytyrosol and tyrosol [13,45,46] and their secoiridoid derivatives oleocanthal (**1**) [20,24,29,31,47–49], oleacein (**2**) [19,26,50], oleuropein aglycone (**3a,b**) [17,27,51] and ligstroside aglycone (**4a,b**) [23,52,53]. No studies have been performed with the other polyphenols available today in pure form (i.e., oleokoronal (**5a,b,c**), oleomissional (**6a,b,c**) and oleocanthalic acid (**7**) [34,40]). Moreover, all these studies have been conducted with only one or a maximum of two compounds on one or up to very few cancer cell lines from one or a maximum of two cancer types by the same research team. Interestingly, all studies on the bioactivity of OOPs on cancer cell lines have been performed under the atmospheric O2 tension (20% (*v*/*v*)) used in cell culture and traditionally applied in drug screening. The O2 tensions, which in the body tissues and tumors are <5% (*v*/*v*) (tissue normoxia and hypoxia), have important effects in the bioenergetic metabolism of the cells affecting the sensitivity/response of tumors to anti-cancer agents [54–63].

The present study contributes to the current knowledge and fills gaps in the understanding of the potential antiproliferative/cytotoxic properties of OOPs by studying six different representatives of these phenols. These in turn are produced in the same laboratory specializing in the isolation and characterization of OOPs from olive oil or olive leaves coming from Greek varieties of olive trees, ensuring thereby the reproducibility of results for each tested compound [34,40,64,65]. Moreover, herein are presented results from the analysis of the antiproliferative/cytotoxic effects of six OOPs on a large number of cancer cell lines from eight different tissue origins and at conditions resembling the tumor microenvironment (i.e., hypoxia and serum starvation). This allows a systematic and reliable comparative analysis of their bioactivity on different tumors since the same methodology was applied and the OOPs were isolated by the same laboratory following standardized protocols. Finally, the test results of the antiproliferative/cytotoxic OOPs' action on non-tumorigenic cell lines are presented, contributing thereby to the search for the most effective ones with the fewest side effects on non-cancer cells.
