Dissertation committee:
Σκαλτσόυνης Αλέξιος - Λέανδρος, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Μητάκου Σοφία, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Φωκιαλάκης Νικόλαος, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Πουλή Νικολαΐς, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Μαράκος Παναγιώτης, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Κωστάκης Ιωάννης, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Ζωΐδης Γρηγόριος, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
Various epidemiological studies have shown a low frequency of cardiovascular and neurodegenerative disease, diabetes and cancer, in the Mediterranean region. This low incidence is thought to be linked wth the comsumption of olives and olive oil, main constituents of South European diet. Phytochemical analysis of leaves of Olea europaea (Oleaceae), olive oil and Olive Mill Waste Waters (OMWW), has revealed the presence of a number of secondary metabolites, more specifically phenolic compounds, such as oleuropein, hydroxytyrosol, tyrosol, ligstroside, oleocanthal and oleacein.
The above mentioned phenolic compounds have been considered to be potently responsible for the biological activity of various olive products, especially due to their antioxidant properties. However, olive oil and edible olives’ production, leads to the formation of tones of waste, including leaves, table olives’ and olive mill waste waters. This waste is extremely toxic for the environment, consisting, nevertheless, a source of bioactive compounds.
During the preparation of this dissertation, extracts of olive leaves and drupes of Olea europaea, an olive oil sample and an OMWW extract, where phytochemicaly explored, using, mainly, chromatographical techniques and aiming at the isolation, identification and biological evaluation of secondary metabolites included.
Among these metabolites, oleuropein, a phenolic secoiridoid glucoside and main constituent of leaves and unprocessed drupes of Olea europaea, with a number of, mentioned in bibliography, biological activities, was used as a high purity raw material, for the semi-synthesis of secoiridoidic analogs, with variety in structure. Until recently, most bibliographical data regarding oleuropein, highlighted the phenolic part of oleuropein’s structure, as the part to be blamed for the molecule’s activity, while there is no study to date, concerning the enhancement of biological activity, through semi-synthesis of analogs, in the context of structure-activity relationship. Eventually, there is a significant interest in a study of this kind, given that, in the Mediterranean region, there is a high availability in olive leaves, thus in high purity oleuropein.
During this thesis, 37 semi-synthetic analogs of oleuropein where synthesized and evaluated, in a first screening, for their in vitro cytotoxicity against cancer cell line SKBR3 of breast adenocarcinoma. The analogs that showed the best activity in this first test, were further evaluated for their activity in vitro against six other cancer cell lines. The analog that showed the best cytotoxicity among the analogs tested, was then evaluated for its in vivo activity against cancer cell line B16.F1 of melanoma, in mouse models, as well as for its toxicity against normal cells too, illustrating impressive results.
Furthermore, triggered by biological data referring especially to the anti-inflammatory activity of oleacein and oleocanthal, 13 analogs were synthesized, among which, 9 were evaluated for their neuroprotective activity against Alzheimer’s disease, however without significant results.
Finally, in the synthetic part of the present study, based on bibliographical data regarding the antioxidant activity of the phenolic compound nuzhenide and taking as granted the low yield of this compound’s isolation from olive drupes’ cores, its total synthesis was attempted.
Αmong the synthesized analogs of oleacein and oleocanthal, 8 were evaluated for their activity agains enzymes cyclooxygenase-1 and 2 (COX-1 and COX-2), showing, in general, better inhibition of COX-2 than of COX-1, whithout illustrating, in total, any remarkable activity.
Last but not least, olive leaves’ and olive drupes’ extracts of Olea europaea, total extract of OMWW and the secondary metabolites isolated from this, were also evaluated for their anti-inflammatory activity against Alzheimer’s disease and against COX-1 and COX-2 enzymes. Among the samples tested, olive drupes’ extract showed the best activity against Alzheimer’s disease, inducing tight junction proteins and upregulating the amyloid beta transporter P-gp, thus this extract is appropriate for further phytochemical analysis.
