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Bouloukostae 2015.Pdf - 1 - “Ιf you do not change direction you may end up where you are heading. “ Lao Tzu - 2 - Thesis Title (in English): “Investigation into the biological properties of Origanum onites essential oil against colon carcinoma growth” Τίτλος Πτυχιακής Εργασίας (στα Ελληνικά): “Διερεύνηση της βιολογικής δράσης αιθέριου ελαίου Origanum onites στην ανάπτυξη καρκινώματος παχέος εντέρου” - 3 - Ευχαριστίες (Acknowledgments) Με την ολοκλήρωση της παρούσας διπλωματικής ολοκληρώνεται ο κύκλος των προπτυχιακών μου σπουδών. Τα τέσσερα αυτά χρόνια αποτέλεσαν μια συναρπαστική διαδρομή, γεμάτη όμορφες αναμνήσεις και εμπειρίες, οι οποίες συνέβαλαν στην διαμόρφωση και στην ωρίμανση μου σαν άνθρωπο και σαν επιστήμονα, και για αυτό νιώθω την ανάγκη να ευχαριστήσω όσους συνέβαλαν σε μικρό ή μεγάλο βαθμό σε αυτή την πορεία. Η παρούσα διπλωματική εργασία πραγματοποιήθηκε στο εργαστήριο Μοριακής Ανοσοβιολογίας, του Τμήματος Μοριακής Βιολογίας και Γενετικής του Δημοκρίτειου Πανεπιστημίου Θράκης, υπό την επίβλεψη της Επίκουρης καθηγήτριας κ. Αικατερίνης Χλίχλια. Θα ήθελα λοιπόν, να της απευθύνω θερμές για την εμπιστοσύνη και την πολύτιμη υποστήριξη που μου έδειξε. Στα πλαίσια της διπλωματικής μου μου δόθηκε η ευκαιρία να συνεργαστώ με εξαιρετικούς ανθρώπους, σε ένα ευχάριστο και συνεργάσιμο περιβάλλον. Πρώτα από όλα, θα ήθελα να ευχαριστήσω την υποψήφια διδάκτωρ, Σπυριδοπούλου Κατερίνα, τον μεταπτυχιακό φοιτητή Αϊνδελή Γεώργιο και την Τιπτιρή-Κουρπέτη Αγγελική για την καθοδήγηση, την εκμάθηση, την υπομονή και την άμεση βοήθεια που μου παρείχαν. Ακόμη, αισθάνομαι ιδιαίτερη ανάγκη να ευχαριστήσω όλα τα παιδιά του εργαστηρίου, την Τομπουλίδου Ευγενία, Θεοφανίδου Θεοδώρα, Κουκουλάκη Αικατερίνη, Αραβίδου Ταμάρα, Γεωργαλλή Μαρία για την άψογη συνεργασία και την φιλική διάθεση. Σημαντική ήταν και η συμβολή των μελών γειτονικών εργαστηρίων με τους οποίους ανέπτυξα σχέσεις και είχα την χαρά να συνεργαστώ. Επίσης, χρωστώ ένα βαθύ ευχαριστώ στην πολύ καλή φίλη Τοκαμάνη Μαρία για την συνεχή συμπαράσταση και τις συμβουλές που μου έχει δώσει. Θα ήθελα, επίσης, να ευχαριστήσω τους συνεργάτες μας, την Παπαβασιλοπούλου Ελένη για την παροχή του ελαίου Origanum onites και την ανάλυση της σύστασης του, τον Κοτσιανίδη Ιωάννη και την Λαμπριανίδου Ελευθερία για την βοήθεια τους σχετικά με την κυτταρομετρία ροής, και τον Βασιλειάδη Σταύρο για την φροντίδα των μυών (Mus Musculus). Τίποτα όμως από όλα αυτά δεν θα ήταν εφικτό χωρίς την ανεκτίμητη υποστήριξη των γονιών μου, Μαργαρίτα και Αθανάσιο. Σας ευχαριστώ για τον άνθρωπο που με βοηθήσατε να εξελιχθώ, για την αμέριστη υποστήριξη σας σε κάθε μου βήμα μέχρι σήμερα και τη δυνατότητα που μου παρείχατε για να ολοκληρώσω τις σπουδές μου. Ελπίζω να σας κάνω υπερήφανους. - 4 - Περίληψη (Abstract) Στο ερευνητικό πεδίο της φαρμακευτικής υπάρχει έντονη η τάση μελέτης διαφόρων φυτοχημικών για τη διερεύνηση των θεραπευτικών τους ιδιοτήτων. Το αιθέριο έλαιο που απομονώνεται από το φυτό ρίγανης Origanum onites L., είδος που συναντάται σε μέρη της Σικελίας ως την ανατολική Τουρκία, έχει διεγείρει το επιστημονικό ενδιαφέρον έχοντας, μεταξύ άλλων, αντιπαρασιτική, αντιμυκητιακή και αντιβακτηριακή δράση έναντι ποικίλων στελεχών. Παρ’ όλα αυτά, οι πιθανές αντικαρκινικές του ιδιότητες δεν έχουν πλήρως εξεταστεί in vitro, ενώ δεν υπάρχουν επίσημα προσβάσιμα δεδομένα σε in vivo δοκιμές. Στη παρούσα μελέτη έγινε μια αρχική εκτίμηση της πιθανής κυτταροτοξικής επίδρασης του ελαίου έναντι του καρκίνου του παχέος εντέρου στις δύο κολονικές καρκινικές κυτταρικές σειρές, την CT26, απομονωμένη από τον Mus musculus και την HT-29, ανθρώπινης προέλευσης με τη μέθοδο SRB, καταλήγοντας στις τιμές EC50 0.0077± 0.0003 % (v/v) και 0.0106 ± 0.0002 % (v/v), αντίστοιχα. Συμπληρωματικά, διερευνήθηκε η δυνατότητα επαγωγής της αντι-πολλαπλασιαστικής του ιδιότητας μέσω του μηχανισμού της απόπτωσης και της αναστολής της μετακίνησης (διήθησης) στα CT26 κύτταρα. Ακόμη, ελέγχθηκε η χημειοπροφυλακτική του δράση με την εκ στόματος του χορήγηση με τη μέθοδο gavage σε BALB/c ποντικούς που δέχτηκαν εκτοπική υποδόρια ένεση συνγονικών CT26 κυττάρων. Σημειώθηκε μείωση 55,6% του όγκου των ανεπτυγμένων απομονωμένων όγκων in vivo, ενώ παράλληλα, εξετάστηκαν τα επίπεδα κυτταροκινών σε δείγματα ορού των ζώων. Καταληκτικά, τέθηκαν οι βάσεις για την μελέτη in vitro και in vivo αντι- νεοπλασματικής δράσης του αιθέριου ελαίου Origanum onites σε καρκίνωμα του κόλονος. Ωστόσο, κρίνεται αναγκαίο να συνεχιστεί η - 5 - έρευνα στο συγκεκριμένο χώρο με σκοπό τον προσδιορισμό του υποκείμενου μηχανισμού δράσης του, αλλά και την αναζήτηση πιθανών τοξικών παρενεργειών με επίδραση φυσιολογικών κολονικών κυττάρων και με μακροχρόνια χορήγηση του in vivo. Μελλοντικά, θα ήταν δυνατή η εφαρμογή του στην αγορά ως πρόσθετο για την παραγωγή βιολειτουργικών τροφίμων, παρέχοντας έτσι στους καταναλωτές τις ευεργετικές του ιδιότητες. - 6 - Abstract Natural plant products have been used throughout human history for various purposes, such as in medicine. Recently, there is a noticeable progress on the discovery of plant-derived compounds playing a pivotal role in treating and/or preventing various ailments, including cancer. The essential oil isolated from the plant Origanum onites L. (OOEO), which is found in parts of Greece, Turkey and Sicily, has stimulated the scientific interest as it exhibits antiparasitic, antifungal and antibacterial activity against various strains. However, in vitro observations related to its anticancer and antitumor activity are limited and there are no published data describing in vivo experiments. In the current work, we investigated the OOEO cytotoxic effect against two colon cancer cell lines; CT26, isolated from Mus musculus and HT-29, of human origin using the SRB assay. The EC50 values are 0.0077± 0.0003 % (v/v) and 0.0106 ± 0.0002 % (v/v), for CT26 and HT-29 cells respectively. Additionally, the induction of apoptotic cell death as well as the inhibition of migration by OOEO were examined on CT26 cells. Moreover, its chemopreventive activity was evaluated after oral administration of oregano oil to BALB/c mice that subsequently were inoculated with syngeneic CT26 cells, resulting in a significant decrease of tumor volume (55,6%) in OOEO- treated mice. In parallel, IL—6 and IL-12 cytokines were detected in mouse serum. To conclude, the performed preliminary experiments examined the in vitro and in vivo anti-neoplastic effect of Origanum onites essential oil on colon carcinoma. However, further experiments are needed in order to determine the underlying molecular mechanisms and potential side effects after treating normal colon cells with OOEO or long-term in vivo OOEO administration. In - 7 - future, oregano oil could be a promising health-promoting additive for the production of functional foods. - 8 - CONTENTS 1. INTRODUCTION ....................................................................................... - 11 - 1.1 Natural Products In Treating Diseases .............................................................................- 12 - 1.1.1 Natural Products From Plants......................................................................................- 13 - 1.1.2 The Linkage Of Natural Product Chemistry And Metabolomics For The Drug Discovery ...............................................................................................................................- 13 - 1.1.3 Introduction to Essential Oils .......................................................................................- 14 - 1.2 Family Lamiaceae - A Large Family Of Aromatic Herbs And Shrubs ..........................- 16 - 1.2.1 Introduction to the genus Origanum ............................................................................- 16 - 1.2.1.1. General information about Origanum .................................................................- 17 - 1.2.1.2 Properties of its members ......................................................................................- 18 - 1.2.2 Characteristics of Origanum onites ................................................................................- 20 - 1.2.2.1 Properties of Origanum onites essential oil (OOEO) ...........................................- 22 - 1.2.2.2 Studies of Origanum onites essential oil (OOEO) against cancer .......................- 23 - 1.3 Colon cancer ........................................................................................................................- 24 - 1.3.1. Colon anatomy .............................................................................................................- 25 - 1.3.2 The stages of cancer development ...............................................................................- 25 - 1.3.3 Factors contributing to colon cancer development .....................................................- 26 - 1.3.4 Inflammation markers associated with colon cancer..................................................- 27 - 1.4 Chemoprevention ...............................................................................................................- 28 - 1.4.1 Identified Natural Products for contributing to treating colon cancer ......................- 29 - 1.4.2 The Role Of Essential Oils In Treating Colon Cancer .................................................- 30 - 1.5 Purpose .................................................................................................................................- 32 - 2. MATERIAL & METHODS ......................................................................... - 33 - 2.1 Cell Cultures ........................................................................................................................-
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