ΕΡΕΥΝΗΤΙΚΟ ΠΡΟΓΡΑΜΜΑ: ΑΡΧΙΜΗΔΗΣ ΙΙΙ "Ενίσχυση Ερευνητικών Ομάδων ΤΕΙ"

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1 ΕΛΛΗΝΙΚΗ ΔΗΜΟΚΡΑΤΙΑ ΤΕΧΝΟΛΟΓΙΚΟ ΕΚΠΑΙΔΕΥΤΙΚΟ ΙΔΡΥΜΑ (Τ.Ε.Ι.) ΑΘΗΝΑΣ ΕΡΕΥΝΗΤΙΚΟ ΠΡΟΓΡΑΜΜΑ: ΑΡΧΙΜΗΔΗΣ ΙΙΙ "Ενίσχυση Ερευνητικών Ομάδων ΤΕΙ" Υποέργο:23 Τίτλος: ««ΣΥΝΘΕΣΗ ΑΝΑΛΟΓΩΝ ΤΗΣ ΥΔΡΟΞΥΤΥΡΟΣΟΛΗΣ (ΗΤ), ΜΕΛΕΤΗ ΤΗΣ ΑΝΤΙΟΞΕΙΔΩΤΙΚΗΣ ΔΡΑΣΗΣ ΚΑΙ ΤΩΝ ΦΥΣΙΚΟΧΗΜΙΚΩΝ ΙΔΙΟΤΗΤΩΝ ΤΟΥΣ»» Επιστημονικός Υπεύθυνος: Χάρης Ε. Σεμιδαλάς ΠΑΡΑΔΟΤΕΟ ΕΠΙΣΤΗΜΟΝΙΚΗ ΕΡΓΑΣΙΑ ΣΕ ΔΙΕΘΝΕΣ ΕΠΙΣΤΗΜΟΝΙΚΟ ΣΥΝΕΔΡΙΟ 8 th International Symposium on Computational Methods in Toxicology and Pharmacology Integrating Internet Resources, June 2-25, 205, Chios, Greece Τίτλος Παραδοτέου: «5.2 Μια παρουσίαση σε επιστημονικό συνέδριο» Τίτλος επιστημονικής εργασίας: «Membrane interaction studies of hydroxytyrosol (HT) and HT-analogues using differential scanning calorimetry, Raman spectroscopy and molecular dynamic simulations» στο Πλαίσιο του Πακέτου Εργασίας 5: Μελέτη της αλληλεπίδρασης των αναλόγων της υδροξυτυροσόλης με βιολογικές μεμβράνες μέσω διαφορικής θερμιδομετρίας σαρώσεως και φασματοσκοπίας Raman. Ιούνιος 205

2 Συντάκτες:. Χάρης Ε. Σεμιδαλάς, Επίκ. Καθηγητής, ΕΥ, Μέλος ΚΕΟ 2. Νικόλαος Ζόγκζας, Καθηγητής, Μέλος ΚΕΟ 3. Δήμητρα Μάργαρη, Τεχνολόγος Τροφίμων, Μέλος ΟΕΣ 4. Πέτρος Χατζηγεωργίου, Χημικός, Μέλος ΟΕΣ 5. Κυριάκος Βύρας, Ομότιμος Καθηγητής ΕΚΠΑ, Μέλος ΚΕΟ 6. Κώστας Ποταμίτης, Ερευνητής ΕΙΕ 7. Παναγιώτης Ζουμπουλάκης, Ερευνητής ΕΙΕ, Μέλος ΚΕΟ

3 Πίνακας περιεχομένων Σύντομη παρουσίαση της επιστημονικής εργασίας που παρουσιάστηκε στο συνέδριο... 3 Απόσπασμα προγράμματος συνεδρίου... 4 Η επιστημονική εργασία όπως δημοσιεύτηκε στα πρακτικά του συνεδρίου... 8 Διαφάνειες ή Poster παρουσίασης

4 Σύντομη παρουσίαση της επιστημονικής εργασίας που παρουσιάστηκε στο συνέδριο Α. Γενικά στοιχεία συνεδρίου Το 8 th International Symposium on Computational Methods in Toxicology and Pharmacology Integrating Internet Resources, πραγματοποιήθηκε στη Χίο κατά το διάστημα 2-25 Ιουνίου 205 στο ξενοδοχείο Chandris Hotel. Το διεθνές συνέδριο διοργανώθηκε από την Φαρμακευτική Σχολή του Αριστοτελείου Πανεπιστημίου Θεσσαλονίκης ( Β. Σύντομη παρουσίαση της επιστημονικής εργασίας που παρουσιάστηκε στο συνέδριο Η υδροξυτυροσόλη (ΗΤ) είναι ένα φυσικό αντιοξειδωτικό που εμφανίζει ένα ευρύ φάσμα βιολογικών δράσεων όπως αντιοξειδωτική, αντιμικροβιακή και αντιφλεγμονώδη για την αναστολή της οξείδωσης της LDL καθώς επίσης παρουσιάζει την ικανότητα να μειώνει τον κίνδυνο στεφανιαίας νόσου και αθηροσκλήρωσης. Ο σκοπός της παρούσης μελέτης ήταν να διερευνηθεί η αλληλεπίδραση μεταξύ ΗΤ και των αναλόγων της με μοντέλα μεμβρανών από DPPC χρησιμοποιώντας τη διαφορική θερμιδομετρία σαρώσεως (DSC), τη φασματοσκοπία Raman και προσομειώσεις μοριακής δυναμικής. 3

5 Απόσπασμα προγράμματος συνεδρίου 4

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7 ΕΡΕΥΝΗΤΙΚΟ ΠΡΟΓΡΑΜΜΑ: ΑΡΧΙΜΗΔΗΣ ΙΙΙ 6

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9 Η επιστημονική εργασία όπως δημοσιεύτηκε στα πρακτικά του συνεδρίου Membrane interaction studies of Hydroxytyrosol (HT) and HT-analogues using Differential Scanning Calorimetry, Raman spectroscopy and Molecular Dynamics simulations. Charis Semidalas, Nikolaos Zogkzas, 2 Dimitra Margari, Petros Chatzigeorgiou 3, Kyriakos Viras 3, Costas Potamitis 4, Panagiotis Zoumpoulakis 4 Laboratory of Organic Chemistry, Department of Food Technology, Technological Educational Institute of Athens 2 Laboratory of Food Engineering, Department of Food Technology, Technological Educational Institute of Athens 3 Laboratory of Physical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens 4 Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation Corresponding author chsemid@teiath.gr Hydroxytyrosol (HT) is a natural antioxidant exhibiting a broad spectrum of biological activities from antioxidant, antimicrobial and anti-inflammatory to LDL oxidation inhibition and capacity to reduce the risk of coronary heart disease and atherosclerosis.the aim of the present study was to investigate the interaction between HT and its analogues with DPPC model membranes using DSC, Raman spectroscopy and MD simulations. The thermal scan attributed to,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) bilayers shows two characteristic endothermic peaks referring to the pre-transition and the main transition respectively. In the presence of the hydroxytyrosol (HT) and its analogs the following observations have been made. At only 95:5 ratio of DPPC:HT (or analog) the pre-transition is suppressed indicating an effect in the head group regime of the DPPC. Furthermore, with increasing the guest molecule concentration the main transition temperature (T m ) and the transition cooperativity decrease monotonously. This shows that guest molecules exert an additional effect in the alkyl chains, when the concentration is increased. The peak height Raman intensity ratios I 090 /I 30, I 2850 /I 2880 and I 2935 /I 2880 at thirteen temperatures in the range of C have been calculated in order to study the intrachain and interchain interactions of the mentioned compounds with DPPC bilayers. Also, the inflection points of the graphs of the above ratios vs temperature have been determined. These points are close to the T m found from DSC thermograms and they correspond to the prepared liposomes of DPPC with the above compounds. The interaction mode of HT and its analogues with DPPC bilayers was further investigated with Molecular Dynamics (MD) simulations using Desmond 3.6 and OPLS 2005 force field. In order to study the partitioning of the studied compounds at the DPPC bilayer, several molecules of each compound were positioned (a) in the aqueous phase at a distance of 5-6 Å from the polar headgroups of DPPC and (b) 8

10 inside the lipid core of the lipid membrane. The merged system was solvated with TIP4P water molecules and the final system was subjected to MD simulations for 40 ns. Acknowledgement: This research has been co-financed by the European Union (European Social Fund ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: ARCHIMEDES III. Investing in knowledge society through the European Social Fund. 9

11 Διαφάνειες ή Poster παρουσίασης Membrane interaction studies of Hydroxytyrosol (HT) and HT-analogues using Differential Scanning Calorimetry, Raman spectroscopy and Molecular Dynamics simulations. Charis Semidalas, Nikolaos Zogzas, 2 Dimitra Margari, Petros Chatzigeorgiou 3, Kyriakos Viras 3, Costas Potamitis 4, Panagiotis Zoumpoulakis 4 Laboratory of Organic Chemistry, 2 Laboratory of Food Engineering, Department of Food Technology, Technological Educational Institute of Athens 3Laboratory of Physical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens 4Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation Hydroxytyrosol (HT) is a natural antioxidant exhibiting a broad spectrum of biological activities from antioxidant, antimicrobial and anti-inflammatory to LDL oxidation inhibition and capacity to reduce the risk of coronary heart disease and atherosclerosis.the aim of the present study was to investigate the interaction between HT and its analogs with DPPC model membranes using DSC, Raman spectroscopy and MD simulations. Ι: Hydroxytyrosol (HT), II: Homovanillyl alcohol, III: 4-(3,4-dimethoxyphenyl)butan--ol, IV: 4-(3,4-dihydroxyphenyl)butan--ol (HBT). Scheme : Hydroxytyrosol and it s analogs 0

12 Differential scanning calorimetry (DSC) measurements were performed in a Perkin- Elmer Pyris 6 calorimeter at a temperature range o C. The molar ratios of DPPC:compound were 95:5, 90:0, and 85:5 and the fully hydrated multilamellar dispersions were prepared by adding deionized water directly to the DSC pans containing weighed amounts of the dried samples, then sealing the pans. After the pans were sealed, repeated scanning and cooling was made at a rate 2.5 oc/min until identical thermograms were obtained. The thermal scan attributed to,2- Dipalmitoyl-3-sn-phosphatidylcholine (DPPC) bilayers shows two characteristic endothermic peaks referring to the pre-transition and the main transition respectively. At only 95:5 ratio of DPPC:HT (or analog) the pre-transition is suppressed indicating an effect in the head group regime of the DPPC. Furthermore, with increasing the guest molecule concentration the main transition temperature (T m ) and the transition cooperativity decrease monotonously. This shows that guest molecules exert an additional effect in the alkyl chains, when the concentration is increased (Schemes 2 and 3). Scheme 2: DSC scans of fully hydrated pure DPPC bilayers and bilayers of DPPC mixed with HT at DPPC:HT molar ratios 95:5, 90:0, and 85:5.

13 Scheme 3: DSC scans of fully hydrated pure DPPC bilayers and bilayers of DPPC mixed with HBT at DPPC:HBT molar ratios 95:5, 90:0, and 85:5. The full width at halfmaximum (FWHM) of the main phase transition is greater than of the corresponding of HT. The Perkin-Elmer spectrometer GX-FT Raman was used for taking the spectra of Scheme with a source of laser Nd:YAG at 064 nm, with a laser power 400 mw at the sample. In order to obtain a good signal to noise ratio, 00 scans were co-added for the spectra. The peak height Raman intensity ratios I 090 /I 30, I 2850 /I 2880 and I 2935 /I 2880 at thirteen temperatures in the range of C have been calculated in order to study the intrachain and interchain interactions of the mentioned compounds with DPPC bilayers. Also, the inflection points of the graphs of the above ratios vs temperature have been determined. These points are close to the temperature T m of the main transition found from DSC thermograms and they correspond to the prepared liposomes of DPPC with the compounds I-V (Schemes 4, 5 and 6). 2

14 Scheme 4: Raman intensity ratio I 090 /I 30 of the compound IV with DPPC at thirteen temperatures. The value of this ratio shows the proportion of gauche and trans conformers of lipid bilayer chains and intramolecular interactions. The larger the size of that ratio, the more structures gauche prevail, the smaller the intermolecular interactions and thus the lipid bilayer shows greater liquidity. Scheme 5: Raman intensity ratio I 2850 /I 2880 of the compound IV with DPPC at thirteen temperatures. The value of this ratio is a measure of the interactions prevailing in lipid bilayers. The smaller the value of the ratio, the greater these. 3

15 Scheme 6: Raman intensity ratio I 2935 /I 2880 of the compound IV with DPPC at thirteen temperatures. The value of the ratio is a measure of disorder of lipid bilayers. Smaller size of that ratio means a stronger organization and greater value reveals loose packing of the chains. The initial system of the hydrated DPPC bilayer was retrieved from the preset lipid bilayers of Desmond (Schrodinger Suite 204). In order to study the partitioning of the two compounds (HT and HBT) at the DPPC bilayer, three molecules of each compound (6 in total) were placed in the aqueous phase, at a distance of 5-6 Å from the polar headgroups of DPPC and the same number (6) of molecules were placed inside the lipid core. The merged system (Fig. ) was solvated with 5059 TIP4P water molecules and the final system, consisted of atoms, was subjected to MD simulations for 40 ns. 4

16 Fig.. The initial system explicitly solvated with water molecules The energy of the system reached to a plateau in 5 ns. Compounds which were initially placed in the hydrophobic core moved outwards and stabilized at the polar headgroups forming H-bonds with the polar heads of choline groups and the diffused water molecules. Molecules which were initially placed in the aqueous phase also tend to move towards the polar head-groups of DPPC bilayers (Fig. 2). This effect is more pronounced for HBT since it bears a longer hydrocarbon chain with higher affinity for the aliphatic chains of the DPPC. 5

17 Fig. 2. The initial and final frame of the system along with the total energy (E) and Potential Energy (E_p) plots vs simulation time. 6

18 The disappearance of the pretransition phase bilayers in the DSC thermograms even in the smallest proportion (95:5) means interaction with the polar heads of choline bilayer. This is expected due the polarity of hydroxytyrosol and its analogs. The symmetry of the DSC peaks in the different proportions of the compounds due to good and complete homogeneity that confirms their admission to aliphatic bilayers. The peak height Raman intensity ratios reflect the transition behaviour of the different bilayers studied and in particular describe the fluidity of the membranes. MD simulations revealed the localisation of the two molecules in the mesophase of the lipid bilayers towards the polar headgroups. H-bond formation was observed for HT and HBT with phoshatidilocholine head groups and diffused water molecules in the bilayer. MD results are in agreement with the experimental ones of DSC thermograms. This research has been co-financed by the European Union (European Social Fund ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: ARCHIMEDES III. Investing in knowledge society through the European Social Fund. 7