Study of natural bioactive compounds with high resolution NMR spectroscopy of cryogenic technology and evaluation of their antitumor and antiangiogenic activity

The main objective of the current PhD thesis was the study of flavonoids with specific structural characteristics and substitutions which confer to their biological proeperties under investigation.Firstly, the structural characteristics of a group of selected flavonoids were studied by the use of 1D and 2D NMR spectroscopy. Furthermore, by the use of diffusion spectroscopy DOSY NMR a new methodology was developed for the separation and the identification of flavonoids in mixtures. Then, it was studied the mechanism of the antioxidant activity of flavonoids and their ability to chelate two essential metal ions, Zn2+ and Fe2+. NMR spectroscopic studies and DFT theoretical calculations indicate sites 3-4 and 4-5 as coordination sites of Zn2+ through the deprotonation of OH-3 and OH-5 for quercetin and luteolin, respectively. Also, it was observed that the complexation of quercetin with Zn2+ facilitates chelation with Fe2+ due to OH-3 deprotonation. This conclusion can effectively justify the improved antioxidant activities of flavonoids in in vitro studies when they are already complexed to metal ions. Moreover, we studied their antiproliferative activity against two human cancer cell lines and their structure-activity relationship. The presence of a strong intermolecular hydrogen bond in combination with two methoxy groups in positions 3 and 4’ as well as hydroxyl groups in positions 5, 7 and 3’ were found to be essential structural parameters for a potent antiproliferative activity.5,7,3’-trihydroxy-3,4’-dimethoxyflavone, which demonstrated the strongest anticancer activity, was selected in order to study its antiangiogenic activity. This flavonoid was able to inhibit VEGF-induced angiogenesis in HUVECs endothelial cells with a very low IC50 value. Thus, dimethoxyflavone should be promoted for further anticancer and antiagiogenic evaluation studies and can be a lead compound for the synthesis of more potent compounds.The flavonoid quercetin was selected in order to study its ability to induce apoptosis in T-leukemic cells. By the use of a multidisciplinary approach, in vitro experiments, biochemical and biophysical studies as well as docking calculations, it was suggested that quercetin induces apoptosis by directly binding the antiapoptotic proteins Bcl-2 and Bcl-xL in BH3 domain. A state-of-the-art technique was used in order to study the interaction of water-soluble quercetin derivatives with Bcl-2 protein. In-cell NMR spectroscopy indicated that this analogue interacts directly with Bcl-2 in intact Jurkat leukemic cancer cells that overexpress Bcl-2. This is the first report of the interaction of a mitochondrial membrane protein with a small molecule at a cellular level. Thus, in-cell NMR spectroscopy is a highthrouput and robust technique for the screening of drug-protein receptors interactions with promishing future applications.Σκοπός της παρούσας διδακτορικής διατριβής αποτελεί η μελέτη πρότυπων φλαβονοειδών που φέρουν συγκεκριμένα δομικά χαρακτηριστικά και υποκαταστάσεις ώστε να διαθέτουν βελτιωμένες βιολογικές ιδιότητες. Αρχικά, μελετήθηκαν τα δομικά χαρακτηριστικά και οι φυσικοχημικές ιδιότητες επιλεγμένων φλαβονοειδών με χρήση φασματοσκοπίας NMR 1D και 2D. Στη συνέχεια με χρήση φασματοσκοπίας διάχυσης DOSY NMR αναπτύχθηκε μια νέα μεθοδολογία για τον ποιοτικό διαχωρισμό και την ταυτοποίηση φλαβονοειδών σε μίγματα. Έπειτα, μελετήθηκε ο μηχανισμός της αντιοξειδωτικής δραστικότητας των φλαβονοειδών και η ικανότητά τους να συμπλοκοποιούνται με δύο σημαντικά μεταλλικά ιόντα, τον Zn2+ και τον Fe2+. Μελέτες φασματοσκοπίας 1D NMR και θεωρητικοί υπολογισμοί σε επίπεδο DFT υπέδειξαν ως θέσεις συμπλοκοποίησης του Zn2+ τις 3-4 και 4-5 για την κερσετίνη και την λουτεολίνη, αντίστοιχα, με αποπρωτονίωση των ΟΗ-3 για την πρώτη περίπτωση και του ΟΗ-5 για την δεύτερη. Επίσης, διαπιστώθηκε ότι η συμπλοκοποίηση της κερσετίνης με Zn2+ υποβοηθά τη σύμπλεξη με τον Fe2+ λόγω αποπρωτονίωσης. Η διαπίστωση αυτή μπορεί να αποτελέσει μια ικανή επεξήγηση του φαινομένου της βελτιωμένης αντιοξειδωτικής ικανότητας των φλαβονοειδών όταν είναι ήδη σε συμπλοκοποιημένη μορφή που έχει παρατηρηθεί σε προηγούμενες in vitro μελέτες.Στη συνέχεια πραγματοποιήθηκε η μελέτη της αντιπολλαπλασιαστικής τους δράσης ενάντια σε δύο ανθρώπινες καρκινικές κυτταρικές σειρές και η συσχέτιση των δομικών χαρακτηριστικών με τη δραστικότητα. Διαπιστώθηκε ότι τα φλαβονοειδή με τον ισχυρότερο ενδομοριακό δεσμό έχουν βελτιωμένη δραστικότητα, ενώ η παρουσία μεθόξυ υποκαταστατών στις θέσεις 3 και 4’ και υδροξυλομάδων στις 5,7 και 3’ είναι απαραίτητη για ισχυρή αντιπολλαπλασιαστική δράση.Για την αντιαγγειογενετική δραστικότητα επιλέχθηκε να μελετηθεί το φλαβονοειδές 5,7,3’-τριυδροξυ-3,4’-διμεθοξυφλαβόνη, το οποίο παρουσίασε και τη σημαντικότερη αντικαρκινική δράση in vitro. Από τη μελέτη αυτή προέκυψε ότι το φλαβονοειδές αυτό αναστέλλει την επαγώμενη από τον VEGF αγγειογένεση στα ενδοθηλιακά κύτταρα HUVECs και αποτελεί ένα προτεινόμενο μηχανισμό. Η διμεθοξυφλαβόνη, συνεπώς, μπορεί να αποτελέσει ένωση «οδηγό» και να προωθηθεί για μελλοντικές μελέτες αντικαρκινικών και αντιαγγειογενετικών ιδιοτήτων τόσο της ίδιας όσο και πιο δραστικών αναλόγων της. Το φλαβονοειδές κερσετίνη επιλέχθηκε για την μελέτη της ικανότητάς του να επιφέρει απόπτωση σε Τ λευχαιμικά κύτταρα. Mε τη χρήση in vitro πειραμάτων αλλά και βιοχημικών και φυσικοχημικών μελετών και υπολογισμών πρόσδεσης, διαπιστώθηκε ότι η κερσετίνη αλληλεπιδρά άμεσα με τις αντιαποπτωτικές πρωτεΐνες Bcl-2 και Bcl-xL στον τομέα ΒΗ3 επάγωντας απόπτωση. Με τη χρήση της φασματοσκοπίας in-cell NMR επιτεύχθηκε η μελέτη της αλληλεπίδρασης ενός συνθετικού υδατοδιαλυτού αναλόγου της κερσετίνης με την Bcl-2 πρωτεΐνη μέσα σε ζωντανά κύτταρα Jurkat που υπερεκφράζουν την πρωτεΐνη αυτή. Η μελέτη αλληλεπίδρασης της Bcl-2 με μικρά μόρια αποτελεί την πρώτη σε διεθνές επίπεδο μελέτη μιτοχονδριακής μεμβρανικής πρωτεΐνης σε κυτταρικό επίπεδο. Συνεπώς, η φασματοσκοπία in-cell NMR αποτελεί ένα δυναμικό εργαλείο σάρωσης αλληλεπιδράσεων φαρμάκων και πρωτεϊνικών στόχων με δυνατότητα εξέλιξης και ιδιαίτερα υποσχόμενες μελλοντικές εφαρμογές

Structural Basis of Artemisinin Binding Sites in Serum Albumin with the Combined Use of NMR and Docking Calculations

Artemisinin is known to bind to the main plasma protein carrier serum albumin (SA); however, there are no atomic level structural data regarding its binding mode with serum albumin. Herein, we employed a combined strategy of saturation transfer difference (STD), transfer nuclear Overhauser effect spectroscopy (TR-NOESY), STD–total correlation spectroscopy (STD-TOCSY), and Interligand Noes for PHArmacophore Mapping (INPHARMA) NMR methods and molecular docking calculations to investigate the structural basis of the interaction of artemisinin with human and bovine serum albumin (HSA/BSA). A significant number of inter-ligand NOEs between artemisinin and the drugs warfarin and ibuprofen as well as docking calculations were interpreted in terms of competitive binding modes of artemisinin in the warfarin (FA7) and ibuprofen (FA4) binding sites. STD NMR experiments demonstrate that artemisinin is the main analyte for the interaction of the A. annua extract with BSA. The combined strategy of NMR and docking calculations of the present work could be of general interest in the identification of the molecular basis of the interactions of natural products with their receptors even within a complex crude extract

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis–Menten kinetics with Km and Vmax values of 21 ± 1.6 μM and 44.8 ± 4.0 U × mg−1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development

DFT Calculations of 1H- and 13C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution

A density functional theory (DFT) study of the 1H- and 13C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution high resolution nuclear magnetic resonance (NMR). The experimental 1H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The “inside” olefinic protons of the conjugated double bonds are deshielded than those of the “outside” protons. Furthermore, in the cis/trans isomers, the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental 1H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest energy DFT optimized single conformer, for all functionals and basis sets used. The other low energy conformers have negligible effects on the computational 1H-NMR chemical shifts. We conclude that proton NMR chemical shifts are more discriminating than carbon, and DFT calculations can provide a valuable tool for (i) the accurate prediction of 1H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution

Valorization of carob fruit residues for the preparation of novel bi-functional polyphenolic coating for food packaging applications

The food industry has become interested in the development of innovative biomaterials with antioxidant and antimicrobial properties. Although several biopolymers have been evaluated for food packaging, the use of polyphenolic coatings has been unexplored. The purpose of this work was to develop an antioxidant and antimicrobial coating for food packaging through the polymerization of carob phenolics. At first, the polyphenolic coatings were deposited in glass surfaces polymerizing different concentrations of carob extracts (2 and 4 mg mL−1) at three pH values (7, 8 and 9). Results demonstrated that the coating produced at pH 8 and at a concentration of 4 mg mL−1 had the most potent antioxidant and antimicrobial potential. Then, the coating was applied directly on the salmon fillet (coating) and on the plastic container (active packaging). Peroxide and thiobarbituric acid-reactive substances (TBARS) methods were used to measure the potency to inhibit lipid oxidation in salmon fillets. Furthermore, the anti-Listeria activity of coatings was also assessed. Results showed a significant decrease of lipid oxidation during cold storage of salmon fillets for both treatments; the superiority of applied coating directly on the salmon fillets was also highlighted. Regarding the antimicrobial potency, the polyphenolic coating depleted the growth of Listeria monocytogenes after 10 days storage; while the active packaging had no effect on Listeria monocytogenes. Overall, we describe the use of low-cost carob polyphenols as precursors for the formation of bifunctional coatings with promising applications in food packaging

Unprecedented Ultra-High-Resolution Hydroxy Group ¹H NMR Spectroscopic Analysis of Plant Extracts

A general method is demonstrated for obtaining ultra-high resolution in the phenolic hydroxy group ¹H NMR spectroscopic region, in DMSO-<i>d</i>₆ solution, with the addition of picric acid. Line-width reduction by a factor of over 100 was observed, which resulted in line-widths ranging from 1.6 to 0.6 Hz. This unprecedented resolution, in combination with the shielding sensitivity of the hydroxy group absorptions to substituent effects at least up to 11 bonds distant and the application of 2D ¹H–¹³C HMBC techniques, allows the unequivocal structure analysis of natural products with phenolic hydroxy groups in complex plant extracts

Vitamin D in asthma and future perspectives

Haidong Huang,1 Konstantinos Porpodis,2 Paul Zarogoulidis,2,3 Kalliopi Domvri,2 Paschalina Giouleka,2 Antonis Papaiwannou,2 Stella Primikyri,2 Efi Mylonaki,2 Dionysis Spyratos,2 Wolfgang Hohenforst-Schmidt,4 Ioannis Kioumis,2 Konstantinos Zarogoulidis2 1Department of Respiratory Diseases, Changhai Hospital/First Affiliated Hospital of the Second Military Medical University, Shanghai, People&rsquo;s Republic of China; 2Pulmonary Department, &ldquo;G Papanikolaou&rdquo; General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece; 3Department of Interventional Pneumology, Ruhrlandklinik, West German Lung Center, University Hospital, University Duisburg&ndash;Essen, Essen, Germany; 4II Medical Clinic, &ldquo;Coburg&rdquo; Hospital, University of W&uuml;rzburg, Coburg, Germany Abstract: Humans have the ability to synthesize vitamin D during the action of ultraviolet (UV) radiation upon the skin. Apart from the regulation of calcium and phosphate metabolism, another critical role for vitamin D in immunity and respiratory health has been revealed, since vitamin D receptors have also been found in other body cells. The term &ldquo;vitamin D insufficiency&rdquo; has been used to describe low levels of serum 25-hydroxyvitamin D that may be associated with a wide range of pulmonary diseases, including viral and bacterial respiratory infection, asthma, chronic obstructive pulmonary disease, and cancer. This review focuses on the controversial relationship between vitamin D and asthma. Also, it has been found that different gene polymorphisms of the vitamin D receptor have variable associations with asthma. Other studies investigated the vitamin D receptor signaling pathway in vitro or in experimental animal models and showed either a beneficial or a negative effect of vitamin D in asthma. Furthermore, a range of epidemiological studies has also suggested that vitamin D insufficiency is associated with low lung function. In the future, clinical trials in different asthmatic groups, such as infants, children of school age, and ethnic minorities are needed to establish the role of vitamin D supplementation to prevent and/or treat asthma. Keywords: vitamin D, asthma, immunomodulation, anti-inflammatio

Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS

Cp40 is a 14-amino acid cyclic analog of the peptidic complement inhibitor compstatin that binds with sub-nanomolar affinity to complement component C3 and has already shown promise in various models of complement-related diseases. The preclinical and clinical development of this compound requires a robust, accurate, and sensitive method for quantitatively monitoring Cp40 in biological samples. In this study, we describe the development and validation of an ultra-high performance liquid chromatography electrospray mass spectrometry method for the quantitation of Cp40 in human and non-human primate (NHP) plasma. Isotope-labeled Cp40 was used as an internal standard, allowing for the accurate and absolute quantitation of Cp40. Labeled and non-labeled Cp40 were extracted from plasma using reversed phase-solid phase extraction, with recovery rates exceeding 80%, indicating minor matrix effects. The triply charged states of Cp40 and isotope-labeled Cp40 were detected at m/z 596.60 and 600.34, respectively, via a Q-TOF mass spectrometer and were used for quantitation. The method was linear in the range of 0.18-3.58μg/mL (r(2)≥0.99), with precision values below 0.71% in NHP and 0.77% in human plasma. The accuracy of the method ranged from -2.17% to 17.99% in NHP and from -0.26% to 15.75% in human plasma. The method was successfully applied to the quantitation of Cp40 in cynomolgus monkey plasma after an initial intravenous bolus of 2mg/kg followed by repetitive subcutaneous administration at 1mg/kg. The high reproducibility, accuracy, and robustness of the method developed here render it suitable for drug monitoring of Cp40, and potentially other compstatin analogs, in both human and NHP plasma samples during pharmacokinetic and pharmacodynamic studies