Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Karkoula Evangelia
Dissertation committee:
Τσαρμπόπουλος Αντώνης, Αναπληρωτής Καθηγητής, Ιατρική, ΕΚΠΑ
Σκαλτσούνης Αλέξιος-Λέανδρος, Καθηγητής, Φαρμακευτική, ΕΚΠΑ
Γκίκας Ευάγγελος, Επίκουρος Καθηγητής, Φαρμακευτική, ΕΚΠΑ
Πάντος Κωνσταντίνος, Καθηγητής, Ιατρική, ΕΚΠΑ
Bergquist Jonas, Καθηγητής, Τμήμα Χημείας, Πανεπιστήμιο της Ουψάλα
Σκαρμέας Νικόλαος, Αναπληρωτής Καθηγητής, Αιγινήτειο Νοσοκομείο, ΕΚΠΑ
Δάλλα Χριστίνα, Επίκουρη Καθηγήτρια, Ιατρική, ΕΚΠΑ
Original Title:
Ανάπτυξη και ολιστική αξιολόγηση του βιολογικού ρόλου βιοδραστικών ουσιών από το φυτό Crocus sativus L. σε νευροεκφυλιστικές νόσους
Translated title:
Crocus sativus L. – derived bioactive compounds: Development and holistic evaluation of their biological role in neurodegenerative diseases
Summary:
Crocus sativus is a species of flowering plant of the Crocus genus in the Iridaceae family. Saffron, the dried stigmas of the flower Crocus sativus L. is considered to be among the most expensive spices in the world, and it is mainly used as a traditional herbal medicine but also as a flavoring and food coloring agent in everyday life. The main saffron constituents include crocins, which are mono- and bis-esters of crocetin with glucose and/or gentiobiose. Saffron is considered to be a potential weapon against neurodegenerative diseases since saffron, crocin, crocetin and saffranal have shown remarkable anti oxidant activity. Moreover, saffron has exhibited significant activity against Parkinson’s and Alzheimer’s disease.
The overview of the current study was to isolate the most abundant crocin, trans-crocin-4 (TC4) from Cr. Sativus in sufficient quantities in order to perform a Pharmacokinetic study in mice and subsequently assess the bioavailability of TC4, in mice plasma after i.p. administration, as well as its capability to cross the Blood Brain Barrier (BBB). The final goal of this study was to explore the alterations to the metabolic fingerprint after the i.p. administration of TC4 and to annotate the features that were significantly altered.
Initially, this study demonstrates a simple and effective method for one-step isolation of the main secondary metabolites of a Methanol-Water 50:50 v/v saffron extract employing step-gradient centrifugal partition chromatography (CPC) performed both in dual mode and elution−extrusion mode, using five biphasic solvent systems. In total, from all the CPC experiments performed, 6185 mg of pure TC4 were isolated, 15 saffron constituents have been isolated and characterized leading to the establishment of the developed CPC methodology, as a suitable approach for the saffron stigmas analysis in the laboratory as well as the natural products industry.
The subsequent step of the current study included the development and full validation of two UPLC-PDA methodologies, according to U.S FDA and EMA guidelines, for the rapid and accurate quantification of TC4 and crocetin (CRC) in mice plasma and brain after i.p. administration. The two UPLC-PDA methods were successfully applied for the determination of CRC and TC4 in mouse plasma and brain after i.p. administration of TC4 (50 and 150 mg/kg) in a time range of 0-240 minutes. Due to the selection of i.p. administration route, the first-pass metabolism and/or gastric hydrolysis were bypassed, a fact that enhanced the bioavailability of TC4. Furthermore, TC4 was found to be capable of crossing the Blood Brain Barrier (BBB) and build up levels in the mouse brain, regardless of its highly hydrophilic character. Interestingly, CRC was not detected in any plasma or brain sample, although it has been reported that TC4 quickly hydrolyzes to CRC after p.o. administration. Therefore i.p. administration could be used in the case of TC4 for the accurate determination of its biological role.
In the last step of the current study, the metabolic fingerprint and its associated alterations following i.p. administration of TC4 in male and female mice were determined by an untargeted UPLC-HRMS metabolomics approach. Statistical evaluation of the results was achieved by multivariate analysis (MVA), i.e., Principal Component Analysis (PCA), and (sparse) Partial Least Squares–Discriminant Analysis (sPLS-DA). Due to the high variability imposed by various factors e.g. sex, administration dose, and time points, the ML-(s)PLS–DA has proven to be the only effective approach. Furthermore, a preliminary sex-related effect on the metabolome has been proven to exist, denoting that the administration in both genders is indispensable in order to acquire safe conclusions as reliable metabolome pictures. Finally, this UPLC-HRMS-based methodology clearly demonstrated that the time sequence of metabolome changes was due to the administration of TC4. In accordance with the results provided by various statistical softwares, the selected features were further implemented to online databases so as to annotate them and to identify potential metabolites.
Main subject category:
Health Sciences
Keywords:
Centrifugal partition chromatography, Crocetin, Crocin, Saffron, UPLC, Bioavailability, Mice plasma, Mice brain, Metabolomics, Neurosteroids, Sex differences
Number of references:
280
