| Περίληψη: | The present research study included the development of analytical methodology based on chromatographic and mass spectrometric techniques for the determination and fingerprinting of bioactive compounds derived from Crocus sativus L. and also their interaction with biomolecules, with the aim to investigate their role in human health. The main bioactive analyzed compounds were picrocrocin, crocetin and analogues thereof, which are unusually glucosylated carotenoids (ranging from one up to a total five glucosyl-residues) and specifically Gentiobiosyl-gentiotriosyl-E-crocetin (trans-crocin-5, TC-5), Gentiobiosyl-neapolitanosyl-E-crocetin (trans-crocin-5′, TC-5′) bis-Gentiobiosyl-E-crocetin (trans-crocin-4, TC-4), Gentiobiosyl-glucosyl-E-crocetin (trans-crocin-3, TC-3), bis-Glucosyl-E-crocetin (trans-crocin-2, TC-2), (4) bis-Gentiobiosyl-13-Z-crocetin (cis-crocin-4, CC-4), Gentiobiosyl-glucosyl-13-Z-crocetin (cis-crocin-3, CC-3), Crocetin (CRT) and Picrocrocin (PICR) with ultimate objective the assessment of their biological effect on the prevention and treatment of various neurodegenerative diseases like Parkinson's and Alzheimer's disease. Because of their particular complicated structural motif and the extendeed degree of symmetry which constitutes a rather complex mixture of glycoconjugates it became imperative to isolate and characterize their structures mainly with state-of-the-art chromatographic and mass spectrometric techniques. The developed methodologies included the use of the following modern analytical techniques and instrumentation: i) Size-exclusion chromatography (SEC), Medium pressure liquid chromatography (MPLC), semi-preparative High Performance Liquid Chromatography (semi-Prep HPLC) and Fast Centrifugation Partition Chromatography (FCPC) for the separation and isolation of aforementioned bioactive compounds, ii) various tandem mass spectrometric techniques such as MALDI-TOF/RTOF-MS in combination with collision-induced dissociation, ESI-QqRTOF-MS/MS, IP-MALDI Q-TOFMSn (n = 1 or 2), ESI-LTQ-orbitrap-MS/MS and ESI-IT-MS/MS for detailed structure elucidation, characterization and diffentiation of bioactive compounds isolated from Crocus sativus L. and iii) ESI-QqRTOF-MS and ESI-Ion Mobility Spectrometry-MS (ESI IMS-MS) for the screening of the noncovalent interaction between beta amyloid peptide Aβ(1-40) and Crocus-derived bioactive compounds natural products (such as crocins with different number of carbohydrate moieties). In conclusion, the application of MALDI high energy CID to structure elucidation of Crocus-derived bioactive compounds turned out to be very helpful particular because low energy CID often fails to provide structural relevant fragmentation whereas for the differentiation of selected positional crocetin glycoside isomers ESI-ion trap with low energy CID was more efficient due to the capability of multistage MSn. UHPLC-HRMSn with the aid of an orbitrap analyzer was the best configuration for the identification and structure elucidation of crocins from complex saffron extract with high confidence and the exploration and finding of minor compounds and new compounds. ESI MS methodology mainly quadrupole reflectron time-of-flight (QqTOF) instrumentation managed to detect noncovalent complexes between Aβ (1-40) and crocins (with different number of glucose moieties) in a series of studies focused on screening the ability of several bioactive phytochemicals in terms of complexating Aβ (1-40) and ‘‘locking’’ them in a non-toxic conformation, thus acting as potential antiamyloidogenic agents offering an ideal protective alternative against Aβ (1-40) toxicity. The aforementioned natural products and derivatives thereof can be eventually exploited, in the form of nutraceuticals towards the prevention and/or treatment of AD.
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