Supervisors info:
Αλέξιος-Λέανδρος Σκαλτσούνης Καθηγητής ΤΟΜΕΑΣ ΦΑΡΜΑΚΟΓΝΩΣΙΑΣ ΚΑΙ ΧΗΜΕΙΑΣ ΦΥΣΙΚΩΝ ΠΡΟΙΟΝΤΩΝ ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΕ ΕΚΠΑ
Σοφία Μητάκου Καθηγήτρια ΤΟΜΕΑΣ ΦΑΡΜΑΚΟΓΝΩΣΙΑΣ ΚΑΙ ΧΗΜΕΙΑΣ ΦΥΣΙΚΩΝ ΠΡΟΙΟΝΤΩΝ ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΕ ΕΚΠΑ
Μαρία Χαλαμπαλάκη Επίκουρη Καθηγήτρια ΤΟΜΕΑΣ ΦΑΡΜΑΚΟΓΝΩΣΙΑΣ ΚΑΙ ΧΗΜΕΙΑΣ ΦΥΣΙΚΩΝ ΠΡΟΙΟΝΤΩΝ ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΕ ΕΚΠΑ
Summary:
The subject of this study is the application of the Counter-Current Chromatography, which is a modern technique of process and separation complex extracts and isolation pure metabolites of both primary and secondary metabolism of plants. By applying this technique, it has been possible to develop an appropriate method for obtaining pure metabolites depending on the properties of the extract and the metabolite.
Two different plant materials, Gentiana lutea and Olea europaea, were chosen, as they are rich in bioactive metabolites with remarkable pharmacological properties.
The primary objective of this thesis was the processing of methanol extract of gentian root for the rapid and efficient isolation of the active metabolite gentiopicroside using the most current laboratory methods. Thus, the extract rich in gentiopicroside, was first analyzed by the C.P.E. method to reduce processing time and a total of 10 enriched fractions were obtained. Further analysis of the enriched fractions with preparative HPLC was obtained gentiopicroside in high purity. These two techniques use a different mechanism and their combination in the same separation process can ensure a more efficient purification of the target. Along with the achievement of the primary objective, a more detailed phytochemical analysis of the total extract was happened, from which it was possible to obtain and identify 6 major metabolites of the extract. (Gentisin, Isogentisin, Isogentisin-3-O-Primeveroside, Isogentisin-7-O-Primeveroside, Swertiamarin and Loganic Acid)
An equally important aim of the present work was the rapid and efficient isolation of Oleanolic and Maslinic Acid from olive products using modern chromatographic methods. Oleanolic and maslinic acid are two bioactive triterpenic acids with a variety of biological activities. The separation procedure included the use of Centrifugal Partition Chromatography as the main step by applying the pH-Zone refining method. This method exploited the acidic nature of the two triterpenes and, in particular, their ability to dramatically modification the coefficient partition in the two phases of a two-phase system according to the pH of the system. The acetonic extract of the olive leaves, in addition to the fatty compounds and chlorophylls, is also a rich source of oleanolic acid. The separation involved treating of the extract with charcoal to remove the large volume of chlorophylls and then, the fraction was analyzed by pH-Zone refining C.P.E., which led to the isolation of pure oleanolic acid. The total polyphenolic fraction of olive oil contains important bioactive substances of various chemical categories and is a rich source of maslinic acid. The isolation of maslinic acid from the olive oil fraction involved the use of Chromatography Partition Chromatography by applying two different methods. Initially, the extract was fractionated with dual-mode CPE and then the fraction enriched in the metabolite was analyzed by pH-Zone refining C.P.E to obtain pure maslinic acid.
Furthermore, a significant part of this thesis was the analysis of the Total Polyphenol Fraction (T.P.F.) by gradient mode C.P.C., which ensured the intake of enriched fractions with high chemical interest. The treatment of these fractions led to the isolation of three new metabolites. Analysis of fraction 19 by the preparative TLC method led to the isolation of a compound with a similar chemical structure to oleocanthal, which was identified as Oleocanthalic Acid. By applying preparative HPLC, fraction 21 was analyzed to give a compound similar in structure to oleacein and was elucidated as Oleaceinic Acid. Finally, a similar procedure in fraction 27 led to the isolation of Eda Acid, which, while had been isolated from olive oil products, is the first time to be mentioned in olive oil.
The last experimental part of this thesis was the control of the antioxidant activity of the isolated metabolites of olive oil through the application of the DPPH protocol. The results obtained remarkable conclusions linking the action of the compound with the presence of the catechol moiety in the molecule.
Keywords:
CounterCurrent Chromatography, Isolation of Bioactive Compounds, Gentiana, Olive Products, DPPH
