Stabilnost hlorofila na oksidacioni stres u vodenom medijumu i u lipozomima

Abstract

The stability of chlorophyll on chosen oxidative stresses in disorganized aqueous medium and more organized liposomal structures is a topic of this doctoral thesis. The unique role of chlorophyll, based on its specific and very diverse chemical reactivity justifies its application in industrial technologies, food processing and pharmaceutical and cosmetic industry as well as in medicine. The question about chlorophyll stability is set as one of the preconditions for its potential applications in various areas of science and technology. In vitro oxidative stresses applied to the chlorophyll are: chemical oxidation by Fenton reagent and thermal initiator, enzyme catalyzed oxidation and photo-oxidation by continuous visible and UV-B irradiation. The stability of chlorophyll in the aqueous medium after oxidative stress was studied using absorption spectroscopy while the corresponding changes in the structure of Chl-molecules was followed by electro-spray mass spectrometry combined with UHPLC chromatography, in order to get the possible insight into the degradation-oxidation mechanisms. Changes in the physical conditions of the liposome interior (organized lipid structures), which affect the behavior of incorporated chlorophyll penetration and therefore a different degradation degree, were investigated using absorption and fluorescence spectroscopy. In addition to the presented methods, in order to evaluate some of the possible mechanisms involved in oxidative stress, two peroxide tests were performed: test of conjugated dienes and TBA-MDA test. The results show that the stability of chlorophyll depends not only on chlorophyll concentration but also the nature of the applied oxidative stress. Generally, the chlorophyll in liposomes, the same as in the aqueous medium, shows a low stability degree, which resulted in its modifications, creating oxidation-degradation products. The destructiveness of the applied oxidative stresses on chlorophyll could be ranked as follows: the effect of Fenton reagent < effect of the thermal initiator < VIS < UV-B radiation, at precisely defined conditions. Chlorophyll is more stable in liposome dispersions, which supports the conclusion that it is better to keep it in liposomes than in water. xiv Based on these results it can be concluded that the more accurate knowledge of the chlorophyll behavior under the oxidative stress, as well as its optimal concentration and stability in the potential formulations, is crucial to its further use.