De Marco, Veronica (2008) Sviluppo di un modello cinetico e sua applicazione allo studio dell'inibizione della perossidazione lipidica da parte del plasma e dei fitofenoli in diversi sistemi. [Ph.D. thesis]
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The term "antioxidant" has several meanings and so it is necessary specifying the antioxidant mechanism we intend to study. We focused the attention on the role of antioxidants in the inhibition of lipid peroxidation process. In literature, the mechanisms at the base of the inhibition process have been discussed more or less rigorously and several model systems reproducing conditions of accelerated oxidation have been used and described. The differences in the used substrates, in the composition of the systems and in the analytical methods produced data difficult to compare.
In the order to overcome these problems, we set up a method for the study of the lipid peroxidation, both by developing the kinetic model of the reactions and by setting up the experimental micelle system. The rigorous approach, based on the variation of oxygen consumption during the various phases of the process, permits to evaluate two fundamental parameters characterizing a radical chain-breaking antioxidant in the inhibition of lipid peroxidation: the "capacity" (n), that is the number of free radicals scavenged by each molecule of inhibitor, and the "efficiency" (IC50-1), that is the ability of an inhibitor to scavenge peroxyl radicals. This method permits to obtain both parameters correctly and simultaneously from a single kinetic run.
The elaborated kinetic model evaluates the reactions occurring in the inhibition of lipid peroxidation process with particular attention to the termination reactions between peroxyl radicals besides the propagation reactions. The termination reactions compete with the inhibition reactions of antioxidants. Indeed, the number of peroxyl radicals scavenged by an inhibitor molecule decreases with its consumption, but the number of peroxyl radicals disappearing by mutual interaction increases. A rigorous kinetic analysis permits to obtain a complex function containing the rate of oxygen consumption which is linearly correlated with the time. This linear relationship is characterized by the "C" parameter. The "C" value permits to obtain both "n" and "IC50-1" values concerning the inhibition process. In the case of pure antioxidants "n" is related to the stoichiometry of the inhibition process and "IC50-1" is related to the kinetic rate constant of the trapping process. The validity of the kinetic model and of the analytical method were verified measuring the inhibition of lipid peroxidation by different antioxidants at several concentrations and in various micelle systems. We tested many plant polyphenols such as the derivatives of benzoic acid, hydroxycinnamic acids, flavanols, flavonols and anthocyanins. For these compounds it was calculated the values of "n" and "IC50-1" characterizing their antioxidant activity in the inhibition of lipid peroxidation process.
On the base of the good results, we verified the possibility to applicate the method for the study of complex systems such as foods and human plasma.
In the case of foods, characterized by the presence of a significant amount of antioxidants, which have a protecting action on the human health as described by recent epidemiological studies, the developed method correctly explains the experimental data and permits to evaluate simultaneously both capacity (PRTC: Peroxyl Radical Trapping Capacity) and efficiency (PRTE: Peroxyl Radical Trapping Efficiency) for each tested food. It is important to point out that many foods, although they contain various antioxidants, show a kinetic behaviour like a single inhibitor, peculiarity that permits to obtain typical values of PRTC and PRTE for these foods. These values depend on the food composition and then they are good indicators of the antioxidant properties of each food.
The application of the method to measure the human plasma activity in the scavenging of peroxyl radicals shows its adaptability to study complex systems. Indeed, in the case of plasma, it permits to demonstrate the presence of two inhibition phases related to the properties of contained antioxidants. The first phase of inhibition is characterized by a zero-order process with respect to antioxidant concentration that means 100% efficiency in the scavenging of peroxyl radicals, while the second phase is characterized by a first-order process with respect to the antioxidant concentration. To understand this behaviour we have analyzed both the contribution of the high and low molecular weight components of plasma in the inhibition process and the action of the single antioxidants. In particular, we have verified that the high molecular weight (HMW) fraction of plasma, containing vitamin E and albumin-bound bilirubin, inhibits lipid peroxidation by a zero-order process and so we have obtained only the antioxidant capacity. The antioxidants present in the low molecular weight (LMW) fraction, which are mainly ascorbic acid and uric acid, inhibit the lipid peroxidation by a first-order process with respect to the inhibitor concentration. For this reason, it is possible to obtain the values of capacity and efficiency of LMW. The plasma analysis has shown the high synergy existing between the high and low molecular weight components. Indeed, the analysis of the oxygraphic trace by means of an ulterior refining of our method displayed that a part of antioxidant activity of the low molecular weight component, characterized by relatively low efficiency and high capacity, is transferred to the high molecular weight component with a remarkable increase of the plasma efficiency. This method appears useful to study different situation of human plasma antioxidant activity.
In conclusion, the proposed method can be applied both to evaluate single antioxidants and to study complex systems such as food and human plasma and permits to light particularly interesting aspects such as synergistic and antisynergistic phenomena that the other currently used methods do not show.
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