Studio dell'attività antiossidante di metaboliti secondari da piante medicinali nella prevenzione del danno vascolare

Oxygen and nitrogen-derived free radicals play an important role in the pathogenesis of several human diseases including neurodegeneration, chronic inflammation, cancer and cardiovascular disorders such as atherosclerosis, ischemic heart disease and hypertension. Epidemiological and experimental studies suggest the hypothesis that oxidative modifications of the endothelium and low-density lipoproteins (ox-LDLs) are crucial targets for the lipid-induced initiation and progression of atherogenesis; therefore, the preservation of the endothelial antioxidant capacity and the inhibition of LDL peroxidation by specific antioxidants are an attractive strategy to prevent and possibly to treat atherosclerosis and other related cardiovascular disorders. Baicalein (from Scutellaria baicalensis G.), (-)-bilobalide (Ginkgo biloba L.), eupatorin (Eupatorium semiserratum DC.), galangin (propolis), magnolol (Magnolia officinalis L.), myricetin (Myrica rubra S.Z.), oleuropein (Olea europaea L.) and silybinin (Silybum marianum L.) are plant-derived compounds widely used in traditional medicine: the aim of this study was to investigate their radical-scavenging capacity, in order to prevent ox-LDLs generation and endothelial dysfunction, using different in vitro and ex vivo assays. Antiradical properties were firstly analysed using both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) scavenging assays, in that these methods rely on either single electron transfer or hydrogen atom transfer reaction kinetics. The DPPH assay was set in a final concentration range of 10^-7–10^-2 M, confirming a remarkable reducing capacity for each phenolic compound, especially for myricetin, oleuropein and baicalein. The ORAC detection proved that oleuropein, myricetin and magnolol are the most active compounds. In both DPPH and ORAC assays, the compound (-)-bilobalide did not show any noteworthy antioxidant activity. Oxidation of LDLs may provide the crucial link between plasma LDL and atherogenesis: for this reason, the antioxidant effect of each compound was studied on the susceptibility of human LDLs to oxidation. A determination of the extent of LDL oxidation was pointed out spectrophotometrically by measuring the formation of thiobarbituric acid reactive substances (TBARS), after incubation with 10 microM CuSO4, and by monitoring the oxidation kinetics profile of LDL particles at 234 nm. Free radical damage to lipids results in the production of malondialdehyde (MDA), which reacts with thiobarbituric acid under conditions of high temperature and acidity generating a chromogen; the lowest levels of TBARS, measured as nMol MDA/mg LDLs, were observed for oleuropein 2.5 microM and baicalein 5.0 microM. The kinetic of LDL oxidation was studied using the lag time index. Magnolol, oleuropein and myricetin (0.5 microM) exhibited a significantly higher LDL protection degree than the other polyphenols, with a considerable prolongation of the lag phase. Besides these evaluations, we used human umbilical vein endothelial cell (HUVEC) primary cultures to investigate ROS production. The HUVECs represent an interesting model which plays a pivotal role in the study of different patophysiological processes, including arterial disease. The protective function of antioxidants towards oxidative stress was measured in a range from 0.01 microM to 5 microM through the dichlorofluorescin assay, a fluorimetric probe used to evaluate the intracellular peroxides generation. ROS detection was carried out both in basal conditions and after 500 microM H2O2 addition, as an external oxidative stimulus. In basal conditions, the phytoconstituents showed a mild antioxidant activity, even at different specific concentrations, highlighting an heterogeneous trend. Under H2O2-induced oxidative stress, galangin 5 microM showed higher antioxidant activity, and myricetin showed a significant antioxidant profile at 0.01 microM, 0.1 microM and 5 microM, even if in a less extent. The results suggest the polyphenolics as a possible strategy against the oxidative stress-related disorders as atherosclerosis. This encourages the future research of plant-derived compounds as effective antioxidant agents, in order to decrease ox-LDLs production in vivo and to provide a suitable basis for novel pharmacological development against free radicals damage