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Browsing Mestrados da ESTM by Subject "6-hydroxi-dopamine"
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- Estudo do efeito neuroprotetor de terpenos isolados da alga Sphaerococcus coronopifolius num modelo in vitro da doença de Parkinson.Publication . Granados, Juan José Córdoba; Pedrosa, Rui Filipe Pinto; Alves, Celso Miguel da Maia; Gaspar, HelenaParkinson´s disease (PD) is a neurodegenerative illness characterized by the progressive loss of dopaminergic cells and the formation of Lewy bodies in the substantia nigra pars compacta, leading to motor system dysfunctions such as resting tremor and bradykinesia, affecting autonomy and cognition. Furthermore, following the growing pattern of old-age population, the discovery of new therapeutic agents able to prevent or treat this condition is of utmost importance. In this context, especially when compared with terrestrial environment, marine environment represents a vast area that still remains widely unexplored, constituting a huge source of interesting secondary metabolites with unusual chemical structures with several biological activities, including neuroprotective activities. Therefore, the objective of this dissertation was to isolate compounds from the red seaweed, Sphaerococcus coronopifolius, aiming to evaluate their antioxidant and neuroprotective activities on an in vitro cellular model of PD. The isolation and purification of compounds was achieved through preparative chromatographic techniques, namely column chromatography and thin layer chromatography. Structural elucidation of compounds was determined by nuclear magnetic resonance (NMR). Regarding biological activities, antioxidant activity of compounds (1 – 100 µM) was evaluated through the 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) methods. The neuroprotective activity was evaluated on SH-SY5Y human neuroblastoma cellular model, which was exposed to the neurotoxin 6-hydroxy-dopamine (6-OHDA- 100 µM), mimicking PD effects, in absence and/or presence of the compounds (0.3, 1, 3 and 10µM; 24 h). Several biomarkers linked to the loss of dopaminergic neuronal cells, namely production of reactive oxygen species (ROS), alterations of mitochondrial membrane potential (MMP), and Caspase 3 activity were evaluated. The cytotoxicity of compounds was evaluated both on SH-SY5Y neuronal cells and on fibroblasts (3T3 cells). In our results, four compounds were isolated and when compared to NMR literature data they were identified as the sesquiterpene alloaromadendrene and the brominated diterpenes (1), sphaerococcenol A (2), 12S-hydroxy-bromosphaerol (3) and 12R-hydroxy- bromosphaerol (4). These terpenes revealed weak antioxidant activity compared to ascorbic acid in all methods tested. Through the ORAC assay, 12R-hydroxy-bromosphaerol revealed the highest capacity to reduce peroxyl radicals compared with the remaining compounds. Regarding the neuroprotective activities, treatment of SH-SY5Y cells with 6- OHDA decreased cell viability in approximately 50%, an effect that was mediated by an increase of ROS production, a depolarization of mitochondrial membrane potential and a stimulation of Caspase-3 activity. On the other hand, when SH-SY5Y cells were exposed to 6-OHDA in the presence of 12R-hydroxy-bromosphaerol there was an increase of SH- SY5Y cell viability of 24%, 23% and 18% at 3, 1 and 0.3 µM, respectively. The treatment with 1 µM 12R-hydroxy-bromosphaerol promoted a significant decrease of ROS levels (9%) and the treatment with 3, 1, and 0.3 µM concentrations reduced the MMP depolarisation induced by 6-OHDA exposition in 13%, 19% and 35%. Furthermore, the treatment with 0.3 µM 12R-Hydroxy-bromosphaerol reduced Caspase-3 activity in 113% to values similar with vehicle situation. Sphaerococcenol A and alloaromadendrene did not revealed capacity to recover the neurotoxicity induced by 6-OHDA. In conclusion, 12R-hydroxy-bromosphaerol exhibited the highest neuroprotective activity that seems to be mediated by the inhibition of ROS production, MMP protection and Caspase-3 activity. Therefore, additional assays should be considered in order to validate the therapeutic potential of this compound in PD.