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Resumo(s)
A doença de Parkinson (DP) é uma patologia neurodegenerativa progressiva caracterizada pela perda de neurónios dopaminérgicos e pela acumulação de corpos de Lewy, conduzindo a défices motores e cognitivos. As terapias atualmente disponíveis, como a Levodopa, são essencialmente sintomáticas e não alteram a progressão da doença. Compostos naturais marinhos, em particular os exopolissacarídeos (EPS), apresentam propriedades antioxidantes e anti-inflamatórias, tornando-se candidatos promissores para estratégias neuroprotetoras.
O principal objetivo da presente dissertação consistiu na caracterização química e na avaliação das atividades antioxidante, neuroprotetora e anti-inflamatória de EPS extraídos de três dinoflagelados marinhos: Protoceratium reticulatum, Gymnodinium catenatum e Prorocentrum lima.
A caracterização incluiu análise da composição química, identificação de grupos funcionais por espetroscopia de infravermelho com transformada de Fourier (FTIR) e determinação estrutural por ressonância magnética nuclear de protão (1H RMN). As atividades biológicas foram avaliadas por ensaios antioxidantes (2,2-difenil-1-picril-hidrazilo (DPPH) (0–100 μg/mL), capacidade redutora férrica (FRAP) (100 μg/mL) e redução do anião superóxido (0–100 μg/mL), efeito neuroprotetor em células SH-SY5Y não diferenciadas e diferenciadas (3–300 μg/mL; 24 h) com ácido retinóico (10 μM) e forbol (TPA; 80 μM) expostas à neurotoxina 6-hidroxidopamina (6-OHDA; 100 μM), e atividade anti-inflamatória (1–100 μg/mL; 24 h) em células de microglia BV-2, com base na produção de óxido nítrico (NO).
Os EPS apresentaram elevada pureza, evidenciada pela presença residual de compostos fenólicos, proteínas ou ácidos nucleicos, na sua composição. P. reticulatum exibiu o maior teor de sulfatos (106,10 ± 7,63 mg eq Na₂SO₄/g EPS). Embora não tenha sido possível determinar o conteúdo total de carboidratos, todas as espécies apresentaram bandas de absorção típicas de exopolissacarídeos (3500–3394 cm⁻¹; 1663–1619 cm⁻¹; 1442–1423 cm⁻¹; 1150–990 cm⁻¹; 873 cm⁻¹; 667–597 cm⁻¹) através dos dados obtidos pela técnica de FTIR. A análise de 1H RMN revelou desvios químicos característicos de estruturas polissacarídicas e a predominância de ligações β-glicosídicas (δ 4,74 ppm), além da deteção de grupos metilo (δ 1,25–1,16 ppm) e acetilo (δ 1,84 ppm). Relativamente à atividade antioxidante, os EPS não demonstraram qualquer efeito significativo.
De igual modo, nos ensaios realizados com células SH-SY5Y expostas à 6-OHDA, não foi possível observar qualquer atividade neuroprotetora, tanto em células não diferenciadas como em células diferenciadas. Em microglia BV-2 não estimulada com lipopolissacarídeos (LPS), os EPS não alteraram significativamente os níveis de NO, indicando ausência de ativação microglial em condições fisiológicas. Contudo, em células BV-2 estimuladas com LPS, os EPS de P. reticulatum, G. catenatum e P. lima reduziram a produção de NO entre 20 e 30%
Concluindo, embora os EPS não tenham apresentado atividade antioxidante ou neuroprotetora, a sua capacidade de reduzir a produção de NO em microglia ativada demonstra o seu potencial anti-inflamatório. Este estudo representa a primeira caracterização e avaliação biológica de EPS de P. reticulatum, G. catenatum e P. lima, fornecendo dados pioneiros que ampliam o conhecimento sobre EPS com origem em dinoflagelados e sustentam a continuação do seu estudo em futuras estratégias neuroprotetoras.
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of Lewy bodies, leading to motor and cognitive deficits. Current available therapies, such as Levodopa, are mainly symptomatic and do not alter disease progression. Marine natural compounds, particularly exopolysaccharides (EPS), exhibit antioxidant and anti-inflammatory properties, making them promising candidates for neuroprotective strategies. The objective of this dissertation was to chemically characterize and evaluate the antioxidant, neuroprotective, and anti-inflammatory activity of EPS extracted from three marine dinoflagellates: Protoceratium reticulatum, Gymnodinium catenatum, and Prorocentrum lima. Their characterization included chemical composition, identification of functional groups by Fourier transform infrared spectroscopy (FTIR), and structural determination by proton nuclear magnetic resonance (1H NMR). Biological activities were assessed through antioxidant assays—2,2-diphenyl-1-picrylhydrazyl (DPPH) (0–100 μg/mL), ferric reducing antioxidant power (FRAP) (100 μg/mL), and superoxide anion radical scavenging (0–100 μg/mL) neuroprotective effects in undifferentiated and differentiated SH-SY5Y cells (3–300 μg/mL; 24 h) differentiated with retinoic acid (10 μM) and phorbol (TPA; 80 μM) and exposed to the neurotoxin 6-hydroxydopamine (6-OHDA; 100 μM), and anti-inflammatory activity (1–100 μg/mL; 24 h) in BV-2 microglial cells based on nitric oxide (NO) production. The EPS exhibited high purity, as evidenced by the residual presence of phenolic compounds, proteins, or nucleic acids in their composition. P. reticulatum exhibited the highest sulfate content (106.10 ± 7.00 mg eq. Na₂SO₄/g EPS). Although total carbohydrate content could not be determined, all species showed characteristic exopolysaccharide absorption bands (3500–3394 cm⁻¹; 1663–1619 cm⁻¹; 1442–1423 cm⁻¹; 1150–990 cm⁻¹; 873 cm⁻¹; 667–597 cm⁻¹) in the FTIR spectra. 1H NMR analysis revealed chemical shifts typical of polysaccharide structures and a predominance of β-glycosidic linkages (δ 4.74 ppm), as well as methyl (δ 1.25–1.16 ppm) and acetyl groups (δ 1.84 ppm). Regarding antioxidant activity, the EPS showed no significant effects Similarly, in assays conducted with SH-SY5Y cells exposed to 6-OHDA, no neuroprotective activity was observed, in either undifferentiated or differentiated cells. In unstimulated BV-2 microglia, EPS did not significantly effect oxide nitric levels, indicating the absence of microglial activation under physiological conditions. However, in lipopolysaccharide (LPS)-stimulated BV-2 cells, EPS from P. reticulatum, G. catenatum and P. lima, reduced NO production by between 20 and 30%. In conclusion, although EPS did not display antioxidant or neuroprotective activity, their ability to reduce NO production in activated microglia demonstrates anti-inflammatory potential. This study represents the first chareacterization and biological evaluation of EPS from P. reticulatum, G. catenatum, and P. lima, providing pioneering data that expand knowledge on of dinoflagellate-derived EPS and support their exploitation in future neuroprotective strategies.
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of Lewy bodies, leading to motor and cognitive deficits. Current available therapies, such as Levodopa, are mainly symptomatic and do not alter disease progression. Marine natural compounds, particularly exopolysaccharides (EPS), exhibit antioxidant and anti-inflammatory properties, making them promising candidates for neuroprotective strategies. The objective of this dissertation was to chemically characterize and evaluate the antioxidant, neuroprotective, and anti-inflammatory activity of EPS extracted from three marine dinoflagellates: Protoceratium reticulatum, Gymnodinium catenatum, and Prorocentrum lima. Their characterization included chemical composition, identification of functional groups by Fourier transform infrared spectroscopy (FTIR), and structural determination by proton nuclear magnetic resonance (1H NMR). Biological activities were assessed through antioxidant assays—2,2-diphenyl-1-picrylhydrazyl (DPPH) (0–100 μg/mL), ferric reducing antioxidant power (FRAP) (100 μg/mL), and superoxide anion radical scavenging (0–100 μg/mL) neuroprotective effects in undifferentiated and differentiated SH-SY5Y cells (3–300 μg/mL; 24 h) differentiated with retinoic acid (10 μM) and phorbol (TPA; 80 μM) and exposed to the neurotoxin 6-hydroxydopamine (6-OHDA; 100 μM), and anti-inflammatory activity (1–100 μg/mL; 24 h) in BV-2 microglial cells based on nitric oxide (NO) production. The EPS exhibited high purity, as evidenced by the residual presence of phenolic compounds, proteins, or nucleic acids in their composition. P. reticulatum exhibited the highest sulfate content (106.10 ± 7.00 mg eq. Na₂SO₄/g EPS). Although total carbohydrate content could not be determined, all species showed characteristic exopolysaccharide absorption bands (3500–3394 cm⁻¹; 1663–1619 cm⁻¹; 1442–1423 cm⁻¹; 1150–990 cm⁻¹; 873 cm⁻¹; 667–597 cm⁻¹) in the FTIR spectra. 1H NMR analysis revealed chemical shifts typical of polysaccharide structures and a predominance of β-glycosidic linkages (δ 4.74 ppm), as well as methyl (δ 1.25–1.16 ppm) and acetyl groups (δ 1.84 ppm). Regarding antioxidant activity, the EPS showed no significant effects Similarly, in assays conducted with SH-SY5Y cells exposed to 6-OHDA, no neuroprotective activity was observed, in either undifferentiated or differentiated cells. In unstimulated BV-2 microglia, EPS did not significantly effect oxide nitric levels, indicating the absence of microglial activation under physiological conditions. However, in lipopolysaccharide (LPS)-stimulated BV-2 cells, EPS from P. reticulatum, G. catenatum and P. lima, reduced NO production by between 20 and 30%. In conclusion, although EPS did not display antioxidant or neuroprotective activity, their ability to reduce NO production in activated microglia demonstrates anti-inflammatory potential. This study represents the first chareacterization and biological evaluation of EPS from P. reticulatum, G. catenatum, and P. lima, providing pioneering data that expand knowledge on of dinoflagellate-derived EPS and support their exploitation in future neuroprotective strategies.
Descrição
Palavras-chave
Doença de Parkinson Potencial terapêutico Atividade antioxidante SH-SY-5Y BV-2 Atividade anti-inflamatória