Bifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1–F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin–Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studies.
Amyloid peptides, Aβ1-40 and Aβ1-42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer's Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1-42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid-Aβ1-42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti-amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1-42 oligomers, which also shows anti-amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD-NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.
a b s t r a c tSalvia sclareoides is an aromatic herb native to Portugal, of which phenolic content (Folin-Ciocalteau method), chemical profile (HPLC/DAD), antioxidant activity (DPPH, b-carotene/linoleic acid assays), acute toxicity (MTT method, adapted for non-adherent cells), genotoxicity (short-term chromosomal aberration assay) and prion binding properties were evaluated in the acetone, water, ethanol, methanol and n-butanol extracts. The latter presented the highest phenolic content and antioxidant activity (DPPH assay), and was the single one with the flavonoids (+)-catechin, kaempferol O-glucoside and quercetin. Vanillic acid was the major component of all extracts but gallic, gentisic, caffeic, syringic, coumaric and ferulic acids were also found in some extracts. Only the n-butanol extract had components binding to the cellular form of human prion protein detected by NMR which showed specificity for two regions of the folded domain and for the unstructured N-terminal region. Extracts were not cytotoxic nor genotoxic, reinforcing the potential of S. sclareoides for nutraceutical purposes.
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