Lucélia Tavares

Antioxidant Properties and Neuroprotective Capacity of Strawberry Tree Fruit (Arbutus unedo)

Berries contain significant amounts of phytochemicals, including polyphenols, which are reported to reduce cancer risk, coronary heart disease and other degenerative diseases. These effects are mainly attributed to the antioxidant capacity of polyphenols found in berries. Strawberry tree (Arbutus unedo) berries are used in folk medicine but seldom eaten as fresh fruits. Their phenolic profile and antioxidant capacity reveal a high potential, but they are not well characterized as a “health promoting food”. The aim of this study was to assess the antioxidant properties of the edible strawberry tree fruit in vitro and in a neurodegeneration cell model. Raspberry (Rubus idaeus), a well documented health-promoting fruit, was used as a control for comparison purposes. A. unedo yielded a similar content in polyphenols and a slightly lower value of total antioxidant capacity in comparison to R. idaeus. Although the chemically-measured antioxidant activity was similar between both fruits, R. idaeus increased neuroblastoma survival in a neurodegeneration cell model by 36.6% whereas A. unedo extracts caused no effect on neuroblastoma viability. These results clearly demonstrate that a promising level of chemically-determined antioxidant activity of a plant extract is not necessarily correlated with biological significance, as assessed by the effect of A. unedo fruit in a neurodegeneration cell model.

Antioxidant capacity of Macaronesian traditional medicinal plants.

The use of many traditional medicinal plants is often hampered by the absence of a proper biochemical characterization, essential to identify the bioactive compounds present. The leaves from five species endemic to the Macaronesian islands with recognized ethnobotanical applications were analysed: Apollonias barbujana (Cav.) Bornm., Ocotea foetens (Ainton) Baill, Prunus azorica (Mouill.) Rivas-Mart., Lousã, Fern. Prieto, E. Días, J.C. Costa & C. Aguiar, Rumex maderensis Lowe and Plantago arborescens Poir. subsp. maderensis (Dcne.) A. Hans. et Kunk.. Since oxidative stress is a common feature of most diseases traditionally treated by these plants, it is important to assess their antioxidant capacity and determine the molecules responsible for this capacity. In this study, the antioxidant capacity of these plants against two of the most important reactive species in human body (hydroxyl and peroxyl radicals) was determined. To trace the antioxidant origin total phenol and flavonoid contents as well as the polyphenolic profile and the amount of trace elements were determined. There was a wide variation among the species analysed in what concerns their total leaf phenol and flavonoid contents. From the High Performance Liquid Chromatography (HPLC) electrochemically detected peaks it was possible to attribute to flavonoids the antioxidant capacity detected in A. barbujana, O. foetens, R. maderensis and P. azorica extracts. These potential reactive flavonoids were identified for A. barbujana, R. maderensis and P. azorica. For R. maderensis a high content (7 mg g-1 dry weight) of L-ascorbic acid, an already described antioxidant phytomolecule, was found. A high content in selenomethionine (414.35 μg g-1 dry weight) was obtained for P. arborescens subsp. maderensis extract. This selenocompound is already described as a hydroxyl radical scavenger is reported in this work as also possessing peroxyl radical scavenging capacity. This work is a good illustration of different phytomolecules (flavonoids, organic acids and selenocompounds), presents in leaves of the five traditional medicinal plants endemic to Macaronesia, all exhibiting antioxidant properties.

Neuroprotective effects of digested polyphenols from wild blackberry species

PurposeBlackberry ingestion has been demonstrated to attenuate brain degenerative processes with the benefits ascribed to the (poly)phenolic components. The aim of this work was to evaluate the neuroprotective potential of two wild blackberry species in a neurodegeneration cell model and compare them with a commercial variety.MethodsThis work encompasses chemical characterization before and after an in vitro digestion and the assessment of neuroprotection by digested metabolites. Some studies targeting redox/cell death systems were also performed to assess possible neuroprotective molecular mechanisms.ResultsThe three blackberry extracts presented some quantitative differences in polyphenol composition that could be responsible for the different responses in the neurodegeneration cell model. Commercial blackberry extracts were ineffective but both wild blackberries, Rubus brigantinus and Rubus vagabundus, presented neuroprotective effects. It was verified that a diminishment of intracellular ROS levels, modulation of glutathione levels and activation of caspases occurred during treatment. The last effect suggests a preconditioning effect since caspase activation was not accompanied by diminution in cell death and loss of functionality.ConclusionsThis is the first time that metabolites obtained from an in vitro digested food matrix, and tested at levels approaching the concentrations found in human plasma, have been described as inducing an adaptative response.

(Poly)phenols protect from α-synuclein toxicity by reducing oxidative stress and promoting autophagy

Parkinsons disease (PD) is the most common movement neurodegenerative disorder and is associated with the aggregation of α-synuclein (αSyn) and oxidative stress, hallmarks of the disease. Although the precise molecular events underlying αSyn aggregation are still unclear, oxidative stress is known to contribute to this process. Therefore, agents that either prevent oxidative stress or inhibit αSyn toxicity are expected to constitute potential drug leads for PD. Both pre-clinical and clinical studies provided evidence that (poly)phenols, pure or in extracts, might protect against neurodegenerative disorders associated with oxidative stress in the brain. In this study, we analyzed, for the first time, a (poly)phenol-enriched fraction (PEF) from leaves of Corema album, and used in vitro and cellular models to evaluate its effects on αSyn toxicity and aggregation. Interestingly, the PEF promoted the formation of non-toxic αSyn species in vitro, and inhibited its toxicity and aggregation in cells, by promoting the autophagic flux and reducing oxidative stress. Thus, C. album (poly)phenols appear as promising cytoprotective compounds, modulating central events in the pathogenesis of PD, such as αSyn aggregation and the impairment of autophagy. Ultimately, the understanding of the molecular effects of (poly)phenols will open novel opportunities for the exploitation of their beneficial effects and for drug development.

Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

BackgroundRNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression.FindingsIn this work, a comparative analysis of the RNA quality obtained from SK-N-MC cells was performed using six commonly used RNA isolation kits: two phenol-based kits and four non-phenol based kits. The non-phenol based kits tested AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini, EasySpin and Ilustra RNAspin Mini RNA Isolation, all performed well and resulted in the isolation of high quality RNA, as evaluated by A260/A280. The RNA extracted with AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini and EasySpin provided the highest RNA yields. In particular, the RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit did not show any detectable genomic DNA contamination even without previous DNase treatment or after RNA direct PCR amplification using universal 18S primers.ConclusionsThe RNA extracted from SK-N-MC cells with AxyPrep Multisource Total RNA Miniprep Kit was superior with respect to the RNA quality and concentration. This kit does not use aggressive organic solvents and RNA free of genomic DNA was isolated without the need for DNase treatment.

The neuroprotective potential of phenolic-enriched fractions from four Juniperus species found in Portugal

The increase in population lifespan has enhanced the incidence of neurodegenerative diseases, for which there is, as yet, no cure. We aimed to chemically characterize phenolic-enriched fractions (PEFs) from four wild Juniperus sp. found in Portugal (Juniperus navicularis, Juniperus oxycedrus badia, Juniperus phoenicea and Juniperus turbinata) and address their potential as sources of natural products for treatment of neurodegenerative diseases. Leaves from the four Juniperus sp. evaluated contained a range of phenolic components which differed quantitatively between the species. The PEFs obtained were rich sources of phenolic compounds, exhibited acetylcholinesterase (AChE) inhibitory activity and also displayed effective intracellular radical scavenging properties in neurons submitted to oxidative injury but showed a different order of effectiveness compared to AChE inhibition. These properties made them good candidates for testing in a neurodegeneration cell model. Pre-incubation with J. oxycedrus badia PEF for 24h protected neurons from injury in the neurodegeneration cell model.

Polyphenols journey through blood-brain barrier towards neuronal protection.

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Bioactive compounds from endemic plants of Southwest Portugal: inhibition of acetylcholinesterase and radical scavenging activities.

Context: Natural products are reported to have substantial neuroprotective activity due to their radical scavenging capacity, and also acetylcholinesterase (AChE) inhibitory capacity, both activities important in neurodegeneration. Objective: The undesirable side effects of compounds in pharmacological use make it important to identify natural neuroprotective molecules. This work assesses the potential of five endemic Portuguese plants as sources of neuroprotective compounds. Materials and methods: Antioxidant capacity for peroxyl radical was determined by Oxygen Radical Absorbance Capacity method and for hydroxyl by Electron Paramagnetic Resonance, as well as AChE inhibitory capacity of the plant hydroethanolic extracts. The molecules responsible for these valuable properties were also tentatively identified by HPLC. Results and discussion: Armeria rouyana and Thymus capitellatus presented some of the highest phenolic contents (76.60 ± 7.19 and 12.82 ± 0.24 mg GAE g−1 dw, respectively) and antioxidant capacities (592 ± 116 and 449 ± 57 μmol TE g−1 dw, respectively). The flavonoids were identified as the phytomolecules related to the antioxidant capacity of these plant extracts; in the case of A. rouyana, l-ascorbic acid also made an important contribution (3.27 ± 0.26 mg g−1 dw). Plant extracts clearly demonstrated effective AChE inhibitory activity (480 ± 98 and 490 ± 46 μg mL−1, respectively), that could be associated to polyphenols. Conclusions: The extracts of A. rouyana and T. capitellatus and their active components, especially polyphenols, demonstrate interesting neuroprotective potential. They, therefore, deserve further study as their phytomolecules are promising sources of either natural neuroprotective products and/or novel lead compounds.

Daily polyphenol intake from fresh fruits in Portugal: contribution from berry fruits

Abstract Fresh fruits, particularly berries, are rich in polyphenols. These bioactive compounds are important in the prevention of chronic inflammatory diseases. The present study aimed to assess polyphenol intake from fresh fruit in Portugal and the relative contribution of berries to overall intake, using an online semi quantitative food frequency questionnaire. Consumption of processed berry products was also studied. Mean fresh fruit consumption was 365.6 ± 8.2 g/day. Berries accounted for 9% of total fresh fruit intake, from which 80% were due to strawberries. Total polyphenol intake from fresh fruits was 783.9 ± 31.7 mg of Gallic Acid Equivalents (GAE) per day, from which 14% were from berries. Within berries, strawberries accounted for 11% of total polyphenol intake, with the other consumed berries accounting for 3% of the total polyphenol intake per day. Main reasons reported for relative low consumption of berries were market availability and price. The most consumed processed berry product was yogurt.

Elucidating phytochemical production in Juniperus sp.: seasonality and response to stress situations

Phenolic-enriched extracts from Portuguese junipers were described as potential neuroprotective natural products. However, evaluation of plants for pharma/nutraceutical industry is based on secondary metabolite content, which is influenced by the environmental conditions. Therefore, it became important to elucidate the metabolic response of the junipers to seasonality and to stress conditions with regard to polyphenol production. Seasonal conditions modulated total phenolic and flavonoids contents of the four juniper species. Higher levels of phytochemicals were obtained when plants were not actively growing. However, only a few differences were registered in the relative contents of psydrin and a procyanidin dimer, according to species. Salt stress and methyl jasmonate elicitation promote different responses within the species. They were effective in enhancing phenolic accumulation, with just a few alterations in the relative contents of procyanidins, epicatechin, and quercetin derivatives. These differences were also reflected in gene expression of important enzymes from biosynthetic pathways.