In vitro study of the blood-brain barrier transport of bioactives from Mediterranean foods.

The Mediterranean diet (MD), characterized by olive oil, olives, fruits, vegetables, and wine intake, is associated with a reduced risk of dementia. These foods are rich in bioactives with neuroprotective and antioxidant properties, including hydroxytyrosol (HT), tyrosol (TYRS), serotonin (SER) and protocatechuic acid (PCA), a phenolic acid metabolite of anthocyanins. It remains to be established if these molecules cross the blood-brain barrier (BBB), a complex interface that strictly controls the entrance of molecules into the brain. We aimed to assess the ability of tyrosine (TYR), HT, TYRS, PCA and SER to pass through the BBB without disrupting its properties. Using Human Brain Microvascular Endothelial Cells as an in vitro model of the BBB, we assessed its integrity by transendothelial electrical resistance, paracellular permeability and immunocytochemical assays of the adherens junction protein ß-catenin. The transport across the BBB was evaluated by ultra-high-performance liquid chromatography high resolution mass spectrometry. Results show that tested bioactives did not impair BBB integrity regardless of the concentration evaluated. Additionally, all of them cross the BBB, with the following percentages: HT (∼70%), TYR (∼50%), TYRS (∼30%), SER (∼30%) and PCA (∼9%). These results provide a basis for the MD neuroprotective role.

Hydroxytyrosol and dopamine metabolites: Anti-aggregative effect and neuroprotective activity against α-synuclein-induced toxicity.

The abnormal aggregation of the α-synuclein (αsyn) protein is involved in the formation of Lewy bodies in the brain of patients suffering from Parkinson disease (PD). Hydroxytyrosol (HT), a polyphenolic compound present in olives, olive oil, and wine, has been shown to inhibit aggregation and destabilise the αsyn aggregates, preventing neuronal cell death. However, very limited data have been published on the study of its metabolites. Therefore, this study investigated the capacity of the metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL), 4-hydroxy-3-methoxyphenylethanol (MOPET), and 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL) to prevent the aggregation and toxic effects of αsyn fibrils. In vitro techniques, such as Thioflavin T (ThT), Transmission Electronic Microscopy (TEM), electrophoresis, thiazolyl blue tetrazolium bromide (MTT), and Real-Time PCR (RT-PCR) were used. Our results show that among these three metabolites, DOPAL exerts the greatest effect, preventing aggregation and αsyn-induced neurotoxicity. In fact, DOPAL has the ability to completely inhibit αsyn fibril formation at low doses. Moreover, this metabolite has a potent destabilising effect on the αsyn fibrils. Concerning neuroprotection, DOPAL can counteract the toxicity induced by αsyn. The vitagene expression results show a possible relationship between the neuroprotection mechanism exhibited by DOPAL and the modulation of SIRT-2 and Hsp70.

Hydroxytyrosol in Foods: Analysis, Food Sources, EU Dietary Intake, and Potential Uses.

Hydroxytyrosol (HT) is a phenolic compound with proven biological properties present in a limited number of foods such as table olives, virgin olive oil (VOO) and wines. The present work aims to evaluate the dietary intake of HT in the European (EU) population by compiling scattered literature data on its concentration in foods. The consumption of the involved foods was estimated based on the EFSA Comprehensive European Food Consumption Database. The updated average contents of HT are as follows: 629.1, 5.2 and 2.1 µg/g for olives, olive oil and wine, respectively. The HT estimated intake in the European Union (EU) adult population falls within 0.13-6.82 mg/day/person, with table olives and wine being the main contributors. The estimated mean dietary intake of HT in EU countries is 1.97 ± 2.62 mg/day. Greece showed the highest HT intake (6.82 mg/day), while Austria presented the lowest (0.13 mg/day). Moreover, HT is an authorized novel food ingredient in the EU that can be added to different foods. Since the estimated HT intake is substantially low, the use of HT as a food ingredient seems feasible. This opens new possibilities for revalorizing waste products from olive oil and olive production which are rich HT sources.

Anti-VEGF Effect of Bioactive Indolic Compounds and Hydroxytyrosol Metabolites.

Angiogenesis is a key process involved in both cancer and cardiovascular diseases, the vascular endothelial growth factor (VEGF) and its VEGF receptor-2 (VEGFR-2) being the main triggers. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signaling by hydroxytyrosol (HT) metabolites and indolic compounds and establish a relation between their structure and bioactivity. Experiments involved the evaluation of their potential to inhibit VEGF on human umbilical vein endothelial cells (HUVECs) by ELISA assay and their subsequent effect on the downstream signaling pathway (PLCγ1, Akt, and endothelial nitric oxide synthetase (eNOS)) by Western blot. Respectively, 3,4-dihydroxyphenylacetaldehyde (DOPAL) (100 µM) and indole pyruvic acid (IPy) (1 mM) were capable of inhibiting VEGFR-2 activation with an IC50 value of 119 µM and 1.037 mM. The anti-angiogenic effect of DOPAL and IPy is mediated via PLCγ1. Additionally, DOPAL significantly increases eNOS phosphorylation, while IPy maintained it. These data provide for the first time evidence of the anti-angiogenic effect of DOPAL and IPy for future use as potential bioactive food ingredients.

Melatonin, protocatechuic acid and hydroxytyrosol effects on vitagenes system against alpha-synuclein toxicity.

Preventing the abnormal assembly of α-synuclein (α-Syn) and the correct modulation of vitagenes system exercise strong neuroprotective effects. It has been reported that melatonin (MEL), protocatechuic acid (PCA) and hydroxytyrosol (HT) reduce α-Syn toxicity. Their effect on the vitagenes system of PC12 cells have not been explored yet. These bioactive can cross the blood brain barrier (BBB). Therefore, this work aims to evaluate the inhibitory and destabilising capacities of MEL, PCA, HT, and their combinations on α-Syn kinetics and effects on vitagenes system (sirtuin-1 (SIRT-1), sirtuin-2 (SIRT-2), heme oxygenase (HO-1) and heat shock protein 70 (Hsp-70)). In vitro techniques (Thioflavin T (ThT), Transmission Electronic Microscopy (TEM), electrophoresis, MTT assay and qPCR) were used. Compounds, both individually and simultaneously were able to decrease the toxicity induced by α-Syn. Concurrently, occurrence of PCA (100 µM) +HT (100 µM) showed the highest inhibitory effect against α-Syn fibril formation and destabilisation of α-Syn fibrils (88 and 62%, respectively). Moreover, these compounds increased the expression of SIRT-2, HO-1 and Hsp70, contributing to a neuroprotective effect. In addition, the most important result is the increase on the expression of SIRT-2 caused by the combination of MEL + HT + PCA in the absence of α-Syn fibrils.

Hydroxytyrosol Decreases LPS- and α-Synuclein-Induced Microglial Activation In Vitro.

Neuroinflammation is a common feature shared by neurodegenerative disorders, such as Parkinson's disease (PD), and seems to play a key role in their development and progression. Microglia cells, the principal orchestrators of neuroinflammation, can be polarized in different phenotypes, which means they are able to have anti-inflammatory, pro-inflammatory, or neurodegenerative effects. Increasing evidence supports that the traditional Mediterranean dietary pattern is related to the reduction of cognitive decline in neurodegenerative diseases. A considerable intake of plant foods, fish, and extra virgin olive oil (EVOO), as well as a moderate consumption of red wine, all characteristic of the Mediterranean diet (MD), are behind these effects. These foods are especially rich in polyphenols, being the most relevant in the MD hydroxytyrosol (HT) and their derivatives present in EVOO, which have demonstrated a wide array of biological activities. Here, we demonstrate that HT is able to reduce the inflammation induced by two different stimuli: lipopolysaccharide and α-synuclein. We also study the possible molecular mechanisms involved in the anti-inflammatory effect of HT, including the study of nuclear factor kappa B (NF-кB), mitogen-activated protein kinases (MAPKs), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and inflammasome. Our data support the use of HT to prevent the inflammation associated with PD and shed light into the relationship between MD and this neurological disorder.