Purification, structural elucidation, antioxidant capacity and neuroprotective potential of the main polyphenolic compounds contained in Achyrocline satureioides (Lam) D.C. (Compositae).

Achyrocline satureioides (Lam) D.C (Compositae) is a native medicinal plant of South America traditionally utilized for its anti-inflammatory, sedative and anti-atherosclerotic properties among others. Neuroprotective effects have been reported in vivo and could be associated to its elevated content of flavonoid aglycones. In the present study we performed the isolation and structure elucidation of the major individual flavonoids of A. satureioides along with the in vitro characterization of their individual antioxidant and neuroprotective properties in order to see their putative relevance for treating neurodegeneration. Exact mass, HPLC-MS/MS and 1H NMR identified dicaffeoyl quinic acid isomers, quercetin, luteolin, isoquercitrin, and 3-O-methylquercetin as the mayor polyphenols. Flavonoids intrinsic redox properties were evaluated in the presence of the endogenous antioxidants GSH and Ascorbate. Density Functional Theory (DFT) molecular modeling and electron density studies showed a theoretical basis for their different redox properties. Finally, in vitro neuroprotective effect of each isolated flavonoid was evaluated against hydrogen peroxide-induced toxicity in a primary neuronal culture paradigm. Our results showed that quercetin was more efficacious than luteolin and isoquercitrin, while 3-O-methylquercetin was unable to afford neuroprotection significantly. This was in accordance with the susceptibility of each flavonoid to be oxidized and to react with GSH. Overall our results shed light on chemical and molecular mechanisms underlying bioactive actions of A. satureioides main flavonoids that could contribute to its neuroprotective effects and support the positive association between the consumption of A. satureioides as a natural dietary source of polyphenols, and beneficial health effect.

In silico characterization of cytisinoids docked into an acetylcholine binding protein.

Homology models of nicotinic acetylcholine receptors (nAChRs) suggest that subtype specificity is due to non-conserved residues in the complementary subunit of the ligand-binding pocket. Cytisine and its derivatives generally show a strong preference for heteromeric alpha4beta2* nAChRs over the homomeric alpha7 subtype, and the structural modifications studied do not cause large changes in their nAChR subtype selectivity. In the present work we docked cytisine, N-methylcytisine, and several pyridone ring-substituted cytisinoids into the crystallographic structure of the Lymnaea stagnalis acetylcholine binding protein (AChBP) co-crystallized with nicotine (1UW6). The graphical analysis of the best poses showed that cytisinoids have weak interactions with the side chains of the non-conserved amino acids in the complementary subunit justifying the use of PDB 1UWB as a surrogate for nAChR. Furthermore, we found a high correlation (R(2)=0.96) between the experimental pIC(50) values at alpha4beta2* nAChR and docking energy (S) of the best cytisinoid poses within the AChBP. Due to the quality of the correlation we suggest that this equation might be used as a predictive model to propose new cytisine-derived nAChRs ligands. Our docking results also suggest that further structural modifications of these cytisinoids will not greatly alter their alpha4beta2*/alpha7 selectivity.

Nicotine-Induced Neuroprotection in Rotenone In Vivo and In Vitro Models of Parkinson's Disease: Evidences for the Involvement of the Labile Iron Pool Level as the Underlying Mechanism.

Parkinson's disease (PD) is characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinical and experimental evidence suggest that the activation of the nicotinic acetylcholine receptor (nAChR) could be protective for PD. In this study, we investigated the neuroprotective capacity of nicotine in a rat PD model. Considering that iron metabolism has been implicated in PD pathophysiology and nicotine has been described to chelate this metal, we also studied the effect of nicotine on the cellular labile iron pool (LIP) levels. Rotenone (1 µg) was unilaterally injected into the median forebrain bundle to induce the degeneration of the nigrostriatal pathway. Nicotine administration (1 mg/K, s.c. daily injection, starting 5 days before rotenone and continuing for 30 days) attenuated the dopaminergic cell loss in the SNpc and the degeneration of the dopaminergic terminals provoked by rotenone, as assessed by immunohistochemistry. Furthermore, nicotine partially prevented the reduction on dopamine levels in the striatum and improved the motor deficits, as determined by HPLC-ED and the forelimb use asymmetry test, respectively. Studies in primary mesencephalic cultures showed that pretreatment with nicotine (50 µM) improved the survival of tyrosine hydroxylase-positive neurons after rotenone (20 nM) exposure. Besides, nicotine induced a reduction in the LIP levels assessed by the calcein dequenching method only at the neuroprotective dose. These effects were prevented by addition of the nAChRs antagonist mecamylamine (100 µM). Overall, we demonstrate a neuroprotective effect of nicotine in a model of PD in rats and that a reduction in iron availability could be an underlying mechanism.

In vivo modulation of dopaminergic nigrostriatal pathways by cytisine derivatives: implications for Parkinson's Disease.

Nicotinic acetylcholine receptor agonists are considered potential pharmacological agents for Parkinson's disease treatment, due to their ability to improve experimental Parkinson symptomatology, reduce 3,4-dihydroxy-L-phenylalanine-induced dyskinesias and stop the neurodegenerative process at an experimental level. In the present work, the ability of the nicotinic agonist cytisine and two halogenated derivatives (3-bromocytisine and 5-bromocytisine) to induce striatal dopamine release was characterized in vivo by microdialysis. Cytisine, 5-bromocytisine and nicotine were much more efficacious than 3-bromocytisine in eliciting dopamine release in response to their local application through the microdialysis probe. Moreover, the agonists were intermittently administered before and after an intranigral injection of 6-hydroxydopamine (6-OHDA), and striatal dopamine tissue levels were assessed 8 days after the lesion. Both cytisine and its 5-bromo derivative (but not the 3-bromo derivative) significantly prevented the decrease of striatal dopamine tissue levels induced by 6-OHDA. These results suggest that the efficacy of nicotinic agonists to stimulate dopamine release in vivo through presynaptic nicotinic receptors could be related to their potential to induce striatal protection.

Reduction of ischemic brain damage and increase of glutathione by a liposomal preparation of quercetin in permanent focal ischemia in rats.

Oxidative stress is implicated in the pathogenesis of cerebral ischemia injury, and the flavonoids have shown to be neuroprotective in experimental models of cerebral ischemia. Previously, we have shown that an aqueous preparation of quercetin did not reach the brain while a liposomal preparation produced measurable cerebral amounts of quercetin that reduced significantly the cerebral damage provoked by permanent middle cerebral artery occlusion (pMCAo) of rats. In this context, the protective effects of liposomal quercetin (LQ) were investigated in the same model after 1 and 4 hours of arterial occlusion. LQ was administered in a single dose (30 mg/kg), at 30 min, 1 and 4 h after pMCAo, and the brain was studied 24 h later. Cerebral damage and the oedema volume were assessed with a tetrazolium salt (TTC). The status of brain tissue, the neuronal population, the global motor behaviour as well as the antioxidant, endogenous reduced glutathione (GSH), were also assessed in the brain. Thirty min after LQ there was a significantly protective effect against ischemic lesion demonstrated by a significant increase in numbers of cells in striatum and cortex, together with a partial reversal of motor deficits. GSH levels decreased after ischemia in ipsilateral striatum and cortex, and the LQ preparation reversed these effects 24 h after the occlusion. Our results suggest that endogenous brain GSH is critical in the defense mechanisms after ischemia, as a significant mediator of the protective effects of the LQ preparation.

Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: a study on human leukocytes.

AIM OF THE STUDY: Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of "Marcela". Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). MATERIALS AND METHODS: Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. RESULTS: In PBMCs, Achyrocline satureioides infusion in the 0.06-0.24microg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-gamma and interleukin (IL)-4. Lower concentrations of the infusion (0.006-0.03microg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-gamma and IL-4 and decreased the ratio IFN-gamma/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations > or =0.06microg/ml QE. On the contrary, in the 0.0012-0.03microg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. CONCLUSIONS: The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.

C3-halogenation of cytisine generates potent and efficacious nicotinic receptor agonists.

Neuronal nicotinic acetylcholine receptors subserve predominantly modulatory roles in the brain, making them attractive therapeutic targets. Natural products provide key leads in the quest for nicotinic receptor subtype-selective compounds. Cytisine, found in Leguminosae spp., binds with high affinity to alpha4beta2* nicotinic receptors. We have compared the effect of C3 and C5 halogenation of cytisine and methylcytisine (MCy) on their interaction with native rat nicotinic receptors. 3-Bromocytisine (3-BrCy) and 3-iodocytisine (3-ICy) exhibited increased binding affinity (especially at alpha7 nicotinic receptors; Ki approximately 0.1 microM) and functional potency, whereas C5-halogenation was detrimental. 3-BrCy and 3-ICy were more potent than cytisine at evoking [3H]dopamine release from striatal slices (EC50 approximately 11 nM), [3H]noradrenaline release from hippocampal slices (EC50 approximately 250 nM), increases in intracellular Ca2+ in PC12 cells and inward currents in Xenopus oocytes expressing human alpha3beta4 nicotinic receptor (EC50 approximately 2 microM). These compounds were also more efficacious than cytisine. C3-halogenation of cytisine is proposed to stabilize the open conformation of the nicotinic receptor but does not enhance subtype selectivity.

From ligand design to therapeutic efficacy: the challenge for nicotinic receptor research.

S-Nicotine, the principal psychoactive constituent of Nicotiana tabacum, underpins addiction to tobacco smoking. Although tobacco consumption is a leading cause of death worldwide, nicotine itself is also proposed to have potential therapeutic benefits for a diverse range of conditions. Nicotine interacts with its cognate receptors in the central nervous system to exert a predominantly modulatory influence, making neuronal nicotinic receptors attractive therapeutic targets. Here, we focus on three natural products as lead compounds for drug discovery programs, nicotine, epibatidine and cytisine, and consider the aims and limitations that shape these drug discovery endeavors.

Intravenous nanosomes of quercetin improve brain function and hemodynamic instability after severe hypoxia in newborn piglets.

Perinatal asphyxia is a major cause of death and neurological morbidity in newborns and oxidative stress is one of the critical mechanisms leading to permanent brain lesions in this pathology. In this context we have chosen quercetin, a natural antioxidant, known also by its brain protective effects to study its potential as a therapy for brain pathology provoked by severe hypoxia in the brain. To overcame the difficulties of quercetin to access the brain, we have developed lecithin/cholesterol/cyclodextrin nanosomes as a safe and protective vehicle. We have applied the nanosomal preparation intravenously to newborn piglets submitted to a severe hypoxic or ischemic/hypoxic episode and followed them for 8 or 72 h, respectively. Either towards the end of 8 h after hypoxia or up to 72 h after, electroencephalographic amplitude records in animals that received the nanosomes improved significantly. Animals receiving quercetin also stabilized blood pressure and recovered spontaneous breathing. In this experimental group mechanical ventilation assistance was withdrawn in the first 24 h while the hypoxic and vehicle groups required more than 24 h of mechanical ventilation. Three days after the hypoxia the suckling and walking capacity in the group that received quercetin recovered significantly compared with the hypoxic groups. Pathological studies did not show significant differences in the brain of newborn piglets treated with nanosomes compared with hypoxic groups. The beneficial effects of quercetin nanosomal preparation after experimental perinatal asphyxia show it as a promising putative treatment for the damaged brain in development.

Antioxidant activity, cellular bioavailability, and iron and calcium management of neuroprotective and nonneuroprotective flavones.

Few studies have been undertaken on the relationship of the structure of flavones and neuroprotection. Previously, we described the structural determinants of the neuroprotective activity of some natural flavones in cerebellar granule neurons in culture against an oxidative insult (H2O2). In the present work, we analyzed anti-oxidant activity, cellular iron, and Ca(2+) levels and cellular bioavailability of neuroprotective and nonneuroprotective flavones in the same experimental paradigm. Oxidative cellular damage produced by H2O2 was prevented by all of the studied flavones with rather similar potency for all of them. Labile Iron Pool was neither affected by protective nor nonprotective flavones. Intracellular Ca(2+) homeostasis was not affected by protective flavones either. Nonetheless, fisetin, the nonprotective flavone, decreased Ca(2+) levels modifying Ca(2+) homeostasis. Methylation of the catechol group, although weakens anti-oxidant capacity, keeps the neuroprotective capacity with less degradation and lower toxicity, constituting promising structural alternatives as leads for the design of neuroprotective molecules.

Quercetin in brain diseases: Potential and limits.

Quercetin is a ubiquitous flavonoid present in beverages, food and plants that has been demonstrated to have a role in the prevention of neurodegenerative and cerebrovascular diseases. In neuronal culture, quercetin increases survival against oxidative insults. Antioxidation appears to be a necessary but not sufficient condition for its neuroprotective action and modulation of intracellular signaling and transcription factors, increasing the expression of antioxidant and pro survival proteins and modulating inflammation, appears as important for neuronal protection. Quercetin also regulates the activity of kinases, changing the phosphorylation state of target molecules, resulting in modulation of cellular function and gene expression. Concentrations of quercetin higher than 100 µM consistently show cytotoxic and apoptotic effects by its autoxidation and generation of toxic quinones. In vivo, results are controversial with some studies showing neuroprotection by quercetin and others not, requiring a drug delivery system or chronic treatments to show neuroprotective effects. The blood and brain bioavailability of free quercetin after ingestion is a complex and controversial process that produces final low concentrations, a fact that has led to suggestions that metabolites would be active by themselves and/or as pro-drugs that would release the active aglycone in the brain. Available studies show that in normal or low oxidative conditions, chronic treatments with quercetin contributes to re-establish the redox regulation of proteins, transcription factors and survival signaling cascades that promote survival. In the presence of highly oxidative conditions such as in an ischemic tissue, quercetin could become pro-oxidant and toxic. At present, evidence points to quercetin as a preventive molecule for neuropathology when administered in natural matrices such as vegetables and food. More research is needed to support its use as a lead compound in its free form in acute treatments, requiring new pharmaceutical formulations and/or structural changes to limit its pro-oxidant and toxic effects.

Some aspects of the in vivo neuroprotective capacity of flavonoids: bioavailability and structure-activity relationship.

On the basis of previous work showing that flavonoids structurally related to quercetin are neuroprotective for cells in culture, this work was directed towards determining if several flavonoids (quercetin, fisetin and catechin) could acutely and by an intraperitoneal (IP) route reach significant cerebral concentrations and either prevent or facilitate recovery from a brain lesion induced by focal ischemia in rats. Aqueous and liposomal preparations of quercetin, fisetin and catechin were administered IP in a single dose and assessed in the brain by HPLC at 30 min, 1 h, 2 h and 4 h. Ischemic damage from focal middle cerebral artery occlusion was assessed spectrophotometrically with 2,3,5,-triphenylltetrazolium chloride (TTC). Infarct volume was assessed by an image analysis system following perfusion with TTC. The status of the cerebral tissue was evaluated by hematoxylin-eosin. Flavonoids administered in aqueous preparations were undetected in the brain. Cerebral concentrations of catechin (10.5 ng/g), fisetin (8.23 ng/g) and quercetin (509 ng/g) were detected in the brain only after IP injection of the liposomal preparations. Spectrophotometric analysis of brain tissue with the TTC-technique showed that liposomal quercetin reduced ischemic damage and infarct volume after permanent occlusion of the middle cerebral artery (ischemic: 41.3 mm3 vs liposomal quercetin: 17 mm3). In liposomal quercetin-treated animals there was also recovery of the cytoarchitecture in ischemic areas of striatum and cortex. Although a liposomal preparation of fisetin had similar effects, catechin failed to protect brain tissue. In conclusion, early administration of liposomal preparations of quercetin and structurally related flavonoids are beneficial and neuroprotective in experimental focal ischemia.

Cell culture protection and in vivo neuroprotective capacity of flavonoids.

Flavonoids are an important group of recognized antioxidants ubiquitous in fruits, vegetables and herbs. There are epidemiological evidences for the stroke-protecting capacity of flavonoids and while the neuroprotective power of complex extracts rich in flavonoids like those of Ginkgo biloba, green tea or lyophilized red wine have been demonstrated in several studies, neuroprotection by individual flavonoids has been poorly studied in vivo. The neuroprotective capacity of individual flavonoids was studied in PC12 cells in culture and in a model of permanent focal ischemia (permanent Middle Cerebral Artery Occlusion - pMCAO). In the in vivo experiments, flavonoids were administered in lecithin preparations to facilitate the crossing of the blood brain barrier. The simultaneous incubation of PC12 cells with 200 micro M hydrogen peroxide (H2O2) and different flavonoids for 30 min resulted in a conspicuous profile: quercetin, fisetin, luteolin and myricetin significantly increased cell survival while catechin, kaempherol and taxifolin did not. Quercetin was detected in brain tissue 30 min and 1 h after intraperitoneal administration. When one of the protective flavonoids (quercetin) and one of those that failed to increase PC12 cell survival (catechin) were assessed for their protective capacity in the pMCAO model, administered i.p. 30 min after vessel occlusion, quercetin significantly decreased the brain ischemic lesion while catechin did not. It is concluded that when administered in liposomal preparations, flavonoids structurally related to quercetin could become leads for the development of a new generation of molecules to be clinically effective in human brain ischemia.

Cytoprotection by neutral fraction of tannat red wine against oxidative stress-induced cell death.

Some of the beneficial effects of the Mediterranean diet on human pathologies have been attributed to red wine polyphenols. It has been postulated that the antioxidant activity of the latter would be also responsible for the cytoprotective capacity of red wine that has been reported in a few papers. Nevertheless, red wine shows a complex composition, and the active fraction is not known yet. In this context, the protective capacity of total lyophilized extracts of red wine and anthocyanin, neutral, or acidic fractions, was explored in PC12 cells in culture after a hydrogen peroxide insult. Although all fractions showed high antioxidant activity, only the neutral fraction was cytoprotective. The analysis of this active fraction showed that it was rich in the aglycons quercetin and myricetin as well as the glycosides of kaempferol, isorhamnetin, epicatechin, and catechin, some of which are known to be cytoprotective. This is the first paper to reveal the active fraction of total wine responsible of its cytoprotection.

Neuroprotective actions of flavones and flavonols: mechanisms and relationship to flavonoid structural features.

Epidemiological studies have shown positive preventive action of flavonoids on cardiovascular and neurodegenerative events. Among the six groups in which flavonoids are classified, the flavones and flavonols, based on the backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one) are the most commonly encountered within the families and genera of the higher plants. Numerous studies support a neuroprotective activity of flavones such as luteolin and flavonols such as kaempherol and quercetin in experimental focal ischemia and models of neurodegeneration. Antioxidation, modulation of signaling cascades and gene expression as well as anti-inflammation appear as the main protective mechanisms and mitochondria are a likely main target mediating the preventive actions against oxidative stress. Flavones and flavonols re-establish the redox regulation of proteins, transcription factors and signaling cascades that are otherwise inhibited by elevated oxidative stress. The final survival or death of the neuron depends on flavone and flavonol concentrations, time of exposure and, mainly, metabolic and oxidative neuronal circumstances. Neuroprotection appears to be linked to specific structural motifs, beyond those involved in antioxidation. By themselves or as templates for synthetic compounds, flavone and flavonol molecules show potential as multi-targeted therapeutic tools for protecting the brain. Nonetheless, more research needs to be done on the correlation of potential beneficial effects of flavones and flavonols and their mechanisms of action.

Life or death: neuroprotective and anticancer effects of quercetin.

ETHNOPHARMACOLOGICAL RELEVANCE: Quercetin is a ubiquitous flavonoid that is present in numerous plants that are utilized in many different cultures for their nervous system and anticancer effects. To better understand the neuroprotective and antiproliferative activities of quercetin, we present a comprehensive review of the divergent actions that contribute to the ethnopharmacological profile of these plants. RESULTS: The pharmacological activities of quercetin that modulate antioxidation/oxidation/kinase-signaling pathways might be differentially elicited in neurons compared with malignant cells, ultimately promoting cell survival or death in a cell type- and metabolism-specific manner. Whereas the broad antioxidation and anti-inflammatory activities of quercetin are important for neuronal survival, the oxidative, kinase- and cell cycle-inhibitory, apoptosis-inducing effects of quercetin are essential for its anticancer effects. The diverse mechanistic interactions and activities of quercetin that modulate the phosphorylation state of molecules as well as gene expression would alter the interconnected and concerted intracellular signaling equilibrium, either inhibiting or strengthening survival signals. These mechanisms, which have been mainly observed in in vitro studies, cannot be easily translated into an explanation of the divergent simultaneous neuroprotective and anticancer effects observed in vivo. This is in part due to low bioavailability in plasma and in the brain, as well as the nature of the actual active molecules. CONCLUSIONS: Numerous studies have demonstrated the beneficial effects of chronic quercetin intake, which is ethnopharmacologically meaningful, as many plants that are chronically ingested by people contain quercetin. Although quercetin and quercetin-containing plants exhibit potential as therapeutic modalities in neuropathology and in cancer, the data collectively highlight the need to elucidate issues such as bioavailability as well as its correlation with effectiveness at biomarkers in vivo. There would be an increased potentential of these plants for chemoprevention and neuropathology prevention.

Pretreatment with natural flavones and neuronal cell survival after oxidative stress: a structure-activity relationship study.

Quercetin shows structural features that have been related to the antioxidant potency of flavonoids and also shows neuroprotection in different models of oxidative death. Because only a few studies have focused on the flavonoid structural requirements for neuroprotection, this work evaluated the protective capacity of 13 flavones structurally related to quercetin, isolated from Kenyan plants, to rescue primary cerebellar granule neurons from death induced by a treatment with 24 h of hydrogen peroxide (150 microM). Each flavone (0-100 microM) was applied 24 h prior to the oxidative insult, and neuronal viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results suggest that the o-dihydroxy substitution in the B-ring is not necessary to afford neuroprotection and could be partly responsible for neurotoxic effects. Furthermore, the hydroxy substitutions in the positions C3 (C-ring) in C5 and C7 (A-ring) would be important for neuroprotection in this model.

Increase in locomotor activity after acute administration of the nicotinic receptor agonist 3-bromocytisine in rats.

Nicotinic acetylcholine receptors influence striatal dopaminergic activity and its outcome on motor behavior. For these reasons, nicotinic receptors have been considered as therapeutically relevant targets for Parkinson's disease, in which a dramatic loss of dopamine affects motor functions. The aim of the present work was to compare the effects on locomotor activity induced by the nicotinic agonist cytisine and two brominated derivatives, 5- and 3-bromocytisine (5-BrCy and 3-BrCy) using nicotine for comparison. After acute systemic administration of the agonists only 3-BrCy induced an increase in locomotor activity. To study the mechanism of action involved in this increase we co-administered 3-BrCy with the nicotinic antagonist mecamylamine and also examined 3-BrCy's effects in rats pre-treated with the long acting nicotinic antagonist chlorisondamine, administered directly in the dorsal and ventral striatum. We studied the role of the dopaminergic system by co-administration of the D2 dopamine receptor antagonist, haloperidol. The results indicate that the increase in motor activity elicited by 3-BrCy was mediated by nicotinic receptors in the dorsal and ventral striatum and depends on the interaction of nicotinic receptors with the dopaminergic system. We conclude that 3-BrCy might be a new tool to study the modulation of the dopaminergic system by nicotinic receptors and their behavioral implications.

After cellular internalization, quercetin causes Nrf2 nuclear translocation, increases glutathione levels, and prevents neuronal death against an oxidative insult.

In this work we describe the protective effects of quercetin against H(2)O(2) in 24-h-pretreated neuronal cultures. We explored quercetin availability and subcellular fate through the use of HPLC-Diode Array Detection (DAD), epifluorescence, and confocal microscopy. We focused on quercetin modulation of thiol-redox systems by evaluating changes in mitochondrial thioredoxin Trx2, the levels of total glutathione (GSH), and the expression of the gamma-glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH synthesis, by the use of Western blot, HPLC, and real-time PCR techniques, respectively. We further explored the activation of the protective NF-E2-related factor 2 (Nrf2)-dependent signaling pathway by quercetin using immunocytochemistry techniques. Our results showed rapid quercetin internalization into neurons, reaching the nucleus after its addition to the culture. Quercetin pretreatment increased total GSH levels, but did not increase Trx2. Interestingly it caused Nrf2 nuclear translocation and significantly increased GCLC gene expression. At the moment of H(2)O(2) addition, intracellular quercetin or related metabolites were undetectable in the cultures although quercetin pretreatment prevented neuronal death from the oxidant exposure. Our findings suggest alternative mechanisms of quercetin neuroprotection beyond its long-established ROS scavenging properties, involving Nrf2-dependent modulation of the GSH redox system.