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The purpose of this work was to evaluate the total phenol content and antioxidant activity of different types of handcrafted beers (Ego, Alter, Fiat Lux, Triplo Malto, Ubi, and Maior), as well as the starting materials (malts, hops, and yeast), the intermediate products, and the waste products (spent malts, hops, and yeast), in view of their use in innovative cosmetic formulations. Extractions from starting and spent samples were taken from water or 70° alcohol. The total phenol content (Folin Ciocalteau Essay) of all the brewing products depended on the specific product under investigation. The highest values were found in starting hops (ranging from approximately 93 to 155 mg GAE/g, according to the extraction solvent), intermediate ones in starting malt and starting yeast, and the lowest values in wort. The total phenol content in the final beers originates from the phenols that were extracted from the different ingredients, namely the starting malts, hops and yeast, but non-negligible values were still observed in spent products. The method used for the evaluation of the antioxidant activity, trolox equivalent antioxidant capacity (DPPH), ferric-ion reducing antioxidant parameter (FRAP), and radical cation scavenging activity and reducing power (ABTS) strongly influenced the results. In general, the results reflected the trend observed for the total phenol content: that beers are progressively enriched by phenols originating from all the starting ingredients, and that spent products still possess non-negligible antioxidant activity. It is interesting to note that waste yeast frequently showed higher values than those of the starting material, it can be inferred that yeast is able to absorb phenols from the beer during brewing. By considering the interest in exploiting waste derived from processing foods, the biological activity of waste Alter brewery products has been evaluated on a cell culture of keratinocytes (spent products of malt, hop, and yeast). Preliminary in vitro assays in keratinocyte HaCaT cells were carried out to assess the potential bioactivity of spent extracts. Among the spent extracts, the spent hop and yeast extracts showed the ability to improve the mitochondrial activity and prevent oxidative stress in HaCaT cells, two features in skin ageing. In conclusion, this study offers evidence that waste from handcrafted beers can be an interesting source of phenols for the preparation of skin anti-aging cosmetics.

The important functions of the endothelium and the relationship between cardiovascular risk factors and endothelial dysfunction suggest the primary role of this tissue as a target for dietary strategies aimed at the prevention from related diseases. Cereals are key component of a healthy and balanced diet, and the presence of non-specific lipid-transfer protein 2 (nsLTP2) in wheat represents an added value to contribute to maintain the functionality of the vascular endothelium and consequently of the cardiovascular system. Indeed, nsLTP2 downregulates the expression of the main cell adhesion molecules induced by a pro-inflammatory cytokine and, meanwhile, upregulates heme oxigenase-1, exerting a cytoprotective/anti-inflammatory activity. Therefore, nsLTP2 might represent a food-derived tool to protect the vascular system against several pathological conditions.

One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.

Neurodegenerative diseases are driven by several mechanisms such as inflammation, abnormal protein aggregation, excitotoxicity, mitochondrial dysfunction and oxidative stress. So far, no therapeutic strategies are available for neurodegenerative diseases and in recent years the research is focusing on bioactive molecules present in food. In particular, extra-virgin olive oil (EVOO) phenols have been associated to neuroprotection. In this study, we investigated the potential antioxidant and neuroprotective activity of two different EVOO extracts obtained from Quercetano cultivar trees grown in two different areas (plain and hill) of the Tuscany region (Italy). The different geographical origin of the orchards influenced phenol composition. Plain extract presented a higher content of phenyl ethyl alcohols, cinnammic acids, oleacein, oleocanthal and flavones, meanwhile, hill extract was richer in lignans. Hill extract was more effective in protecting differentiated SH-SY5Y cells from peroxide stress thanks to a marked upregulation of the antioxidant enzymes heme oxygenase 1, NADPH quinone oxidoreductase 1, thioredoxin Reductase 1 and glutathione reductase. Proteomic analysis revealed that hill extract plays a role in the regulation of proteins involved in neuronal plasticity and activation of neurotrophic factors such as BDNF. In conclusion, these data demonstrate that EVOOs can have important neuroprotective activities, but these effects are strictly related to their specific phenol composition.

Acmella oleracea (L.) R.K. Jansen, also named jambu, is an edible and medicinal plant native to Brazil but extensively cultivated all over the world due to its diverse utilizations in food, cosmetics, pharmaceutics, and pest management science. Most of applications are related to the presence of active ingredients, namely alkylamides, among which spilanthol is the most important one. On the other hand, the plant biomass resulting from the large-scale cultivation of A. oleracea may yield an essential oil (EO), which can be used for some industrial purposes, for instance as larvicide against vector mosquitoes. This EO is mainly characterized by mono- and sesquiterpenes, however also spilanthol can be detected in the mixture. To maximize the content of spilanthol in the A. oleracea EO, A. oleracea aerial parts obtained from a cultivation in central Italy were subjected to microwave-assisted extraction (MAE). This procedure allowed to obtain a higher yield and spilanthol content when compared with traditional hydrodistillation (0.47 v 0.22 %, and 13.31 vs 2.24 %, respectively). The EO terpene fraction was mainly represented by β-pinene (10.8 %), myrcene (12.3 %), (E)-caryophyllene (19.4 %) and α-humulene (1.1 %). The A. oleracea EO was encapsulated into a nanoemulsion (NE) containing 6 % EO using a high-energy method. The formulation physical stability was assessed by optical microscope and DLS analyses at different timepoints showing stability up to one year of storage. The A. oleracea EO and its NE, along with the isolated spilanthol, were evaluated for acute toxicity against the 3rd instar larvae of Culex quinquefasciatus, a filariasis and arbovirus vector of public health importance, achieving LC50 values of 16.1, 407.5, and 3.1 μL/L, respectively. The larvicidal activity seemed to be not dependent on the interaction with the cholinergic system, as shown by the high IC50 values of A. oleracea EO and spilanthol exhibited in the anti-acetylcholinesterase (AChE) assay. Testing LC30 values, the A. oleracea-borne products showed a significant impact on larval development, fecundity, fertility and natality of C. quinquefasciatus. Testing LC30 values, the A. oleracea EO-NE provided a higher efficacy than spilanthol, reducing the adult fertility, in terms of egg hatchability (%) and the overall abundance of F1 larvae. These results can be considered as promising for the development of new mosquito larvicides. The safety use of A. oleracea EO, NE and spilanthol was demonstrated in assays on mammalian fibroblasts and microglia cells, showing low level of cytotoxicity coupled with protective effects against inflammation.

Neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, are caused by the progressive death of neurons in different regions of the nervous system. Their etiology is multifactorial with oxidative stress as one of the most impacting factors. The brain, due to its high oxygen consumption and high lipid content, is highly vulnerable to the effects of reactive oxygen species. The increase in the generation of free radicals damages the cellular biomolecules and induces necrosis or apoptosis. Sweet cherry fruits are a nutritionally dense food rich in anthocyanins, quercetin, hydroxycinnamates, potassium, fiber, vitamin C, and carotenoids. In the present study, we investigated the antioxidant activity of different sweet cherries (Prunus avium L.), obtained through a natural breeding program at the University of Bologna, in neuron-like SH-SY5Y cells differentiated with retinoic acid. The phytochemical pattern of five different cherry extracts has been characterized by HPLC-DAD/FL analysis. To study the protective effects of the extracts, SH-SY5Y cells were treated with different concentrations of the extracts for 24 h before H2O2 exposure. All the extracts, except one, were able to significantly increase cell viability and decrease ROS levels in respect to cells exposed to H2O2 as measured by MTT and DCFH-DA assays, respectively. Three of the five extracts significantly increased GSH levels in respect to H2O2 exposed cells as measured by monochlorobimane assay. The same extracts that significantly reduced ROS production were also able to up-regulate some important phase II antioxidant enzymes, such as GR and NQO1, as measured by RT-PCR. Our findings support the idea that these new sweet cherries can be considered a new functional food with a high antioxidant and neuroprotective effect and these effects seem to be related to the specific phenolic pattern of the different cherry extracts. This work was supported by MIUR-PRIN 2015 (No. 20152HKF3Z).

Neurodegenerative diseases, such as Alzheimer’s Disease (AD), are characterized by a multifactorial etiology and one of the most impacting factors is represented by oxidative stress, a biochemical process that is given by an imbalance between reactive oxygen species (ROS) and endogenous antioxidant defenses. Cherries, and in particular sweet cherries, are a nutritionally dense food rich in anthocyanins, quercetin, hydroxycinnamates, potassium, fiber, vitamin C, carotenoids, and melatonin. In the present study, we investigated the antioxidant activity of different Sweet Unibo cherries (Prunus avium L.), obtained through a natural breeding program at the University of Bologna, in neuron-like SH-SY5Y cells differentiated with Retinoic Acid (RA). Five different cherry extracts were characterized by HPLC-DAD/FL analysis. Cells were treated with the different cherry extracts for 24 h before H2O2 exposure. All the extracts, except one, were able to significantly increase cell viability, measured by MTT assay, in respect to cells exposed to H2O2. Moreover, the same four extracts reduced ROS production as measured by the dichlorofluorescein diacetate assay meanwhile only three of five extracts significantly increased GSH levels in respect to H2O2 exposed cells as measured by monochloro bimane assay. The same extracts that significantly reduced ROS production were also able to up-regulate phase II antioxidant enzymes glutathione reductase and NQO1 as measured by RT-PCR. Our findings support the idea that cherries can have a beneficial health effect in counteracting oxidative stress in neurons and these effects seem to be related to the specific phenolic pattern of the different cherry extracts. This work was supported by MIUR-PRIN 2015 (No. 20152HKF3Z).

Increasing experimental data suggests that the regular intake of polyphenols represents a potential way to improve the quality of life in aging. Pterostilbene is a derivative of the polyphenols resveratrol that has higher bioavailability and neuroprotective activity than resveratrol itself. The present study was carried out to investigate the anti-aging and anti-inflammatory effects of a lifelong dietary supplementation of pterostilbene in Drosophila melanogaster focusing on the expression of genes related to lifespan and senescence regulation (SIRT1; FOXO; p16; NOTCH) or to inflammatory response (DOME and Eiger1), after 2 weeks (T1) or 2 months (T2) of pterostilbene supplementation. To test the anti-neuroinflammatory effect of pterostilbene, the expression of IL1 and iNOS has been investigated by RT-PCR in BV2 microglial cells stimulated with LPS. Pterostilbene supplementation significantly increased the mean life span of both male and female fruit flies. Consistently, pterostilbene supplementation significantly increased NOTCH and SIRT1 expression in female flies at T1 and T2, whereas only at T2 in males. FOXO and p16 were up-regulated at T1 in females and at T2 in males. Eiger and DOME were reduced only in females at T1 and T2, respectively. The anti-inflammatory effect was also observed in BV-2 cells were PTS significantly reduced the up-regulation of IL1 and iNOS induced by LPS. These data confirmed the the anti-inflammatory activity of pterostilbene and suggest that it is influenced by gender.

Castanea sativa cultivation has been present in Mediterranean regions since ancient times. In order to promote a circular economy, it is of great importance to valorize chestnut groves’ by-products. In this study, leaves and spiny burs from twenty-four Castanea trees were analyzed by 1H NMR metabolomics to provide an overview of their phytochemical profile. The Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) performed on these data allowed us to distinguish ‘Marrone’ from ‘Castagna’, since the latter were generally more enriched with secondary metabolites, in particular, flavonoids (astragalin, isorhamnetin glucoside, and myricitrin) were dominant. Knowing that microglia are involved in mediating the oxidative and inflammatory response of the central nervous system, the potential anti-inflammatory effects of extracts derived from leaves and spiny burs were evaluated in a neuroinflammatory cell model: BV-2 microglia cells. The tested extracts showed cytoprotective activity (at 0.1 and 0.5 mg/mL) after inflammation induction by 5 µg/mL lipopolysaccharide (LPS). In addition, the transcriptional levels of IL-1β, TNF-α, and NF-kB expression induced by LPS were significantly decreased by cell incubation with spiny burs and leaves extracts. Taken together, the obtained results are promising and represent an important step to encourage recycling and valorization of chestnut byproducts, usually considered “waste”.

Sweet cherries (Prunus avium L.) are highly appreciated fruits for their taste, color, nutritional value, and beneficial health effects. In this work, seven new cultivars of sweet cherry were investigated for their main quality traits and nutraceutical value. The phytochemical profile of three classes of phenolic compounds and the antioxidant activity of the new cultivars were investigated through high-performance liquid chromatography with diode array detection (HPLC-DAD) and spectrophotometric assays, respectively, and compared with those of commonly commercialized cultivars. Cyanidine-3-O-rutinoside was the main anthocyanin in all genotypes, and its levels in some new cultivars were about three-fold higher than in commercial ones. The ORAC-assayed antioxidant capacity was positively correlated with the total anthocyanin index. The nutraceutical value of the new cultivars was investigated in terms of antioxidant/neuroprotective capacity in neuron-like SH-SY5Y cells. Results demonstrated that the new cultivars were more effective in counteracting oxidative stress and were also able to upregulate brain-derived neurotrophic factor (BDNF), a pro-survival neurotrophin, suggesting their potential pleiotropic role in counteracting neurodegenerations.

Neurodegenerative diseases include a variety of pathologies such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and so forth, which share many common characteristics such as oxidative stress, glycation, abnormal protein deposition, inflammation, and progressive neuronal loss. The last century has witnessed significant research to identify mechanisms and risk factors contributing to the complex etiopathogenesis of neurodegenerative diseases, such as genetic, vascular/metabolic, and lifestyle-related factors, which often co-occur and interact with each other. Apart from several environmental or genetic factors, in recent years, much evidence hints that impairment in redox homeostasis is a common mechanism in different neurological diseases. However, from a pharmacological perspective, oxidative stress is a difficult target, and antioxidants, the only strategy used so far, have been ineffective or even provoked side effects. In this review, we report an analysis of the recent literature on the role of oxidative stress in Alzheimer’s and Parkinson’s diseases as well as in amyotrophic lateral sclerosis, retinal ganglion cells, and ataxia. Moreover, the contribution of stem cells has been widely explored, looking at their potential in neuronal differentiation and reporting findings on their application in fighting oxidative stress in different neurodegenerative diseases. In particular, the exposure to mesenchymal stem cells or their secretome can be considered as a promising therapeutic strategy to enhance antioxidant capacity and neurotrophin expression while inhibiting pro-inflammatory cytokine secretion, which are common aspects of neurodegenerative pathologies. Further studies are needed to identify a tailored approach for each neurodegenerative disease in order to design more effective stem cell therapeutic strategies to prevent a broad range of neurodegenerative disorders.

Purpose of review This review summarises the most recent evidence regarding the effects of diet in preventing and reducing age-related cognitive decline and neurodegenerative diseases. Recent findings Recent evidence indicates that nutraceuticals and whole diet approaches may protect against the development of age-related cognitive decline and pathological neurodegeneration. The neuroprotective effects are diverse depending on the nutrient employed and may involve a reduction of neuroinflammation, an activation of the endogenous antioxidant defence system and a modulation of the gut microbiota structure and function. Summary This review summarises the existing evidence in favour of diet as a viable alternative approach to directly impact cognitive decline and neurodegenerative diseases. The single nutrient (polyphenols, B vitamins, long-chain polyunsaturated fatty acids) versus whole diet approach (Mediterranean diet, Dietary Approaches to Stop Hypertension, MIND, Nordic, ketogenic) is presented and discussed. Potential mechanisms of action underlying the beneficial effects of these diets are also described. Implementation of large-scale preventive interventions based on dietary patterns identified as being beneficial to brain health should be a research and public health priority, ideally in conjunction with other health-promoting lifestyle factors.

Natural antioxidants have been proposed to have beneficial effects on health and on different disease states, such as neurodegenerative and cardiovascular diseases, diabetes and cancer. The use of natural plant antioxidant products to handle different diseases has very ancient roots; well before the development of modern medicine with synthetic drugs and antioxidants. A lot of the biological activities of natural antioxidants have been ascribed to their ability to scavenge reactive oxygen species (ROS) that counteract oxidative stress. In the last years, a multitude of studies have suggested that their classical hydrogen-donating antioxidant activity is unlikely to be the sole explanation for their effects. First of all, natural antioxidants are subjected to an extensive metabolism in vivo that modifies their redox potentials. Moreover, the concentration of natural antioxidants and their metabolites in vivo are lower than that usually utilized in vitro. Accumulating evidence suggests that the cellular effects of natural antioxidants may also be mediated by their interactions with specific proteins central to intracellular signaling cascades, their modulation of the expression and activity of key proteins, their influencing of epigenetic mechanisms or their modulation of the gut microbiota. This special issue, concerning new mechanisms in the action of natural antioxidants in health and disease, contains nine contributions, seven research articles and two reviews, and details recent advances on this topic.

Background: There is a growing awareness that nutritional habits may influence risk of several inflammatory and immune-mediated disorders, including autoimmune diseases, through various mechanisms. The aim of the present study was to investigate dietary habits and their relationship with redox homeostasis in the setting of thyroid autoimmunity. Materials and Methods: Two hundred subjects (173 females and 27 males; median age, 37 years) were enrolled. None were under any pharmacological treatment. Exclusion criteria were any infectious/inflammatory/autoimmune comorbidity, kidney failure, diabetes, and cancer. In each subject, serum thyrotropin (TSH), free thyroxine, antithyroid antibodies, and circulating oxidative stress markers were measured. A questionnaire on dietary habits, evaluating the intake frequencies of food groups and adherence to the Mediterranean diet, was submitted to each participant. Results: Among the 200 recruited subjects, 81 (71 females and 10 males) were diagnosed with euthyroid Hashimoto's thyroiditis (HT); the remaining 119 (102 females and 17 males) served as controls. In questionnaires, HT subjects reported higher intake frequencies of animal foods (meat, p = 0.0001; fish, p = 0.0001; dairy products, p = 0.004) compared with controls, who reported higher intake frequencies of plant foods (legumes, p = 0.001; fruits and vegetables, p = 0.030; nuts, p = 0.0005). The number of subjects who preferentially consumed poultry instead of red/processed meat was lower in HT subjects than in controls (p = 0.0141). In logistic regression analysis, meat consumption was associated with increased odds ratio of developing thyroid autoimmunity, while the Mediterranean diet traits were protective. In HT subjects, serum advanced glycation end products (markers of oxidative stress) were significantly higher (p = 0.0001) than in controls, while the activity of glutathione peroxidase and thioredoxin reductase, as well as total plasma antioxidant activity, were lower (p = 0.020, p = 0.023, and p = 0.002, respectively), indicating a condition of oxidative stress. Stepwise regression models demonstrated a significant dependence of oxidative stress parameters on consumption of animal foods, mainly meat. Conclusions: The present study suggests a protective effect of low intake of animal foods toward thyroid autoimmunity and a positive influence of such nutritional patterns on redox balance and potentially on oxidative stress-related disorders.

Aging and age-related neurodegeneration are among the major challenges in modern medicine because of the progressive increase in the number of elderly in the world population. Nutrition, which has important long-term consequences for health, is an important way to prevent diseases and achieve healthy aging. The beneficial effects of Vigna unguiculata on metabolic disorders have been widely documented. Here, we show that an aqueous extract of V. unguiculata beans delays senescence both in Saccharomyces cerevisiae and Drosophila melanogaster, in a Snf1/AMPK-dependent manner. Consistently, an increased expression of FOXO, SIRT1, NOTCH and heme oxygenase (HO) genes, already known to be required for the longevity extension in D. melanogaster, is also shown. Preventing ?-synuclein self-assembly is one of the most promising approaches for the treatment of Parkinson's disease (PD), for which aging is a risk factor. In vitro aggregation of ?-synuclein, its toxicity and membrane localization in yeast and neuroblastoma cells are strongly decreased in the presence of bean extract. In a Caenorhabditis elegans model of PD, V. unguiculata extract substantially reduces the number of the age-dependent degeneration of the cephalic dopaminergic neurons. Our findings support the role of V. unguiculata beans as a functional food in age-related disorders.

The petals of the saffron crocus (Crocus sativus L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2&prime, 7&prime, dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC50 = 0.66 mg/mL versus EC50 = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases.

Neurodegenerative diseases are among the most serious health problems affecting millions of people worldwide, and their incidence is dramatically growing together with increased lifespan [...]

Sulforaphane, a biologically active isothiocyanate compound extracted from cruciferous vegetables, has been shown to exert cytotoxic effects on many human cancer cells, including leukemia. However, the exact molecular mechanisms behind the action of sulforaphane in hematological malignancies are still unclear. Like other cancer cells, leukemia cells produce high level of reactive oxygen species; in particular, hydrogen peroxide derived from Nox family is involved in various redox signal transduction pathways, promoting cell proliferation and survival. Recent evidence show that many tumour cell types express elevated level of aquaporin isoforms, and we previously demonstrated that aquaporin-8 acts as H2O2 transport facilitator across the plasma membrane of B1647 cells, a model of acute myeloid human leukemia. Thus, the control of AQP8-mediated H2O2 transport could be a novel strategy to regulate cell signalling and survival. To this purpose, we evaluated whether sulforaphane could somehow affect aquaporin-8-mediated H2O2 transport and/or Nox-mediated H2O2 production in B1647 cell line. Results indicated that sulforaphane inhibited both aquaporin-8 and Nox2 expression, thus decreasing B1647 cells viability. Moreover, the data obtained by coimmunoprecipitation technique demonstrated that these two proteins are linked to each other; thus, sulforaphane has an important role in modulating the downstream events triggered by the axis Nox2-aquaporin-8. Cell treatment with sulforaphane also reduced the expression of peroxiredoxin-1, which is increased in almost all acute myeloid leukemia subtypes. Interestingly, sulforaphane concentrations able to trigger these effects are achievable by dietary intake of cruciferous vegetables, confirming the importance of the beneficial effect of a diet rich in bioactive compounds.

Stevia rebaudiana Bertoni possess a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of the study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in a cell model expressing the insulin dependent glucose transporter, Glut 4. The potential antioxidant activity of steviol glycosides was also assessed, as oxidative stress is associated with diabetes. The insulin-mimetic effect and the antioxidant property exerted by steviol glycosides suggest their potential beneficial role in the co-treatment of diabetes and in health maintenance.

The effect of broccoli extract (BE)-enriched diet was studied in order to evaluate its ability to counteract liver cholesterol oxidation products (COPs) induced by acute strenuous exercise in rats. Thirty-two female Wistar rats were randomly divided into four groups: control diet without exercise (C), BE-enriched diet without exercise (B), control diet with acute exhaustive exercise (S) and BE-enriched diet with acute exhaustive exercise (BS). The study lasted 45 days and on the last day, rats of S and BS groups were forced to run until exhaustion on a treadmill. Glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and cholesterol oxidation products (COPs) were determined in liver. Exhaustive exercise was clearly responsible for tissue damage, as evidenced by the increase of lactate dehydrogenase (LDH) plasma activity increase in the S group rats. Moreover, the exercise protocol reduced CAT activity in liver, while it did not affect GST, GR and GPx. The BE-enriched diet increased raised GST, GR and CAT activities in rats of BS group. The main COPs found were 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholestanetriol, 24-hydroxycholesterol and 27-hydroxycholesterol. The BE-enriched diet led to reduced cholesterol oxidation following exhaustive exercise; the highest level of COPs was found in the S group, whereas the BS rats showed the lowest valueamount. This study indicates that the BE-enriched diet increases antioxidant enzyme activities and exerts an antioxidant effect towards cholesterol oxidation in rat liver, suggesting the use of phytochemicals in the prevention of oxidative damage and in the modulation of the redox environment.

Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

The modulation of H2 O2 production by NADPH oxidase (Nox), on vascular endothelial growth factor (VEGF) stimulation, affects the redox signaling linked to cancer cell proliferation. H2 O2 signal transduction involves reversible oxidation of thiol proteins, leading to the formation of cysteine sulfenic acids, responsible for the temporary inactivation of many phosphatases. These events imply that H2 O2 reaches its intracellular targets. As Aquaporin-8 (AQP8) has been demonstrated to funnel Nox-produced H2 O2 across the plasma membrane, this study aims to elucidate the role of AQP8 in the redox signaling occurring in human leukaemia B1647 cells that constitutively produce VEGF. AQP8 overexpression or silencing resulted in the modulation of VEGF ability of increasing or decreasing, respectively, H2 O2 intracellular level. Moreover, data obtained by a dimedone-based immunochemical method for sulfenic acid detection demonstrate that the expression of AQP8 can modulate the amplitude of downstream events, altering the activity of redox-sensitive targets. In particular, AQP8 affected VEGF-induced redox signaling by increasing the sulfenation of the tumor suppressor PTEN, which resulted in its inactivation and, in turn, caused Akt activation. Therefore, the dimedone-based method for easily monitoring cellular protein sulfenation allowed to demonstrate, for the first time, the role of AQP8 on the fine tune of cysteine oxidation in target proteins involved in leukaemia cell proliferation pathways. © 2016 BioFactors, 43(2):232-242, 2017.

The efficient use of stem cells for transplantation is often limited by the relatively low number of stem cells collected. The

Neurodegenerative diseases are a heterogeneous group of disorders characterized by a progressive dysfunction and death of neural cells which lead to compromised motor or cognitive function. Currently, there are no therapeutic approaches to treat or even halt the inexorable progression of these debilitating disorders. In this context, great attention has been paid to phytochemicals because of their broad spectrum of pharmacological and biological activities. Among phytochemicals, olive oil phenols are getting more scientific attention due to their interesting biological activities. In particular, inside the secoiridoid family of olive oil constituents, oleocanthal, also known as (-)decarboxymethyl ligstroside aglycone , has been mainly investigated for its anti-inflammatory activity and its ability to counteract abnormal protein aggregation and deposition involved in Alzheimer’s disease. Oleocanthal demonstrated to possess a strong pleiotropic activity against neurodegeneration as, besides its known biological effects like its anti-inflammatory activity, and its ability to reduce Aβ and neurofibrillary tangles production, new mechanisms of action are emerging like its ability to counteract oxidative stress and to enhance neuron energy metabolism. Moreover, promising results suggest a potential role of this compounds in inhibiting the deleterious effect of protein glycation.

Neuroinflammation is one of the main contributors to the onset and progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Microglial and astrocyte activation is a brain defense mechanism to counteract harmful pathogens and damaged tissues, while their prolonged activation induces neuroinflammation that can trigger or exacerbate neurodegeneration. Unfortunately, to date there are no pharmacological therapies able to slow down or stop the progression of neurodegeneration. For this reason, research is turning to the identification of natural compounds with protective action against these diseases. Considering the important role of neuroinflammation in the onset and development of neurodegenerative pathologies, natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Marine organisms represent a huge source of natural compounds, and among them, algae are appreciated sources of important bioactive components such as antioxidants, proteins, vitamins, minerals, soluble dietary fibers, polyunsaturated fatty acids, polysaccharides, sterols, carotenoids, tocopherols, terpenes, phycobilins, phycocolloids, and phycocyanins. Recently, numerous anti-inflammatory compounds have been isolated from marine algae with potential protective efficacy against neuroinflammation. This review highlights the key inflammatory processes involved in neurodegeneration and the potential of specific compounds from marine algae to counteract neuroinflammation in the CNS.

Referees are essential for the correct execution of each soccer match and national soccer associations support them with physical training guidelines in order to improve agility, aerobic and sprint capacity, to reduce the risk of injury and to meet the minimum required performance levels. Despite the importance given to performance skills no specific nutritional guideline is provided to support the performance required. This study was aimed to investigate soccer field and assistant referees knowledge and consumption of dietary supplements. Seventy-one European referees participated to this survey by filling a short and quick anonymous questionnaire designed to collect information about their refereeing category, educational level, frequency of supplement consumption, type of supplement consumed. Moreover, a sub-group of 20 participants wore a training watch to record energy expenditure, distance, and heart rate during training sessions and matches. Our data show that referees frequently consume supplements especially those refereeing in lower categories, moreover they often follow a friend’s suggestion when deciding to take a supplement instead of trusting a professional prescription. According to our survey, national and international soccer federations should care more about their referee nutritional education.

Introduction: This review is an update on recent progress in proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues, which open the opportunity to investigate diseases and research potential biomarkers, particularly when availability of fresh/frozen tissues is low. Areas covered: We described improvement of existing protocols or the new ones regarding deparaffinization and protein extraction of FFPE samples published from 2014 to today. Moreover, the growing interest to use FFPE tissues for mass spectrometry imaging approach is presented together with the search of post-translational modifications. Expert opinion: In the last few years, the number of papers using FFPE tissues in proteomic analysis is growing. The interest to apply proteomic analysis to FFPE tissues lies in the easy accessibility of a great number of samples from archives. Nevertheless, standardization in the approach among the different researchers is not achieved, making essentially incomparable the results obtained. This limit should be overcome.

This study was carried out to characterize extracts from nine samples of the apple 'Mela Rosa dei Monti Sibillini' (MR) and to assess the antioxidant and anti-inflammatory activities. The extracts were analysed by High Performance Liquid Chromatography coupled with photodiode array detector and mass spectrometry (HPLC-DAD-MS) for 20 phytochemicals. The extracts from the lyophilized material (ELM) were richer in polyphenolic compounds than the dried ones (EDM). The MR extracts contained noteworthy amounts of the investigated analytes compared to one sample of the commercial varieties Annurca, Golden Delicious and Granny Smith used as reference. Principal component analysis (PCA) revealed that the part of the fruit seems to have a significant influence on the chemical composition of the final extract; thus, the peel extracts exhibited higher levels of phenolic compounds, especially epicatechin, procyanidin B2 and phloridzin, and triterpenes than the pulp ones. In general, the lyophilized material showed higher antioxidant activity than the dried material. The strong antioxidant capacity of the MR has also been revealed by the DPPH, ABTS, FRAP and Folin-Ciocalteau assays. The ELM of MR significantly reduced reactive oxygen species in lipopolysaccharide (LPS)-activated mouse brain microglia cells (BV-2 cells). Real-time polymerase chain reaction (RT-PCR) analysis demonstrated that the EDM and ELM of MR were effective in reducing pro-inflammatory cytokines and enzymes in BV-2 and peripheral blood mononuclear cells (PBMC). These results contribute to the exploitation of this ancient variety as a source of nutraceuticals.

Cancer cells are characterized by an increase in spatially localized reactive oxygen species (ROS) production and by an altered redox environment compared to normal cells. Consequently, signalling pathways that promote cell proliferation, survival, angiogenesis and metastasis are hyper-activated. In particular, hydrogen peroxide (H2O2) derived from NOX family is involved in various redox signal transduction pathways and the aquaporin 8 (AQP8) has been identified as a H2O2 transport facilitator across the plasma membrane. Recent evidence demonstrated that many tumor cell types express elevated level of aquaporin isoforms and highlighted a positive correlation between histological tumor grade and the AQP expression. Sulforaphane (SFN), an isothiocyanate compound present in abundance in cruciferous vegetables, has been found to induce therapeutic effects against a wide array of malignancies in both experimental and epidemiological studies . Therefore, this study aimed at the evaluation of the potential effect of SFN on the modulation of AQP8, NOX2 and p-VEGFR-2 expression in B1647 cell line, a model of acute myeloid leukemia. In fact, we previously demonstrated that AQP8 funnels NOX-derived H2O2, triggered by endogenously generated VEGF, which, in turn, provokes VEGFR-2 phosphorylation and the consequent modulation of many cellular activities, resulting in cell survival and proliferation. As peroxiredoxin (Prx1) represents a member of the so called thiol-based antioxidant system that acts as redox switches to modulate redox signalling pathways, the effect of SFN on Prx1 expression was also evaluated. Unravelling the role of AQP8 in cancer redox signalling, and the effect exerted by SFN on its modulation, can offer new potential target for anti-cancer therapy, suggesting the importance of dietary co-treatment.

Human amniotic fluid stem cells (AFSCs) are a powerful source of cells with a plethora of trophic and immunomodulatory functions. These cells can be appropriately expanded ex-vivo to be differentiated in many cell types, in order to dissect developmental pathways, unravel pathogenic mechanisms and replace defective tissues. Traditionally, in vitro AFSCs are exposed to non-physiological concentrations of O2 increasing the production of reactive oxygen species (ROS). The unbalance in the redox signals may impair the AFSC stemness machinery1. In this context the use of natural antioxidants could be proposed as a strategy to prevent the loss of self-renewal and the extensive cellular damage due to abnormal ROS production. To this aim, AFSCs were exposed to sulphorafane (SF) and epigallo-3-catechingallate (EGCG) to counteract ROS deleterious effects. AFSCs were co-treated with 1 M SF and 10 M EGCG. After 72 h a slight increase in their metabolic activity was observed. However, cell population doubling over a cell culture period of 25 days was not different between untreated and co-treated samples. Interestingly, the co-treatment with SF and EGCG synergistically reduced intracellular ROS level in respect to SF or EGCG treatment alone, as measured by DCFH-DA assay. In addition, co-treated AFSCs displayed higher total GSH level in respect to the untreated samples. Self-renewal capacity was investigated evaluating the expression of pluripotent gene markers such as OCT4, NANOG and SOX2 by RT-PCR. Interestingly, the co-treatment was able to up-regulate the expression of all the three genes. Moreover, the chronic treatment for 21 days with SF and EGCG counteracted the expected decrease of OCT4 and NANOG expression during cell passaging. In conclusion, we propose the co-treatment with SF and EGCG as a strategy to counteract oxidative stress and maintain the functionality of AFSCs.

Different studies demonstrated that female gender is associated with improved heart failure survival, and estrogens seem to play a fundamental role in this protection. It has been suggested that 17 β-Estradiol (E2) reduces apoptosis in animal model of myocardial infarction through the modulation of MAPKs involved in oxidative stress. Moreover, recent evidences suggest that gender can influence the response to cardiovascular medications. Therefore, we hypothesized that sex hormones could also modulate the cardioprotective effects of nutraceutical compounds, such as the isothiocyanate sulforaphane (SF), present in Brassica vegetables. In a previous study we evidenced that low concentrations of SF, in the presence of E2, counteracted oxidative damage better ttna SF alone. This effect seems to be related to the up-regulation of several antioxidant enzymes (GST,HO-1,GR, Trx, NQO1) and to the activation of pro-survival pathways (Pi3K/Akt adn ERK 1/2). Aim of this study was to better clarify the mechanisms of E2-mediated potentiation of SF effects. H9c2 cardiomyoblasts were treated with SF in the absence/presence of E2 for different times. After 6h treatment, the co-treatment with SF and E2 induced a higher activation of Nrf2 transcription factor in respets to SF treatment alone. To study the involvement of E2 in SF induced Akt activation specific agonists for estrogen receptors were used. GPR30 (G protein-cooupled receptor 30) and ERβ (estrogen receptor β) emerged to be involved in Akt actovation. Moreover the co-treatment increased free [Ca++] evaluated by Fura-2 probe suggesting its involvement in E2 mediated effects. Our results increase the knowledge on E2 mediated potentiation of SF effects in cardiac cells.

The research of value-added applications for coffee silverskin (CSS) requires studies to investigate potential bioactive compounds and biological activities in CSS extracts. In this study, different ultrasound‐assisted extraction (UAE) methods have been tested to extract bioactive compounds from CSS. The obtained extracts, were characterized using a new HPLC-MS/MS method to detect and quantify 30 bioactive compounds of 2 classes: alkaloids and polyphenols (including phenolic acids, flavonoids, and secoiridoids). CSS extracts obtained with ethanol/water (70:30) as extraction solvent showed the highest levels (p ≤ 0.05) of bioactive compounds (4.01 ± 0.34% w/w). High content of caffeine was observed with levels varying from 1.00% to 3.59% of dry weight of extract (dw). 18 phenolic compounds were detected in CSS extracts with caffeoylquinic acids (3-CQA, 5-CQA and 3,5-diCQA) as the most abundant polyphenols (3115.6 µg g to –5444.0 µg g−1). This study is also one of the first to characterize in-depth flavonoids in CSS revealing the levels of different flavonoids compounds such as rutin (1.63–8.70 µg g−1), quercetin (1.53–2.46 µg g−1), kaempferol (0.76–1.66 µg g−1) and quercitrin (0.15–0.51 µg g−1). Neuroprotective activity of silverskin extracts against H2O2-induced damage was evaluated for the first time suggesting for methanol and ethanol/water (70:30) extracts a potential role as protective agents against neurodegeneration due to their ability to counteract oxidative stress and maintain cell viability. Silverskin extracts were not inhibiting the growth of anyone of the bacterial species included in this study but data obtained by water extract might deserve a deeper future investigation on biofilm-related activities, such as quorum sensing or virulence factors’ expression. From their composition and their evidenced biological activities, CSS extracts could represent valuable ingredients in nutraceutical formulations.

Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.

Cardiovascular diseases (CVDs) are the leading cause of mortality in the Western world. Multiple factors are involved in CVD, including genetic factors and modifiable factors such as diet, physical activity, and smoking. CVD incidence and prevalence increase progressively with age, and it is estimated that over 80% of men and women older than 75 years have clinically manifest CVD. To reduce the gap between life expectancy (LE) and healthy life expectancy is one of the main challenges of the 21st century. Lifestyle improvement appears to be the only sustainable approach to face the dramatic chronic-degenerative disease burden of an aging population. A healthy lifestyle, represented by avoiding smoking, following a healthy diet, and practicing physical activity, protects from chronic-degenerative disease onset and progression. A healthy dietetic approach specifically formulated for elderly people, with a defined pattern of nutraceutical bioactive compounds, may represent a key strategy to improve the aging process and increase the life span. This short review summarizes the biochemical mechanisms underpinning the cardiovascular protective effects of some nutraceutical compounds such as quercetin and sulforaphane.

Introduction Many aspects of cardiovascular diseases are similar in man and women, but some differences on risk profile, age of onset and response to medical treatments are emerging. The incidence of ischemic heart disease is significantly lower in women than in men until menopause. This observation suggests that female sex hormones, such as estrogen, may have a cardioprotective role. Our hypothesis is that sex hormones could modulate the protective effects of some nutraceutical compounds, such as the isothiocyanate sulforaphane, present in Brassica vegetables. This study was designed to explore the protective role of low concentrations of sulforaphane in the presence/absence of 17β-estradiol against oxidative stress in cardiomyocytes. Methods Primary cultures of cardiomyocytes were obtaind from 1-2 days old Sprague-Dawley rats. Cell viability was evaluated by MTT and LDH assays, ROS production by 2,7-dichlorodihydrofluorescein diacetate assay and immunofluorescence staining with anti-8-hydroxyguanosine, GSH levels by the monochlorobimane assay, antioxidant enzyme expression by RT-PCR. Transmission electron Microscopy analysis was performed to investigate the ultrastructure of cells. Results Cardiomyocytes were treated with sulforaphane and/or 17β-estradiol. Sulforaphane and 17β-estradiol co-treatment led to a higher level of protection against H2O2 induced oxidative stress than sulforaphane or 17β-estradiol alone. The co-treatment significantly reduced intracellular ROS levels in respect to 17β-estradiol or sulforaphane and increased the expression of different antioxidant enzymes in respect to sulforaphane or 17β-estradiol alone. As expected. The co-treatment increased GSH intracellular levels. Cells under oxidative stress showed a damaged cytoplasmic morphology, with several vacuoles, dilated rough endoplasmic reticulum and mitochondrial cristae. Co-treatment protected cells from oxidative stress damage, showing well preserved cytoplasmic organelles and nuclear morphology. Conclusions Our results demonstrated, for the first time, that estrogens could modify sulforaphane protective effects, suggesting that nutraceutical efficacy might be different in male and female gender.

Neurological disorders such as stroke, Alzheimer’s and Parkinson’s diseases are associated with high morbidity and mortality, and few or no effective options are available for their treatment. These disorders share common pathological characteristics like the induction of oxidative stress, abnormal protein aggregation, perturbed Ca2+ homeostasis, excitotoxicity, inflammation and apoptosis. A large body of evidence supports the beneficial effects of the Mediterranean diet in preventing neurodegeneration. As the Mediterranean diet is characterized by a high consumption of extra-virgin olive oil it has been hypothesized that olive oil, and in particular its phenols, could be responsible for the beneficial effect of the Mediterranean diet. This review provides an updated vision of the beneficial properties of olive oil and olive oil phenols in preventing/counteracting both acute and chronic neurodegenerative diseases.

In order to examine potential differences in genomic stability, we have challenged fibroblasts derived from five different mammalian species of variable longevity with the genotoxic agents, etoposide and neocarzinostatin. We report that cells from longer-lived species exhibit more tumor protein p53 binding protein 1 (53BP1) foci for a given degree of DNA damage relative to shorter-lived species. The presence of a greater number of 53BP1 foci was associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. These data suggest that cells from longer-lived species have an enhanced DNA damage response. We propose that the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms.

The lower incidence of ischemic heart disease in female with respect to male gender suggests the possibility that female sex hormones could have specific effects in cardiovascular protection. 17β-Estradiol is the predominant premenopausal circulating form of estrogen and has a protective role on the cardiovascular system. Recent evidences suggest that gender can influence the response to cardiovascular medications; therefore, we hypothesized that sex hormones could also modulate the cardioprotective effects of nutraceutical compounds, such as the isothiocyanate sulforaphane, present in Brassica vegetables. This study was designed to explore the protective effects of sulforaphane in the presence of 17β-estradiol against H2O2-induced oxidative stress in primary cultures of rat cardiomyocytes. Interestingly, 17β-estradiol enhanced sulforaphane protective activity against H2O2-induced cell death with respect to sulforaphane or 17β-estradiol alone as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide and lactate dehydrogenase assays. Moreover, 17β-estradiol boosted sulforaphane ability to counteract oxidative stress, reducing intracellular reactive oxygen species and 8-hydroxy-2'-deoxyguanosine levels and increasing the expression of phase II enzymes. Using specific antagonists of estrogen receptor α and β, we observed that these effects are not mediated by estrogen receptors. Otherwise, ERK1/2 and Akt signaling pathways seem to be involved, as the presence of specific inhibitors of these kinases reduced the protective effect of sulforaphane in the presence of 17β-estradiol. Sulforaphane and 17β-estradiol co-treatment counteracted cell morphology alterations induced by H2O2 as evidenced by transmission electron microscopy. Our results demonstrated, for the first time, that estrogens could enhance sulforaphane protective effects, suggesting that nutraceutical efficacy might be modulated by sex hormones.

Traumatic brain injury (TBI) is a mechanical insult to the brain caused by external forces and associated with inflammation and oxidative stress. The patients may show different profiles of neurological recovery and a combination of oxidative damage and inflammatory processes can affect their courses. It is known that an overexpression of cytokines can be seen in peripheral blood in the early hours/days after the injury, but little is known about the weeks and months encompassing the post-acute and chronic phases. In addition, no information is available about the antioxidant responses mediated by the major enzymes that regulate reactive oxygen species levels: superoxide dismutase, catalase, peroxidases, and GSH-related enzymes. This study investigates the 6-month trends of inflammatory markers and antioxidant responses in 22 severe TBI patients with prolonged disorders of consciousness, consecutively recruited in a dedicated neurorehabilitation facility. Patients with a high degree of neurological impairment often show an uncertain outcome. In addition, the profiles of plasma activities were related to the neurological recovery after 12 months. Venous peripheral blood samples were taken blindly as soon as clinical signs and laboratory markers confirmed the absence of infections, 3 and 6 months later. The clinical and neuropsychological assessment continued up to 12 months. Nineteen patients completed the follow-up. In the chronic phase, persistent high plasma levels of cytokines can interfere with cognitive functioning and higher post-acute levels of cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL1b, IL6] are associated with poorer cognitive recoveries 12 months later. Moreover, higher IFN-γ, higher TNF-α, and lower glutathione peroxidase activity are associated with greater disability. The results add evidence of persistent inflammatory response, provide information about long-term imbalance of antioxidant activity, and suggest that the over-production of cytokines and the alteration of the redox homeostasis in the post-acute phase might adversely affect the neurological and functional recovery. Inflammatory and antioxidant activity markers might offer a feasible way to highlight some of the processes opposing recovery after a severe TBI.

Currently, the majority of cell-based studies on neurodegeneration are carried out on two-dimensional cultured cells that do not represent the cells residing in the complex microenvironment of the brain. Recent evidence has suggested that three-dimensional (3D) in vitro microenvironments may better model key features of brain tissues in order to study molecular mechanisms at the base of neurodegeneration. So far, no drugs have been discovered to prevent or halt the progression of neurodegenerative disorders. New therapeutic interventions can come from phytochemicals that have a broad spectrum of biological activities. On this basis, we evaluated the neuroprotective effect of three phytochemicals (sulforaphane, epigallocatechin gallate, and plumbagin) alone or in combination, focusing on their ability to counteract oxidative stress. The combined treatment was found to be more effective than the single treatments. In particular, the combined treatment increased cell viability and reduced glutathione (GSH) levels, upregulated antioxidant enzymes and insulin-degrading enzymes, and downregulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 in respect to peroxide-treated cells. Our data suggest that a combination of different phytochemicals could be more effective than a single compound in counteracting neurodegeneration, probably thanks to a pleiotropic mechanism of action.

The purpose of this study was the development of multifunctional liposomes for nasal administration of tacrine hydrochloride. Liposomes were prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol and/or Omega3 fatty acids. This approach was chosen in order to obtain at the same time two positive results: an enhanced drug permeation through nasal mucosa and a concomitant neuroprotective effect. Several liposome formulations were prepared using the Reverse Phase Evaporation technique followed by membrane filter extrusion. In particular, liposome capacity to enhance drug permeation was evaluated by means of membrane permeation and cellular uptake studies. Furthermore, liposome effect on neuronal viability and intracellular ROS production was evaluated as well as their cytoprotective effect against oxidative stress. All liposome formulations showed a mean diameter in the range of 175 nm to 219 nm with polydispersity index lower than 0.22, a lightly negative zeta potential and excellent encapsulation efficiency. Moreover, along with good mucoadhesive properties, multifunctional liposomes showed a markedly increase in tacrine permeability, which can be related to liposome fusion with cellular membrane, a hypothesis, which was also supported by cellular uptake studies. Finally, the addition of α-tocopherol without Omega3 fatty acids, was found to increase the neuroprotective activity and antioxidant properties of liposomes.

Oxidative stress, excitotoxicity, inflammation, misfolded proteins, and neuronal loss are common characteristics of a wide range of chronic neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. For these disorders, the current healthcare outcomes are considered inadequate; in fact these pathologies are treated after onset of the disease, frequently at near end-stages, and pessimistic prognosis considers pandemic scenario for these disorders over the next 10–20 years. Phytochemicals have been regarded as an alternative and preventive therapeutic strategy to control the occurrence and progression of neurodegenerative diseases. Recent research has shown that dietary phytochemicals have pleiotropic behaviors, exerting antioxidant, anti-inflammatory, and cytoprotective effects in neuronal and glial cells. In particular, isothiocyanates, the activated form of glucosinolates present in Brassica vegetables, have shown neuroprotective activity in several experimental paradigms due to their peculiar ability to activate the Nrf2/ARE pathway, playing a role in boosting the neuronal natural phase 2 enzyme antioxidant defense system and functioning as a powerful indirect antioxidant. This chapter summarizes the preventive glucosinolate-derived isothiocyanates effects in neurodegeneration and underscores the powerful preventive role that these compounds play in assisting the body to help fend off a variety of neurodegenerative diseases.

The isothiocyanate sulforaphane (SF), abundant in Cruciferous vegetables, is known to induce antioxidant/detoxification enzymes in many cancer cell lines, but studies focused on its cytoprotective action in nontransformed cells are just at the beginning. Since we previously demonstrated that SF elicits cardioprotection through an indirect antioxidative mechanism, the aim of this study was to analyze the signaling pathways through which SF exerts its protective effects. Using cultured rat cardiomyocytes, we investigated the ability of SF to activate Akt/protein kinase B (PKB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways, which are implicated in cardiac cell survival, and to increase the phosphorylation of Nuclear factor E2-related factor 2 (Nrf2) and its binding to the antioxidant response element. By means of specific inhibitors, we demonstrated that the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway represents a mechanism through which SF influences both expression and activity of glutathione reductase, glutathione-S-transferase, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase-1, analyzed by western immunoblotting and spectrophotometric assay, respectively, and modulates Nrf2 binding and phosphorylation resulting in a cytoprotective action against oxidative damage. Results of this study confirm the importance of phase II enzymes modulation as cytoprotective mechanism and support the nutritional assumption of Cruciferous vegetables as source of nutraceutical cardioprotective agents.

Oxidative stress plays a major role in the pathophysiology of cardiac disorders, but the experimental data on the protective effects of exogenous antioxidants are controversial. A promising cardioprotective strategy may be through the induction of the endogenous antioxidants and phase II enzymes by chemical inducers. Sulforaphane is an isothiocyanate derived from cruciferous vegetables, and it has gained attention mainly as a potential chemopreventive agent in part through the induction of detoxifying enzymes. Accordingly, this study was undertaken to investigate the time-dependent induction of gene transcription, protein expression, and enzyme activity of antioxidant and phase II enzymes [glutathione reductase, glutathione-S-transferase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase-1, thioredoxin reductase] by sulforaphane in cultured rat neonatal cardiomyocytes. The potential cardioprotective action of sulforaphane was confirmed by the decrease in intracellular reactive oxygen species production, the increase in cell viability, and the decrease in DNA fragmentation after long-term treatment accompanied by the induction of antioxidants and phase II enzymes in cardiomyocytes.

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism.

Apha-1-adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha-1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.

In a single-dosing crossover study, we investigated the ability of apple fruit consumption to protect human lymphocytes against peroxide-induced damage to DNA. Six healthy, non-smoking male volunteers were placed for 2 d on an antioxidant-poor (AP) diet. After 48 h of AP diet, the volunteers were required to consume a homogenate obtained from 600 g of red delicious unpeeled apples or water (500 ml); blood samples were collected 0, 3, 6 and 24 h post-consumption. To evaluate whether the apple intake was sufficient to restore resistance of DNA to oxidative damage, for each subject at any time point the plasma total antioxidant activity, reactive oxygen species (ROS) formation and induction of micronuclei (MN) in isolated lymphocytes following hydrogen peroxide (H2O2) treatment were measured. Results indicated a significant inhibition (58 %, P 2O2-induced MN frequency in the plasma samples collected at 3 h after apple consumption, as compared with plasma samples collected at 0 h (4·17 (sd 1·83) v. 9·85 (sd 1·87) MN/1000 binucleated (BN) cells, respectively). A gradual return towards the value observed at 0 h was recorded starting from 6 to 24 h. MN frequency induced by H2O2 was significantly influenced by plasma total antioxidant activity (r = –0·95, P r = 0·88, P

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved.