Abstract: Aim of this study was to evaluate in vitro the protective and antioxidant properties of the natural compounds lutein and astaxanthin on human primary corneal epithelial cells (HCE-F) exposed to high intensities of blue light. To this purpose, HCE-F cells were irradiated with a blue-light lamp (415-420 nm) at different energies (0 – 20 – 50 - 80 J/cm²). Lutein and astaxanthin at doses found to be nontoxic (50 – 100 – 250 mM) were added to HCE-F right before blue-light irradiation at the selected dose of 50 J/cm². In all experiments, viability was evaluated by the MTT assay and by the production of reactive oxygen species (ROS) using the H2DCF-DA assay. Viability of HCE-F cells progressively decreased from 20 J/cm² to 80 J/cm², and ROS production dramatically increased at 50 and 80 J/cm². The presence of lutein or astaxanthin protected the cells from phototoxicity, with lutein slightly more efficient than astaxanthin also on the blunting of ROS. The association of lutein and astaxanthin was equally effective, and apparently did not give an advantage over the use of lutein alone likely because of the higher efficiency of lutein in blunting blue light radiation, while astaxanthin is more efficient towards the higher wavelengths.

Oxidative stress is often the cause of a wide range of human chronic pathologies including but not limited to stroke, cardiovascular and neurodegenerative diseases. In this setting, increasing efforts are currently being made to the design and synthesis of new antioxidant compounds with enhanced efficacy. Among all the potential molecules of interest, synthetic nitrone spin-traps have attracted a great deal of research attention, particularly due to their dual function: effective inhibitors of oxidative stress and damage, and actual analytical tool for the detection and characterization of free radicals using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, two derivatives of benzoxazinic nitrones (3-aryl-2H-benzo[1,4]oxazin-N-oxides) bearing an electron-withdrawing methyl-acetate group on the benzo moiety (in para and meta positions respect to the nitronyl function) were synthesized. Their in vitro antioxidant activity was evaluated by means of the α,α-diphenyl-β-picrylhydrazyl radical (DPPH•) scavenging assay, and their inhibitory effects on the erythrocyte hemolysis induced by the water soluble free radical initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) compared. In addition, EPR was employed to monitor the decay profiles of DPPH• to evaluate the kinetic behavior of the different antioxidants tested. Results showed that the newly synthesized benzoxazinic derivatives, when compared to the unsubstituted parent compound, act as more effective antioxidants both in cell and cell-free systems. In particular, the para-substituted derivative was able to quickly scavenge DPPH to a greater extent in comparison with the meta-substituted isomer. Overall, these results clearly demonstrate that the introduction of an electron-withdrawing group in the phenyl moiety significantly increased the antioxidant capacity of benzoxazinic nitrones, thus showing exciting opportunities as new therapeutic agents in the treatment of diseases associated with oxidative stress.

The cocoa shell is a by-product generated by the cocoa processing industry that could be used as a nutraceutical owing to the significant amounts of bioactive compounds it contains. Nonetheless, determining the biological potential of the cocoa shell by evaluating the bioaccessibility of the phenolics present therein and their potential antioxidant capacity is still necessary. This work aimed to study the bioaccessibility of phenolic compounds present in the flour (CSF) and an aqueous extract (CSE) from cocoa shells through an in vitro simulated digestion and to assess their antioxidant capacity in vitro and by using intestinal and hepatic cell culture models (IEC-6 and HepG2 cells). A simulated in vitro digestion model (INFOGEST) was used to determine the bioaccessibility of phenolic compounds. Total phenolic compounds and in vitro antioxidant activity were measured by the Folin-Ciocalteu and ABTS assays, respectively. Reactive oxygen species (ROS) were evaluated in IEC-6 and HepG2 cells upon stimulation with t-BOOH. Phenolic compounds present in CSE were more bioaccessible than phenolic compounds present in CSF. During digestion, the bioaccessibility of phenolic compounds from CSF fluctuated in the gastric (2.8 mg/g), intestinal phase (7.6 mg/g), and the colonic (5.7 mg/g) phases. Similarly, for the phenolics of CSE, the bioaccessibility increased from 50.6 mg/g (gastric) to 53.4 mg/g (intestinal) and decreased in the colonic phase (37.2 mg/g). The in vitro antioxidant capacity followed a similar behavior, increasing throughout the digestion in CSF (8.8 to 10.6-fold) and CSE (6.0 to 7.4-fold). Digested CSF and CSE were not cytotoxic for IEC-6 and HepG2 cells and protected their viability under oxidative stress conditions (93-100%). t-BOOH-induced ROS were prevented by CSF (72-88 %) and CSE (81-94 %) bioaccessible fractions in both intestinal and hepatic cells. In conclusion, cocoa shells are a source of potentially bioavailable antioxidant phenolic compounds that may protect cells from oxidative stress.

Plant phenolic compounds are metabolites with a strong antioxidant capacity. Despite the fact that plants synthesize them mainly for their own defense against oxidative stress, these compounds retain the ability to act as ex-plant antioxidants and, therefore, they contribute greatly to the dietary and pharmaceutical properties of plant-food. These compounds have a wide range of structures and functions. Generally, they possess an aromatic ring with one or more hydroxyl substituents and have been classified into at least 10 different classes based on their basic chemical structure. The aim of this work was to carry out the characterization of the phenolic content and the determination of the antioxidant capacity of 4 plant matrices. Firstly, the plant matrices Lippia citriodora, Hibiscus sabdariffa, Olea europaea and Silybum marianum were chosen due to their high content of phenolic compounds. After that, a qualitative characterization of the phenolic profile from the selected plant extracts wasperformed using a high-performance liquid chromatography coupled to mass spectrometry. Finally, determination of the total content of polyphenols was carried out using the Folin-Ciocalteu method and the antioxidant capacity using the electron transfer methods, FRAP and TEAC, and hydrogen donation, ORAC. A comprehensive characterization and biological antioxidant properties of selected sources have been obtained. After the chromatographic analysis of the phenolic profile of each matrix, it was observed that in L. citriodora the abundance of the group of phenylpropanoids and iridoids stands out, in H. sabdariffa the groups of anthocyanins and flavonoids, in O. europaea secoiridoids and terpenes while in S. marianum the group of flavonoids. Regarding the antioxidant capacity, L. citriodora showed better results for the FRAP assay, while for the TEAC and ORAC assays the highest values were for S. marianum.

Introduction: Glyoxalase II (Glo2), the second enzyme of Glyoxalase system, is an antioxidant glutathione-dependent enzyme, that catalyzes the hydrolysis of S-D-lactoylglutathione (SLG) to form D-lactic acid and glutathione (GSH). GSH is the most important thiol reducing agent inside the cell. For its chemistry features GSH plays a crucial role not only in the cellular redox state but also in various cellular processes including protein S-glutathionylation. S-glutathionylation a process that involves the reversible formation of a mix disulphide-bridge between specific cysteine residues and GSH can be spontaneous or catalyzed by enzyme. S-Glutathionylation, also it is involved in protection of protein thiol groups from irreversible oxidation and play key role in redox regulation by activation/inactivation of different enzyme.

Methods: Glo2 and SLG were incubated with different proteins that are known to be glutathionylated, like malate dehydrogenase, actin or cytochrome c purified proteins.

Results: During the hydrolysis of SLG, in the active site of Glo2 there is unprotonated glutatione molecule (GS-) which can be transferred to protein target. These in vitro studies demonstrated high propensity of Glo2 to aggregate with other proteins through its catalytic site leading to an enzymatic regulation of S-glutathionylation in proteins of different origin and cellular compartmentalization.

Conclusion: Identification of active involvement of Glo2 in glutathionylation of different proteins. In this perspective, Glo2 can play a new important regulatory role in S-glutathionylation, acquiring further significance in cellular post-translational modifications of protein.

Ochratoxin A (OTA) is a mycotoxin which represents an emerging problem for both animal and human health, due to its high presence in feed and foods. Exposure to OTA is associated with oxidative stress-induced nephrotoxicity. Therefore, the identification of new antioxidant or adsorbent substances with protective action constitutes one of the main challenges to reduce the negative effects induced by mycotoxins. For this purpose, we investigated the effect of two innovative feed additives, a bio-organoclay (CHS) and a mixture of a tri-octahedral Na-smectite with a ligno-cellulose based material (MIX) alone or in combination with OTA in kidneys of treated chickens. Real-Time PCR analyses for NADPH oxidase 4 (NOX) and p47-phox were performed to evaluate oxidative stress. Our results demonstrated an increase in NOX and p47-phox levels in OTA-treated chickens. Moreover, CHS, more than MIX, was able to reduce OTA-induced toxicity, restoring NOX levels. Taken together, these findings highlight the potential beneficial role of CHS in reverting OTA-induced nephrotoxicity in chickens and could lead to the production of healthier foods with beneficial consequences for human and animal health.

In view of the rising global death rate associated with inflammatory diseases, a cost-friendly, more effective and safer drug with lesser side effect is needed. The aim of this study was to evaluate membrane stabilization, albumin denaturation, protease inhibition and antioxidant activities as possible mechanisms for the anti-inflammatory effects observed in stem-bark of flavonoid-rich extract of Peltophorum pterocarpum (FREPP). The antioxidant activities were assessed using three various assays hydrogen peroxide, Nitric oxide (NO) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. The mechanisms for the anti-inflammatory effect of FREPP was determined by membrane stabilization models, test of albumin denaturation, protease inhibitor assay, while the total flavonoid was examined using the aluminium chloride colorimetric test. The ability of FREPP to inhibit membrane lyses of RBC was used to evaluate the anti-inflammatory activity. At 0.1, 0.4, 0.8 mg/ml, FREPP inhibited hypotonicity-induced haemolysis by 61.03, 76.84 and 83.84% respectively. FREPP was effective in inhibiting albumin denaturation in a concentration dependent manner having the highest inhibition of 69.89% at 50µg/ml. Protease activity was significantly (P< 0.05) reduced across varying concentrations as against the control. It can be inferred from these results that FREPP has antioxidant and anti-inflammatory activities and the mechanisms of the activity may be attributed to its richness in flavonoids known to inhibit lysis of the membrane, albumin denaturation and protease activity.

Circumstantial evidence link the high levels of environmental pollutants to increased incidence of different chronic-degenerative diseases. Among these, the levels of heavy metals (HM) can certainly play a key role. On the other hand, a qualified literature highlights how different phytochemicals, normally present in the diet are able to interfere with HM metabolism helping to reduce tissue concentrations and their negative effects on health. Curcumin appears of particular interest for its protective properties against cadmium (Cd) toxicity.

HL-60 cells, undifferentiated and differentiated by vitamin D analogue EB1089, were pre-incubated with low doses of curcumin (1 microM) and treated for 24 h with different concentrations of Cd. Cell viability was measured using CyQuant fluorescent dye. Autophagy was detected by Cyto-ID assay and measuring the expression of specific markers (LC3-II; p62). ROS (reactive oxygen species) variation was assayed by dichlorofluorescein- diacetate method. The bioavailability of curcumin in HL-60 cells was successfully performed using RP-HPLC coupled with UV-Vis, MS/MS or MS/MSn.

In both differentiated and undifferentiated HL-60 cells, the pre-incubation with 1 microM curcumin determined a significant 20% protection towards the cytotoxicity induced by Cd. The investigation of the mechanism of action evidenced that curcumin lowered ROS increase caused by Cd. The antioxidant activity of curcumin was strengthened by the detection of free curcumin inside the cells after 5 min of treatment. In parallel, we measured the ability of both curcumin and Cd to activate autophagy, as a stress response.

The antioxidant effect induced by curcumin, possibly through Keap1/Nrf2 system and GSH synthesis, is capable to re-modulate the autophagic flux converting the lethal autophagy triggered by Cd to a protective form. Overall, these preclinical results substantiate the design of nutritional interventional studies to investigate the capacity of curcumin to lower the concentration of HM in subjects exposed to environmental pollutants.

The adoption of high-fat diets (HFD) is a major contributor for the increasing prevalence of obesity worldwide. The adoption of unhealthy food habits happens at ever earlier age, raising concerns regarding the deleterious effects of HFD in male reproductive function and the consequences for health outcomes in the progeny. Indeed, obesity is associated to pro-inflammatory states, notably in adipose tissue. Herein we study the impact of HFD from early age in testicular physiology and sperm parameters in two generations of mice, with focus on the testicular oxidative status.

Mice of the diet-challenged generation (F0; n=36) were randomly fed after weaning with standard chow (CTRL) [Carbohydrate: 61.6%, Protein: 20.5%, Fat: 7.2% (16.3% Kcals)] or high-fat diet (HFD) [Carbohydrate: 35.7%, Protein: 20.5%, Fat: 36.0% (59.0% Kcals)] for 200 days or transient high-fat diet (HFDt) (60 days of HFD+140 days of standard chow). The offspring generation (F1; n=36) were obtained by mating with normoponderal females with 120 days post-weaning and fed with chow diet. Glucose homeostasis was evaluated and animals (F0 and F1) were sacrificed 200 days post-weaning, and tissues collected. Epididymal sperm parameters and endocrine parameters were evaluated. Testicular metabolites were extracted and characterized by ¹H-NMR and GC-MS. Testicular mitochondrial and antioxidant activity were evaluated. Data were analyzed by Univariate ANOVA and Pearson’s correlation.

In generation F0, HFD reduced testicular catalase and glutathione reductase (GR) activities. Testicular content of betaine and GSH have increased in HFD too. Testicular lipid content has also elevated in HFD mice, especially in the pro-inflammatory ω6 polyunsaturated fatty acids (PUFA). HFDt partially reversed the consequences of HFD but, interestingly, both HFD and HFDt mice showed poorer sperm parameters than CTRL (lower viability and motility). In generation F1, the offspring of HFD inherited decreased catalase activity, and displayed lower activities of mitochondrial complexes I and IV, in testis. HFD offspring had lower testicular myo-inositol, and HFDt, besides this presented lower 3-hydroxibutyrate. Our data suggests that the adoption of HFD promotes a pro-inflammatory state in testis characterized by decreased activity of antioxidant enzymes and increased content in ω6 PUFAs. The adoption of HFD, even transiently, inhibits testicular antioxidant defences on itself and in the progeny, and induces specific metabolic and lipid signatures correlated to sperm quality.

Epidemiological evidences indicate that there is an inverse association between olive oil intake and bladder cancer risk and several data suggest that a key role to support these beneficial effects is played by its phenolic fraction. Bladder cancer is one of the most common cancers in Western countries. In particular, the transitional cell carcinoma histotype shows an aggressive behavior and the current therapies are ineffective. The anti-proliferative effects of an Extra Virgin Olive Oil phenolic Extract (EVOOE) has been investigated on RT112 and J82, two human bladder cancer cell lines employed as models of superficial and invasive bladder cancer, respectively.
EVOOE reduces cell viability in both cell lines triggering different processes. In RT112 cells, EVOOE triggers a non-protective autophagic response, evidenced by vacuoles formation and LC-3 lipidation (45%) causing a delay in cell growth (132microg/ml induces 30% reduction). Instead, in J82, the invasive transitional cell carcinoma, EVOOE treatment induces a rapid and remarkable decrease of cell viability (33 microg/ml for 24 h induces 40% reduction) triggering an apoptotic process, evidenced with Annexin V positivity and the increased activity of caspases 3 and 9. EVOOE exerts an antioxidant activity in both cell lines reducing ROS (30% in J82 and 15% in RT112) and increasing GSH level (20% in J82 and 40% in RT112). However, comparing the effects of EVOOE with those of other well-known antioxidants, the absence of correlation between antioxidant effects and reduced cell viability was evidenced. Moreover, using an inhibitor of GSH synthesis, we demonstrated that EVOOE acts independently from GSH modulation.
Data presented show that EVOOE possesses pleiotropic activities that intercept different pathways resulting in anti-proliferative effects independently of its antioxidant property