Antioxidant effects of an olive oil total polyphenolic fraction from a Greek Olea europaea variety in different cell cultures.

BACKGROUND: Numerous studies have been carried out concerning the advantageous health effects, especially the antioxidant effects, of olive oil's (OO) individual biophenolic compounds, but none until now for its total phenolic fraction (TPF). Plenty of evidence, in research about nutrition and healthiness, points out that it is the complex mixture of nutritional polyphenols, more than each compound separate, which can synergistically act towards a health result. PURPOSE: The aim of the present study was to examine the antioxidant properties of an extra virgin olive oil (EVOO) total polyphenolic fraction, from a Greek endemic variety of Olea europaea in cell lines. METHODS: EVOO from a Greek endemic variety was used for the extraction of a total polyphenolic fraction, using a green CPE­based method. The redox status [in terms of ROS, GSH, TBARS, protein carbonyls] was assessed at a cellular level, particularly in EA.hy926 endothelial, HeLa, HepG2 hepatic cells and C2C12 myoblasts. Moreover, the levels of glutamate-cysteine ligase catalytic subunit (γ-GCLc) of GSH, one of the most important antioxidant enzymes, were assessed by western blot. RESULTS: According to the results, TPF improves the redox profile of all cell lines, mainly by increasing GSH and its catalytic subunit, while at low, not cytotoxic TPF concentrations there was a decrease in TBARS and carbonyls. Regarding ROS levels a reduction was observed only in the HepG2 cell line, contrary to the other cell lines, that there is no statistically significant difference. CONCLUSION: The TPF appeared to protect cells from oxidative stress due to the strong antioxidant activity of its polyphenols. This could have interesting implications in development of new products based on this olive oil to provide protection and treatment against harmful effects of free radicals.

Toxicity of plant extracts containing pyrrolizidine alkaloids using alternative invertebrate models.

Pyrrolizidine alkaloids (PAs) are a widespread class of hepatotoxic heterocyclic organic compounds found in approximately 3% of world flora. Some PAs have been shown to have genotoxic and carcinogenic effects. The present study focuses on the toxicity effects of four dry extracts obtained from medicinal plants (Senecio vernalis, Symphytum officinale, Petasites hybridus and Tussilago farfara), on two aquatic organisms, Artemia salina and Daphnia magna, and the correlation with their PAs content. A new GC­MS method, using a retention time (TR)­5MS type capillary column was developed. PAs Kovats retention indices, for this type of column were computed for the first time. The lethal dose 50% (LC50) values for the two invertebrate models were correlated (Pearson 's coefficient, >0.9) and the toxicity was PA concentration-dependent, for three of the four extracts. All tested extracts were found to be toxic in both aquatic organism models. The results can be used to develop a GC­MS validated method for the assay of PAs in medicinal plants with a further potential application in the risk assessment study of PAs toxicity in humans.

Anti-inflammatory effects of soyasapogenol I-αa via downregulation of the MAPK signaling pathway in LPS-induced RAW 264.7 macrophages.

The crude extract of soyasaponins was reported to possess anti-inflammatory activity. We determined the new purity group I saponin, I-αa and I-γa that was isolated from wild soybean (Glycine soja) in terms of its efficacy in protecting RAW 264.7 macrophages from lipopolysaccharide (LPS)-stimuli. Cells were treated with soyasaponin I-αa/I-γa (30-300 µΜ) and LPS (0.1 µg/mL) for 24 h. Soyasaponin I-αa inhibited nitric oxide (NO) production at 100 µg/mL, while soyasaponin I-γa demonstrated this effect at a higher concentration (200 µg/mL). The expression levels of iNOS and COX-2 enzymes were downregulated by both soyasaponins. Soyasaponin I-αa exerted its effect via the TNF-α and IL-1ß cytokines. However, soyasaponin I-γa only inhibited the expression of TNF-α. The inflammatory effect of group I soyasaponin was mainly mediated via the phosphorylation of the p38 and JNK proteins. Collectively, these results suggested the potential anti-inflammatory effects of soyasaponins.

Quercetin attenuates the hyperoxic lung injury in neonatal mice: Implications for Bronchopulmonary dysplasia (BPD).

Quercetin (QU) is one of the most common flavonoids that are present in a wide variety of fruits, vegetables, and beverages. This compound possesses potent anti-inflammatory and anti-oxidant properties. Supplemental oxygen is routinely administered to premature infants with pulmonary insufficiency. However, hyperoxia is one of the major risk factors for the development of bronchopulmonary dysplasia (BPD), which is also termed chronic lung disease in premature infants. Currently, no preventive approaches have been reported against BPD. The treatment of BPD is notably limited to oxygen administration, ventilatory support, and steroids. Since QU has been shown to be effective in reducing inflammation and oxidative stress in various disease models, we hypothesized that the postnatal QU treatment of newborn mice will protect against hyperoxic lung injury by the upregulation of the phase I (CYP1A/B) and/or phase II, NADPH quinone reductase enzymes. Newborn C57BL/6J mice within 24 h of birth with the nursing dams were exposed to either 21% O2 (air) and/or 85% O2 (hyperoxia) for 7 days. The mice were treated, intraperitoneally (i.p.) once every other day with quercetin, at a concentration of 20 mg/kg, or saline alone from postnatal day (PND) 2-6. The mice were sacrificed on day 7, and lung and liver tissues were collected. The expression levels of CYP1A1, CYP1B1, NQO1 proteins and mRNA as well as the levels of MDA-protein adducts were analyzed in lung and liver tissues. The findings indicated that QU attenuated hyperoxia-mediated lung injury by reducing inflammation and improving alveolarization with decreased number of neutrophil and macrophage infiltration. The attenuation of this lung injury correlated with the upregulation of CYP1A1/CYP1B1/NQO1 mRNA, proteins and the down regulation of NF-kB levels and MDA-protein adducts in lung and liver tissues. The present study demonstrated the potential therapeutic value of quercetin in the prevention and/or treatment of BPD.

Effects of sheep/goat whey protein dietary supplementation on the redox status of rats.

The purpose of the present study is to estimate the effects of sheep/goat whey protein dietary supplementation on the redox status of blood and tissues of rats. Twelve male Wistar rats were divided into the control group (standard commercial diet) and whey group [standard commercial diet + sheep/goat whey protein (1 g kg b.w/day)] (6 rats/group). The animals were maintainted on their respective diet for 28 days. At the end of the experimental period, reduced glutathione, catalase activity, total antioxidant capacity, thiobarbituric reactive substances, protein carbonyls and the decomposition rate of H2O2 were measured in blood and tissues of rats. According to the results, the rats fed with the sheep/goat whey protein exhibited improved antioxidant status and decreased free radical­induced toxic effects on lipids and proteins. Specifically, in blood, GSH and CAT levels were significantly increased while TBARS and protein carbonyl levels were significantly decreased compared to the control group. Regarding the effects on tissues, it was observed that GSH levels were significantly increased in small intestine, quadriceps muscle, pancreas and lung tissue compared to the control group. The decomposition rate of H2O2 was significantly decreased in liver, brain and quadriceps muscle, but was significantly increased in spleen tissue compared to the control group. TBARS levels were significantly decreased in liver, brain, quadriceps muscle, pancreas, lung and spleen tissue compared to the control group. Finally, protein carbonyl levels were significantly decreased in brain, small intestine, kidney, pancreas and spleen tissue compared to the control group. Thus, the present findings show the beneficial effects of sheep/goat whey protein, a by­product of cheese manufacturing, on the redox status in an in vivo model.

Tissue specific effects of feeds supplemented with grape pomace or olive oil mill wastewater on detoxification enzymes in sheep.

The aim of the present study was to investigate the effects of livestock feed supplemented with grape pomace (GP) or olive oil mill wastewater (OMW) byproducts on the enzymatic activity and protein expression of antioxidants enzymes, in liver and spleen tissue of sheep. Thus, 36 male sheep of Chios breed were divided into 3 homogeneous groups, control group (n = 12), GP group (n = 12) and OMW group (n = 12), receiving standard or experimental feed. Liver and spleen tissues were collected at 42 and 70 days post-birth. The enzymatic activity of superoxide dismutase (SOD) and glutathione-s-transferase (GST) and also the protein expression of γ-synthase glutamyl custeine (γ-GCS) were determined in these tissues. The results showed GP group exhibited increased enzymatic activity of GST and protein expression of γ-GCS in liver compared to control group. In GP group's spleen, GST activity was increased compared to control but γ-GCS expression was not affected. In OMW group's liver, GST activity was increased and γ-GCS expression was reduced compared to control. In OMW group's spleen, GST activity was increased but GCS expression was not affected. SOD activity was not affected in both tissues either in GP or OMW group.

Glutamate­mediated effects of caffeine and interferon­Î³ on mercury-induced toxicity.

The molecular mechanisms mediating mercury­induced neurotoxicity are not yet completely understood. Thus, the aim of this study was to investigate whether the severity of MeHg­ and HgCl2­mediated cytotoxicity to SH­SY5Y human dopaminergic neurons can be attenuated by regulating glutamate­mediated signal­transmission through caffeine and interferon­Î³ (IFN­Î³). The SH­SY5Y cells were exposed to 1, 2 and 5 µM of either MeHgCl2 or HgCl2 in the presence or absence of L­glutamine. To examine the effect of adenosine receptor antagonist, the cells were treated with 10 and 20 µM caffeine. The total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined in the 1 ng/ml IFN­Î³­ and glutamate­stimulated SH­SY5Y cells. Following exposure to mercury, the concentration­dependent decrease in mitochondrial metabolic activity inversely correlated with oxidative stress intensity. MeHg was more toxic than HgCl2. Mercury­induced neuronal death was dependent on glutamate­mediated excitotoxicity. Caffeine reduced the mercury­induced oxidative stress in glutamine-containing medium. IFN­Î³ treatment decreased cell viability and increased oxidative stress in glutamine­free medium, despite caffeine supplementation. Although caffeine exerted a protective effect against MeHg-induced toxicity with glutamate transmission, under co­stimulation with glutamine and IFN­Î³, caffeine decreased the MeHg­induced average oxidative stress only by half. Thereby, our data indicate that the IFN­Î³ stimulation of mercury­exposed dopaminergic neurons in neuroinflammatory diseases may diminish the neuroprotective effects of caffeine.

Development of an assay to assess genotoxicity by particulate matter extract.

The current study describes a method for assessing the oxidative potential of common environmental stressors (ambient air particulate matter), using a plasmid relaxation assay where the extract caused single-strand breaks, easily visualised through electrophoresis. This assay utilises a miniscule amount (11 µg) of particulate matter (PM) extract compared to other, cell­based methods (~3,000 µg). The negative impact of air pollution on human health has been extensively recognised. Among the air pollutants, PM plays an eminent role, as reflected in the broad scientific interest. PM toxicity highly depends on its composition (metals and organic compounds), which in turn has been linked to multiple health effects (such as cardiorespiratory diseases and cancer) through multiple toxicity mechanisms; the induction of oxidative stress is considered a major mechanism among these. In this study, the PM levels, oxidative potential, cytotoxicity and genotoxicity of PM in the region of Larissa, Greece were examined using the plasmid relaxation assay. Finally, coffee extracts from different varieties, derived from both green and roasted seeds, were examined for their ability to inhibit PM-induced DNA damage. These extracts also exerted an inhibitory effect on xanthine oxidase and catalase, but had no effect against superoxide dismutase. Overall, this study highlights the importance of assays for assessing the oxidative potential of widespread environmental stressors (PM), as well as the antioxidant capacity of beverages and food items, with the highlight being the development of a plasmid relaxation assay to assess the genotoxicity caused by PM using only a miniscule amount.

Comparison of antioxidant activity between green and roasted coffee beans using molecular methods.

Coffee is one of the most popular and widely consumed beverages worldwide due to its pleasant taste and aroma. A number of studies have been performed to elucidate the possible beneficial effects of coffee consumption on human health and have shown that coffee exhibits potent antioxidant activity, which may be attributed mainly to its polyphenolic content. However, there is also evidence to suggest that coffee roasting (the procedure which turns green coffee beans to the dark, roasted ones from which the beverage derives) may alter the polyphenolic profile of the beans (e.g., via the Maillard reaction) and, concomitantly, their antioxidant activity. In the present study, the antioxidant activity of 13 coffee varieties was examined in both green and roasted coffee bean extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+)- radical scavenging assays. In addition, 5 selected varieties were also examined for their protective effects against peroxyl and hydroxyl radical­induced DNA strand cleavage. Finally, C2C12 murine myoblasts were treated with non­cytotoxic concentrations of the most potent extract in order to examine its effects on the cellular redox status by measuring the glutathione (GSH) and reactive oxygen species (ROS) levels by flow cytometry. Our results revealed that, in 8 out of the 13 coffee varieties, roasting increased free radical scavenging activity as shown by DPPH and ABTS•+ assays. Moreover, we found that when one coffee variety was roasted for different amounts of time, the increase in the antioxidant activity depended on the roasting time. By contrast, in 5 varieties, roasting reduced the antioxidant activity. Similar differences between the roasted and green beans were also observed in the free radical­induced DNA strand cleavage assay. The observed differences in the antioxidant activity between the different coffee varieties may be attributed to their varying polyphenolic content and composition, as well as to the different molecules produced during roasting. In addition, in the cell culture assay, the tested coffee extract led to increased GSH levels in a dose-dependent manner, indicating the enhancement of cellular antioxidant mechanisms.

Assessment of polyphenolic content, antioxidant activity, protection against ROS-induced DNA damage and anticancer activity of Vitis vinifera stem extracts.

Grape extracts and wine have been studied widely due to their beneficial effects on human health. However, there are only few studies from grape stems extracts. Therefore, the main objective of the present study was the assessment in stem extracts from Greek Vitis vinifera varieties of the total polyphenolic content (TPC), the identification of the polyphenols present in them, and the evaluation of their antioxidant activity, protection against ROS-induced DNA damage and inhibition of liver (HepG2) and cervical (HeLa) cancer cell growth. The range of the TPC in grape stem extracts was from 345 to 584 mg GAE/g dry weight. Moreover, stem extracts contained different classes of polyphenols as flavonols, flavanols, procyanidins, phenolic acids and stilbenes. In DPPH and ABTS assays, the IC50 values of the stem extracts had an average of 7.8 ± 2.8 and 5.4 ± 2.6 µg/mL respectively. Also, all stem extracts inhibited OH- and ROO-induced DNA damage dose dependent with average IC50 values of 478 ± 217 and 1.15 ± 0.85 µg/mL respectively. Furthermore, stem extracts inhibited at low concentrations the growth of HepG2 and HeLa cancer cells with average IC50 values of 50 ± 12 and 32 ± 16 µg/mL respectively. The above activities of grape stem extracts were comparable to those of seed extracts.