Alexandros Priftis

Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells

The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem extracts affects their antioxidant capacity. For example, the results suggested that trans-resveratrol, gallic acid, (+)-catechin, ferulic acid, caffeic acid, quercetin, coumaric acid and kaempferol may be essential for the antioxidant activity of grape stem extracts.

Antioxidant effects of whey protein on muscle C2C12 cells

In the present study, the in vitro scavenging activity of sheep whey protein against free radicals, as well as its reducing power were determined and compared with that of beef protein, soy protein and cow whey protein. Moreover, the possible protective effects of sheep whey protein from tert-butyl hydroperoxide (tBHP)-induced oxidative stress in muscle C2C12 cells were determined by assessing oxidative stress markers by flow cytometry and spectrophotometry. The results showed that sheep whey protein scavenged DPPH, ABTS(+) and OH radicals with IC50 values of 3.1, 4.1 and 1.8 mg of protein/ml. Moreover, the reducing power activity assessed with potassium ferricyanide of sheep whey protein was 1.3mg/ml. As regards to the antioxidant effects in muscle cell line, sheep whey protein at 0.78, 1.56, 3.12 and 6.24 mg of protein/ml increased GSH levels up to 138%, lowered TBARS levels up to 25% and decreased ROS levels up to 41.4%.

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-eth-ylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical scavenging assays. In addition, 5 selected varieties were also examined for their protective effects against peroxyl and hydroxyl radical-induced DNA strand cleavage. Finally, C2C12 murine myoblasts were treated with non-cytotoxic 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 radical-induced 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.

Variations in oxidative stress markers in elite basketball players at the beginning and end of a season

The aim of the present study was to examine the changes occuring in the redox status in male basketball players at the beginning and end of a highly competitive season. For this purpose, the redox status of 14 professional athletes of a European basketball club was examined at 2 different time points, at the beginning (phase 1) and at the end of the season (phase 2). The redox status was assessed in blood using conventional oxidative stress markers, such as thiobarbituric acid reactive substances (TBARS), protein carbonyls (CARB) and the total antioxidant capacity (TAC) in plasma, as well as glutathione (GSH) levels and catalase (CAT) activity in erythrocytes. Moreover, a new static oxidation-reduction potential marker (sORP) was assessed in plasma. Our results revealed that sORP was significantly increased by 9.6% and GSH levels were significantly decreased by 35.0% at phase 2 compared to phase 1, indicating the induction of oxidative stress due to excessive exercise. Moreover, TAC was significantly increased by 12.9% at phase 2 compared to phase 1, indicating the activation of adaptive responses for counteracting oxidative stress. The CARB and TBARS levels were not significantly altered between the 2 phases, although there was a significant correlation (r=0.798) between the sORP and CARB levels. Furthermore, the variations in these markers between athletes were examined. We found that 3 markers exhibited a similar response between athletes, that is, sORP was increased in all 14 athletes, TAC was increased in 13 and the GSH levels were decreased in 14. However, the other 3 markers (i.e., TBARS, CARB and CAT) exhibited marked variations between the athletes, suggesting that the optimal approach with which to counteract (e.g., antioxidant supplementation) the observed increase in oxidative stress is the individualized examination of the redox status of athletes using a series of markers. This would allow the identification of athletes affected by severe oxidative stress and inflammation, and would thus indicate when necessary intervention measures are required to improve their health and performance.

Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

Roasted and green coffee extracts show antioxidant and cytotoxic activity in myoblast and endothelial cell lines in a cell specific manner

Coffee is one of the most highly consumed beverages with potential beneficial health implications, however its molecular mechanism of action has not been completely elucidated yet. To that cause, the polyphenolic composition of different coffee extracts (from Light, Medium and Dark roasts as well as green beans) was examined by UHPLC-HRMS analysis, indicating chlorogenic acids isomers as the main constituents. In the following step, the toxicity of the extracts was tested in myoblasts and endothelial cells and differential toxicity of green and roasted samples was displayed as the myoblasts were more sensitive to green coffee extracts, in contrast to the endothelial cells. Subsequently, biologically relevant, non-cytotoxic extract concentrations were administered to explore their potential effect on cell redox status using flow cytometry and spectrophotometric assays. The results indicated that all coffee extracts improved cell redox status, however differences were observed between the two different cell lines tested, implying that coffee compounds display cell- and tissue-specificity. Glutathione levels were increased in almost all cases up to 70%, while the roasting degree affected the free radical scavenging potential of the extracts and their ability to protect from macromolecular oxidation as exhibited by the differences in ROS, CARB and TBARS levels, especially in the myoblasts.

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, cell-based 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.

Cell‑specific and roasting‑dependent regulation of the Keap1/Nrf2 pathway by coffee extracts

Coffee is a popular beverage that contains various bioactive compounds. However, its molecular mechanism of action is not fully elucidated. In this context, two previously characterized coffee extracts, a lightly roasted and the corresponding green one, were investigated for their effect on nuclear factor erythroid 2-related factor 2 (Nrf2) target gene expression in myoblasts and endothelial cells using quantitative PCR. The tested concentrations were non-cytotoxic and led to improved redox cell status, as was evident by increased reduced glutathione (GSH) levels. In both cell lines, the roasted extract upregulated gene expression more readily than its green counterpart leading to increased NAD(P)H quinone dehydrogenase 1 and γ-glutamyl cysteine ligase catalytic subunit, among others. The green extract had a mixed effect on the endothelial cells, while, as regards the myoblasts it caused the downregulation of some Nrf-target genes. Therefore, a potential dose- and roasting-dependent mechanism is proposed in the current study, accounting for coffees antioxidant activity.

Effect of polyphenols from coffee and grape on gene expression in myoblasts

Coffee and grape contain various bioactive compounds like polyphenols that may exert beneficial effects, especially antioxidant activity, on human health upon consumption. However, the molecular mechanisms through which these effects are achieved are not fully elucidated. Thus, in the present study in order to investigate these mechanisms, a whole genome expression DNA microarray analysis was carried out in myoblasts treated with polyphenols of coffee and grape pomace at concentrations that improved the redox status. Grape was composed of catechin, epicatechin, cyanidin, malvidin, delphinidin, petunidin, myrtillin, kuromanin, oenin, peonidin, quercetin, gallic acid and caftaric acid as LC-MS revealed, with a total polyphenolic content (TPC) of 648 mg of gallic acid equivalents/g of dry matter. Coffee had a TPC of 42.61 mg GAE/g coffee and was composed of 3-chlorogenic acid (16.61 mg/g), 4- and 5-chlorogenic acids (13.62 mg/g), as UHPLC-HRMS revealed. According to the results, grape polyphenols altered mainly the expression of cytoskeleton and differentiation-associated genes, while coffee compounds had a more profound effect, on the expression levels of many metabolic and antioxidant genes possibly through the nuclear factor (erythroid-derived 2) like-2 (Nrf2) pathway.

Exercise-Induced Reductive Stress Is a Protective Mechanism against Oxidative Stress in Peripheral Blood Mononuclear Cells

Eccentric exercise is a well-studied modality that induces oxidative stress and muscle damage. Furthermore, it promotes inflammatory response in which peripheral blood mononuclear cells (PBMCs) are the major mediators. Although free radicals are necessary in a specific range of concentrations, yet unknown, it remains unclear whether reductive redox status (i.e., increased antioxidant defenses and impaired free radical generation) is beneficial or not. Thus, the aim of the present investigation was to examine the effects of reductive stress and the impact of reduced glutathione (GSH) baseline values on the ability of PBMCs to counteract oxidative stress induced by a potent oxidative agent. PBMCs were isolated from the blood of subjects who performed eccentric exercise and treated with t-BOOH for 24 h. The subjects were clustered in the reductive and the oxidative group on the basis of increased or decreased GSH concentration postexercise compared to preexercise values, respectively. According to our results in PBMCs, lipid peroxidation levels as depicted by thiobarbituric acid reactive substances (TBARS) remained unchanged in the reductive group contrary to the observed enhancement in the oxidative group. In addition, GSH concentration and catalase activity increased in the reductive group, whereas they were not affected in the oxidative group. In conclusion, the effects of an oxidizing agent on the redox status of PBMCs isolated from the blood of athletes after acute eccentric exercise are dependent on the baseline values of GSH in erythrocytes. Otherwise, reductive stress defined by increased GSH levels is a protective mechanism, at least when followed by an oxidative stimulus.