Antioxidants

23 pages, 1042 KiB

Open AccessReview

Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa

by Fernando J. Peña, Cristian O’Flaherty, José M. Ortiz Rodríguez, Francisco E. Martín Cano, Gemma L. Gaitskell-Phillips, María C. Gil and Cristina Ortega Ferrusola

Antioxidants 2019, 8(11), 567; https://doi.org/10.3390/antiox8110567 - 19 Nov 2019

Cited by 64 | Viewed by 5837

Abstract

Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a [...] Read more.

Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an “on–off” switch controlling sperm function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress, leading to malfunction and ultimately death of the spermatozoa. Stallion spermatozoa are “professional producers” of reactive oxygen species due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also characteristic of the spermatozoa in the horse. As a result, and combined with the fact that embryos can easily be collected in this species, horses are a good model for the study of redox biology in the spermatozoa and its impact on the embryo. Full article

(This article belongs to the Special Issue Reactive Oxygen Species and Male Fertility)

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20 pages, 3020 KiB

Open AccessArticle

Fibroblasts to Keratinocytes Redox Signaling: The Possible Role of ROS in Psoriatic Plaque Formation

by Victoria Barygina, Matteo Becatti, Francesca Prignano, Torello Lotti, Niccolò Taddei and Claudia Fiorillo

Antioxidants 2019, 8(11), 566; https://doi.org/10.3390/antiox8110566 - 18 Nov 2019

Cited by 22 | Viewed by 5252

Abstract

Although the role of reactive oxygen species-mediated (ROS-mediated) signalling in physiologic and pathologic skin conditions has been proven, no data exist on the skin cells ROS-mediated communication. Primary fibroblasts were obtained from lesional and non-lesional skin of psoriatic patients. ROS, superoxide anion, calcium [...] Read more.

Although the role of reactive oxygen species-mediated (ROS-mediated) signalling in physiologic and pathologic skin conditions has been proven, no data exist on the skin cells ROS-mediated communication. Primary fibroblasts were obtained from lesional and non-lesional skin of psoriatic patients. ROS, superoxide anion, calcium and nitric oxide levels and lipoperoxidation markers and total antioxidant content were measured in fibroblasts. NADPH oxidase activity and NOX1, 2 and 4 expressions were assayed and NOX4 silencing was performed. Fibroblasts and healthy keratinocytes co-culture was performed. MAPK pathways activation was studied in fibroblasts and in co-cultured healthy keratinocytes. Increased intracellular calcium, •NO and ROS levels as well as an enhanced NADPH oxidase 4 (NOX4)–mediated extracellular ROS release was shown in lesional psoriatic vs. control fibroblasts. Upon co-culture with lesional fibroblasts, keratinocytes showed p38 and ERK MAPKs pathways activation, ROS, Ca²⁺ and •NO increase and cell cycle acceleration. Notably, NOX4 knockdown significantly reduced the observed effects of lesional fibroblasts on keratinocyte cell cycle progression. Co-culture with non-lesional psoriatic and control fibroblasts induced slight cell cycle acceleration, but notable intracellular ROS accumulation and ERK MAPK activation in keratinocytes. Collectively, our data demonstrate that NOX4 expressed in dermal fibroblasts is essential for the redox paracrine regulation of epidermal keratinocytes proliferation. Full article

(This article belongs to the Special Issue Redox Regulation of Cell Signalling)

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Contactless keratinocytes-fibroblasts co-incubation protocol. Keratinocytes (KER) were grown on cover-glasses (1.5 cm2) until semi-confluence (generally for 24 h) and put in Petri dish (diameter 10 cm) with semi-confluent fibroblasts. After 48 h of co-incubation, the KER–containing cover-glasses were removed from the Petri dishes, KER were trypsinized and examined. Full article ">Figure 2
Fibroblasts were obtained from the skin of healthy volunteers (CTR) and from unaffected (nLES) and lesional (LES) skin areas of psoriatic patients; (a) Confocal microscopy analysis confirmed the purity of obtained fibroblasts cultures which were positive to anti-vimentin and negative to pan-cytokeratin immunocytochemical staining. (b) The cell cycle was analyzed by FACS Analysis in LES, nLES and CTR fibroblasts on their second passage. (b’) Cells distribution quantification in G0-G1, G2-M and S phases of the cell cycle. In dot plot graphs, each patient/control is identified by a different color (see <a href="#antioxidants-08-00566-t003" class="html-table">Table 3</a>). Values are presented as means ± SD. Each experiment was performed in triplicate. * p < 0.05 vs. CTR. Scale bar = 20 μm. Full article ">Figure 3
Assessment of redox markers in human psoriatic fibroblasts. For the assessment of the intracellular levels of ROS, Ca2+, mitochondrial superoxide anion production and •NO, CTR, nLES and LES fibroblasts were stained with the fluorescent markers H2DCFDA, Fluo-3, MitoSOX, and DAF-FM, respectively. (a) Confocal microscope analysis, and (b,b’) FACS analysis revealed signs of oxidative/nitrosative stress in LES fibroblasts. (c) Thiobarbituric acid reactive substance (TBARS) assay revealed increased lipid peroxidation in LES with respect to nLES and CTR fibroblasts. (d) Decreased total antioxidant capacity measured by oxygen radical antioxidant capacity (ORAC) assay and expressed in Trolox equivalents was evident in psoriatic versus CTR fibroblasts. In dot plot graphs, each patient/control is identified by a different color (see <a href="#antioxidants-08-00566-t003" class="html-table">Table 3</a>). Values are presented as means ± SD. Each experiment was performed in triplicate. & p < 0.05, && p < 0.01. * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR. Scale bar = 20 μm. Full article ">Figure 4
NADPH oxidase activity, expression and intracellular localization in human fibroblasts. NADPH oxidase activity was measured in intact living cells by luminometric assay following NADPH stimulation (arrow 1). Superoxide dismutase (SOD) addition to the reaction mixture (arrow 2) brought to immediate and significant luminescence decrease. (a) The diphenylene iodonium chloride (DPI), a potent flavoenzyme inhibitor, abolished NADPH-triggered NADPH oxidase activity in fibroblasts suggesting that extracellular ROS production stimulated by NADPH was NADPH oxidase-dependent. (a’) Area Under Curve (AUC) values quantified from the NADPH oxidase activity curves. (b) Hydrogen peroxide fluorometric detection in CTR, nLES and LES cell culture medium. (c) Western blot analysis of NOX1 (the band detected at 65 kDa, additional band observed at 50 kDa), NOX2 (the band detected at 91 kDa) and NOX4 (the band detected at 67 kDa, additional bands observed at 50 and 20 kDa) expression in fibroblasts. (d) Down-regulation of NOX4 with siNOX4 RNAs abolished the differences in NOX4 expression between LES, nLES and CTR fibroblasts. Moreover, (e) NADPH oxidase activities were similar in LESsiNOX4 and CTRscRNA fibroblasts. (e’) Quantification analysis of AUC values of NADPH oxidase activity. In dot plot graphs, each patient/control is identified by a different color (see <a href="#antioxidants-08-00566-t003" class="html-table">Table 3</a>). Values are presented as means ± SD. Each experiment was performed in triplicate. && p < 0.01, &&& p < 0.001. * p < 0.05 vs. CTR, ** p < 0.01 vs. CTR, *** p < 0.001 vs. CTR. Full article ">Figure 5
Co-culture with fibroblasts alters MAPK signalling, redox homeostasis and cell cycle in primary human keratinocytes (K). (a) Western blot analysis revealed ERK1/2 and p38 pathways activation in K co-cultured with control fibroblasts (K+CTR) and psoriatic non-lesional (K+nLES) and lesional (K+LES) fibroblasts, respectively, with respect to K not in co-culture. Co-culture with fibroblasts didn’t bring to any significant alteration in the JNK pathway in K in our experimental conditions. (b) Following the co-culture with fibroblasts, K were stained with H2DCFDA, DAF-FM and MitoSOX markers in order to estimate the total intracellular ROS, •NO and Ca2+ levels, respectively, by flow cytometry. (b’) The quantification of emitted fluorescence assayed by flow cytometer showed that co-culture with fibroblasts significantly increased intracellular ROS production in K. Only co-culture with LES dramatically increased the intracellular •NO and Ca2+ content vs. K. (c) Co-incubation with fibroblasts and, especially, with LES, altered the distribution of K in G0-G1, S and G2-M phases of cell cycle measured by FACS Analysis. (d) Transfection of fibroblasts with siNOX4 RNAs induced the accumulation of K in G0-G1 phase of the cell cycle. In dot plot graphs, each patient/control is identified by a different color (see <a href="#antioxidants-08-00566-t003" class="html-table">Table 3</a>). Values are presented as means ± SD. Each experiment was performed in triplicate. & p < 0.05, && p < 0.01, &&& p < 0.001; * p < 0.05 vs. K, ** p < 0.01 vs. K, *** p < 0.001 vs. K, **** p < 0.0001 vs. K. Full article ">Figure 6
Schematic representation of the proposed mechanism of NOX4 contribution in psoriatic plaque formation. Healthy primary fibroblasts, as well as fibroblasts obtained from visibly unaffected skin of psoriatic patients, activate ERK1/2, increase intracellular total ROS level and accelerate cell cycle progression in healthy primary keratinocytes in co-culture through the redox signalling mediated by NOX4. Fibroblasts obtained from unaffected and lesional skin of psoriatic patients differ from healthy fibroblasts by elevated mitochondrial superoxide anion production and decreased total antioxidant capacity. Moreover, lesional fibroblasts display significantly higher levels of intracellular •NO, Ca2+ and lipid peroxidation as well as elevated NOX4 activity and extracellular ROS production. Lesional fibroblasts induce significantly higher levels of intracellular ROS and ERK activation in healthy keratinocytes with respect to healthy and unaffected psoriatic fibroblasts. Moreover, co-culture with lesional fibroblasts induced •NO and Ca2+ accumulation in keratinocytes and significant activation of p38 molecular pathway. The result of these metabolic alterations is keratinocytes hyper-proliferation. Hence, dermal fibroblasts may trigger keratinocytes proliferation through NOX4-mediated redox signalling. Full article ">

18 pages, 271 KiB

Open AccessArticle

The Nutritional Value of Non-Traditional Gluten-Free Flakes and Their Antioxidant Activity

by Kristýna Šťastná, Martina Mrázková, Daniela Sumczynski, Betül Cındık and Erkan Yalçın

Antioxidants 2019, 8(11), 565; https://doi.org/10.3390/antiox8110565 - 16 Nov 2019

Cited by 5 | Viewed by 3735

Abstract

Nowadays, there is a growing interest for foods with a lower sugar content and rich in fiber and biologically active substances. The main purpose of this study was to prepare flakes from non-traditional pigmented cereals (Oryza sativa, Chenopodium quinoa, and Eragrostis [...] Read more.

Nowadays, there is a growing interest for foods with a lower sugar content and rich in fiber and biologically active substances. The main purpose of this study was to prepare flakes from non-traditional pigmented cereals (Oryza sativa, Chenopodium quinoa, and Eragrostis tef) and to analyze their fibre, sugar, and in vitro digestibility values. Regarding phenolic antioxidants (soluble, soluble conjugated, and insoluble bound fractions), their content and antioxidant activity were measured using spectrophotometry and high performance liquid chromatography (HPLC) methods. Hydrothermally treated grains resulted in flakes with higher total dietary fibre contents (11.1–24.4%), quinoa and teff flakes were rich in maltose (up to 42.0 mg/g). Non-traditional flakes had lower in vitro digestibility, but conversely, they exhibited the highest phenolic contents corresponding with the highest antioxidant activity values (up to 2.33 mg Gallic acid equivalent/g of total phenolic content and 1.59 mg Trolox equivalent/g for 2,2-diphenyl-1-picrylhydrazyl (DPPH) in case of brown teff). Among free phenolics, the main contributors to an antioxidant activity were p-coumaric, o-coumaric, and gallic acids (r > 0.8186); among the soluble conjugated fractions, they were epigallocatechin, epicatechin, caffeic, and vanillic acids (r > 0.5935); while caffeic, protocatechuic, and ferulic acids (r > 0.5751) were the main contributors among the insoluble bound phenolics. Full article

23 pages, 3107 KiB

Open AccessArticle

Genome–Scale Metabolic Networks Shed Light on the Carotenoid Biosynthesis Pathway in the Brown Algae Saccharina japonica and Cladosiphon okamuranus

by Delphine Nègre, Méziane Aite, Arnaud Belcour, Clémence Frioux, Loraine Brillet-Guéguen, Xi Liu, Philippe Bordron, Olivier Godfroy, Agnieszka P. Lipinska, Catherine Leblanc, Anne Siegel, Simon M. Dittami, Erwan Corre and Gabriel V. Markov

Antioxidants 2019, 8(11), 564; https://doi.org/10.3390/antiox8110564 - 16 Nov 2019

Cited by 16 | Viewed by 4880

Abstract

Understanding growth mechanisms in brown algae is a current scientific and economic challenge that can benefit from the modeling of their metabolic networks. The sequencing of the genomes of Saccharina japonica and Cladosiphon okamuranus has provided the necessary data for the reconstruction of [...] Read more.

Understanding growth mechanisms in brown algae is a current scientific and economic challenge that can benefit from the modeling of their metabolic networks. The sequencing of the genomes of Saccharina japonica and Cladosiphon okamuranus has provided the necessary data for the reconstruction of Genome–Scale Metabolic Networks (GSMNs). The same in silico method deployed for the GSMN reconstruction of Ectocarpus siliculosus to investigate the metabolic capabilities of these two algae, was used. Integrating metabolic profiling data from the literature, we provided functional GSMNs composed of an average of 2230 metabolites and 3370 reactions. Based on these GSMNs and previously published work, we propose a model for the biosynthetic pathways of the main carotenoids in these two algae. We highlight, on the one hand, the reactions and enzymes that have been preserved through evolution and, on the other hand, the specificities related to brown algae. Our data further indicate that, if abscisic acid is produced by Saccharina japonica, its biosynthesis pathway seems to be different in its final steps from that described in land plants. Thus, our work illustrates the potential of GSMNs reconstructions for formalizing hypotheses that can be further tested using targeted biochemical approaches. Full article

(This article belongs to the Special Issue Marine Algal Antioxidants)

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19 pages, 1852 KiB

Open AccessArticle

The Influence of In Vitro Gastrointestinal Digestion on the Chemical Composition and Antioxidant and Enzyme Inhibitory Capacities of Carob Liqueurs Obtained with Different Elaboration Techniques

by Raquel Rodríguez-Solana, Natacha Coelho, Antonio Santos-Rufo, Sandra Gonçalves, Efrén Pérez-Santín and Anabela Romano

Antioxidants 2019, 8(11), 563; https://doi.org/10.3390/antiox8110563 - 16 Nov 2019

Cited by 27 | Viewed by 4301

Abstract

Carob liqueur is a traditional Mediterranean alcoholic beverage obtained via a wide range of production techniques contributing to the different organoleptic attributes of the final product. The aim of this research was to evaluate the stability of the chemical composition and biological capacities [...] Read more.

Carob liqueur is a traditional Mediterranean alcoholic beverage obtained via a wide range of production techniques contributing to the different organoleptic attributes of the final product. The aim of this research was to evaluate the stability of the chemical composition and biological capacities (antioxidant and enzyme inhibition) under in vitro simulated gastrointestinal digestion of liqueurs prepared by flavouring the fig spirit with carob pulp by maceration, distillation, percolation, or aqueous and hydro-alcoholic infusions. For this purpose, the phenolic and furanic compositions, the total phenolic (TPC) and flavonoid (TFC) contents, antioxidant capacity (AC), and enzyme inhibitory potential against acethylcholinesterase, tyrosinase, α-glucosidase and α-amylase enzymes were evaluated. The content of gallic acid decreased after gastrointestinal digestion, while TPC, TFC, and AC significantly increased after each digestion phase. Overall, no significantly different enzyme inhibitions (p < 0.05) were observed among digested liqueurs, with moderate inhibition against acethylcholinesterase and tyrosinase (enzymes related with neurodegenerative diseases), and potent and low inhibitory capacities for α-glucosidase and α-amylase, respectively (ideal conditions employed in antidiabetic therapy). The study indicates that hydro-alcoholic infusion and maceration were the most appropriate methods to obtain liqueurs with higher values of the aforementioned parameters and safe levels of toxic furanics. Full article

(This article belongs to the Special Issue Phenolic Profiling and Antioxidant Capacity in Plants)

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Schematic representation of the liqueur production, by different extraction methods, and the gastric and intestinal phases of the in vitro digestion process, with the respective constituents (such as simulated gastric (SGF) and intestinal (SIF) fluids) and conditions used. Full article ">Figure 2
Carob liqueurs obtained by different extraction methods: hydro-alcoholic infusion (a), maceration (b), percolation (c), aqueous infusion (d) and distillation (e). Full article ">Figure 3
Total phenolic (TPC) and flavonoid (TFC) contents and antioxidant capacity measured by Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays of carob liqueurs obtained with different extraction methods and subjected to gastrointestinal digestion. In each graph (TPC, TFC, TEAC or ORAC), values marked with different letters indicate a significant difference (p < 0.05). Full article ">Figure 4
Enzyme [acetylcholinesterase (AchE), tyrosinase (tyr), α-amylase (α-amyl) and α-glucosidase (α-gluc)] inhibitory capacities of undigested carob liqueurs obtained with different extraction methods. For each enzyme (AchE, tyr, α-amyl, or α-gluc) inhibition method, values marked with different letters indicate a significant difference (p < 0.05). Full article ">Figure 5
Enzyme [acetylcholinesterase (AchE), tyrosinase (tyr), α-amylase (α-amyl) and α-glucosidase (α-gluc)] inhibitory capacities of gastrointestinal digested carob liqueurs obtained with different extraction methods. For each enzyme (AchE, tyr, α-amyl, or α-gluc) inhibition method, values marked with different letters indicate a significant difference (p < 0.05). Full article ">Figure 6
Principal component (PC) analysis plot of gallic acid, furfural, 5-hydroxymethyl furfural (HMF), antioxidant capacity [Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorption Capacity (ORAC) assays], total phenolic (TPC) and flavonoid (TFC) contents and inhibition of enzymatic (α-amylase, α-glucosidase, acetylcholinesterase (AChE) and tyrosinase) capacities of carob liqueurs (circles) obtained by aqueous (AI) and hydro-alcoholic (HI) infusions, distillation (D), maceration (M) and percolation (P), and subjected to gastric (squares) and gastrointestinal (triangles) digestions. Full article ">

14 pages, 273 KiB

Open AccessArticle

Polyphenols from Lycium barbarum (Goji) Fruit European Cultivars at Different Maturation Steps: Extraction, HPLC-DAD Analyses, and Biological Evaluation

by Andrei Mocan, Francesco Cairone, Marcello Locatelli, Francesco Cacciagrano, Simone Carradori, Dan C. Vodnar, Gianina Crișan, Giovanna Simonetti and Stefania Cesa

Antioxidants 2019, 8(11), 562; https://doi.org/10.3390/antiox8110562 - 16 Nov 2019

Cited by 47 | Viewed by 6224

Abstract

Goji berries are undoubtedly a source of potentially bioactive compounds but their phytochemical profile can vary depending on their geographical origin, cultivar, and/or industrial processing. A rapid and cheap extraction of the polyphenolic fraction from Lycium barbarum cultivars, applied after homogenization treatments, was [...] Read more.

Goji berries are undoubtedly a source of potentially bioactive compounds but their phytochemical profile can vary depending on their geographical origin, cultivar, and/or industrial processing. A rapid and cheap extraction of the polyphenolic fraction from Lycium barbarum cultivars, applied after homogenization treatments, was combined with high-performance liquid chromatography (HPLC) analyses based on two different methods. The obtained hydroalcoholic extracts, containing interesting secondary metabolites (gallic acid, chlorogenic acid, catechin, sinapinic acid, rutin, and carvacrol), were also submitted to a wide biological screening. The total phenolic and flavonoid contents, the antioxidant capacity using three antioxidant assays, tyrosinase inhibition, and anti-Candida activity were evaluated in order to correlate the impact of the homogenization treatment, geographical origin, and cultivar type on the polyphenolic and flavonoid amount, and consequently the bioactivity. The rutin amount, considered as a quality marker for goji berries according to European Pharmacopeia, varied from ≈200 to ≈400 µg/g among the tested samples, showing important differences observed in relation to the influence of the evaluated parameters. Full article

(This article belongs to the Special Issue Antioxidant Properties of Natural Products: A Themed Issue in Honor of Professor Isabel C.F.R. Ferreira)

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19 pages, 13321 KiB

Open AccessArticle

Penta-1,2,3,4,6-O-Galloyl-β-d-Glucose Inhibits UVB-Induced Photoaging by Targeting PAK1 and JNK1

by Ji-An Kim, Jae-Eun Lee, Ji Hye Kim, Hyo-Jeong Lee and Nam Joo Kang

Antioxidants 2019, 8(11), 561; https://doi.org/10.3390/antiox8110561 - 15 Nov 2019

Cited by 13 | Viewed by 4764

Abstract

Penta-O-galloyl-β-d-glucose (PGG) is a gallotannin polyphenolic compound that occurs naturally in fermented Rhus verniciflua. The present study aimed to examine the effect of PGG on UVB-induced skin aging and its molecular mechanisms in HaCaT human keratinocytes and SKH-1 [...] Read more.

Penta-O-galloyl-β-d-glucose (PGG) is a gallotannin polyphenolic compound that occurs naturally in fermented Rhus verniciflua. The present study aimed to examine the effect of PGG on UVB-induced skin aging and its molecular mechanisms in HaCaT human keratinocytes and SKH-1 hairless mice models. PGG suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in HaCaT cells by inhibiting phosphorylation of RAF/MEK/ERK, MKK3/6/p38, and c-Jun. UVB-induced ERK and p38 signaling pathways that induce the MMP-1 expression were mediated by PAK1 in HaCaT cells. PGG suppressed PAK1 and JNK1 kinase activities, and directly bound both PAK1 in an ATP-competitive manner and JNK1 in an ATP-noncompetitive manner. Consistently, PGG decreased UVB-induced wrinkle formation, epidermal thickness, type 1 collagen and MMP-13 expression in mouse skin. Overall, these results indicate that PGG exhibits anti-photoaging effects in vitro and in vivo by the suppression of PAK1 and JNK1 kinase activities, and may be useful for the prevention of skin aging. Full article

(This article belongs to the Special Issue Antioxidants in Cosmetics)

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16 pages, 2731 KiB

Open AccessArticle

In Vitro Antioxidant Activity and FTIR Characterization of High-Molecular Weight Melanoidin Fractions from Different Types of Cocoa Beans

by Joanna Oracz and Dorota Zyzelewicz

Antioxidants 2019, 8(11), 560; https://doi.org/10.3390/antiox8110560 - 15 Nov 2019

Cited by 61 | Viewed by 6196

Abstract

Melanoidins from real foods and model systems have received considerable interest due to potential health benefits. However, due to the complexity of these compounds, to date, the exact structure of melanoidins and mechanism involved in their biological activity has not been fully elucidated. [...] Read more.

Melanoidins from real foods and model systems have received considerable interest due to potential health benefits. However, due to the complexity of these compounds, to date, the exact structure of melanoidins and mechanism involved in their biological activity has not been fully elucidated. Thus, the aim of this study was to investigate the total phenolic content, antioxidant properties, and structural characteristics of high-molecular weight (HMW) melanoidin fractions isolated by dialysis (>12.4 kDa) from raw and roasted cocoa beans of Criollo, Forastero, and Trinitario beans cultivated in various area. In vitro antioxidant properties of all studied HMW cocoa fractions were evaluated by four different assays, namely free radical scavenging activity against DPPH^• and ABTS^•+ radicals, ferric reducing antioxidant power (FRAP), and metal-chelating ability. Additionally, the structure–activity relationship of isolated HMW melanoidin fractions were analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The results show that roasting at a temperature of 150 °C and a relative air humidity of 0.3% effectively enhances the total phenolics content and the antioxidant potential of almost all HMW cocoa melanoidin fractions. The ATR-FTIR analysis revealed that the various mechanisms of action of HMW melanoidins isolates of different types of cocoa beans related to their structural diversity. Consequently, the results clearly demonstrated that HMW cocoa fractions isolated from cocoa beans (especially those of Criollo variety) roasted at higher temperatures with the lower relative humidity of air possess high antioxidant properties in vitro. Full article

(This article belongs to the Special Issue Antioxidants in Cocoa)

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Total phenolics content of melanoidin fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups. Results are presented as means ± SD from triplicate assays. Bars with the same lowercase letter (a–g) within each variety do not differ significantly according to Tukey’s HSD test at p < 0.05. Full article ">Figure 2
The weighted average antioxidant capacity of HMW fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups. Results are presented as means ± SD from triplicate assays. Bars with the same lowercase letter (a–f) within each variety do not differ significantly according to Tukey’s HSD test at p < 0.05. Full article ">Figure 3
Total phenolics content of HMW fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups. Results are presented as means ± SD from triplicate assays. Bars with the same lowercase letter (a–h) within each variety do not differ significantly according to Tukey’s HSD test at p < 0.05. Full article ">Figure 4
Fourier transform infrared spectroscopy (FTIR) spectra of HMW fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups. Full article ">

20 pages, 1850 KiB

Open AccessArticle

From the Field to the Pot: Phytochemical and Functional Analyses of Calendula officinalis L. Flower for Incorporation in an Organic Yogurt

by Graziela Bragueto Escher, Lorena do Carmo Cardoso Borges, Jânio Sousa Santos, Thiago Mendanha Cruz, Mariza Boscacci Marques, Mariana Araújo Vieira do Carmo, Luciana Azevedo, Marianna M. Furtado, Anderson S. Sant’Ana, Mingchun Wen, Liang Zhang and Daniel Granato

Antioxidants 2019, 8(11), 559; https://doi.org/10.3390/antiox8110559 - 15 Nov 2019

Cited by 38 | Viewed by 4980

Abstract

Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of [...] Read more.

Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of water and ethyl alcohol, and the lyophilized extract with higher content of antioxidant compounds was incorporated into an organic yogurt. Results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolic content (TPC), flavonoids, and antioxidant activity (ferric reducing antioxidant power (FRAP), total reducing capacity (TRC), and Cu²⁺/Fe²⁺ chelating ability). Phenolic acids and flavonoids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract (LME) also inhibited 12% of Wistar rat’s brain lipid oxidation in vitro, inhibited α-amylase, and α-glucosidase activities, but showed no cytotoxicity towards cancerous cells (HCT8 and A549). However, marigold flower extract protected human erythrocytes against mechanical stress. When added into an organic yogurt model (0 to 1.5%), LME increased TPC and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and TRC), and the sensory analysis showed that the organic yogurt had an acceptance of 80.4%. Our results show that the use of LME may be a technological strategy to increase the content of bioactive compounds in yogurts. Full article

(This article belongs to the Special Issue Phenolic Profiling and Antioxidant Capacity in Plants)

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Full article ">Figure 1
Principal component analysis based on physicochemical properties and antioxidant activity of marigold flower extracts (Calendula officinalis L.). Extract 1 = 100% H2O:0% EtOH; extract 2 = 75% H2O:25% EtOH; extract 3 = 50% H2O:50% EtOH; extract 4 = 25% H2O:75% EtOH; and extract 5 = 0% H2O:100% EtOH. TPC = total phenolic content; FCRC = Folin–Ciocalteau reducing capacity. Full article ">Figure 2
In vitro antihemolytic effect of lyophilized marigold extract (C. officinalis). (A) Effects on hemolysis of human erythrocytes at different concentrations of NaCl; (B) effects of different extract concentrations on hemolysis of human erythrocytes at 0.3% (w/v) NaCl. Different letters (a–d) represent statistically significant differences, p < 0.0001 (B). Full article ">Figure 3
Inhibition of lipid oxidation of Wistar rat brain treated with lyophilized marigold extract (C. officinalis) in comparison to quercetin. Probability value was obtained using unpaired Student t text. Full article ">Figure 4
In vitro inhibitory effects of different concentrations of lyophilized marigold extract (C. officinalis) on the activities of α-amylase (A) and α-glucosidase (B) enzymes. Different letters (a–e) represent statistically significant differences (p < 0.0001). Full article ">Figure 5
Cell viability of A549, HCT8, and IMR90 cells in relation to different concentrations of lyophilized marigold extract (C. officinalis). (A) IC50: The concentration of the agent that inhibits growth by 50% is the concentration at which (T/C) × 100 = 50, where T = number of cells, at time t of treatment; C = control cells at time t of treatment. (B) GI50: The concentration of the agent that inhibits growth by 50%, relative to untreated cells, is the concentration at which ([T−T0]/[C−[]T0]) × 100 = 50, where T and C are the number of treated and control cells, respectively, at time t of treatment and T > T0; T0 is the number of cells at time zero. (C) LC50: The concentration of the agent that results in a net loss of 50% cells, relative to the number at the start of treatment, is the concentration at which ([T−T0]/T0) × 100 = −50; T < T0. Full article ">Figure 6
Results of intracellular ROS measurement in A549 (A) and IMR90 (B) cells by spectrofluoremetry. Treatment = lyophilized marigold extract (C. officinalis) at 10–100 μg/mL. Quantitative data are the mean ± standard deviation. Different letters (a–c) comparing the treatments indicate significant differences (p < 0.05). Full article ">

15 pages, 1697 KiB

Open AccessCommunication

Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi

by Álvaro Mourenza, Natalia Bravo-Santano, Inés Pradal, Jose A. Gil, Luis M. Mateos and Michal Letek

Antioxidants 2019, 8(11), 558; https://doi.org/10.3390/antiox8110558 - 15 Nov 2019

Cited by 8 | Viewed by 4046

Abstract

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, [...] Read more.

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives. Full article

(This article belongs to the Section Antioxidant Enzyme Systems)

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Figure 1
Macrophage infection assays. Intracellular survival in J774.A macrophages of the wild type R. equi 103S+ strain, the virulence plasmid cured R. equi 103S− strain, the triple Δmrx1Δmrx2Δmrx3 mutant, and R. equi Δmrx1Δmrx2Δmrx3 strain individually complemented with each mrx gene. Bacterial viability was measured by quantifying the number of colony-forming units (CFUs) of each strain at 48 h, and data were normalized by the percentage of R. equi 103S+ CFUs. Data are expressed as means ± SD of three independent experiments. One-way ANOVA and post hoc Tukey´s multiple comparison tests were performed to assess for statistical significance related to the wild type strain. ** p-value < 0.01. Full article ">Figure 2
In vitro viability of R. equi strains after 3 h in trypticase soy broth (TSB) supplemented with either 10 mM H2O2 (A) or 5 mM sodium hypochlorite NaClO (B). Control: R. equi 103S+ cultured on plain TSB. Bacterial viability was measured by quantifying the number of CFUs of each strain at 3 h, and data were normalized by the percentage of R. equi 103S+ CFUs recovered from plain TSB. Data are expressed as means ± SD of three independent experiments. One-way ANOVA and post hoc Tukey´s multiple comparison tests were performed to assess for statistical significance across conditions. p-value < 0.05 (*) or p-value < 0.01 (**). Full article ">Figure 3
DETA NONOate susceptibility test. Analysis of the susceptibility to the oxidative agent DETA NONOate of the triple R. equi Δmrx1Δmrx2Δmrx3 mutant and its mrx-complemented derivative strains in comparison to the wild type strain. Results are expressed as means ± SD of three independent experiments. One-way ANOVA and post hoc Tukey’s multiple comparison tests were performed to assess for statistical significance across conditions. p-value < 0.01 (**) or < 0.001 (***). Full article ">Figure 4
Fluorescence analysis of R. equi strains expressing the reduction-oxidation sensitive green fluorescent protein (roGFP2) biosensor under the control of the constitutive promoter Pkan. All Mrxs-roGFP2 fusions were expressed in R. equi Δmrx1Δmrx2Δmrx3. The 405/490 ratio fluorescence of ≈200 cells was evaluated at different time points during treatments with 5 mM H2O2 (A), 5 mM NaClO (B), or 5 mM DETA NONOate (C), and during macrophage infection (D); fluorescence 405/490 ratio was calculated by confocal microscopy at different time points. Data are expressed as means ± SD of ≈200 cells from three independent experiments. Full article ">

16 pages, 3295 KiB

Open AccessArticle

Dietary Flavonoids Luteolin and Quercetin Inhibit Migration and Invasion of Squamous Carcinoma through Reduction of Src/Stat3/S100A7 Signaling

by Jhen-Jia Fan, Wen-Hsien Hsu, Kuen-Haur Lee, Ku-Chung Chen, Cheng-Wei Lin, Yu-Lin A Lee, Tzu-Ping Ko, Lang-Ta Lee, Ming-Ting Lee, Mau-Sun Chang and Chia-Hsiung Cheng

Antioxidants 2019, 8(11), 557; https://doi.org/10.3390/antiox8110557 - 15 Nov 2019

Cited by 74 | Viewed by 4731

Abstract

Flavonoids are well-known antioxidants and have shown the ability to prevent tumor formation and recurrence. Especially in dietary flavonoids, they have provided convenience and consistence of intake for long-term prevention of tumor formation. Previous reports suggested that S100 calcium-binding protein A7 (S100A7) might [...] Read more.

Flavonoids are well-known antioxidants and have shown the ability to prevent tumor formation and recurrence. Especially in dietary flavonoids, they have provided convenience and consistence of intake for long-term prevention of tumor formation. Previous reports suggested that S100 calcium-binding protein A7 (S100A7) might activate epithelial–mesenchymal transition (EMT) signaling and promote the metastasis of tumor cells; however, the regulatory signaling was unclear. In this study, we found that S100A7 was highly expressed in cancer cells and could be reduced by luteolin (Lu) and quercetin (Qu) through Src/Stat3 signaling. We found that the protein levels of S100A7, phosphorylated Src (p-Src), and p-Stat3 were increased in A431-III cells. Flavonoids Lu and Qu reduce protein levels of p-Src, p-Stat3 and S100A7 in A431-III cells. Treatment of A431-III cells with Src inhibitor SU6656 and Stat3 inhibitor S3I-201 also reduced the protein levels of S100A7. Transactivation activity of 5′-upstream regions of S100A7 was activated by Stat3 but was reduced by treatment with Lu, Qu, SU6656 and S3I-201. The treatment also reduced the migratory and invasive abilities of A431-III cells. In a further analysis of EMT markers, the protein level of E-cad increased and that of Twist decreased after treatment with the inhibitors and flavonoids. Overexpression of S100A7 decreased the protein level of E-cad and increased the Twist level, whereas knockdown of S100A7 had the opposite effects. Treatment with S3I-201, Lu and Qu, compared to the control, were found to decrease metastasis of tumor cells in zebrafish larvae. These results suggest that Lu and Qu may inhibit Src/Stat3/S100A7 signaling to reduce tumorigenesis of cancer cells. Full article

(This article belongs to the Special Issue Dietary Antioxidants in Cancer Chemoprevention)

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S100A7 is more-highly expressed in cervical cancer patients and A431-III cells accompanied by activation of Src/Stat3 signaling. (A) The mRNA levels of S100A7 in A431-P and A431-III cells, analyzed by microarray. (B) The protein levels of S100A7, Src, phosphorylated (p)-Src, Stat3 and p-Stat3 in A431-P and A431-III cells. Full article ">Figure 2
Luteolin (Lu) and quercetin (Qu) inhibit S100A7 in A431-III cells by suppressing Src/Stat3 signaling. (A) Cell viability assay of A431-III cells treated with Lu, Qu, Su6656 and S3I-301 using an (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. (B) Western blot analysis of the protein levels in A431-III cells after treatment with 10 and 20 μM of Lu and 20 and 40 μM of Qu. (C) Same as (B), but with 1, 5 and 10 μM of Su6656. (D) Same as (B), but with 100, 200 and 400 μM of S3I-201. Full article ">Figure 3
Transactivation activity of S100A7 is regulated by Src/Stat3 signaling. (A) Western blot analysis of the protein level of Stat3 after transfection with pcDNA3-Stat3-HA plasmid in A431-III cells. (B) Transactivation activity of the 5′-upstream regions of S100A7, analyzed by luciferase assay in A431-III cells. Transactivation activity of the 5′-upstream regions of S100A7 was activated by Stat3 (C), but inhibited by luteolin (D), quercetin (E), Su6656 (F), and S3I-201 (G). Statistical significance between groups was analyzed by a one-way ANOVA with Tukey’s test (* p < 0.05, ** p < 0.01, and *** p < 0.001). Full article ">Figure 4
Src/Stat3 signaling regulates the migratory ability of A431-III cells, which was reduced by the effectors. A wound-healing assay was conducted, and the cells were observed to migrate into the wound area. (A) A431-III cells were treated with dimethyl sulfoxide (DMSO), 10 and 20 μM of luteolin, 20 and 40 μM of quercetin, 1, 5 and 10 μM of Su6656, and 100, 200 and 400 μM of S3I-201. (B) The numbers of migrating cells treated with luteolin (a), quercetin (b), SU6656 (c) and S3I-201 (d) were measured from (A) and analyzed using ImageJ software (NIH, Bethesda, MA, USA). Statistical significance between groups were analyzed by a one-way ANOVA with Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001). Full article ">Figure 5
Src/Stat3 regulates the invasive ability of A431-III cells. (A) A trans-well assay was used to analyze the invasive ability of A431-III cells after pretreatment with 0.1% of DMSO (Aa), 10 μM of Su6656 (Ab), and 400 μM of S3I-201 (Ac) for 24 h prior to seeding. (B) The number of invasive cells were calculated and analyzed by ImageJ software. Statistical significance between groups was analyzed by a one-way ANOVA with Tukey’s test (*** p < 0.001). Full article ">Figure 6
Src/Stat3/S100A7 signaling activates the epithelial-mesenchymal transition in A431-III cells. The protein levels of E-cadherin and Twist were analyzed by Western blot after treatment with 10, 20 μM of luteolin and 20, 40 μM of quercetin (A); 1, 5, 10 μM of SU6656 (B); or 100, 200, 400 μM of S3I-201 (C); and after overexpressing S100A7 in A431-P cells (D); or knockdown of S100A7 in A431-III cells by S100A7 shRNAs (E). Full article ">Figure 7
Metastasis of A431-III cells were reduced by suppression of Src/Stat3 signaling in zebrafish. (A) A431-III cells were stained with Nile red and microinjected into the pericardiac space of zebrafish larvae at 2 days post-fertilization (dpf). (a) A431-III cells were stained with Nile-Red. (b) The bright field view of 5-dpf zebrafish larvae. Bright field view (c), bright field combined with fluorescent view (d) and fluorescent view (e) of migrative tumor cells enlarged from figure b. (f) Fluorescent view of migrative tumor cells (white arrow) enlarged view from figure e. The number of migrative tumor cells were measured by fluorescent microscope. (B) Measurement of metastatic tumor cell numbers pretreated with 0.1% DMSO (DMSO), 400 μM S3I-201 (S3I-201), 20 μM luteolin, and 40 μM quercetin in zebrafish larvae. Statistical significance between groups were analyzed by a one-way ANOVA with Tukey’s test (** p < 0.01; *** p < 0.001). BF: bright field. Full article ">

31 pages, 5644 KiB

Open AccessReview

Nitric Oxide-Releasing Polymeric Materials for Antimicrobial Applications: A Review

by Fan Rong, Yizhang Tang, Tengjiao Wang, Tao Feng, Jiang Song, Peng Li and Wei Huang

Antioxidants 2019, 8(11), 556; https://doi.org/10.3390/antiox8110556 - 15 Nov 2019

Cited by 122 | Viewed by 11107

Abstract

Polymeric materials releasing nitric oxide have attracted significant attention for therapeutic use in recent years. As one of the gaseous signaling agents in eukaryotic cells, endogenously generated nitric oxide (NO) is also capable of regulating the behavior of bacteria as well as biofilm [...] Read more.

Polymeric materials releasing nitric oxide have attracted significant attention for therapeutic use in recent years. As one of the gaseous signaling agents in eukaryotic cells, endogenously generated nitric oxide (NO) is also capable of regulating the behavior of bacteria as well as biofilm formation in many metabolic pathways. To overcome the drawbacks caused by the radical nature of NO, synthetic or natural polymers bearing NO releasing moiety have been prepared as nano-sized materials, coatings, and hydrogels. To successfully design these materials, the amount of NO released within a certain duration, the targeted pathogens and the trigger mechanisms upon external stimulation with light, temperature, and chemicals should be taken into consideration. Meanwhile, NO donors like S-nitrosothiols (RSNOs) and N-diazeniumdiolates (NONOates) have been widely utilized for developing antimicrobial polymeric agents through polymer-NO donor conjugation or physical encapsulation. In addition, antimicrobial materials with visible light responsive NO donor are also reported as strong and physiological friendly tools for rapid bacterial clearance. This review highlights approaches to delivery NO from different types of polymeric materials for combating diseases caused by pathogenic bacteria, which hopefully can inspire researchers facing common challenges in the coming ‘post-antibiotic’ era. Full article

(This article belongs to the Special Issue Delivery of Gaseous Signal Molecules)

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9 pages, 837 KiB

Open AccessArticle

The Effect of Thiamine Concentration on the Antioxidative Activity Indices in Tea Extracts

by Justyna Piechocka, Krystyna Szymandera-Buszka, Joanna Kobus-Cisowska, Anna Gramza-Michałowska and Anna Jędrusek-Golińska

Antioxidants 2019, 8(11), 555; https://doi.org/10.3390/antiox8110555 - 15 Nov 2019

Cited by 9 | Viewed by 4408

Abstract

The aim of the study was to determine correlations between the concentration of thiamine in systems and indicators of the antioxidative activity of ethanol tea extracts in the presence of soybean oil. Variability of the thiamine form was assumed by comparison of the [...] Read more.

The aim of the study was to determine correlations between the concentration of thiamine in systems and indicators of the antioxidative activity of ethanol tea extracts in the presence of soybean oil. Variability of the thiamine form was assumed by comparison of the influence of thiamine hydrochloride or thiamine pyrophosphate and fermentation of ethanol tea extracts. The study provides practical knowledge about the antioxidative activity of ethanol tea extracts in products containing fat and thiamine. The study showed that all tea extracts exhibited higher antioxidative activity in the presence of thiamine amounts of 0.1 and 0.8 mg/100 g. The antioxidative activity of ethanol tea extracts was significantly reduced when the concentrations were higher than the natural level for foods (over 1.0 mg/100 g). The systems containing white tea extract were the most vulnerable, whereas those with black tea were the least vulnerable. The presence of thiamine pyrophosphate in the system was more strongly correlated with reduced activity of the extracts than the presence of thiamine hydrochloride. Full article

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17 pages, 3686 KiB

Open AccessArticle

Preparation and Evaluation of Resveratrol-Loaded Composite Nanoparticles Using a Supercritical Fluid Technology for Enhanced Oral and Skin Delivery

by Eun-Sol Ha, Woo-Yong Sim, Seon-Kwang Lee, Ji-Su Jeong, Jeong-Soo Kim, In-hwan Baek, Du Hyung Choi, Heejun Park, Sung-Joo Hwang and Min-Soo Kim

Antioxidants 2019, 8(11), 554; https://doi.org/10.3390/antiox8110554 - 14 Nov 2019

Cited by 44 | Viewed by 5829

Abstract

We created composite nanoparticles containing hydrophilic additives using a supercritical antisolvent (SAS) process to increase the solubility and dissolution properties of trans-resveratrol for application in oral and skin delivery. Physicochemical properties of trans-resveratrol-loaded composite nanoparticles were characterized. In addition, an in [...] Read more.

We created composite nanoparticles containing hydrophilic additives using a supercritical antisolvent (SAS) process to increase the solubility and dissolution properties of trans-resveratrol for application in oral and skin delivery. Physicochemical properties of trans-resveratrol-loaded composite nanoparticles were characterized. In addition, an in vitro dissolution–permeation study, an in vivo pharmacokinetic study in rats, and an ex vivo skin permeation study in rats were performed. The mean particle size of all the composite nanoparticles produced was less than 300 nm. Compared to micronized trans-resveratrol, the trans-resveratrol/hydroxylpropylmethyl cellulose (HPMC)/poloxamer 407 (1:4:1) nanoparticles with the highest flux (0.792 μg/min/cm²) exhibited rapid absorption and showed significantly higher exposure 4 h after oral administration. Good correlations were observed between in vitro flux and in vivo pharmacokinetic data. The increased solubility and flux of trans-resveratrol generated by the HPMC/surfactant nanoparticles increased the driving force on the gastrointestinal epithelial membrane and rat skin, resulting in enhanced oral and skin delivery of trans-resveratrol. HPMC/surfactant nanoparticles produced by an SAS process are, thus, a promising formulation method for trans-resveratrol for healthcare products (owing to their enhanced absorption via oral administration) and for skin application with cosmetic products. Full article

(This article belongs to the Special Issue Natural Phenolic Compounds for Health, Food and Cosmetic Applications)

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15 pages, 2075 KiB

Open AccessArticle

Piperlongumine Induces Cell Cycle Arrest via Reactive Oxygen Species Accumulation and IKKβ Suppression in Human Breast Cancer Cells

by Chang Hee Jeong, Haram Ryu, Do Hyun Kim, Wei Nee Cheng, Jee Eun Yoon, Sukyung Kang and Sung Gu Han

Antioxidants 2019, 8(11), 553; https://doi.org/10.3390/antiox8110553 - 14 Nov 2019

Cited by 38 | Viewed by 5802

Abstract

Piperlongumine (PL), a natural product derived from long pepper (Piper longum L.), is known to exhibit anticancer effects. However, the effect of PL on cell cycle-regulatory proteins in estrogen receptor (ER)-positive breast cancer cells is unclear. Therefore, we investigated whether PL can modulate [...] Read more.

Piperlongumine (PL), a natural product derived from long pepper (Piper longum L.), is known to exhibit anticancer effects. However, the effect of PL on cell cycle-regulatory proteins in estrogen receptor (ER)-positive breast cancer cells is unclear. Therefore, we investigated whether PL can modulate the growth of ER-positive breast cancer cell line, MCF-7. We found that PL decreased MCF-7 cell proliferation and migration. Flow cytometric analysis demonstrated that PL induced G2/M phase cell cycle arrest. Moreover, PL significantly modulated the mRNA levels of cyclins B1 and D1, cyclin-dependent kinases 1, 4, and 6, and proliferating cell nuclear antigen. PL induced intracellular reactive oxygen species (hydrogen peroxide) accumulation and glutathione depletion. PL-mediated inhibition of IKKβ expression decreased nuclear translocation of NF-κB p65. Furthermore, PL significantly increased p21 mRNA levels. In conclusion, our data suggest that PL exerts anticancer effects in ER-positive breast cancer cells by inhibiting cell proliferation and migration via ROS accumulation and IKKβ suppression. Full article

(This article belongs to the Section Natural and Synthetic Antioxidants)

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15 pages, 2892 KiB

Open AccessArticle

Silver Nanoparticles Induce Mitochondrial Protein Oxidation in Lung Cells Impacting Cell Cycle and Proliferation

by Reetta J. Holmila, Stephen A. Vance, Stephen Bruce King, Allen W. Tsang, Ravi Singh and Cristina M. Furdui

Antioxidants 2019, 8(11), 552; https://doi.org/10.3390/antiox8110552 - 14 Nov 2019

Cited by 51 | Viewed by 4675

Abstract

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects [...] Read more.

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a panel of lung cell lines (A549, BEAS-2B, Calu-1 and NCI-H358). The exposure to AgNPs caused cell cycle arrest and decreased cell proliferation in A549, BEAS-2B and Calu-1, but not in NCI-H358. The mitochondrial reactive oxygen species (ROS) and protein oxidation increased in a time- and dose-dependent manner in the more sensitive cell lines with the AgNP exposure, but not in NCI-H358. While ionizing radiation also induced changes in the mitochondrial redox profiles, in general, these were not synergistic with the effects of AgNPs with the exception of NCI-H358 and only at a higher dose of radiation. Full article

(This article belongs to the Special Issue When the Anticancer Strategies Modulating Oxidative Stress Meet Nanomedicine: New Perspectives)

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16 pages, 1314 KiB

Open AccessArticle

Influence of Cooking and Ingredients on the Antioxidant Activity, Phenolic Content and Volatile Profile of Different Variants of the Mediterranean Typical Tomato Sofrito

by Ana Beltrán Sanahuja, Saray López De Pablo Gallego, Salvador E. Maestre Pérez, Arantzazu Valdés García and María Soledad Prats Moya

Antioxidants 2019, 8(11), 551; https://doi.org/10.3390/antiox8110551 - 14 Nov 2019

Cited by 14 | Viewed by 5098

Abstract

In this study, six different sofrito formulations were compared with the raw recipe for total phenolic content (TPC), antioxidant activity tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) methods. The volatile profile was also obtained by [...] Read more.

In this study, six different sofrito formulations were compared with the raw recipe for total phenolic content (TPC), antioxidant activity tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) methods. The volatile profile was also obtained by the headspace solid-phase microextraction-gas chromatography mass spectrometry (HS-SPME-GC–MS) procedure. The cooking process and the addition of herbs, and garlic improved the final content of antioxidant compounds compared to the basic recipe and the raw ingredients. The total volatile content was higher in the samples that contained rosemary and thymus. Some of the volatiles had proven antioxidant properties and for that reason the sofrito with rosemary with the higher volatile content was also the one with the higher antioxidant capacity and TPC. In conclusion, as well as the processing technique, the addition of selected typical Mediterranean herbs apart from given flavour can contribute to improving the nutritional antioxidant profile of dishes and be used as a natural method to increase the shelf-life of preparation. Full article

(This article belongs to the Special Issue Phenolic Profiling and Antioxidant Capacity in Plants)

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16 pages, 1582 KiB

Open AccessArticle

Novel Polymeric Nanocarriers Reduced Zinc and Doxycycline Toxicity in the Nematode Caenorhabditis elegans

by Manuel Toledano, Manuel Toledano-Osorio, María D. Navarro-Hortal, Alfonso Varela-López, Raquel Osorio and José L. Quiles

Antioxidants 2019, 8(11), 550; https://doi.org/10.3390/antiox8110550 - 14 Nov 2019

Cited by 15 | Viewed by 4700

Abstract

The objective was to evaluate the toxicity of zinc- and doxycycline-loaded polymeric nanoparticles (NPs) using Caenorhabditis elegans as a model organism. These NPs are composed of ethylene glycol dimethacrylate, 2-hydroxyethyl methacrylate and methacrylic acid. NPs were loaded with doxycycline (D-NPs) and zinc (Zn-NPs) [...] Read more.

The objective was to evaluate the toxicity of zinc- and doxycycline-loaded polymeric nanoparticles (NPs) using Caenorhabditis elegans as a model organism. These NPs are composed of ethylene glycol dimethacrylate, 2-hydroxyethyl methacrylate and methacrylic acid. NPs were loaded with doxycycline (D-NPs) and zinc (Zn-NPs) by chemical adsorption, and loading efficacy was demonstrated. Worm death rate in a concentration-response curve basis was calculated for lethality. Metabolism was evaluated through pharyngeal pumping assay. Body length measurements, brood size and egg lays were used to gauge growth, reproduction and fertility respectively. Intracellular hydrogen peroxide levels were determined to assess the reactive oxygen species production. One-way ANOVA and Bonferroni were used for comparisons (p < 0.05). Tested NPs at the highest dosage did not affect lethality or worm metabolism, expressed in terms of death rate and pharyngeal pumping per minute, respectively. Zn-NPs slightly increased worm growth. The concentration of the intracellular hydrogen peroxide levels was the lowest in the D-NPs group. The distinct NPs and concentrations employed were shown to be non-toxic for in situ administration of zinc and doxycycline, reducing the harmful effects of these compounds. Full article

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Figure 1
Images of nanoparticles observed under TEM (A), AFM (B) and FESEM (C and D). NPs are spherical and homogeneous in shape. NPs do not agglomerate, and they are approximately 250 nm in diameter. Full article ">Figure 2
Zinc adsorption kinetics on nanoparticles as a function of time (A), Zinc adsorption kinetics on nanoparticles as a function of zinc concentration in initial solutions (B) and doxycycline adsorption kinetics on nanoparticles as a function of time and doxycycline concentration in initial solutions (C). Full article ">Figure 3
Lethal toxicity expressed as a percentage of surviving worms, after the nematodes were exposed to different nanoparticles and concentrations. Results are presented as mean and standard error of three different assays (A–C). Each independent assay included three NGM plates with ten worms in each one. A total of 90 worms were then tested for each experimental group. No differences in worm surviving percentages were found after ANOVA analysis (p > 0.05). D-NPs: doxycycline-doped nanoparticles. NPs: undoped nanoparticles. Zn-NPs: zinc-doped nanoparticles. Full article ">Figure 4
Mean and standard error of pharyngeal pumping (A), body length (B), fertility (C) and reproduction (D) of Caenorhabditis elegans exposed to different NPs, at 10 mg/mL concentration. One-way ANOVA was not significant for pharyngeal pumping (p > 0.05). ANOVA tests were significant for body length, fertility and reproduction (p < 0.05). For each graph, similar letters (a,b) indicate no significant differences between the NPs experimental groups, after Bonferroni post hoc testing at p < 0.05. D-NPs: doxycycline-doped nanoparticles. NPs: undoped nanoparticles. Zn-NPs: zinc-doped nanoparticles. Full article ">Figure 5
Intracellular hydrogen peroxide production, expressed as the ratio of in oxidized/reduced Hyper, when Caenorhabditis elegans is exposed to the different nanoparticles at 10 mg/mL concentration. Results are presented as mean and standard error. Different lowercase letters (a,b) indicate significant differences (p < 0.05) between the distinct experimental groups at base line. Different capital letters (A–C) represent the existence of significant difference (p < 0.05) between the experimental groups induced with 20 mM of hydrogen peroxide. The asterisk (*) represents the existence of statistically significant differences (p < 0.05) between the baseline and the induced with hydrogen peroxide levels within the same experimental group (A). Confocal microscopy images (5×) representing worm’s fluorescence at same dosages of NPs and H2O2 as for <a href="#antioxidants-08-00550-f005" class="html-fig">Figure 5</a>A D-NPs: doxycycline-doped nanoparticles. NPs: undoped nanoparticles. Zn-NPs: zinc-doped nanoparticles (B). Full article ">

4 pages, 186 KiB

Open AccessEditorial

Antioxidant and Anti-Inflammatory Properties of Plants Extract

by Mario Allegra

Antioxidants 2019, 8(11), 549; https://doi.org/10.3390/antiox8110549 - 14 Nov 2019

Cited by 31 | Viewed by 6727

Abstract

Inflammation is an adaptive response triggered by noxious stimuli and conditions such as infection and tissue injury [...] Full article

(This article belongs to the Special Issue Antioxidant and Anti-inflammatory Properties of Plants Extract)

16 pages, 9833 KiB

Open AccessArticle

Oxidative Stress Increases Endogenous Complement-Dependent Inflammatory and Angiogenic Responses in Retinal Pigment Epithelial Cells Independently of Exogenous Complement Sources

by Timon-Orest Trakkides, Nicole Schäfer, Maria Reichenthaler, Konstanze Kühn, Ricardo J. M. G. E. Brandwijk, Erik J. M. Toonen, Florian Urban, Joachim Wegener, Volker Enzmann and Diana Pauly

Antioxidants 2019, 8(11), 548; https://doi.org/10.3390/antiox8110548 - 13 Nov 2019

Cited by 31 | Viewed by 5462

Abstract

Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE [...] Read more.

Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1–48 h to H₂O₂ had reduced cell–cell contact and increased markers for epithelial–mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration. Full article

(This article belongs to the Special Issue Oxidative Stress and Inflammation as Targets for Novel Preventive and Therapeutic Approches in Non Communicable Diseases)

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ARPE-19 cells reduced tight junctions and circumvented apoptosis under oxidative stress. (A,D) ARPE-19 cells untreated (without (w/o)) and stressed with H2O2 for (B,C) 4 h or (E,F) 24 h translocated the zonula occludens protein 1 (ZO-1, green) time-dependently from the (A,D) cell membrane to the (B,E) cytoplasm. (C,F) ARPE-19 cells treated with oxidative stress showed a minimal TUNEL-positive (light blue) apoptotic reaction after (F) 24 h. Full article ">Figure 2
Oxidative stress increased the expression of complement receptor subunit CD11b and C5aR1 in ARPE-19 cells. (A) Cd11b mRNA expression was increased 4 h after H2O2 treatment. This effect was confirmed on a protein level by immunohistochemistry using (B,C) anti-CD11b (red) antibodies. (D) C5ar1 mRNA also increased on (D) mRNA and (E–G) protein level (anti-C5aR1, green) in H2O2 treated cells. (H) Western blots of ARPE-19 cell lysates detected C5aR1 between 40 and 60 kDa after 4–24 h H2O2 treatment (full immunoblots are shown in the <a href="#app1-antioxidants-08-00548" class="html-app">Supplementary Materials, Figure S3A,B</a>; n = 1) (I) Quantitatively, C5aR1 expression was increased in H2O2-treated cells in the Western blots. (A,D) Mean with standard deviation is shown, * p ≤ 0.05, ** p ≤ 0.01. The dotted line depicts the untreated control; (B,E,H,I) w/o untreated control. Full article ">Figure 3
Oxidative stress induced complement component accumulation in ARPE-19 cells. (A) Properdin mRNA levels were increased 24 h following H2O2 treatment. This did not affect (B) apical properdin secretion, but was confirmed in the protein level by immunohistochemistry using an (C–E) anti-properdin (red) antibody. (F) C3 mRNA and (G) apical C3 protein secretion were not altered in stressed ARPE-19 cells. Immunohistochemistry using (H–J) anti-C3 (green) antibodies showed an increase of cell-associated (I,J) C3 after oxidative stress treatment. (K) Cfh mRNA and (L) CFH apical protein concentration were decreased following H2O2 treatment. (M–O) Immunohistochemistry using anti-complement factor H (CFH, purple) antibodies showed an increase in cell-associated (N,O) CFH after oxidative stress treatment. Mean with standard deviation is shown, ** p ≤ 0.01, **** p ≤ 0.0001; dotted line depicts untreated control (A,F,K); w/o untreated control (G,G,L); ELISA control standard curves and protein concentrations in the basal supernatants are shown in the <a href="#app1-antioxidants-08-00548" class="html-app">Supplementary Materials, Figure S4D–I</a>. Full article ">Figure 4
C3 and complement receptor CD11b were colocalized in ARPE-19. (A) Unstressed (w/o) and (B) H2O2-treated ARPE-19 cells were stained with anti-C3 (green) and anti-CD11b (red) antibodies. Overlapping staining signals (yellow) suggested a colocalization of C3 and CD11b. (C) C3 and activation products (C3b α’ and C3d) were detected in untreated and H2O2-treated ARPE-19 cells using a Western blot under reducing conditions (controls: native C3, C3b, human serum (NHS), and C3-depleted human serum (NHS C3dpl)). Full immunoblots are shown in the <a href="#app1-antioxidants-08-00548" class="html-app">Supplementary Materials, Figure S3C,D</a>; immunoblots were repeated twice. Full article ">Figure 5
The expression of intracellular proteases was increased by oxidative stress in ARPE-19. (A) Ctsb and (B) ctsl mRNA expression increased 24 h after H2O2 treatment. This effect was confirmed on the protein level in immunostainings using an (C,D) anti-CTSL (green) antibody. (A,B) Mean with standard deviation is shown, * p ≤ 0.05, ** p ≤ 0.01, dotted line depicts untreated control; (C) w/o untreated control. Full article ">Figure 6
Increased nlrp3 and foxp3 mRNA expression correlated with proinflammatory and proangiogenic factor secretion. (A) Nlrp3, (B) foxp3, and (C) il1β mRNA levels increased either (A,B) 4 h or (C) 24 h and 48 h following H2O2 treatment. The proinflammatory cytokine release of (D) Interleukin (IL)-1β and (E) IL-6 was detected in stressed ARPE-19 cells. This was correlated with an enhanced secretion of the proangiogenic factors (F) IL-8 and (G) vascular endothelial growth factor (VEGF)-α in H2O2-treated cells. MFI: mean fluorescence intensity. Mean with standard deviation is shown, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; (A,B,C) dotted line depicts untreated control; (D–G) w/o untreated control; protein concentrations in the basal supernatants are shown in the <a href="#app1-antioxidants-08-00548" class="html-app">Supplementary Materials, Figure S4J–L</a>. Full article ">Figure 7
Olaparib enhanced oxidative stress-dependent expression changes in ARPE-19 cells. ARPE-19 cells were treated for 4 h with H2O2, and the effect of simultaneously added olaparib on transcription was investigated. (A) Cd11b, (B) c5aR1, and (C) nlrp3 transcripts were significantly increased in olaparib-treated, stressed cells compared to unstressed cells. Olaparib also increased the expression of (D) properdin, (E) ctsb, and (F) cfd. (G) Foxp3 mRNA levels were not changed in stressed ARPE-19 cells following olaparib addition. Mean with standard deviation is shown, * p ≤ 0.05; dotted line depicts untreated control. Full article ">

12 pages, 2042 KiB

Open AccessArticle

Salicylic Acid and Melatonin Alleviate the Effects of Heat Stress on Essential Oil Composition and Antioxidant Enzyme Activity in Mentha × piperita and Mentha arvensis L.

by Milad Haydari, Viviana Maresca, Daniela Rigano, Alireza Taleei, Ali Akbar Shahnejat-Bushehri, Javad Hadian, Sergio Sorbo, Marco Guida, Caterina Manna, Marina Piscopo, Rosaria Notariale, Francesca De Ruberto, Lina Fusaro and Adriana Basile

Antioxidants 2019, 8(11), 547; https://doi.org/10.3390/antiox8110547 - 13 Nov 2019

Cited by 55 | Viewed by 6071

Abstract

The aim of this study was to evaluate changes in the chemical profile of essential oils and antioxidant enzymes activity (catalase CAT, superoxide dismutase SOD, Glutathione S-transferases GST, and Peroxidase POX) in Mentha × piperita L. (Mitcham variety) and Mentha arvensis L. [...] Read more.

The aim of this study was to evaluate changes in the chemical profile of essential oils and antioxidant enzymes activity (catalase CAT, superoxide dismutase SOD, Glutathione S-transferases GST, and Peroxidase POX) in Mentha × piperita L. (Mitcham variety) and Mentha arvensis L. (var. piperascens), in response to heat stress. In addition, we used salicylic acid (SA) and melatonin (M), two brassinosteroids that play an important role in regulating physiological processes, to assess their potential to mitigate heat stress. In both species, the heat stress caused a variation in the composition of the essential oils and in the antioxidant enzymatic activity. Furthermore both Salicylic acid (SA) and melatonin (M) alleviated the effect of heat stress. Full article

(This article belongs to the Special Issue Natural Phenolic Compounds for Health, Food and Cosmetic Applications)

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18 pages, 2267 KiB

Open AccessArticle

Assessing the Efficacy of Dietary Selenomethionine Supplementation in the Setting of Cardiac Ischemia/Reperfusion Injury

by Leila Reyes, David P. Bishop, Clare L. Hawkins and Benjamin S. Rayner

Antioxidants 2019, 8(11), 546; https://doi.org/10.3390/antiox8110546 - 13 Nov 2019

Cited by 14 | Viewed by 3429

Abstract

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes [...] Read more.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes as well as readily react with MPO-derived oxidants. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and an in vivo rat model of cardiac I/R injury. Exposure of the H9c2 cardiac myoblast cell line to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation and was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation. This protection was credited primarily to the direct oxidant scavenging ability of SeMet, with a minor contribution arising from the ability of SeMet to bolster cardiac myoblast glutathione peroxidase (GPx) activity. In vivo, a significant increase in selenium levels in the plasma and heart tissue were seen in male Wistar rats fed a diet supplemented with 2 mg kg⁻¹ SeMet compared to controls. However, SeMet-supplementation demonstrated only limited improvement in heart function and did not result in better heart remodelling following I/R injury. These data indicate that SeMet supplementation is of potential benefit within pathological settings where excessive HOCl is known to be generated but has limited efficacy as a therapeutic agent for the treatment of heart attack. Full article

(This article belongs to the Special Issue Anti-Inflammatory and Antioxidant Effects of Dietary Supplementation and Lifestyle Factors)

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Figure 1
Selenomethionine (SeMet) supplementation protects against H9c2 cellular dysfunction elicited by HOCl. H9c2 cells (1 × 105 cells mL−1) serum-starved and supplemented with (hatched bars) or without (solid bars) SeMet (25 µM) for 24 h were exposed to HOCl for 1 h with or without SeMet (25 µM) and analysed immediately for ΔΨm and intracellular Ca2+ accumulation using JC-1 and the fluorescent Ca2+ indicator Fluo,4-AM, respectively and the extent of cell death measured using Annexin-V/propidium iodide (PI) staining. (A) Representative dot plots of JC-1 staining with (B) quantification of ΔΨm immediately following exposure to HOCl and SeMet supplementation or CCCP (0.1 mM) as a positive control (+ve). (C) Representative histogram flow plot of Fluo,4-AM staining with (D) quantification of intracellular Ca2+ accumulation immediately following exposure to HOCl and SeMet supplementation. (E) Representative dot plots of Annexin-V/PI staining with (F) quantification of necrotic cells and (G) late apoptotic cells immediately following exposure to HOCl and SeMet supplementation. Data are expressed as a percentage of the whole cell populations as mean ± S.E.M. from n = 3 biological repeats performed in triplicate. (B,D) Data are expressed as a fold change over respective controls assayed in the absence of oxidant and SeMet as mean ± S.E.M. from n = 3 biological repeats performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Hank’s buffered salt solution (HBSS) control (0 µM), # p < 0.05 vs. control cells assayed in the absence of SeMet (0 µM) as determined by two-way ANOVA with Bonferroni post-hoc testing. Full article ">Figure 2
SeMet supplementation alters the redox status of H9c2 cells exposed to HOCl insult resulting in protection. (A–C) H9c2 cells (1 × 105 cells mL−1) were exposed to either increasing concentration of SeMet in serum-free media for 24 h, (D) supplemented with SeMet (25 µM) prior to and during oxidative exposure and exposed to HOCl as described in <a href="#antioxidants-08-00546-f001" class="html-fig">Figure 1</a> or (E–G) exposed to HOCl alone (control) and subjected to different SeMet supplementation conditions (SeMet, supplementation throughout the whole treatment period; pre-treatment, supplementation prior to HOCl exposure; co-treatment, supplementation during HOCl exposure). (B,D,G) Quantification of glutathione peroxidase (GPx) activity immediately post treatment, with data expressed as a percentage fold change over control cells (0 µM) expressed as mean ± S.E.M (n ≥ 3). (C) mRNA gene expression of GPx1, Trx1 and Trxrd1 measured immediately post treatment using qPCR, with data expressed as a fold change of their respective controls as mean ± S.E.M (n = 3). Viability assessed (A) immediately or (F) 24 h post treatment using the lactate dehydrogenase (LDH) release assay or by using the (E) MTS assay immediately post treatment, with data expressed as a percentage viability relative to the control cells (0 µM) as mean ± S.E.M from n ≥ 3 biological repeats performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HBSS controls; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. control cells assayed in the absence of SeMet as determined by two-way ANOVA with Bonferroni post-hoc testing. Full article ">Figure 3
Selenium tissue levels in rats fed SeMet supplemented chow. Male Wistar rats (100–125 g) were randomly assigned into groups receiving either normal chow (control) or normal chow supplemented with SeMet (2 mg kg−1) ad libitum for 8 weeks. Quantification of selenium in the (A) plasma, (B) heart, (C) brain, (D) kidney and (E) liver was performed using inductively coupled plasma-mass spectrometry (ICP-MS) with (F) total GPx activity measured within rat plasma. Data expressed as mean ± S.E.M. from n = 5 (Control) and n = 6 (SeMet). * p < 0.05, *** p < 0.001, **** p < 0.0001 difference between control and SeMet-supplemented group as determined by two-tailed unpaired Student’s t-test. Full article ">Figure 4
SeMet supplementation is not protective against cardiac ischemia/reperfusion (I/R) injury. Male Wistar rats (100–125 g) were fed either normal chow (control, open bars) or SeMet-supplemented chow (checked bars) as described in <a href="#antioxidants-08-00546-f003" class="html-fig">Figure 3</a> and subjected to 30 min ischemia or sham surgery followed by reperfusion and recovery for either 24 h or 4 weeks. (A) Representative images of 24 h heart left ventricle (LV) stained with triphenyl tetrazolium chloride (TTC) to differentiate infarcted tissue (white) from viable, muscle tissue (red) and (B) quantification of the infarcted region at 24 h expressed as the percentage area to the total area of the LV as mean ± S.E.M (n ≥ 5). (C) Representative echocardiogram images in M-mode and quantification (D) fractional shortening (FS) and (E) ejection fraction (EF) of control and SeMet-supplemented animals subjected to cardiac I/R injury. Data presented as mean ± S.E.M from control sham n = 5; control I/R n = 8; SeMet sham n = 5; SeMet I/R n = 8 rats per group, performed in triplicate. (F) Representative images of 4 week heart LV stained with Milligan’s trichrome to differentiate fibrotic tissue (blue) from muscle tissue (purple) and (G) quantification of the fibrotic region expressed as the percentage area to the total area of the LV as mean ± S.E.M sham n = 5; control n = 8; SeMet n = 8 rats per group, performed in triplicate. * p < 0.05, **** p < 0.0001 vs. sham, ## p < 0.01 vs. control, no significant (ns) changes between control and SeMet-supplemented group as determined by two-way ANOVA with Holm–Sidak post-hoc testing. Full article ">

12 pages, 5126 KiB

Open AccessArticle

Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR)

by Kathleen Fontana, Nneamaka Onukwue, Bei-Lei Sun, Cristina Lento, Leslie Ventimiglia, Sahar Nikoo, James W. Gauld, Derek J. Wilson and Bulent Mutus

Antioxidants 2019, 8(11), 545; https://doi.org/10.3390/antiox8110545 - 13 Nov 2019

Cited by 3 | Viewed by 3453

Abstract

Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for [...] Read more.

Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill–Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn²⁺-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen–deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme. Full article

(This article belongs to the Special Issue NO(NOx) and H2S)

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S-nitrosoglutathione reductase (GSNOR) steady-state kinetics. (A) Varying amounts of S-nitrosoglutathione (GSNO) (0 to 200 μM) plus a constant amount of NADH (80 μM) were added to a 1-mL cuvette along with 400 μL of phosphate-buffered saline (PBS). The absorbance was monitored for 15 s to establish the blank rate at which time a constant volume of purified recombinant GSNOR (5 μL corresponding to a final concentration of 20 nM) was rapidly added to the assay mixture with the aid of a plumper. The change in absorbance was monitored for a further 60 s. The net enzymatic initial rates were calculated for each GSNO (red circles). The steady-state kinetic parameters were estimated from a fit of the data to the Michaelis–Menten (dashed blue line) or the Hill–Langmuir (red line) algorithms. The error bars represent SD, n = 4. (B) The same data as in <a href="#antioxidants-08-00545-f001" class="html-fig">Figure 1</a>A displayed with a narrower GSNO range to emphasize the sigmoidal behavior of the data. Full article ">Figure 2
Putative allosteric site near the structural zinc, as obtained from MD simulations. (A) GSNOR A Chain (template structure– PDB ID: 3QJ5 [<a href="#B21-antioxidants-08-00545" class="html-bibr">21</a>]) with GSNO bound to Asn185 and Gly321 Lys188 and Lys323; (B) Close up of the interactions between GSNOR residues and GSNO. The protein structure was visualized with a UCSF Chimera 1.11.2. Full article ">Figure 3
Hydrogen–deuterium exchange (HDX)-MS heat map of GSNOR. GSNOR crystal structure (PDB ID: 3QJ5 [<a href="#B21-antioxidants-08-00545" class="html-bibr">21</a>]) with D-uptake superimposed color-code based on Δ4s + Δ2s data (column 11 <a href="#app1-antioxidants-08-00545" class="html-app">Table S2</a>) data: red: >Δ1%; cyan: 0% to Δ –0.5%; sky blue: Δ –0.5% to Δ −1%; blue: >−1%. The protein structure was visualized with UCSF Chimera 1.11.2. Full article ">Figure 4
Steady-state kinetics of GSNOR wt and mutants. Varying amounts of GSNO (0 to 200 μM) plus a constant amount of NADH (80 μM) were added to a 1-mL cuvette along with 400 μL of phosphate-buffered saline (PBS). The absorbance was monitored for 15 s to establish the blank rate at which time a constant volume of purified recombinant GSNOR (wt or mutants) (5 μL corresponding to a final concentration of 20 nm) was rapidly added to the assay mixture with the aid of a plumper. The change in absorbance was monitored for a further 60 s. The net enzymatic initial rates were calculated for each GSNO. Wt (●); K188A (X); K323A (▼); K188A/K323A (X). The steady-state kinetic parameters were estimated from a fit of the data (red or black lines) to the Hill–Langmuir algorithm. The error bars represent SD, n = 4. The table below the figure summarizes the kinetic parameters extracted from the data in the figure. Full article ">

10 pages, 774 KiB

Open AccessReview

Caveats for the Good and Bad of Dietary Red Meat

by Anthony T. Omaye and Stanley T. Omaye

Antioxidants 2019, 8(11), 544; https://doi.org/10.3390/antiox8110544 - 12 Nov 2019

Cited by 11 | Viewed by 5540

Abstract

Red meat and its constituents of heme iron or free iron have been the target of scrutiny related to their purported association to many chronic diseases. However, in contrast, red meat provides a rich source of nutrition. In 2007, Al Tappel hypothesized that [...] Read more.

Red meat and its constituents of heme iron or free iron have been the target of scrutiny related to their purported association to many chronic diseases. However, in contrast, red meat provides a rich source of nutrition. In 2007, Al Tappel hypothesized that the mechanistic explanation for the adverse impact of iron and heme iron could be the strong influence these substances have in initiating and promoting oxidative stress. Also, there is an emphasis on the importance of dietary antioxidants in the modulation of these adverse effects. The goal of this argumentative review is to provide an update of the importance of dietary red meat for health, and the hypothesis that oxidative stress initiated by dietary iron and heme iron may be related to chronic diseases, with a particular emphasis on recent research that impacts the paradigm. We also examine potential dietary changes that could substantially modify the potential adverse outcomes of chronic diseases initiated by heme iron mechanisms, e.g., consumption of antioxidant-rich fruits and vegetables. Full article

(This article belongs to the Special Issue Protein and Lipid Oxidation in Meat and Meat Products)

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12 pages, 870 KiB

Open AccessReview

Nitrosative Stress in Retinal Pathologies: Review

by Antolin Cantó, Teresa Olivar, Francisco Javier Romero and María Miranda

Antioxidants 2019, 8(11), 543; https://doi.org/10.3390/antiox8110543 - 11 Nov 2019

Cited by 39 | Viewed by 4559

Abstract

Nitric oxide (NO) is a gas molecule with diverse physiological and cellular functions. In the eye, NO is used to maintain normal visual function as it is involved in photoreceptor light transduction. In addition, NO acts as a rapid vascular endothelial relaxant, is [...] Read more.

Nitric oxide (NO) is a gas molecule with diverse physiological and cellular functions. In the eye, NO is used to maintain normal visual function as it is involved in photoreceptor light transduction. In addition, NO acts as a rapid vascular endothelial relaxant, is involved in the control of retinal blood flow under basal conditions and mediates the vasodilator responses of different substances such as acetylcholine, bradykinin, histamine, substance P or insulin. However, the retina is rich in polyunsaturated lipid membranes and is sensitive to the action of reactive oxygen and nitrogen species. Products generated from NO (i.e., dinitrogen trioxide (N₂O₃) and peroxynitrite) have great oxidative damaging effects. Oxygen and nitrogen species can react with biomolecules (lipids, proteins and DNA), potentially leading to cell death, and this is particularly important in the retina. This review focuses on the role of NO in several ocular diseases, including diabetic retinopathy, retinitis pigmentosa, glaucoma or age-related macular degeneration (AMD). Full article

(This article belongs to the Special Issue Antioxidants and Retinal Disease)

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13 pages, 11899 KiB

Open AccessReview

Antioxidants versus Food Antioxidant Additives and Food Preservatives

by Rafael Franco, Gemma Navarro and Eva Martínez-Pinilla

Antioxidants 2019, 8(11), 542; https://doi.org/10.3390/antiox8110542 - 11 Nov 2019

Cited by 61 | Viewed by 12875

Abstract

Natural and processed foods are fragile and can become unpalatable and/or rotten. The processed food industry uses preservatives to enable distribution, even to different continents, and to extend the useful life of their products. Preservatives impede oxidation, a mandatory step in rotting, either [...] Read more.

Natural and processed foods are fragile and can become unpalatable and/or rotten. The processed food industry uses preservatives to enable distribution, even to different continents, and to extend the useful life of their products. Preservatives impede oxidation, a mandatory step in rotting, either by aerobic or anaerobic mechanisms. From a functional point of view, these compounds are antioxidants, and, therefore, a kind of contradiction exists when a preservative is considered “bad” for human health while also thinking that antioxidants provide benefits. The basis of antioxidant action, the doses required for preservation, and the overall antioxidant action are revisited in this work. Finally, the bad and the good of food additives/preservatives are presented, taking into account the main mediator of antioxidant beneficial actions, namely the innate mechanisms of detoxification. Foods that strengthen such innate mechanisms are also presented. Full article

(This article belongs to the Special Issue Oxidative Stress in Food Additives and Other Exposomes)

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9 pages, 1205 KiB

Open AccessArticle

Quantitative Analysis of Bioactive Phenanthrenes in Dioscorea batatas Decne Peel, a Discarded Biomass from Postharvest Processing

by Minyoul Kim, Myeong Ju Gu, Joon-Goo Lee, Jungwook Chin, Jong-Sup Bae and Dongyup Hahn

Antioxidants 2019, 8(11), 541; https://doi.org/10.3390/antiox8110541 - 10 Nov 2019

Cited by 15 | Viewed by 3814

Abstract

Dioscorea batatas Decne (Chinese yam) has been widely cultivated in East Asia for the purposes of food and medicinal uses for centuries. Along with its high nutritional value, the medicinal value of D. batatas has been extensively investigated in association with phytochemicals such [...] Read more.

Dioscorea batatas Decne (Chinese yam) has been widely cultivated in East Asia for the purposes of food and medicinal uses for centuries. Along with its high nutritional value, the medicinal value of D. batatas has been extensively investigated in association with phytochemicals such as allantoin, flavonoids, saponins and phenanthrenes. Phenanthrenes are especially considered the standard marker chemicals of the Chinese yam for their potent bioactivity and availability of analysis with conventional high performance liquid chromatography with ultraviolet detection (HPLC-UV) methods. In order to investigate how much the contents of phenanthrenes are in the actual food products provided for consumers, D. batatas tuber was peeled and separated into its peel and flesh as in the conventional processing method. A quantitative analysis using the HPLC-UV method revealed that phenanthrenes are concentrically present in the D. batatas peel, while phenanthrenes are present in the flesh under the limit of detection. The difference in the contents of phenanthrenes is estimated to have arisen the considerable difference of antioxidant potential between the peel and the flesh. The results from this study suggest the high value of the discarded biomass of the Chinese yam peel and the necessity for the utilization of the Chinese yam peel. Full article

(This article belongs to the Special Issue Polyphenolic Antioxidants from Agri-Food Waste Biomass)

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15 pages, 1007 KiB

Open AccessArticle

Phytochemical Characterization of Commercial Processed Blueberry, Blackberry, Blackcurrant, Cranberry, and Raspberry and Their Antioxidant Activity

by Zoriţa Diaconeasa, Cristian I. Iuhas, Huseyin Ayvaz, Dumitriţa Rugină, Andreea Stanilă, Francisc Dulf, Andrea Bunea, Sonia Ancuța Socaci, Carmen Socaciu and Adela Pintea

Antioxidants 2019, 8(11), 540; https://doi.org/10.3390/antiox8110540 - 10 Nov 2019

Cited by 42 | Viewed by 7132

Abstract

Being delicious and containing strong disease-fighting agents, berries represent an increasing proportion of fruits consumed nowadays in our diet. However, berries are highly perishable as fresh and, therefore, they are usually processed into various products to extend their shelf-life and availability throughout the [...] Read more.

Being delicious and containing strong disease-fighting agents, berries represent an increasing proportion of fruits consumed nowadays in our diet. However, berries are highly perishable as fresh and, therefore, they are usually processed into various products to extend their shelf-life and availability throughout the year. Among the fruit-containing products, jam is one of the most common due to its nourishing properties, its low production costs, and its accessibility for a lengthy period. Rather than home preparation, consumers nowadays increasingly prefer to purchase commercial jams from markets due to its convenience. Although fresh berries have been extensively studied for their phenolic compounds, a limited number of studies investigating commercially manufactured jams have been conducted so far. Considering this, the objective of this study was to assess the total phenolic, flavonoid, and anthocyanin content and the antioxidant activity of five commonly consumed commercial berry jams (blueberry (Vaccinium myrtillus), blackberry (Rubus fruticosus) and blackcurrant (Ribes nigrun) mixture, blackcurrant (Ribes nigrun), cranberry (Vaccinium macrocarpon) and raspberry (Rubus idaeus)) collected from the market. Even though a possible loss of phenolics, anthocyanins, and a decrease of radical scavenging activity may occur during jam processing and subsequent storage, our data indicated that the selected commercial jams remained good sources of nutritive molecules with antioxidant properties based on the high levels of total phenolics, flavonoids, anthocyanins, and elevated antioxidant activities determined in this study. Additionally, the samples were characterized by GC-MS for their volatile profiles, and terpenes were found to be the dominating class covering more than 74% of volatile compounds in the jams. Full article

(This article belongs to the Special Issue Phenolic Profiling and Antioxidant Capacity in Plants)

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13 pages, 863 KiB

Open AccessArticle

Specific Activity of Superoxide Dismutase in Stallion Seminal Plasma Is Related to Sperm Cryotolerance

by Marion Papas, Jaime Catalán, Beatriz Fernandez-Fuertes, Laura Arroyo, Anna Bassols, Jordi Miró and Marc Yeste

Antioxidants 2019, 8(11), 539; https://doi.org/10.3390/antiox8110539 - 9 Nov 2019

Cited by 36 | Viewed by 4004

Abstract

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. [...] Read more.

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability. Full article

(This article belongs to the Section Antioxidant Enzyme Systems)

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20 pages, 1370 KiB

Open AccessArticle

Impact of ApoE Polymorphism and Physical Activity on Plasma Antioxidant Capability and Erythrocyte Membranes

by Rebecca Piccarducci, Simona Daniele, Jonathan Fusi, Lucia Chico, Filippo Baldacci, Gabriele Siciliano, Ubaldo Bonuccelli, Ferdinando Franzoni and Claudia Martini

Antioxidants 2019, 8(11), 538; https://doi.org/10.3390/antiox8110538 - 9 Nov 2019

Cited by 13 | Viewed by 3639

Abstract

The allele epsilon 4 (ε4) of apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease (AD). ApoE protein plays a pivotal role in the synthesis and metabolism of amyloid beta (Aβ), the major component of the extracellular plaques that constitute [...] Read more.

The allele epsilon 4 (ε4) of apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease (AD). ApoE protein plays a pivotal role in the synthesis and metabolism of amyloid beta (Aβ), the major component of the extracellular plaques that constitute AD pathological hallmarks. Regular exercise is an important preventive/therapeutic tool in aging and AD. Nevertheless, the impact of physical exercise on the well-being of erythrocytes, a good model of oxidative stress and neurodegenerative processes, remains to be investigated, particularly depending on ApoE polymorphism. Herein, we evaluate the oxidative status, Aβ levels, and the membrane’s composition of erythrocytes in a cohort of human subjects. In our hands, the plasma antioxidant capability (AOC), erythrocytes membrane fluidity, and the amount of phosphatidylcholine (PC) were demonstrated to be significantly decreased in the ApoE ε4 genotype and non-active subjects. In contrast, erythrocyte Aβ content and lipid peroxidation increased in ε4 carriers. Regular physical exercise was associated with an increased plasma AOC and membrane fluidity, as well as to a reduced amount of erythrocytes Aβ. Altogether, these data highlight the influence of the ApoE genotype on erythrocytes’ well-being and confirm the positive impact of regular physical exercise. Full article

(This article belongs to the Special Issue Redox Signalling and Exercise)

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Figure 1
Plasma AOC levels, erythrocyte Aβ accumulation, and plasma lipid peroxidation amount in active and non-active subjects depending on ApoE genotype. (a) Plasma AOC depends on ApoE genotype and increases with physical activity. High TOSC values are associated with elevated antioxidant capacity. (b) Aβ accumulation in erythrocytes is influenced by ApoE genotype and decreases with physical activity. (c) Lipid peroxidation is higher in ApoE ε4 carriers and is not modulated by physical activity. The data are presented as the mean value ± S.D. and are representative of three independent experiments (n = 3). Difference among groups were assessed by One-way ANOVA. P-values were adjusted with Sidak’s multiple comparison test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between the indicated subgroups. Full article ">Figure 2
Membrane composition and membrane fluidity in active and non-active subjects depending on ApoE genotype. (a,b) The amount of PC and PE is differently affected by ApoE polymorphism and physical activity. The results are expressed in terms of concentration of PC (μM) and PE (μM). (c) Membrane fluidity of erythrocytes is negatively affected by ApoE ε4 polymorphism, but positively modulated by physical exercise. By determining the ratio of the fluorescence of excimer (Ex/Em = 350/470 nm) to the fluorescence of monomer (Ex/Em = 350/370 nm), quantitative monitoring of membrane fluidity is achieved. Elevated levels of the excimer to monomer fluorescence ratio (Ie/Im) reveal a higher membrane fluidity. The data are presented as the mean value ± S.D. and are representative of three independent experiments (n = 3). Differences among groups were assessed by one-way ANOVA. P-values were adjusted with Sidak’s multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001 between the indicated subgroups. Full article ">Figure 3
Correlation between plasma and erythrocytes well-being parameters. Correlation analysis between erythrocytes Aβ (a) or lipid peroxidation (b) and plasma AOC. (c) Correlation analysis between PE and age. Correlation analysis between plasma AOC (d), Aβ (e), or PE concentration (f) and Borg scale. Correlation between variables was determined by simple linear regression analysis, using the StatView program (Abacus Concepts, Inc., SAS Institute, Cary, NC). P and R2 values obtained for each correlation are reported in the respective panel. Full article ">Figure 4
Multiple linear regression analysis in ε4-non carriers and ε4 carriers. The analysis was used to assess the relationship between erythrocytes Aβ concentration and physical activity level in ε4 carriers and non-ε4 carriers. Full article ">Figure 5
Modulation of the Aβ contents, oxidative status, and membrane’s composition of erythrocytes by ApoE polymorphism and physical activity. The presence of the ApoE ε4 genotype enhances Aβ levels and lipid peroxidation, and decreases plasma AOC, membrane fluidity, and PC amount in erythrocytes. Regular physical activity counteracts the negative effects exhibited by the ε4 genotype on Aβ levels, plasma AOC, and membrane fluidity. Full article ">

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Antioxidants, Volume 8, Issue 11 (November 2019) – 63 articles

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