Development of a Holistic In Vitro Cell-Free Approach to Determine the Redox Bioactivity of Agricultural Products.

In recent years, there has been a strong consumer demand for food products that provide nutritional benefits to human health. Therefore, the assessment of the biological activity is considered as an important parameter for the promotion of high-quality food products. Herein, we introduce a novel methodology comprising a complete set of in vitro cell-free screening techniques for the evaluation of the bioactivity of various food products on the basis of their antioxidant capacity. These assays examine the free radical scavenging activities, the reducing properties, and the protective ability against oxidative damage to biomolecules. The adoption of the proposed battery of antioxidant assays is anticipated to contribute to the holistic characterization of the bioactivity of the food product under examination. Consumer motivations and expectations with respect to nutritious food products with bio-functional properties drive the global food market toward food certification. Therefore, the development and application of scientific methodologies that examine the quality characteristics of food products could increase consumers' trust and promote their beneficial properties for human health.

Determination of Redox Status in Different Tissues of Lambs and Kids and Their in-between Relationship.

The objective of this study was to assess the resting values of the physiological oxidative stress exhibited by lambs and kids reared in Greece, and the potential correlations between redox biomarker levels in blood and other tissues (liver, diaphragm, quadriceps, psoas major muscle). For this purpose, lambs and kids at different developmental stages (d.s.) were used. The latter corresponded to four live weight categories (LWC), each representing 25%, 35%, 70% and 100% of mature body weight. In each of the above tissues, the levels of five common redox biomarkers were determined: glutathione (GSH), catalase (CAT), total antioxidant capacity (TAC), thiobarbituric reactive substances (TBARS), and protein carbonyls (CARBS). The results revealed that lambs and kids belonging to the 35% LWC had weaker endogenous antioxidant pools, while animals in the 70% and 100% LWC had elevated intrinsic antioxidant defense systems. Blood redox biomarkers were associated with the respective ones measured in the diaphragm, liver, quadriceps, and psoas major of both species. Importantly, TBARS levels in blood of animals in the 25% and 100% LWC are correlated with the TBARS levels in all other tissues tested. Blood antioxidant parameters might be used as potential biomarkers to predict the antioxidant status of tissues that affect meat quality. The latter would facilitate quality assessment prior to slaughter, allowing for timely nutritional interventions that can improve meat products.

Redox Status of Pregnant Ewes after Vaccination against Clostridial Diseases.

The redox status shortly after the vaccination of pregnant ewes is rather unexploited. Thus, the present study was designed to evaluate the fluctuation of redox status after the administration of the annual booster dose of a polyvalent clostridial vaccine in pregnant ewes, 3 to 4 weeks before lambing, with or without a simultaneous injection of Vit E/Se. In total, 24 pregnant Lacaune ewes 3-4 weeks before lambing were randomly allocated into four equal groups: the V (vaccinated with a polyvalent clostridial vaccine), VE (vaccinated and injected IM with Vit E/Se), E (injected IM with Vit E and Se), and C (neither vaccinated nor injected with Vit E/Se). The study period lasted for 21 days, starting on the day of administration. Four redox biomarkers, the antioxidant capacity (TAC), the thiobarbituric acid reactive substances (TBARS), the reduced glutathione (GSH), and the catalase (CAT) were evaluated in blood samples collected from all ewes before the injections (0 h) and then at 12 (12 h), 24 (D1), and 48 h (D2), and thereafter on days 4 (D4), 6 (D6), 10 (D10), 14 (D14), and 21 (D21). The results reveal that the TAC was the only biomarker evaluated that was significantly affected by group and significantly lower in vaccinated animals (V and VE groups) compared to non-vaccinated (E and C groups). The absence of an increase in the TBARS values after vaccination in groups V and VE indicates the absence of significant oxidative stress. Overall, it can be assumed that annual booster immunizations against clostridial diseases do not impose acute oxidative stress on pregnant ewes in the last month of pregnancy.

Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status and Immunity of Their Offspring.

In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1ß, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs' blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes' colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs' plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk.

Assessment of Antioxidant and Antimutagenic Properties of Red and White Wine Extracts In Vitro.

Wine is an alcoholic beverage of complex composition obtained through the fermentation of grape must. The consumption of wine has already been associated with a multitude of beneficial effects due to its high polyphenolic content. In this study, four Greek emblematic wines from two red (i.e., Xinomavro and Agiorgitiko) and two white (i.e., Assyrtiko and Malagouzia) varieties were analyzed for the estimation of their antioxidant profiles. To address this question, we assessed their ability to scavenge both synthetic and endogenous free radicals, such as DPPH•, ABTS+•, OH•, O2-, their potential reducing power, and their antimutagenic and antigenotoxic properties. All varieties exhibited potent antioxidant activity, as indicated by the results of methods above, with the red wines appearing more effective than the white ones regarding antioxidant capacity. Our small-scale study is the first to reveal that these wine varieties may have the ability to scavenge the most reactive endogenous radicals. In the future, this finding must be accompanied by larger studies to fill a knowledge gap in the scientific literature concerning a holistic approach of the in vitro antioxidant action of plant polyphenolic compounds. Conclusively, we believe that wines possess high bioactivity that allow them to settle in the industry of food additives and medicinal products.

Redox Biomarker Baseline Levels in Cattle Tissues and Their Relationships with Meat Quality.

Cattle breeds or crossbreds with high productivity traits have been developed to meet a growing demand for food. When intensive farming practices are followed, animals face several challenges which can result in poor performance, compromised welfare and the reduced quality of their products. Our study aims to highlight the resting values of the physiological oxidative stress that three cattle breeds exhibit, and their potential relationship with meat quality. For this purpose, we determined the levels of five common redox biomarkers (glutathione (GSH), catalase (CAT), total antioxidant capacity (TAC), thiobarbituric reactive substances (TBARS) and protein carbonyls (CARBS)) in the tissues of three commonly used beef cattle breeds (Charolais (CHA), Limousin (LIM) and Simmental (SIM)) and their association with specific meat quality traits that depend on color, pH and texture. The results revealed that LIM cattle breed animals have elevated intrinsic antioxidant defense systems in comparison to CHA and SIM cattle breed animals. In addition, the meat quality parameters were associated with the redox biomarkers. We propose that the determination of specific antioxidant parameters in the blood might be used as potential biomarkers to predict meat quality. This would allow farmers to nutritionally intervene to improve the quality of their products.

A novel combined bioactivity / chemoactivity holistic approach for the evaluation of dietary supplements.

There is increasing evidence that the excessive generation of free radicals in the human body plays a major role in the pathophysiology and development of various diseases, closely associated with oxidative damage. In this frame, the consumption of antioxidant nutrients through food or dietary supplements may prevent from the harmful effects of free radicals on human cells. This work proposes a holistic approach consisting of distinct methodologies, suitable to evaluate the antioxidant and chemoprotective activity of three novel dietary supplements, each one containing active substances with complementary properties. In the first step, this approach includes in vitro studies to evaluate the antioxidant activity of the dietary supplements by measuring the parameters of free radical scavenging capacity, of reducing power activity, as well as, their ability to protect biomolecules from oxidation. Furthermore, the evaluation of their antimutagenic and antigenotoxic effects is also presented. SubsequentlySub, the specific effects of the dietary supplements were examined in three cancer cell lines (HepG2, HeLa, MKN45), by measuring redox biomarkers such as glutathione, reactive oxygen species and thiobarbituric acid reactive substances, using flow cytometry and spectrophotometry. Our results indicate that all the dietary supplements exhibit high antioxidant, antimutagenic, antigenotoxic and lipid protective activity. The most prominent result is their capability to induce oxidative damage on cancer cells via the critical decrease of the levels of their intracellular glutathione, as well as the increase of ROS and lipid peroxidation levels after the administration of non-cytotoxic concentrations. We suggest that the proposed methodology could constitute a valuable tool for the characterization of dietary supplements based on their chemical and functional properties.

Interplay between oxidative damage, the redox status, and metabolic biomarkers during long-term fasting.

Obesity and its related metabolic disorders, as well as infectious diseases like covid-19, are important health risks nowadays. It was recently documented that long-term fasting improves metabolic health and enhanced the total antioxidant capacity. The present study investigated the influence of a 10-day fasting on markers of the redox status in 109 subjects. Reducing power, 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation(ABTS) radical scavenging capacity, and hydroxyl radical scavenging capacity increased significantly, and indicated an increase of circulating antioxidant levels. No differences were detected in superoxide scavenging capacity, protein carbonyls, and superoxide dismutase when measured at baseline and after 10 days of fasting. These findings were concomitant to a decrease in blood glucose, insulin, glycated hemoglobin (HbA1c), total cholesterol, low-density lipoprotein (LDL) and triglycerides as well as an increase in total cholesterol/high-density lipoprotein (HDL) ratio. In addition, the well-being index as well as the subjective energy levels increased, documenting a good tolerability. There was an interplay between redox and metabolic parameters since lipid peroxidation baseline levels (thiobarbituric acid reactive substances [TBARS]) affected the ability of long-term fasting to normalize lipid levels. A machine learning model showed that a combination of antioxidant parameters measured at baseline predicted the efficiency of the fasting regimen to decrease LDL levels. In conclusion, it was demonstrated that long-term fasting enhanced the endogenous production of antioxidant molecules, that act protectively against free radicals, and in parallel improved the metabolic health status. Our results suggest that the outcome of long-term fasting strategies could be depending on the baseline values of the antioxidative and metabolic status of subjects.

Biofunctional Feed Supplemented With By-products of Olive Oil Production Improves Tissue Antioxidant Profile of Lambs.

BACKGROUND/AIM: Olive mill wastewater (OMW) is a byproduct of olive oil production. The aim of the study was to estimate the redox profile of lambs' vital organs after consumption of an OMW-supplemented feed. MATERIALS AND METHODS: Twenty-four lambs received breast milk until day 15. Then, they were divided in two groups: control and OMW, n=12 each. The control group received standard ration, while the OMW group received OMW enriched feed along with mother's milk until day 42 and animals (n=6 per group) were sacrificed. The remaining 12 received the feeds until day 70 and sacrificed. Tissue samples were collected at day 42 and 70 and specific redox biomarkers were assessed. RESULTS: Overall, the OMW feed improved tissue redox profile by affecting the glutathione S-transferase (GST) activity and γ-glutamate-cysteine ligase (γ-GCL) expression in all tested tissues. Superoxide dismutase (SOD) activity was not affected. CONCLUSION: The polyphenol-rich byproduct reinforced lamb redox profile and may putatively improve their wellness and productivity.

Effects of Melatonin Administration to Pregnant Ewes under Heat-Stress Conditions, in Redox Status and Reproductive Outcome.

Heat stress is a known promoter of reactive oxygen species generation, which may compromise pregnancy and foetal development. Melatonin is a pleiotropic molecule that regulates various processes including pregnancy. Thus, it could be used to ameliorate the redox status of pregnant heat-stressed ewes and the outcome of their pregnancy. Sixty-eight ewes participated in the study, which were allocated into two equal groups, i.e., Melatonin (M) and Control (C) group. All ewes were exposed to heat stress from D0 to D120. In both groups, after oestrus synchronization of ewes, rams were introduced to them for mating (D16). In M group, starting with sponges' insertion (D0), melatonin implants were administered four-fold every 40 days. Pregnancy diagnosis was performed by means of ultrasonography. Daily evaluation of temperature humidity index (THI), rectal temperature, and breathing rate were performed throughout the study. Blood samples were collected repeatedly from D0 until weaning for assaying redox biomarkers. Milk yield was measured thrice during puerperium. The results showed that melatonin administration throughout pregnancy improved the redox status of heat-stressed ewes and increased the mean number and bodyweight of lambs born per ewe, as well as the milk production. Therefore, melatonin may be used as antioxidant regimen in heat-stressed ewes for improving their reproductive traits.

Effects of Dietary Grape Pomace Supplementation on Performance, Carcass Traits and Meat Quality of Lambs.

BACKGROUND/AIM: A previous study revealed that the inclusion of grape pomace (GP) in the diet for growing lambs had beneficial effects on the redox status and fecal microbiota. Herein, we investigated the effect of GP inclusion on performance, carcass traits and fatty acid composition of meat. MATERIALS AND METHODS: In the experimental trial of 55 days, lambs were fed with standard or diet supplemented with GP. Performance, carcass traits and fatty acid profile of quadriceps muscle were assessed. RESULTS: GP inclusion in the diet improved growth performance, since the average daily gain was significantly increased by 2-fold in GP group. Regarding the fatty acid composition of meat, GP inclusion significantly increased the content of long chain n-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, and reduced the n-6/n-3 ratio compared to the control group. CONCLUSION: GP supplementation in lamb diet may improve performance and may have beneficial effects on meat quality.

Novel Feed Including Olive Oil Mill Wastewater Bioactive Compounds Enhanced the Redox Status of Lambs.

BACKGROUND/AIM: The aim of the present study was to investigate the antioxidant effects of a feed supplemented with polyphenolic additives from olive mill wastewater (OMW) on lambs. MATERIALS AND METHODS: Lambs received breast milk until the postnatal period, and then they were divided into two groups and received control and OMW feed for 55 days. Redox biomarkers were measured in blood and tissues at days 15, 42 and 70 after feeding. RESULTS: Feed supplemented with OMW reduced thiobarbituric acid reactive species and protein carbonyls and increased total antioxidant capacity, glutathione and catalase activity in both blood and tissues. CONCLUSION: The administration of OMW-containing feed reinforced the antioxidant defense of lambs, which may improve their wellbeing and productivity. Additionally, this exploitation of OMW may solve problems of environmental pollution in areas with olive oil industries.

Enhancement of Antioxidant Mechanisms and Reduction of Oxidative Stress in Chickens after the Administration of Drinking Water Enriched with Polyphenolic Powder from Olive Mill Waste Waters.

The aim of the study was to examine the effects of a polyphenolic powder from olive mill wastewater (OMWW) administered through drinking water, on chickens' redox status. Thus, 75 chickens were divided into three groups. Group A was given just drinking water, while groups B and C were given drinking water containing 20 and 50 µg/ml of polyphenols, respectively, for 45 days. The antioxidant effects of the polyphenolic powder were assessed by measuring oxidative stress biomarkers in blood after 25 and 45 days of treatment. These markers were total antioxidant capacity (TAC), protein carbonyls (CARB), thiobarbituric acid reactive species (TBARS) and superoxide dismutase activity (SOD) in plasma, and glutathione (GSH) and catalase activity in erythrocytes. The results showed that CARB and TBARS were decreased significantly in groups B and C, and SOD decreased in group B compared to that in group A. TAC was increased significantly in group C and GSH was increased in group B, while catalase activity was increased in groups B and C compared to that in group A. In conclusion, this is the first study showing that supplementation of chickens with polyphenols from OMWW through drinking water enhanced their antioxidant mechanisms and reduced oxidative stress-induced damage.

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

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

Novel feed including bioactive compounds from winery wastes improved broilers' redox status in blood and tissues of vital organs.

Currently, there is a great interest in the production of animal feed with antioxidant activity. The aim of this study was to examine the potential antioxidant effects of a feed supplemented with grape pomace (GP), a winery by-product with high environmental load, in chickens. Broilers of 15 days post birth were separated into two groups fed either with standard diet or with diet supplemented with GP for 35 days. Blood and tissues collections were performed after feeding for 15 and 35 days with the experimental diet (i.e. at 30 and 50 days post birth). Free radical toxicity markers, namely thiobarbituric acid reactive substances, protein carbonyls, total antioxidant capacity, reduced glutathione, catalase activity and rate of H2O2 decomposition were determined in blood and tissues of vital organs. The results indicated that feed supplemented with GP decreased oxidative stress-induced toxic effects and improved chickens' redox status, and so it may also improve their wellness and productivity. On the other hand, this exploitation of GP may solve problems of environmental pollution in areas with wineries.