*4.1. Effects of Dietary Interventions on Direct ROS Generation*

To date, electron paramagnetic resonance (EPR) technology is the only method that can directly detect ROS generation in in vivo conditions [57]. Short-lived ROS can be added to the spin trap to form a spin-adduct that has a comparatively longer half-life to be detected using EPR spectroscopy [58]. Zeng et al. [42] revealed that consumption of AVA-rich oatmeal before high-intensity interval training (HIIT) significantly mitigates exercise-induced ROS generation compared to the control group, by using the EPR method. AVA, as one of the major components of polyphenolic amides (nonflavonoids), is considered the most important antioxidant found in oats [59,60]. Therefore, it can be speculated that the hydroxyl groups of AVA contribute to antioxidant defense through their ability to trap ROS in vitro [61]. Another assumption is that AVA can activate the nuclear factor erythroid 2-related factor 2 (NRF2) defense system against oxidative stress by attacking the α, β-unsaturated carbonyl moiety [62]. However, the underlying mechanisms for these effects are still unclear.

Indeed, only the study of Zeng et al., [42] applied direct ROS measurements using EPR technology, whereas the other experiments in this review used oxidative stress markers, inflammatory markers and antioxidant activity levels to interpret the changes in ROS production, as will be discussed below.

#### *4.2. Effects of Dietary Interventions on ROS-Induced Macromolecule Damage*

In the majority of studies, F2-isoprostanes, 8-isoprostanes, lipid hydroperoxides (LH), thiobarbituric acid-reactive substances (TBARS) and malondialdehydes (MDA) were used as the oxidative markers, which result from lipoperoxidation by oxidative damage. Similarly, protein carbonylation (PC) was used as a marker of protein damage, and 8- Hydroxydeoxyguanosine (8-oxodG) as a specific marker of 2- -deoxyguanosine damage after ROS attack to DNA. In this narrative review, *n* = 14 articles ([29–32,37,40,46–53]) showed the antioxidant effects diets on oxidative stress markers.

Davison et al. [29], Wiswedel et al., [32] and Allgrove et al., [30] observed the beneficial antioxidant effects of dark chocolate by detecting the plasma levels of F2-isoprostane, while Davison et al., [29] and Wiswedel et al., [32] confirmed the acute antioxidant effects of dark chocolate due to its polyphenolic properties, Allgrove et al., [30] and Taub et al., [31] showed that these beneficial effects can also be seen following long-term dietary interventions. The derivatives of catechin and epicatechin, which can both be defined as monomeric flavanols, are the major antioxidant components in cacao beans (chocolate) [63]. The acute antioxidant effects of flavanols in cocoa were evaluated by Davison et al., [29] who investigated the association between the increased plasma epicatechin levels and F2-isoprostanes and found decreased oxidative stress markers in a group given dark chocolate compared to a control group. In this study, after consuming 100 g dark chocolate or an isomacronutrient control bar, each healthy male subject cycled for 2.5 h at ~60% maximal oxygen uptake. Blood samples were analyzed at pre-exercise and immediately postexercise. Plasma F2 isoprostane, also showed a decline after ingestion of a high-flavanol cocoa drink combined with strenuous cycling exercise in the study of Wiswedel et al., [32].

Despite the acute antioxidant effects, Allgrove et al., [30] found that consuming 40 g of dark chocolate twice daily for two weeks significantly decreased plasma F2-isoprostane levels at exhaustion and after one hour of recovery in a prolonged exercise trial.

Differently, the study of Taub et al., [31] explored the mechanisms underlying the long-term antioxidant effects of dark chocolate by examining human muscle samples (quadriceps femoris). After consuming 20 g of dark chocolate or placebo for three months, the VO2 max and total work of each sedentary subject was assessed on a stationary bicycle. After exercise, the skeletal muscle evidenced significant decreases in PC and increased glutathione (GSH) levels only in the dark chocolate group [31]. Furthermore, the protein levels of liver kinase B1(LKB1), adenosine monophosphate (AMP)-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), and their active forms (phosphorylated AMPK and LKB1), along with citrated synthase

(CS) activity, were found significantly elevated [31]. Accordingly, the dark chocolate might activate upstream control systems and improve mitochondria performance in skeletal muscle, contributing to the improvements in maximum work achieved and VO2 max.

It has to be mentioned that the studies investigating the effects of cocoa had heterogeneous designs, which prevents us from making definite conclusions. Davison et al., [29] used 100 g dark chocolate (39.1 mg catechin, 96.8 mg epicatechin, 58.4 mg Dimer B2, 7.3 mg Dimer B5, 34.7 mg Trimer C and 10.5 mg tetramer D); Wiswedel et al., [32] chose a highflavanol cocoa drink (187 mg flavan-3-ols/100 mL); Allgrove et al. [30] applied the 40 g 70% chocolate; Taub et al., [31] provided the dark chocolate at total of 20 g and ~100 calories per day.

In addition to cocoa, other phenol-rich fruits also exhibited antioxidant effects during exercise by detecting oxidative stress markers, including blueberry [37], cherry [40] and red orange [46]. In the study by McAnulty et al., [37], participants consumed 150g of blueberries in a milkshake every day for one week prior to one session of high-intensity training in hyperthermic conditions. The results showed that the blueberry diet attenuated an increase in LH concentration caused by exercise stress but not F2-isoprostane levels, compared with a blueberry-flavored shake as a placebo [37]. Montmorency cherry juice (30 mL twice per day) was provided for one week before and 48 h after a bout of strength exercise in the study of Bowtell et al., [40]. The recovery of isometric muscle strength after high-intensity exercise was improved. PC were lower in the Montmorency cherry juice group compared with the isoenergetic fruit concentrate (placebo) group. Red oranges are a cultivar of the Citrus sinensis family which are generally rich in vitamin C, anthocyanins, and flavanones. The Sanguinello cultivar red orange juice (ROJ) was provided as the intervention diet in a study by Pittaluga et al., [46] due to the remarkable antioxidant ability of anthocyanin family, such as cyanidine-3-*O*-β-glucoside (C3G). In this elderly human trial, the intervention group (250 mL ROJ thrice a day for 4 weeks) had lower exhausted exercise-induced MDA, lower hypoxanthine/xanthine system activation, and less ascorbic acid consumed.

Purple sweet potato leaves (PSPL), as another phenol-rich diet, showed decreases in oxidative stress markers in an exercise trial [47]. Chang et al., [47] investigated the effects of a 7-day PSPL-diet on running exercise-induced oxidative stress in a nontrained, young male population. PSPL consumption significantly increased total polyphenols concentrations, and significantly decreased plasma PC and TBARS in the PSPL group [47]. PSPLs, botanically identified as Ipomoea batatas (L.) Lam, have the highest levels of polyphenols and flavonoids (33.4 ± 0.5 mg gallic acid/g and 426.8 ± 8.9 μg/g dry wt) [64]. This research group also proved that the PSPL diet could modulate antioxidative status [65] and immune responses [66] in basketball players during a training period.

Besides phenol-rich foods, probiotic-rich dairy also showed promising antioxidant effects on the levels of oxidative stress markers. Mazani et al., [48] described the antioxidant effect of 450 g of probiotic yogurt taken daily for two weeks by young females. Compared with regular yogurt, after intense physical activity, probiotic yogurt consumption significantly decreased serum levels of MDA, and some inflammatory factors (tumor necrosis factor-α (TNF-α), matrix metalloproteinase 2 (MMP2), matrix metallopeptidase 9 (MMP9)), and increased the levels of superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (TAC). This result might be explained by the previous assumptions that some strains of probiotics positively prevent and correct oxidative stress in humans due to their direct antioxidative activity and positive effect on the immune system [67,68].

Lycopene is a carotenoid that is mainly found in tomatoes [69]. Among dietary carotenoids, lycopene is one of the most active antioxidants with a singlet-oxygen-quenching capacity twice as high as that of β-carotene and ten times greater than that of α-tocopherol [70]. However, the underlying mechanism of how it resists oxidative stress in vivo is still unclear. Two major potential hypotheses to explain the antioxidant abilities of lycopene are oxidative and nonoxidative mechanisms [69]. In a human trial by Harms-Ringdahl et al., [49], a daily

intake of tomato juice, equal to 15 mg lycopene per day for five weeks significantly reduced the serum levels of 8-oxodG after extensive physical exercise.

A few dietary strategies that have been described as mixed foods have shown antioxidant effects on markers of oxidative stress. To investigate the effects of mixed antioxidant foods on resistance training-induced oxidative stress, a diet containing salmon flakes, green and yellow vegetable juice, and lingonberry jam, which contain astaxanthin, β-carotene, and resveratrol was provided by Kawamura et al., [50]. This mixed diet was consumed by the intervention group twice a week for 10 weeks. The results showed that serum PC levels tended to be lower immediately after exercise than before exercise in the intervention group only. The mixture of these nutrients might collectively enhance the antioxidant effects in this trial.

However, the combined antioxidant mechanisms of mixed foods are complex, and previous studies have shown inconsistent results for some mixed diets. Sureda et al., [51] demonstrated that a mixed beverage with vitamin C and E reduced the plasma oxidative damage induced by a half-marathon. After this, Carrera-Quintanar et al., [71] proved that ingestion of a mixed beverage that included an excess of polyphenolic antioxidants (*Lippia citriodora*) for 21 days could interfere with antioxidant activities and reduce the gene expression of specific enzymes (e.g., Cu-Zn-SOD, Mn-SOD and glutathione reductase (GRD)) in neutrophils, in a human study without exercise intervention. Recently, Carrera-Quintanar et al., [52] compared the antioxidant effects of two mixed beverages and one control beverage on exercise-induced oxidative damage: a mixed beverage enriched with vitamins C and E; the same beverage with extra *Lippia citriodora* extract; and the control beverage. This study was performed in a 2000-m running exercise trial. However, the results showed that all the oxidative stress markers increased in the control group, plasma PC significantly increased only in the mixed beverage with *Lippia citriodora*, and no significant changes in oxidative stress levels were detected for the mixed beverage which only added vitamins C and E. Accordingly, further studies are needed to explore the mechanisms by which certain antioxidants in the *Lippia citriodora* extract were less effective at combating oxidative stress than their components in isolation.

In contrast, one study did not find positive effects by testing oxidative stress markers [53]. In a four-month study of 216 women, a twice-daily multi-nutrient-fortified milk drink (MFMD), containing added protein, milk fat globule membrane (phospholipids and other bioactives), vitamin D, calcium, and other micronutrients, did not enhance the effects of an exercise program on markers of oxidative stress (marker: 8-isoprostane, PC) and the primary outcome measure of stair climbing ascent power [53]. However, the MFMD did elicit greater improvements in various secondary outcomes of physical functions compared to an energy-matched placebo [53].
