**2. Study on powerful antioxidants**

#### **2.1 Study on resveratrol**

The reducing agent treatment efficacy was observed by evaluation of anion permeability for chloride, lipid peroxidation and hemolysis in RBCs. Anion permeability for chloride is an indicator of membrane protein damage and is evaluated in RBCs by the specific absorption of methemoglobin (CM) at 590 and 635 nm after treatment of heparinized blood with NaNO2. The measurement of the membrane lipid degradation is obtained by the determination of MDA. The lipid peroxidation susceptibility is observed after the oxidative stress induced by AAPH. The hemolysis assays are conducted on blood samples in phosphate buffer saline. To evaluate in human RBC the *in vitro* effects of AAPH and the antioxidant activity of polyphenols from red wine (resveratrol, catechin and naturally red wine), authors start the research evaluating RBC anion permeability for chloride. **Figure 1** shows the values of membrane anion permeability for chloride in both groups (controls and AAPH-treated RBCs). The statistical analysis of data shows that AAPH *in vitro* treatment lowers the parameter in comparison to all controls. Furthermore, the reducing agent treatment (T2-T4) also significantly recovers the activity up to values higher than AAPH treatment. The recovery of anion permeability for chloride is always lower than control data. Anion permeability for chloride values were slightly higher after red wine and resveratrol treatments (as reducing agents) in both experimental groups (controls and AAPHtreated RBCs), corresponding to a preserved architecture and anion permeability for chloride of the human erythrocyte membranes. To confirm the efficacy of oxidant and reducing treatments on human RBC *in vitro* authors evaluated lipid peroxidation, hemolysis and RBC morphology. In **Figure 2**, MDA levels are described in membranes from several varieties of treated and untreated RBC with AAPH, under the action of reducing agents or without treatment. On the contrary, as described in **Figure 2**, AAPH-induced increases of MDA levels are partially recovered by natural reducing agents at levels almost comparable to control the group. C2-C4 results always slow down MDA levels in RBC membranes, but catechin is less effective. Red wine, among them, is almost as powerful as resveratrol alone in both groups (controls and AAPHtreated RBCs). As shown in **Figure 3** time courses of RBC hemolyses are described at several incubation times (60, 120, 180 and 240 min) either under the action of natural oxidative pathways or by treatment with radical donor AAPH (60 mM). This experiment shows also the natural reducing agents partially recover the effect of both oxidative and reducing agent treatments. As described in all experiments, AAPH-

#### **Figure 1.**

*RBC membrane anion permeability for chloride. Data are mean* � *standard error of the mean on 10 determinations. \*P < 0.05 according to one-way analysis of variance and Bonferroni* post-hoc *test coupled data at each time were performed. C = control sample,T = treated sample [8] C1 = control; C2 = control + red wine 5, 2 mM (reducing power equivalents of gallic acid); C3 = control + resveratrol 5 μM; C4 = control + Catechin 50 μM; T1 = AAPH 60 mM; T2 = AAPH 60 mM + red wine 5,2 mM (reducing power equivalents of gallic acid); T3 = AAPH 60 mM + resveratrol 5 μM; T4 = AAPH 60 mM+ Catechin 50 μM.*

#### **Figure 2.**

*Effects of lipid peroxidation and induced by 2.2*<sup>0</sup> *-azobis-2 amidinopropan dihydrochloride (60 mM) (*t *= 60 min) on the malonyldialdehyde levels of human RBC membranes. Data are mean* � *standard error of the mean on 10 determinations. Column labels are according to \**P *< 0.05 and \*\**P *< 0.01 according to one-way analysis of variance and Bonferroni* post-hoc *test for coupled data at each time were performed (Figure 1).*

*Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

#### **Figure 3.**

*Effect of 2.2*<sup>0</sup> *-azobis-2 amidinopropan dihydrochloride-induced hemolysis in sportive human RBCs at several times. Data are mean* � *standard error of the mean on 10 determinations. Column labels are according to incubation times a = (60 min); b = (120 min); c = (180 min); d = (240 min). \**P *< 0.05, \*\** P *< 0.01 and \*\*\** P *< 0,001 according to according to one-way analysis of variance and Bonferroni* post-hoc *test for coupled data at each time were performed (Figure 1). C = Control sample,T = Treated sample (Gallo et al., 2013) C1 = Control; C2 = Control + Red Wine 5, 2 mM (reducing power equivalents of gallic acid); C3 = Control +Resveratrol 5 μM; C4 = Control + Catechin 50 μM; T1 = AAPH 60 mM; T2 = AAPH 60 mM + Red Wine 5,2 mM (reducing power equivalents of gallic acid); T3 = AAPH 60 mM + Resveratrol 5 μM; T4 = AAPH 60 mM+ Catechin 50 μM. Of course C1 and T1 are a = (60 min). And so on, saying the others.*

induced hemolysis is more relevant than in other treated samples. As described in previous experiments (**Figures 1** and **2**) resveratrol and red wine polyphenol treatments are more efficient than catechin in preserving RBC membrane structural recovery at 60 and 120 min of incubation. In the present study, polyphenol (resveratrol and red wine) treatment is associated with an improvement in erythrocyte anion permeability for chloride-related with cell membrane derangements. The cell system exchange of Cl� for HCO3 � represents a target of membrane oxidative damage and is evaluated in RBCs by the specific CM at 590 and 635 nm after treatment of heparinized blood with NaNO2. According to our research, the in vitro action model of artificial agent AAPH on RBC plasma membranes shows that anion permeability for chloride (evaluated by CM [9]) decreases after treatment and is partially recovered after administration of either resveratrol or red wine polyphenols extracts. Our data confirm those on LDL peroxidation [10] with AAPH by MDA, hemolysis and RBC morphology study from our laboratory [8, 11]. Moreover in the present work all previous results are compared, so that a relationship among them is evident and correlate each other, so that the action of red wine polyphenol extracts are demonstrated as effective reducing agents both alone and in mixture. MDA, hemolysis and RBC morphological study confirmed the resveratrol and red wine efficacy on improvement of RBC membrane integrity against in vitro oxidative stress damages. The described damages could be linked to the erythrocyte membrane framework, so a lack of adequate lipid organization can significantly influence ion permeability. The alteration of membrane fluidity consequent to lipid oxidation represented by MDA level increase can reflect the change of the structure and function of membrane lipids and proteins. The permeability is another important property of erythrocyte membrane. Depending on the exchanger (Cl� \$ HCO3 �), that transports anions across the membrane, it is related to the framework of erythrocyte membrane. To sum up, the alteration of anion permeability can reflect the change of erythrocyte membrane protein framework. Moreover, our results could describe the overall effect of the three described alterations together.
