*2.4. Hemolysis Assay*

RBC hemolysis extent was determined spectrophotometrically, according to Tagliafierro et al. [41]. After simultaneous treatment with HgCl2 and *Feijoa* extracts for 24 h, the reaction mixture was centrifuged at 1100× *g* for 5 min, and the released hemoglobin (Hb) in the supernatant was evaluated by measuring the absorption at 540 nm ( *A*). As a positive control, packed RBC were used hemolyzed with ice-cold distilled water at 40:1 *<sup>v</sup>*/*<sup>v</sup>*, and by measuring the A540 of the supernatant obtained centrifuging the suspension at 1500× *g* for 10 min (*B*). The percentage of hemolysis was calculated as the ratio of the readings ( *A*/*B*)×100%.

#### *2.5. Determination of Reactive Oxygen Species*

ROS generation was determined using the dichlofluorescein (DCF) assay, according to Tagliafierro et al. [41]. Using this method, 250 μL of intact RBC (hematocrit 10%) were incubated with the non-polar, non-fluorescent 2-,7--dichlorodihydrofluorescin diacetate (DCFH-DA) at a final concentration of 10 μM for 15 min at 37 ◦C. After centrifuging at room temperature at 1200× *g* for 5 min, the supernatant was removed, and the hematocrit was re-adjusted to 10% with bu ffer A. RBC were then treated concurrently with HgCl2 and *Feijoa* extracts in the dark for 4 h. After the incubation, 20 μL of RBC were diluted in 2 mL of water, and the fluorescence intensity of the oxidized derivative DCF was recorded (λexc502; λem520). The results were expressed as fluorescence intensity/mg of Hb.

#### *2.6. Quantification of Intracellular Glutathione*

Intracellular GSH content was determined spectrophotometrically by reacting with DTNB reagent, according to Van den Berg et al. [47]. After co-incubation with HgCl2 and *Feijoa* extracts for 4 h, the samples (0.25 mL) were centrifuged, and the cells were lysed by the addition of 0.6 mL of ice-cold water. Proteins were precipitated with 0.6 mL ice-cold metaphosphoric acid solution (1.67 g metaphosphoric acid, 0.2 g EDTA, and 30 g NaCl in 100 mL of water). After incubation at 4 ◦C for 5 min, the protein precipitate was removed by centrifugation at 18,000× *g* for 10 min, and 0.45 mL of the supernatant was mixed with an equal volume of 0.3 M Na2HPO4. Then, 100 μL of DTNB solution (20 mg DTNB plus 1% of sodium citrate in 100 mL of water) was then added to the sample, and after a 10 min incubation at room temperature, the absorbance of the sample was read against the blank at 412 nm.

#### *2.7. Estimation of Free Sulfhydryl Groups in Isolated Red Blood Cell Membranes*

Free sulfhydryl groups in membrane proteins (2.5 μg/μ<sup>L</sup> for each sample estimated by Bradford assay) were assayed, according to the method of Ellman [48]. To do this, 650 μL of HgCl2-treated RBC were washed three times in 40 volumes of 5 mM sodium phosphate buffer pH 8.8, centrifuged at 10,000× *g* for 20 min at 4 ◦C, then washed several times with the same buffer, for the complete removal of Hb. Then, 1 mL of 0.1 M Tris–HCl pH 7.5 was added to 50 μL membrane protein. The colorimetric reaction was started by adding 50 μL of 10 mM DTNB in methanol. After 15 min of incubation at room temperature, the absorbance was read against the blank at 412 nm. Blanks were run for each sample in which DTNB was not added to methanol.

#### *2.8. Morphological Analysis of Red Blood Cells*

To investigate the possible protective effect of *Feijoa* extracts on alterations in the Hg-induced erythrocytes' shape, we treated the cells both with 40 μM HgCl2 as well as 20 or 80 μg/mL of *Feijoa* peel and pulp extracts for 4h at 37 ◦C. After incubation, erythrocytes were washed twice with phosphate-buffered saline pH 7.4 (PBS), and counted in a Burker chamber. The confocal laser scanning microscope analyses were performed according to Nguyen [49], with few modifications. In brief, the cells were then fixed with 2% formaldehyde for 1 h at 4◦C, then washed several times and incubated with anti-human anti glycophorin A FITC antibody for 30 min at 4◦C in the dark. Afterwards, the samples were placed on glass slides and air-dried for 1 h. The slides were dipped quickly, and gently washed stepwise with ethanol from 50% to 75%, 90%, and then 100% for dehydration. Finally, cells were fixed in 2% formaldehyde and washed three times with PBS. For confocal laser scanning microscope imaging, several randomly selected frames from each sample were captured for morphological observation and statistical strength. Excitation and emission filters were set at 488 nm and 550–600 nm, respectively.
