**3. Results**

#### *3.1. Feijoa Peel and Pulp Extracts Protect Against Hg-Induced Hemolysis.*

The rescue of Hg-induced hemolysis by *Feijoa* extracts was assayed separately for peel and pulp, and the results are shown in Figures 1 and 2, respectively. It can be seen that 24 h treatment of RBC with 40 μM HgCl2 resulted in approximately 13–17% hemolysis, compared to 1–2% in negative controls, as expected based on our previous work [41]. *Feijoa* peel extract potently reduced Hg-induced hemolysis compared to that of the pulp, with a significant 3% drop in hemolysis at 10 μg/mL, and a steady reduction of about 1% with each doubling of peel extract (Figure 1). *Feijoa* pulp treatment reduced cellular lysis in similar proportions, but the required protective extract concentration to do so was almost eight-fold greater than that of the peel extract (Figure 2). No cytotoxic effect was found by either *Feijoa* extracts up to the maximum concentration utilized in this study (data not shown).

**Figure 1.** *Feijoa* peel acetonic extract reduces Hg-induced hemolysis. Cells were treated with 40 μM HgCl2 for 24 h with increasing acetonic extract concentrations. Data are the means ± standard errors of the mean (SEM) (*n* = 9). Statistical significance was calculated by one-way ANOVA followed by Tukey's test. \*\* (*p* < 0.05) indicates a significant difference from cells lacking HgCl2 treatment. # (*p* < 0.05) and ## (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

**Figure 2.** *Feijoa* pulp extract reduces Hg-induced hemolysis. Cells were treated with 40 μM HgCl2 for 24 h in the presence of increasing concentrations of extract. Data are the means ± SEM (*n* = 9). Statistical significance was calculated with one-way ANOVA followed by Tukey's test. \*\* (*p* < 0.05) indicates a significant difference from cells lacking HgCl2 treatment. # (*p* < 0.05) and ## (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

#### *3.2. Feijoa Peel and Pulp Extracts Reduce Reactive Oxygen Species Production in Red Blood Cells*

The fluorescence probe DCF assay elucidated the protective role of *Feijoa* extracts against oxidative stress in RBC, as reported in Figures 3 and 4. ROS production increased nearly two-fold in Hg-treated RBC compared to the negative control. In contrast, co-incubation with 10, 20, 40, or 80 μg/mL of both peel and pulp acetonic extracts incrementally reduced ROS production in RBC. Similar to hemolysis, *Feijoa* peel extract prevented ROS production more potently than the pulp, and remarkably reduced fluorescence levels by approximately 50% at 10 μg/mL, to near control values at the highest extract concentration. At the same concentrations, *Feijoa* pulp extract also significantly reduced ROS production compared to the non-*Feijoa* protected RBC, reaching a maximum of about 50% at 80 μg/mL.

**Figure 3.** *Feijoa* peel extract protects against Hg-induced reactive oxygen species (ROS) production in red blood cells (RBC). Cells were treated with 40 μM HgCl2 for 4 h in the presence of increasing concentrations of extract. ROS production was determined by fluorescence unit means of the dichlofluorescein (DCF) probe. Data are the means ± SEM (*n* = 9). Statistical significance was calculated with one-way ANOVA followed by Tukey's test. \*\* (*p* < 0.05) indicates a significant difference from cells lacking HgCl2 treatment. # (*p* < 0.05) and ## (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

**Figure 4.** *Feijoa* pulp extract protects against Hg-induced ROS production in RBC. Cells were treated with 40 μM HgCl2 for 4 h in the presence of increasing concentrations of extract. ROS production was determined by fluorescence unit means of the DCF probe. Data are the means ± SEM (*n* = 9). Statistical significance was calculated with one-way ANOVA followed by Tukey's test. \*\* (*p* < 0.05) indicates a significant difference from cells lacking HgCl2 treatment. # (*p* < 0.05) and ## (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

#### *3.3. Peel and Pulp Extracts Prevent Hg-Induced Glutathione and Membrane Thiol Depletion in Red Blood Cells*

GSH depletion is a key mechanism of Hg toxicity due to the weakening of the antioxidant defense system. We therefore evaluated the possible protective effect of *Feijoa* peel and pulp extracts on this specific, Hg-induced metabolic condition. As shown in Figure 5, 4 h treatments of RBC with 40 μM HgCl2 reduce GSH levels by about 40%. Co-incubation with 20, 80, or 100 μg/mL of *Feijoa* peel extract prevented GSH depletion by about 20% with each concentration, such that GSH levels were unchanged from healthy control levels at the latter two peel extract concentrations. For the pulp extract, data indicate that 20 μg/mL had no effect on GSH levels, while significant protection was observed at 80 and 100 μg/mL, to a maximum of about 90% GSH levels compared to controls.

**Figure 5.** *Feijoa* peel and pulp extracts protect against Hg-induced glutathione (GSH) decrease in RBC. Cells were treated with 40 μM HgCl2 for 4 h in the presence of increasing concentrations of *Feijoa* peel or pulp. Data are the means ± SEM (*n* = 9). Statistical significance was calculated with one-way ANOVA followed by Tukey's test. \* (*p* < 0.05) and \*\* (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

Based on these results, it seemed appropriate to evaluate the efficacy of peel and pulp extracts in reducing the Hg-induced depletion of membrane thiols, using membranes obtained from intact RBC after incubation with HgCl2 (Figure 6). Exposure to 40 μM HgCl2 reduced the level of membrane thiols by about 40%. This depletion was significantly counteracted by about 45% and 75% given co-incubation with 40 and 80 μg/mL of peel extract, respectively. Again, the pulp extract was less protective than the peel at the same concentrations, such that membrane thiol depletion was counteracted only at 80 μg/mL, by about 50%.

**Figure 6.** *Feijoa* peel and pulp extracts protect against Hg-induced membrane thiol depletion in RBC. Cells were treated with 40 μM HgCl2 for 4 h in the presence of increasing concentrations of *Feijoa* peel or pulp. Data are the means ± SEM (*n* = 9). Statistical significance was calculated with one-way ANOVA followed by Tukey's test. \* (*p* < 0.05) and \*\* (*p* < 0.01) indicate significant differences from cells lacking *Feijoa* extract treatment.

#### *3.4. Peel and Pulp Extracts of Feijoa Reduce Microvesicles Released from Red Blood Cells*

To investigate the protective role of *Feijoa* extracts on erythrocyte morphological changes and MV formation known to be induced by Hg treatment [41,46], cells treated with HgCl2 and peel or pulp extracts, as described in the Materials and Methods section, were analyzed with confocal microscopy. Hg treatment was associated with loss of the typical erythrocyte biconcave shape, as well as the formation of MV clearly discernible on cell membranes (not observable in the control) (Figure 7, Panel A). Cell treatment with *Feijoa* extracts completely restored the typical biconcave shape at 20 μg/mL and 80 μg/mL for peel and pulp, respectively (Figure 7C–F).

**Figure 7.** Peel and pulp extracts of *Feijoa* reduce microvesicles (MV) released from RBC. Untreated cells are shown in (**A**). Cells were treated with 40 μM HgCl2 for 4 h (**B**) and concurrently treated with 20 or 80 μg/mL of *Feijoa* peel (**C** and **D**, respectively) and pulp (**E** and **F**, respectively) extracts. RBC were stained with Annexin V-FITC.
