**4. Discussion**

Mercury is not only highly toxic, but is an increasingly pervasive dietary heavy metal. As a matter of fact, concerns on the effect of Hg exposure on human health are not only limited to occupationally exposed workers, but also to the general population, mainly via contaminated food ingestion. Although in some European populations the overall Hg daily intake is below the tolerable amount [50,51], appreciable proportions of large fish populations are reported to contain levels of this heavy metal exceeding this amount, up to 2.22 mg/kg wet weight, including anglerfish (*Lophius piscatorius*) and black-bellied angler (*Lophius budegassa*) [52]. Discovering analogous means to simultaneously combat and protect against diet-based Hg toxicity is therefore crucial to public health. In this respect, phytochemicals able to counteract structural and metabolic alterations associated with heavy metal exposure are attractive for the reduction of their toxicity [53–56]. Data from our group indicate that hydroxytyrosol, an olive oil-derived phenolic antioxidant, has the potential to modulate the toxic e ffects exerted by Hg in human RBC [41–43].

To expand data on the potential role of nutrition in heavy metal toxicity, intact human RBC were exposed to 40 μM HgCl2, in line with our previous studies. Several markers of cellular toxicity were then evaluated to test the protective e ffect of *Feijoa* fruit extracts. According to data reported in similar experimental conditions, RBC treatment with 40 μM HgCl2 for 4 h results in a doubling of ROS production, as indicated by DCF fluorescence [41]. Hg-induced ROS generation follows a significant decrease of GSH, which builds up a pro-oxidative microenvironment and renders cells more susceptible to ROS-mediated oxidative damage. A significant decrease in membrane thiols is also detectable in Hg-treated cells. The resulting hemolysis is significantly increased and measurable later, at 24 h.

Here we show the first evidence of *Feijoa* fruit extract protection against HgCl2-induced toxic effects in human RBC. The acetonic extracts of both the pulp and peel were able to counteract oxidative stress and cellular thiol decrease in Hg-treated RBC. The peel extract had a greater protective e ffect compared to the pulp, although to varying extents for the di fferent markers analyzed, which is at least partially due to the greater proportion and diversity of polyphenols in the peel [3,57,58]. Interestingly, the protective e ffect of the peel from ROS production is only two-fold, compared to an eight-fold e ffect against overall cytotoxicity indicated by hemolysis. Whereas Hg sequesters and inactivates GSH by binding to sulfhydryl groups, polyphenols act on the resulting ROS by virtue of their hydrogen and electron transfer abilities. The presence of additional bioactive compounds with di fferent activities in the peel (i.e., chelating properties) can also be hypothesized. Remarkably, as little as 10 μg/mL of *Feijoa* peel extract significantly a ffects all the tested markers, and 80 μg/mL completely prevents Hg-induced ROS production.

The data presented in this paper, although obtained from in vitro studies on human cells, also o ffer significant experimental evidence that *Feijoa* extracts prevent Hg-induced RBC shape alteration in RBC, which could be taken into account for future clinical investigations. In fact, although a particularly high GSH concentration may partially protect RBC from Hg's toxic e ffects, chronic exposure could a ffect RBC viability and induce morphological changes, also a ffecting cardiovascular disease. As mentioned before, Hg exposure enhances pro-coagulant activity of these cells, resulting in a contributing factor for Hg-related thrombotic disease [46]. In our previous studies, we raised the fascinating hypothesis that metabolic and shape modification of RBC may be regarded as a clinical biomarker, indicating increased cardiovascular risk in Hg-exposed individuals [41,42].

Our findings, in agreemen<sup>t</sup> with the literature data, strengthen the nutritional relevance of *Feijoa* bioactive compounds to the claimed health-promoting e ffects of this fruit. There is growing interest in utilizing *Feijoa* fruit for human consumption, due to its appetizing quality and its claimed health benefits. *Feijoa* fruit is an excellent source of vitamins and nonessential nutrients, as well as a variety of bioactive compounds endowed with significant antioxidant, antibacterial, and anti-inflammatory activities [10–13]. In this respect, there is a general agreemen<sup>t</sup> that the health-promoting e ffects of fruit and vegetable intake result from the combined properties and synergistic action of all bioactive constituents, including polyphenols [59,60]. These compounds can improve health due to their strong antioxidant activity, counteracting oxidative stress-induced cellular dysfunctions and modulating key mechanisms implicated in the development of oxidative stress-related human pathologies. Polyphenols are very useful in combating the deleterious e ffects of heavy metals. For example, Sobeh et al. [61] isolated and identified two compounds from the leaves of *Syzygium*

*samarangense* (myricitrin and 3,5-di-O-methyl gossypetin), both showing antioxidant activities [62,63] and strongly reducing intracellular ROS accumulation and carbonyl content, while also protecting the intercellular GSH levels in keratinocytes (HaCaT) after exposure to sodium arsenite, one of the more toxic environmental heavy metals [61].

*Feijoa* has been proposed as an ideal candidate for nutraceutical strategies in the development of functional foods [64]. The data reported in this paper expands upon the known beneficial effects of *Feijoa* fruit, particularly related to chronic human exposure to heavy metal. In this respect, an interesting observation is that the very low active concentrations utilized in our study could be approached in vivo upon daily intake of *Feijoa* fruit. In this respect, some studies indicate that *Feijoa* fruit extracts are well tolerated in animal models. Karami et al. [65] demonstrated the hepatoprotective activity of methanolic extract of *Feijoa* fruit in a concentration range of 10–100 mg/kg, using the isolated rat liver perfusion system. The same group also investigated nephroprotective effects of leaf extracts (10–40 mg/kg) on renal injury induced by acute doses of ecstasy (MDMA) in mice [66]. Moreover, in a recent study, *Feijoa* leaf extract was shown to be devoid of toxicity in rats up to 2 g/Kg, [11]. Finally, we have confirmed by MTT test, on human leucocytes as well (data not shown), that treatment for 24 h with 5, 50, and 500 μg/mL of acetonic extracts of *F. sellowiana* did not induce significant cytotoxic effects, as already demonstrated on either the Caco-2 or HT-29 cell lines [16].

The food industry is increasingly interested in the utilization of non-edible parts of fruits. Phytochemicals are proposed for designing foods with added functional value, aiming to beneficially affect target functions in the body and reduce the risk of diseases. These compounds are present in large quantities in waste products from the agri-food supply chain, especially peels and seeds. Our data, showing a greater protective effect from the *Feijoa* peel on Hg cytotoxicity than from the pulp, corroborates this rationale. Recovering and using such a waste product, normally destined to magnify industrial waste production, would give new life to the less noble part of the fruit. This is further in line with recent studies that propose the potential utilization of *Feijoa* fruit peel for added processing and functional value. As demonstrated by Sun-Waterhouse et al. [64], the extracts produced from *Feijoa* waste, such as the peel, retain high pectin content, which is advantageous for food applications. Moreover, the possibility to utilize *Feijoa* peel-containing food packaging film for the inhibition of foodborne bacteria was recently demonstrated [67].

In conclusion, the novel beneficial properties of *Feijoa* reported in this paper, regarding its efficacy to reduce heavy metal toxicity in human RBC, provide biochemical bases for the use of *Feijoa*-based functional foods or pharmacological preparations in preventing and combating mercury-related illnesses.

**Author Contributions:** Conceptualization, C.M. and M.P.; methodology, F.T., R.N., S.S., and A.B.; investigation, F.T., R.N., and V.M.; data curation, F.T. and R.N.; formal analysis, F.T., R.N., and A.B.; writing—original draft preparation, C.M., M.P., and K.V.G.; writing—review and editing, C.M., M.P., K.V.G., and V.M.; supervision, C.M. and M.P.; project administration, C.M.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
