**1. Introduction**

*Feijoa sellowiana (Feijoa)*, commonly known as pineapple guava, is an evergreen shrub in the Mytraceae family that is native to South America. It is commonly cultivated in tropical and subtropical countries, such as Brazil, Uruguay, Paraguay, and Argentina, but its cultivation has been extended to other countries, including Italy. *Feijoa* fruit, an intensely fragrant dark green oval berry, is commonly eaten fresh or as a variety of commercially available processed foods, such as jam, ice cream, and yoghurt [1,2].

The advances in chemical composition and biological activities of di fferent botanical parts of *Feijoa* have recently been summarized in a mini-review by Fan Zhu [3]. The fruit is the most utilized botanical part of *Feijoa*, and its nutritional value is generally defined by the presence of dietary fiber, essential amino acids, potassium, and vitamins, including vitamin C. Recent studies have added to *Feijoa* nutritional properties, including high folic acid content, and particularly high iodine (3 mg/100 g

wet weight) [4]. This fruit also contains pharmacologically relevant bioactive phytochemicals in the pulp and peel, including polyphenols, the major contributors of its bioactivities [3]. Plant polyphenols are widely known for their antioxidant activity [5], and *Feijoa* contains a variety of these compounds, including epigallocatechin, procyanidin B2, epigallocatechin gallate, myricetin-3-O-galactoside, epicatechin gallate, quercetin-3-galactoside, and quercetin-3-rhamnoside [6]. *Feijoa* extract has demonstrated antioxidant activity in a variety of human blood cells, as well as in vivo in rats [7,8]. Potential mechanisms of this activity have been studied, first by Rossi et al., wherein *Feijoa* extract suppressed nitric oxide production in murine macrophages via either attenuation of the nuclear factor κB (NF-κB) or activation of mitogen-activated protein kinase (MAPK) [9]. Additional activities include anti-microbial, anti-inflammatory, anti-cancer, and anti-diabetic capacities [10–15]. In human intestinal epithelial cells, *Feijoa* fruit improved lactase and sucrose-isomaltase activity and reduced proliferation, but was not cytotoxic, and also prevented lipid peroxidation [16].

The claimed *Feijoa* biological activities sugges<sup>t</sup> that its bioactive components have grea<sup>t</sup> potential to be developed into functional food or natural supplements [17]. In addition to diet, *Feijoa* fruit extract is used for industrial purposes, including cosmetic preparation, due to its emollient, elasticizing, and dermo-purifying properties. Furthermore, *Feijoa* extracts have recently been considered as a potential sunscreen resource, due to their proven high absorption in the UV region [18].

Heavy metals are common environmental pollutants and come from natural and anthropogenic sources [19–21]. In recent decades, their contamination has increased dramatically [22–24]. In this respect, attention has focused on how bioactive phytochemicals may counteract heavy metal-induced body burden [25]. Of particular concern is the heavy metal mercury (Hg), which exists in several redox forms, each endowed with different bioavailabilities and toxicities [19,20]. It reacts adversely within organisms, but also remains and bioaccumulates within aquatic animal tissue, which is the major Hg source to humans. Hg poisoning in humans manifests as neurological, kidney, immune, and respiratory disorders, and is especially toxic in pregnan<sup>t</sup> women [26–30]. Evidence from human and animal studies suggests that mercury also affects reproductive function [31], as do other heavy metals, such as copper [32–34] and cadmium [35]. Moreover, high levels of Hg may induce or exacerbate cardiovascular diseases [36–38]. Hg travels through the bloodstream and binds with high affinity to sulphydryl groups (-SH) [39]. This effectively increases the concentration of reactive oxygen species (ROS) in blood and tissue cells by inactivating or sequestering antioxidants, such as glutathione (GSH) [40]. Recent data from our group showed that hydroxytyrosol, an antioxidant phenol present in high concentrations in virgin olive oil, has the potential to modulate Hg toxicity in red blood cells (RBC), confirming the viability of designing nutritional strategies to counteract the adverse effects of Hg exposure in humans [41–43].

The aim of this study was to extend the relatively new research on the beneficial properties of *Feijoa* by testing the role of its components against Hg toxicity using intact human RBC. These anucleated cells, free of cellular organelles, represent a particularly important cellular model to study heavy metal toxicity [44,45]. First, we evaluate the ability of *Feijoa* peel and pulp acetonic extracts to protect human RBC from Hg-induced hemolysis. Then, the antioxidant properties of *Feijoa* extracts are measured in RBC by their ability to counteract the Hg-induced increase in ROS cellular generation. Protection against GSH, as well as membrane thiol depletion, are also measured. Moreover, confocal laser scanning microscopy was used to determine the protective effect of *Feijoa* extracts on Hg-treated RBC morphological alterations, as well as microvesicle (MV) formation. Exposure, even at low Hg concentrations, induces morphological changes that cause an increase in the procoagulant activity of these cells [46].

#### **2. Materials and Methods**
