Phenolics from monofloral honeys protect human erythrocyte membranes against oxidative damage

Abstract

The aim of the present work was to analyze the phenolic extracts from two monofloral Cuban honeys for their in vitro total antioxidant capacity, phenolic compounds content and free radical scavenging activity. The phenolic extracts, rich in lipophilic compounds, were tested further for their ability to inhibit AAPH-induced oxidative damage (hemolysis, lipid peroxidation and cytosolic depletion of reduced glutathione and decrease of superoxide dismutase activity) in erythrocytes. Results indicate an important total antioxidant capacity measured by TEAC and ORAC assays, as well as a relevant radical scavenging activity performed by EPR. Moreover, 13 phenolic compounds were identified using HPLC–LC/MS with quercetin as the most abundant flavonoid. The results also show that both extracts were able to inhibit erythrocytes oxidative damage, and that this may likely be due to their incorporation into cell membranes and their ability to cross it and reach the cytosol. In fact, flavonoid uptake by erythrocytes was further confirmed by testing quercetin, which efficiently incorporated into erythrocytes. Overall, this study indicates that honey contains relevant antioxidant compounds responsible, at least in part, for its biological activity and that uptake of its flavonoids may provide defense and promote cell functions in erythrocytes.

Introduction

In the long human tradition, honey has been used not only as a nutrient but also as a medicine. During the past decade, the use of honey for therapeutic purposes has been re-evaluated in a more scientific setting and several properties have been identified (Dias et al., 2008). They include antibacterial (Estevinho et al., 2008, Gomes et al., 2010), antifungal (Feás and Estevinho, 2011) and anti-inflammatory effects as well as stimulation of wound and burn healing (Alvarez-Suarez et al., 2010a). Honey also displays a significant antioxidant activity; recent studies demonstrate a strong correlation between the content of phenolic compounds in honeys from various floral sources and their antioxidant capacity (Gheldof and Engeseth, 2002, Blasa et al., 2006, Alvarez-Suarez et al., 2010b, Morais et al., 2011).

Flavonoids are the major functional components of honey and may significantly contribute to its total antioxidant activity and beneficial effects in human health (Hung et al., 2004). One of the potential benefits of flavonoids is to stabilize cell membranes by reducing lipid peroxidation and scavenging free radicals (Chaudhuri et al., 2007). In addition, data from in vitro experiments have recently revealed that the antioxidant properties of flavonoids could lie in their localization in lipoprotein domains and cell membranes, which generally serve as targets for lipid peroxidation, suggesting a protective interaction of flavonoids with lipid bilayers. In fact, certain flavonoids may incorporate into the hydrophobic core of the membrane bilayer, causing a reduction in membrane fluidity and membrane stability (Arora et al., 2000, Chaudhuri et al., 2007). This reduced membrane fluidity may limit diffusion of free radicals and improve the antioxidant effectiveness of flavonoids.

The aim of the present work was to study the antioxidant activity and the protective effect against oxidative damage in red blood cells (RBCs) of two monofloral Cuban honey phenolic extracts (PE). Ether fractions obtained after methanol extraction were analyzed for phenolic contents and ability to scavenge different free radicals. The protective capacity of honey-phenolics against AAPH-induced hemolysis in human erythrocytes was studied. In the same way, the capacity to protect RBCs against cytosolic reduced glutathione (GSH) and superoxide dismutase (SOD) depletion as well as lipid peroxidation were investigated. Finally, since quercetin was found to be the most abundant flavonoid in honey PE, its intracellular accumulation in human RBCs was studied to determine the capacity of this flavonoid to cross the erythrocyte membrane, and to protect the cell against oxidation.