Annurca apple polyphenols prevent mercury-induced phosphatidylserine externalization in human erythrocytes via...

Phosphatidylserine (PS) exposure on the surface of red blood cells (RBC) is a hallmark of membrane asymmetry loss and a prothrombotic signal often induced by oxidative stress and heavy metal toxicity. Mercury (Hg) is known to disrupt cellular redox balance and calcium homeostasis, leading to PS externalization and increased thrombotic risk. Natural antioxidants such as polyphenols may provide protection against these effects. The Annurca apple (Malus pumila Mill. cv. Annurca), a cultivar rich in procyanidins and phenolic compounds, has shown antioxidant and membrane-stabilizing properties.

Human RBC were isolated from healthy donors and pretreated with Annurca apple extracts (0.30 and 1.5 μg catechin equivalents (CAEq)/mL corresponding to 1-5 μM) derived from unripe and ripe fruit (flesh and peel). Cells were then exposed to mercury chloride (HgCl₂, 20 μM). PS exposure was quantified by annexin V-FITC staining and flow cytometry. Activities and expression levels of ATP11C (flippase) and PLSCR1 (scramblase) were assessed by fluorescence assays, immunocytochemistry, and Western blotting. Intracellular ATP and Ca²⁺ levels were measured using luminescent and fluorometric assays.

HgCl₂ treatment markedly increased PS exposure, disrupted ATP11C activity, upregulated PLSCR1, elevated intracellular Ca²⁺, and reduced ATP levels. Pretreatment with Annurca apple extracts significantly decreased PS exposure in a dose-dependent manner, particularly with ripe fruit extracts. This effect was associated with normalization of intracellular Ca²⁺ and downregulation of PLSCR1, but not with restoration of ATP levels or ATP11C activity. Peel extracts displayed stronger protective effects than flesh extracts.

Annurca apple polyphenols protect RBC from Hg-induced membrane damage primarily through modulation of calcium-dependent PLSCR1 activity, thereby preserving membrane asymmetry. Unlike other polyphenols such as hydroxytyrosol, Annurca extracts act independently of ATP restoration, highlighting a distinct mechanism centered on ionic homeostasis. These findings support the potential of Annurca apple as a functional food for mitigating cardiovascular risk associated with heavy metal exposure.