Solvent–Solvent Fractionation of Ora-pro-nobis (Pereskia aculeata) Leaves Enhances Polyphenol Enrichment and Red Blood Cell Protection Against Oxidative and Osmotic Stress

Ora-pro-nobis is a bioactive food plant widely distributed across Latin America and the Caribbean, with its biological activities largely attributed to leaf polyphenols. Solvent-solvent fractionation is an effective approach to enriching these compounds. Dichloromethane (FD), ethyl acetate (FAE), n-butanol (FB), and aqueous (FAq) fractions were obtained from the crude extract (CE), and their phenolic profiles, chemical antioxidant activity (CAA), and red blood cell (RBC) protection were evaluated. FAE contained the highest levels of total phenols (65 mg of GAE/g), flavonoids (56 mg of CE/g), chlorogenic acid (5147 μg/g), p-coumaric acid (10625 μg/g), ferulic acid (18482 μg/g), ellagic acid (36402 μg/g), and quercetin (1491 μg/g). In contrast, FB was the richest in rutin (3889 μg/g), and FAq was the richest in gallic acid (880 μg/g). In CAA assays, FAE exhibited superior activity in DPPH (79 mg of AAE/g), ABTS (114 mg of AAE/g), and FRAP (152 mg of AAE/g), while CE was most effective in Fe2+-chelation (96 mg of EDTAE/g). In TBARS assays, all samples protected RBCs comparably (61.5-68.8% inhibition), except FD (45.3%). All fractions inhibited oxidative hemolysis in a dose-dependent manner and mitigated protein oxidation. They also reduced erythrocyte osmotic fragility by lowering H50 (0.401-0.424%) and osmotic hemolysis (45.2-74.3%). Overall, FAE concentrated the highest load of bioactive compounds and emerged as the most promising fraction for nutraceutical development.