Honey flavonoids as protection agents against oxidative damage to human red blood cells

Honey flavonoids as protection agents against oxidative damage to human red blood cells Abstract Unlike other components of the Mediterranean diet, namely fruits, vegetables, wine and olive oil, that have been intensively studied for the ability to protect vital cell components from oxidative damage and slow the aging process by neutralizing free radicals, honey is relatively unknown for its antioxidant properties. Among natural food antioxidants, polyphenols are ubiquitously distributed in the vegetable kingdom as plant secondary metabolites. Natural polyphenols can range from simple molecules to highly polymerised compounds, with flavonoids representing the most common and widely distributed subgroup. The aim of our work was to assess and compare antioxidant activities of honey phenol extracts, separated by their hydrophobicity. Available literature indicates that no previous studies have been reported on antioxidant properties of polyphenols of italian honeys. Thus, to our knowledge, this is the first report of such an in vitro study. In the present study, raw multifloral honey, collected over the Marche region (central Italy), was used. Phenols were absorbed on an Amberlite XAD-2 column and eluted by methanol. The eluate was vacuum-dried, dissolved with water and partitioned in ethyl ether to fractionate the polar and non-polar compounds. Honey phenol extracts separated on the base of their hydrophobicity were evaluated for the antioxidant content by the FRAP assay, and for the ability to inhibit oxidative damage induced by radical species generated in the water phase or in the membrane of human erythrocytes. The peroxyl radical-scavenger activity was tested by the inhibition of RBC hemolysis induced by 2,20-azobis-(amidinopropane)dihydrochloride (AAPH) which generates such radicals, attacking from the outside of the membrane. The antioxidant efficiency against oxidative stress induced by hydroperoxides was also estimated, utilising water-soluble hydrogen peroxide (H2O2) and the lipophilic tert-butylhydroperoxide (t-BOOH). The water and ether fractions obtained from crude methanol extract of honey exhibited a phenolic content of 5.33 and 2.62 mg caffeic acid equivalents/100 g honey, respectively. These values correlate well with those of total antioxidant power, as assessed by FRAP assay (37.67 vs. 10.65 lmol/100 g honey). Flavonoid contents were 2.57 and 1.64 mg catechin equivalents/100 g honey for ether and water fractions, respectively. Although both honey fractions protect erythrocytes against AAPH -induced lysis, only the ether fraction was found to be active in inhibiting hemolysis but not methemoglobin and ferrylhemoglobin formation caused by H2O2. In addition, the ether fraction prevents t-BOOH -induced lipid peroxidation in whole erythrocytes and in isolated membranes. The significant antioxidant effect against damages induced by both water-soluble and hydrophobic exogenous oxidants suggests that the ether fraction, owing to its lipophilic character, can interact with red blood cell membrane, and the protective effect can be associated with the binding of the flavonoids to the membrane. On the other hand, the water fraction is more hydrophilic than ether fraction and it acts only from the outside of the membrane by scavenging the radicals before they attack the erythrocyte membrane.