Lorena Martínez Zamora¹, Gaspar Ros Berruezo², Gema Nieto Martínez²

(1) NUTRICIÓN Y BROMATOLOGÍA, UNIVERSIDAD DE MURCIA, 30100, España (Región de Murcia)
(2) NUTRICION Y BROMATOLOGIA, IMIB, España

Protein oxidation has been defined as a covalent modification of proteins induced by reactive species during oxidative stress [1]. Citrus fruits (Citrus sinensis), acerola (Malpiguia emarginata), and rosemary (Rosmarinus officinalis L.) have shown promising antioxidant efficiency due to their compoisition rich in phenolic compounds and vitamin C, which also have shown beneficial health effects. The aim of the present work was to investigate the antioxidant activity and mechanisms of phenolic-rich extracts (citrus, acerola, and rosemary), in a meat model oxidized by an hydrophilic or lipophilic radical initiator. Protein oxidation was evaluated by analysis of thiol oxidation and myosin heavy chain protection by SDS-Page.

1.5 g of pork meat were homogenized in 12.5 ml of 0.05 M MES buffer, pH = 5.8, together with phenolic extracts (Citrus, Rosemary, Acerola) using an Ultra Turrax T25 at 11,600 rpm for 30 sec samples were kept on ice to reduce oxidation. Subsequently, the azo-initiators, 2,2’-azobis(2-amidinopropane)dihydrochloride (AAPH) diluted in MilliQ water (0.54 mM final concentration) or 2,2’-azobis(2,4-dimethylvaleronitrile (AMVN) diluted in 99.9 % EtOH (3 mM as final concentration) were added as hydrophilic oxidation initiator (OXHydro), or lipophilic oxidation initiator (OXLip), respectively. After oxidation, thiol groups were quantified [2], and SDS-Page method was performed [3].

Evaluation of the effects of the phenolic extracts on thiol oxidation in the two oxidizing systems, OXHydro or OXLip, showed that Citrus was a more effective antioxidant against protein thiol loss as compared to Acerola and Rosemary, especially in the OXHydro system. Rosemary was found to be slightly, though significantly, prooxidative in the OXHydro system. Evaluation of the radical scavenging activities of the phenolic extracts showed that Rosemary efficiently scavenged radicals to a level similar to the non-oxidized control (C-NoOX). Citrus also showed protective radical scavenging activities being most effective in the OXHydro system. In contrast, Acerola, showed significant prooxidant activities especially in the OXLip system leading to 7-fold increase in radical signal intensity as compared to the C-NoOX (Figure 1). Otherwise, the intensity of MHC in Figure 2 showed as Citrus was the most protective compound against protein oxidation, while phenolics from Rosemary can form thiol-quinone adducts with free thiol groups. This results can be corroborated by previous researchers that showed a similar behavior between phenolics from green tea and free thiol groups [3, 4].

In conclusion, Citrus was found to protect almost fully against protein thiol loss in the meat model system initiated by the hydrophilic initiator, OXHydro and by the lipiphilic initiator, OXLip.. The same component showed also efficient protection of Myosin Heavy Chain (MHC) as determined by SDS-Page. In contrast, Rosemary was found to behave as a prooxidant in the OXHydro system when evaluating protein thiol loss and also MHC. This demonstrated as thiol-quinone adducts are formed among free thiol groups and phenolics from Rosemary. Acerola also acted as a prooxidant, resulting in decreased protection of MHC, especially in the OXLip system, owing proposedly to high concentration of ascorbic acid in the extract.