Antioxidant effects of ‘Mela Rosa dei Monti Sibillini’ on Drosophila melanogaster longevity

Aging could be considered as the result of a time-dependent accumulation of cell damages that inexorably brings to functional cellular impairments and increased vulnerability to chronicdegenerative diseases. The "Free Radical Theory of Aging", developed by Harman in 1956 [1], and also known as 'the oxidative damage theory of aging', has undergone several variations over the last years, including the suggestion of a central role for reactive oxygen species (ROS) produced by both environmental or endogenous factors [2]. Indeed, free radicals, by damaging biomolecules, compromise cellular homeostasis. Therefore, it is clear that natural dietary compounds with high content of antioxidants represent an extraordinary potential tool in the major healthcare challenge of delaying aging. Among fruits, great attention has been paid to apple, as it is a rich source of bioactive compounds, especially polyphenols. In the last years, an ancient variety of apple, whose cultivation had been abandoned for a long time, has been rediscovered: “la mela rosa dei Monti Sibillini” [3]. Aim of this work was to study the effect of the supplementation of two different extracts obtained from mela rosa dei Monti Sibillini on both longevity and the endogenous antioxidant defence system of Drosophila melanogaster (wild type, Canton strain). These fruit flies represent one of the most commonly used animal model for studying the complexity of the aging process because they not only share with humans many metabolic pathways, but also have functional homologs to nearly 65% of human disease-causing genes [4]. In this study, flies, separated by sex (n.200 for both males: M, and females: F; collected in seven tubes/gender/treatment), were lifelong supplemented (0.5% diluted in water) or not (CTR group) with two extracts: one obtained from the whole apples (A group) and the second one from their peels (B group). At two different intervals (at 15 and 45 days for F and at 15 or 30 days for M), some flies (n5/tube for three replicates), after sedation, were collected and, immediately, frozen (-80°C) till the evaluation of the expression of some genes related to antioxidant defences, such as: heme oxidase-1 (HO1); thioredoxin reductase (TRX); superoxide dismutase (SOD) and glutathione peroxidase (GpX) by RT-PCR assay. The longevity in supplemented female flies was significantly increased (by 10 and 20% in A and B groups, respectively) and also the mean half-life of CTR flies was significantly lower than the ones of A and B group. Furthermore, the B groups presented a much higher increase of half-life vs A groups (A vs B; p< 0.0001). In male flies, no significant effects were observed for longevity, although the average half-life of CTR group was significantly lower than the ones of A and B group. Furthermore, in males, the different extract supplementation (A vs B) did not differently modulate the half-life of fruit flies. A different trend over time of antioxidant gene expressions was observed in the two genders following the A or B supplementations. Female flies, physiologically characterized by higher antioxidant defenses [5], in the early stage of supplementation showed a down regulation of all the antioxidant genes here 14 evaluated; whereas, at 45 days, the gene expression increased for GpX, HO1 and TxR in B group. Male flies, instead, showed a modulation of antioxidant gene expression since the early stages, where upregulation of SOD and HO1 were observed in A group at 15 days. At 30 days, the GpX and HO1 gene expressions resulted increased in B group, whereas TxR in A group. The data obtained from this study suggest that the oral supplementation of the extracts of mela rosa dei Monti Sibillini starting from birth is able to prolong the average life of both female and male flies, while an effect on maximum longevity was registered only in females. Also with regard to the modulation of the antioxidant enzymes, different results were obtained in the two genders. In particular, in females, a down regulation of the expression of SOD, GpX, HO1 and TxR genes in the early stages of life was observed, which was not found in males. During aging, however, when the endogenous antioxidant defences decrease also in females, there was an up-regulation of antioxidant genes, modulated by supplementation in both sexes, although with a higher increase in female specimens. These data suggest that the apple is a potential functional food whose chronic consumption during life might prevent the onset of those chronic-degenerative diseases in which oxidative stress plays a key role. Moreover, these results sustain the cultivation of this ancient variety of apple to promote the economic growth in this area.