ANTIOXIDANT PROTECTION OF TUSCAN TOMATO PEEL POLYPHENOLS IN A CELLULAR MODEL OF SARCOPENIA

Background: Tomato by-products contain a great variety of biologically active substances and might represent a significant source of natural antioxidant supplements of the human diet. The preliminary studies were carried out on two ancient Tuscan tomato peel varieties, Rosso di Pitigliano (RED) and Perina a Punta della Valtiberina (PER), obtained by growing plants in normal (-Ctr) or in drought stress conditions (-Ds) present in the Regional Bank of the Germplasm of Tuscany. The variety chosen was Rosso di Pitigliano for the best beneficial effects on vascular related dysfunction. The preliminary aim of the thesis was to create an in vitro model of sarcopenia, induced by dexamethasone using human skeletal muscle myoblasts (HSMM). Sarcopenia is a disease that affects athletes who practice endurance physical activity. In these, an excessive exercise increased reactive oxygen species (ROS) levels, that, if not properly balanced by the endogenous antioxidant system, can compromise the performance of the athletes. Furthermore, in controlling muscle mass an important role is played by serine/threonine kinase and a decreased activation of the Akt-mTOR pathway by sarcopenia contributes to protein synthesis reduction. The main aim of study was to evaluate the cytoprotective properties of tomato peel polyphenols from Rosso di Pitigliano, cultivated in normal or in drought stress conditions, on an in vitro model of sarcopenia. Methods: The antioxidant activity and total polyphenol content (TPC) were measured. The identification of bioactive compounds of several tomato peel was performed by HPLC. HUVEC were pre-treated with different TPC of RED-Ctr or RED-Ds, then stressed with H2O2. Cell viability, ROS production and CAT, SOD and GPx activities were evaluated. Permeation of antioxidant molecules contained in RED across excised rat intestine was also studied. The phenol content of both peel extracts was investigated by Ultra High-Performance Liquid Chromatography (UHPLC) analyses coupled to electrospray ionization high-resolution mass spectrometry (ESI-HR-MS). Morphological sarcopenia induction and treatment with tomato peels extracts were performed. The effector’s expression was evaluated by Real-Time PCR reactions after setting the optimal reaction conditions. Myotubes-differentiated were examined for the expression of Myosin heavy chain-2 (MYH2), Troponin T type 1 (TNNT) and Myogenin (MYOG). Furthermore, Protein kinase B (AKT1) and Forkhead Box O1 (FOXO1) mRNA expression was evaluated. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were performed. Results: RED-Ds tomato peel extract possessed higher TPC than compared to RED-Ctr (361.32 ± 7.204 mg vs. 152.46 ± 1.568 mg GAE/100 g fresh weight). All extracts were non-cytotoxic. Two hours pre-treatment with 5 μg GAE/mL from RED-Ctr or RED-Ds showed protection from H2O2-induced oxidative stress and significantly reduced ROS production raising SOD and CAT activity (* p < 0.05 and ** p < 0.005 vs. H2O2, respectively). The permeation of antioxidant molecules contained in RED-Ctr or RED-Ds across excised rat intestine was high with non-significant difference between the two RED types (41.9 ± 9.6% vs. 26.6 ± 7.8%). Phenolic acids increase in the stressed tomato peel extract, while flavonoids decrease. Data shows a protective effect of 5μg GAE/ml TPC of Red DS extract on the sarcopenia. FOXO1 mRNA expression was significantly increased when cells treated with Dexa, but this expression was significantly decreased in Red Ds+Dexa (p <0.0001 vs control). AKT1 mRNA expression was increased in myotubes pre-treated with Red Ds and Dexa (p <0.0001 vs control). Myosin heavy chain 2 (MYH2), troponin T (TNNT1), miogenin (MYOG), were express in myotubes differentiated (p<0.001 vs Control). DEXA significantly reduces the antioxidant enzyme activity of SOD compared with untreated cells (p < 0.0001), but RED-Ds increased SOD activity. Conclusions: The final results show that the tomato peel extract of Rosso di Pitigliano, grown in conditions of drought stress, represents a good source of bioactive molecules, which protects the endothelium from oxidative stress even at low concentrations. Furthermore, the polyphenols from tomato peel show a cytoprotective effect in the in vitro model of sarcopenia without the use of vehicles for absorption.