Polyphenols from Thinned Young Apples: HPLC-HRMS Profile and Evaluation of Their Anti-Oxidant and Anti-Inflammatory Activities by Proteomic Studies

The qualitative profile of thinned apple polyphenols (TAP) fraction (approximate to 24% of polyphenols) obtained by purification through absorbent resin was fully investigated by LC-HRMS in positive and negative ion mode and using ESI source. A total of 68 polyphenols were identified belonging to six different classes: flavanols, flavonols, dihydrochalchones, flavanones, flavones and organic and phenolic acids. The antioxidant and anti-inflammatory activities were then investigated in cell models with gene reporter for NRF2 and NF-kappa B and by quantitative proteomic (label-free and SILAC) approaches. TAP dose-dependently activated NRF2 and in the same concentration range (10-250 mu g/mL) inhibited NF-kappa B nuclear translocation induced by TNF-alpha and IL-1 alpha as pro-inflammatory promoters. Proteomic studies elucidated the molecular pathways evoked by TAP treatment: activation of the NRF2 signaling pathway, which in turn up-regulates protective oxidoreductases and their nudeophilic substrates such as GSH and NADPH, the latter resulting from the up-regulation of the pentose phosphate pathway. The increase in the enzymatic antioxidant cellular activity together with the up-regulation of the heme-oxygenase would explain the anti-inflammatory effect of TAP. The results suggest that thinned apples can be considered as a valuable source of apple polyphenols to be used in health care products to prevent/treat oxidative and inflammatory chronic conditions.