MASS SPECTROMETRIC STRATEGIES TO IDENTIFY BIOACTIVE COMPONENTS FROM NATURAL EXTRACTS WITH ANTI-INFLAMMATORY ACTIVITIES

Oxidative stress and chronic inflammation are two conditions that can worsen various diseases, spanning metabolic, neurological disorders to tumour formation. In this context, a wide source of bioactive molecules exhibiting anti-inflammatory effects, combined with antioxidant potential, can be found in fruit and vegetables extracts. In particular, it has been reported that certain phytoconstituents are able to target two closely related systems within the cell, namely the NRF2 and NF-κB pathways. While the NRF2 pathway is responsible for regulating an extensive panel of antioxidant enzymes, NF-κB is the main effector pathway involved in inflammation. Phytochemicals in general mediate their effects by modulating the DNA-binding capacity of both these transcriptional factors and, in turn, correcting an unbalanced cellular state related to an uncontrolled inflammatory condition. Whereas previously, as a source of these phytochemicals, the focus was on the 'nobler' parts of the plant (i.e. the fruits, leaves and roots), today much more emphasis is placed on the analysis of industrial by-products. Specifically, this strategy aimed at the reuse of industrial by-products could reduce the impact of cultivation on waste production, minimizing disposal problems while obtaining a potential source of bioactive compounds. Even though there is general knowledge on the possible targets of these compounds, newer techniques combining different -omics approaches could better illustrate the biological impact of these extracts while identifying the phytochemical compounds responsible for it. Combining these different approaches to a concept of circular economy to valorise waste products could be a key to obtain useful health care products to prevent or even treat oxidative and inflammatory conditions. In particular, in this thesis, I will explore the antioxidant and anti-inflammatory activities of different extracts obtained from by-products of grape and apple cultivation. This investigation will employ a multifaceted approach, integrating engineered cell cultures for phenotypic screening of bioactive extracts, Mass Spectrometric-based metabolomics to unravel the primary components of these extracts, and Mass Spectrometric-based proteomic studies to comprehensively analyse their impact on the cell proteome. The exploration of potential mechanisms of action will be conducted through both in-vitro and ex-vivo quantitative proteomic studies. Conclusively, the thesis will showcase the application of a MS-CETSA (Mass Spectrometry – Cellular Thermal Shift Assay) methodology to probe the direct target of a bergamot leaves polyphenols extract.