EXPLOITATION OF ANTIOXIDANT BIOACTIVE COMPOUNDS FROM AGRO-INDUSTRIAL BY-PRODUCTS

This PhD project aimed to investigate how different agro-industrial by-products can be exploited to obtain extracts enriched with bioactive compounds that possess antioxidant and antiradical properties. For this purpose, plant parts that are generally discarded because they are recognized as waste or as a production surplus and are not marketable, were selected among the vegetable productions that are valuable to local producers in Sardinia (Italy). The investigation was focused on four matrices: − Saffron (Crocus sativus L.) petals; − Artichoke (Cynara cardunculus L. var. scolymus) leaf by-products; − Salvia desoleana water residue from essential oil production (plant steam-distillation); − Caper (Capparis spinosa L.) brine residue from salty flower buds’ production. Once the agro-industrial by-products were selected, the study focused on identifying the best extraction techniques according to the principles of green chemistry and sustainability. In fact, the extraction phase is fundamental for the separation and the recovery of bioactive compounds and allows to obtain an appropriate sample for the subsequent analytical investigation. Nowadays, due to their low-cost and environmental-friendliness, innovative and “green” extraction techniques are used. These techniques overcome the problems related to the traditional methods, minimizing energy consumption and sample degradation. Therefore, Ultrasound-Assisted Extraction (UAE), Deep Eutectic Solvents Extraction (DESE), Subcritical Water Extraction (SWE) and Supercritical Carbon Dioxide Extraction (SCO2E) were used. Considering that it is mostly about polar extraction methods, the quali-quantitative analyses were mainly focused on the identification of small polar molecules, and mainly on phenolic compounds. This class of organic molecules has potential health benefits mainly due to the strong antioxidant activity, useful in the prevention of cardiovascular and neurodegenerative diseases. For this reason, the extracts thus obtained have been characterised using analytical techniques such as LC-MS/MS and LC-PDA to determine organic polar compounds, mainly phenolic ones. This is a significant process that allowed to understand which bioactive compounds are extracted and how these green extraction methods can influence the total amount and/or the preferential extraction of some compounds. Finally, biological activities, such as antioxidant and antiradical properties, were investigated with different assays. Antioxidant activity was determined by evaluating total reducing power (FRAP and CUPRAC assays) and free radical scavenging activity (ABTS•+ and DPPH• assays). Also, in vitro analyses on intestinal differentiated Caco-2-cell cultures (cytotoxic activity and determination of intracellular ROS production) were performed to test the ability to scavenge reactive oxygen species. Moreover, molecular docking of target polyphenols was performed within the active sites of Kelch-like ECH-associated protein 1 (Keap1), inducible nitric oxide synthase (iNOS), and NADPH oxidase 2 (NOX2), enzymes that are responsible for the improvement of cellular antioxidant defences, the release of nitric oxide (NO), and the production of ROS. The results obtained in the PhD project have shown that the four selected matrices are worthy of being exploited for their potential application in the health and food industry. In this way, talking about the circular economy, the selected matrices can no longer be considered waste but agro-industrial by-products. In fact, first, it reduced the environmental impact of the by-products; secondly, and most importantly, there is an increase in the value of raw materials as a source of bioactive compounds, which can be used for their features in nutraceutical, pharmaceutical, and cosmetic industries.