Valorisation of medicinal species typical of the biodiversity of the Italian territory for the production of innovative plant products for human health

Stress is a physiological mechanism activated in the body in response to internal or external perturbations, aiming to re-establish homeostasis. It occurs both at the whole-organism and cellular levels, regulating various metabolic pathways. In this context, adaptogenic substances – especially of plant origin – have attracted attention for their ability to modulate stress responses. This work investigates such mechanisms by developing cellular stress models and testing vegetable substances derived from by-products of the agri-food chain. Olive leaf extracts (OLEs) obtained from pruning waste across different Italian regions were studied in vitro on the neuronal cell line SH-SY5Y (human neuroblastoma). The project integrates three major themes: the study of adaptogenic substances, the neuroprotective role of antioxidant activity, and the valorisation of agricultural by-products through sustainable methods. Olive leaves, rich in bioactive compounds, fit these objectives and offer a renewable source for potential therapeutic use. Starting from pruning waste of O. europaea of different varieties from various areas of southern Italy, two types of OLEs were produced: macerated in 70% v/v ethanol and decoctions. Their content in total polyphenols, total triterpenes and oleuropein have been characterized, through colorimetric assays and HPLC-DAD analysis of the phenolic fraction; OLEs were compared with standardized dry extract of the US Pharmacopoeia to verify and confirm their phytochemical potential. From these analyses, we found that extracts produced from pruning waste contain significant levels of oleuropein, polyphenols and triterpenes, and a phytocomplex that is perfectly comparable in terms of quality with that of the Pharmacopoeia extract. Considering that olive leaf antioxidant power is the most consolidated data from the literature, the anti-radical and antioxidant power of the extracts was quantified through DPPH and ORAC assays. The results confirmed that even if produced from by-products, the extracts exhibit an extraordinary antioxidant capacity; that of ethanolic extracts is slightly higher than that of decoctions, reflecting the respective quantities of polyphenols and oleuropein. The pharmacokinetics of the main bioactive compounds of olive leaves were also examined: oleuropein demonstrated over 70% stability after the digestive process, while hydroxytyrosol – its main degradation product, as well as the most antioxidant molecule contained in olive leaves – showed superior absorption at both the intestinal level and across the blood-brain barrier compared to oleuropein, being in fact more bioavailable. Subsequently, OLEs were tested on SH-SY5Y cells to evaluate their cytotoxicity at 4h and 24h and it emerged that they are very well tolerated even at high concentrations. Through the development of a series of in vitro neuronal stress models, this study made it possible to demonstrate that olive leaf extract has notable neuroprotective properties against stress of different types, in particular: oxidative stress, cortisol, glutamate and serum deprivation. This was verified by cytotoxicity assays, quantification of intracellular ROS, and the study of ATP metabolism in relation to stress. Furthermore, the molecular mechanisms linked to the regulation of the endocannabinoid system, heat shock proteins and neurotrophins were explored, leading to the awareness that olive extract modulates all these pathways, promoting a more efficient cellular response to stress. Finally, some preliminary in vivo behavioural tests on the Lymnaea stagnalis animal model allowed us to investigate the protective capacity of olive extract against the Garcia effect (association between a negative stimulus and a new food) in relation to heat-stress, as well as its modulatory action towards the formation of long-term memory.