Phd in BIOCHEMISTRY

Thesis title: Use of olive oil polyphenols to counteract neuroinflammation and neurodegenerative diseases

Neurodegenerative diseases represent a health emergency that we should address in the coming decades. The underlying causes of these diseases are still unclear, but they are probably closely linked to neuroinflammation and oxidative stress processes in the brain. The use of molecules that can counteract these phenomena could be an excellent tool in hampering the progression of these pathologies. Polyphenols are molecules naturally present in plant foods that exert many biological effects, particularly against inflammation and oxidative stress. They seem to be promising molecules in the biomedical field, indeed in recent years some of them have been used in several clinical trials. Olive oil is a good source of polyphenols, particularly of secoiridoids, a class of compounds which comprise oleocanthal and oleacein which are only found in this type of oil. The extraction of these polyphenols generally involves the use of organic solvents, such as methanol and hexane, a process which may lead to risks for the operators and environmental threats, as the oil matrix and the polyphenolic product may retain traces of organic solvents, thus being unsuitable for food use. To solve the extraction problem, alternative extraction techniques can be used such as Ultrasound-assisted extraction (UAE), Microwave-assisted extraction (MAE), Pressurised liquid extraction (PLE) and Supercritical fluid extraction (SFE). These techniques are mainly used not only on oil but also on olive pomace and wastemill water to retrieve polyphenols lost during the process of olive oil production. Moreover they are extremely energy-demanding. An alternative method, certainly simpler, quicker and environmentally friendly is the use of Deep Eutectic Solvents (DES). DES are binary solvents consisting of a hydrogen bond donor (nontoxic quaternary ammonium salts or amino acids) and a hydrogen bond acceptor (polyols, organic acids or carbohydrates). These two chemical families, through thermal, mechano-chemical or freeze-drying treatments, form an eutectic solvent characterised by an intense network of hydrogen bonds. When the eutectic solvent are made of natural compounds that are non toxic and even edible (such as monosaccarides, aminoacids, alcohols), they are named Natural DES (NaDES). In this work, the bioactive capacities of polyphenols extracted from extravirgin olive oil are evaluated on different cellular and animal models of diseases, paying particular attention to inflammation and oxidative stress. In the first section, experiments are presented to evaluate whether there are biological differences between polyphenols extracted by the classical method and those extracted via NaDES, by determining the biological response (inflammation and oxidative stress) on murine BV2 microglia cells following administration of the polyphenols before or after treatment with a pro-inflammatory stimulus. In the second section, polyphenols extracted by classical method (OOEP) were administered for 8 weeks in drinking water (10 mg/kg/day) to C57BL/6J mice under physiological or low-grade inflammation conditions (obtained by intraperitoneal injection of 0.5 mg/kg/week LPS). The mice were assessed in their cognitive capacity (long-term memory, anxiety-like behaviour) and inflammation and oxidative stress status. A broad-spectrum metabolomics study was performed in the liver and intestine. At the same time, we evaluated the impact of LPS and polyphenols on the microbiota. In the third and final section, polyphenols extracted with a NaDES made up with betaine and glycerol in a 1:2.2 molar ratio (OOE-DES) were administered for 12 weeks in drinking water (5 mg/kg/day) to euploid B6EiC3SnF1 and trisomic Ts2Cje mice. The impact of polyphenols on the mice in terms of cognition, oxidative stress and inflammation at the brain cortex and liver level was assessed, and a broad-spectrum lipidomic analysis was conducted on the brain, liver and caecum tissues. The data obtained show that olive oil polyphenols extracted with NaDES, OOE-DES, have the same anti-inflammatory and anti-oxidant capacities as classical extracted OOEP at both the cellular and animal level. From a comprehensive point of view, olive oil polyphenols positively impact the brain directly (by modulating inflammation) and indirectly (by modifying the microbiota and metabolites produced both in the gut and liver), promoting neurodevelopment and synaptogenesis. Thus, olive oil polyphenols are an excellent tool to alleviate neuroinflammation and in this way strike at the base of neurodegeneration. Moreover, olive oil polyphenols manage oxidative stress by modulating the activation of the pathway under the control of nuclear erythroid 2-related factor 2 (Nrf2) and Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), regulating the expression of antioxidant enxymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX).