BIOLOGICAL PROPERTIES OF CITRUS LIMON DERIVATES: INSIGHTS OF THE HEPATOPROTECTIVE EFFECTS OF LEMON ESSENTIAL OIL FRACTIONS AND OF LEMON-DERIVED NANOVESICLES.

The nutraceutical industry is increasingly focused on identifying natural compounds that offer both health benefits and appealing sensory qualities, aiming to develop foods that can be incorporated into a health-conscious diet. Citrus fruits, particularly Citrus limon, are widely regarded as essential in daily nutrition, containing beneficial compounds such as flavonoids, minerals, essential oils, and carotenoids. Nowadays their derivates are seen as a key player in preventive healthcare. In detail, Lemon essential oil (LEO) is renowned for its aromatic and health-enhancing qualities, yet less focus has been placed on the biological properties of the fractions derived from LEO. In the first part of the project, I aim to explore the capacity of a citral-enriched fraction of LEO (Cfr-LEO) to counteract inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) caused by lipopolysaccharide (LPS) in healthy human liver cells. Immortalized human hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS over varying periods. Our findings demonstrate that Cfr-LEO pretreatment inhibits LPS-induced inflammation, oxidative stress, and EMT in THLE-2 cells by reducing NF-κB activation, proinflammatory cytokine release, ROS production, and the expression of NRF2 and p53. Furthermore, Cfr-LEO exhibits a protective effect against LPS-induced EMT. These findings highlight the potential of Cfr-LEO in the nutraceutical field, offering both aromatic benefits and biological activity, with the possibility of developing food and beverage products enriched with Cfr-LEO to prevent or alleviate liver-related conditions.Furthermore, numerous investigations have explored the interaction between plant-derived nanovesicles (PDNVs) and mammalian cells, underlining the capability of these natural nanovesicles to regulate several molecular signaling pathways. Apart from their enhanced bioavailability, stability, and low toxicity, PDNVs possess biological proprieties that make them applicable against pathological conditions, such as hepatic diseases. In the second part of the project, I aim to investigate the antioxidant properties of lemon-derived nanovesicles, produced at laboratory (LNVs) and industrial scale (iLNVs), stemming from an in vitro study in human healthy hepatocytes (THLE-2) to a functional in vivo experimentation in a rat model of hepatic dysmetabolism, i.e. metabolic syndrome induced by a high-fat diet (HFD). Our findings demonstrate that in THLE-2 cells LNVs and iLNVs decrease ROS production and upregulate the expression of antioxidant mediators, Nrf2 and HO1. Furthermore, the in vivo assessment reveals that the oral administration of iLNVs in HFD-fed rats improves glucose tolerance and lipid dysmetabolism, alongside amelioration in biometric parameters and systemic redox homeostasis. In addition to this, iLNVs administration upregulates Nrf2/HO-1 signaling in HFD rat’s liver. In light of our comprehensive outcomes, we support the potential of LNVs/iLNVs as a promising approach for managing hepatic and dysmetabolic disorders. In the end, I preliminarily investigate the antimicrobial properties of LNVs isolated via Size Exclusion Chromatography (SEC) and their potential to stimulate the human immune system. The research determinates whether LNVs can enhance the innate immune response by promoting the activity of macrophages, which are crucial for detecting and eliminating pathogens. In particular, we established a protocol for evaluating if THP1 M0 differentiated cells, pre-treated with LNVs, influence bacterial growth and colony formation. This research holds promise for developing LNV-based treatments that target pathogens while enhancing the body’s natural defenses, providing a dual strategy for combating infectious diseases.In conclusion, this work paves the way for creating specialized products, formulated with scientifically tested citrus essential oils and LNVs, designed to prevent or alleviate chronic conditions related to liver dysfunction or to enhance the immune response against pathogens.