CHEMICAL CHARACTERIZATION AND IN VITRO AND IN VIVO EVALUATION OF ANTIOXIDANT PROPERTIES OF THREE SICILIAN ENDEMIC BRASSICA EXTRACTS

In recent years, scientific interest in natural compounds with antioxidant properties has significantly increased, largely due to the established link between oxidative stress and a range of chronic diseases. The prolonged use of conventional medications to manage chronic conditions associated with oxidative stress can lead to drug resistance and undesirable side effects, making natural alternatives an increasingly attractive option. Numerous studies have already highlighted the beneficial effects of Brassica species in both in vitro and in vivo models of oxidative stress. Sicily, known for its rich biodiversity, is home to more than 10 endemic species of this genus. Since plants have been shown to concentrate phytochemicals in response to environmental factors like salinity, solar radiation, and other stressors, we chose to explore wild Sicilian Brassica species. We hypothesized that, due to the unique climate of Sicily, these wild Brassica species may show specific phytochemical profiles offering distinct and potentially potent biological effects. The aim of this project was to evaluate the biological properties of endemic Brassica species through both in vitro and in vivo analyses. We selected three endemic species from different habitats: B. macrocarpa endemic to Favignana island, B. rupestris subsp. hispida endemic to Ficuzza wood, and B. tardarae endemic to Tardara gorges. methanolic extracts were prepared from the leaves, and the chemical composition was analysed by HPLC-MS/MS. The three Brassica species showed a similar chemical profile, containing significant amounts of glucosinolates and phenolic compounds. Notably, differences were observed in the sinapyl derivatives, which were found in higher concentrations in B. hispida compared to the other two species. Subsequently, their radical scavenging activities were evaluated using the diphenyl-2-picrylhydrazyl (DPPH) assay. All three Brassica species exhibited scavenging activities. In consideration of these results, we continued our investigation into the potential beneficial effects of these extracts in vitro, using the RAW 264.7 murine macrophage cell model. Assessing toxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay we observed a reduction in cell viability were for B. hispida and B. tardarae, while B. macrocarpa proved to be safe at all concentrations tested. Moreover, using the Griess reaction we assessed in RAW 264.7 cells subjected to lipopolysaccharide (LPS)-induced oxidative stress the ability of the extracts to reduce nitric oxide (NO) production. At safe concentrations, B. hispida and B. tardarae were ineffective in reducing NO production, while B. macrocarpa successfully decreased NO levels. Real-time PCR analysis confirmed that B. macrocarpa treatment resulted in a reduction in NOS2 mRNA expression. Furthermore, in the 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe assay, B. macrocarpa, once again, proved to be efficient in reducing the levels of ROS increased by exposure to LPS in a concentration-dependent manner.For in vivo studies, in the zebrafish (Danio rerio) embryo the toxicity, behaviour, and antioxidant properties of B. macrocarpa were evaluated. Zebrafish embryos were subjected to the acute toxicity test (FET). Throughout the 96-hour treatment period, no induced malformations were observed; however, changes in spontaneous movement and heart rate were noted. Behavioural tests did not indicate significant alterations in anxiety-like, aversion, or exploratory behaviours, and all groups displayed normal swimming patterns. Exposure to sub-lethal concentrations for 26 hours did not result in any noticeable changes in the key oxidative stress markers evaluated. By using copper sulphate (CuSO4)-induced oxidative stress model, zebrafish embryos pre-treated with B. macrocarpa extract showed a significant reduction in ROS and NO levels compared to the untreated group. The extract also modulated the activities of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). Additionally, lipid peroxidation and protein oxidation were reduced. However, the extract did not appear to reverse CuSO4-induced DNA damage. In conclusion, our data demonstrated that B. macrocarpa exhibited strong antioxidant properties, proving safe in the in vitro murine macrophage model, while in the FET test conducted on zebrafish embryo, showed to be safe up to 500 μg/mL. B. hispida and B. tardarae showed antioxidant effects solely in the DPPH assay and resulted to induce, in our model, cell toxicity at low concentration, hampering any further studies on their possible effects in our biological setting. Additional research is needed to further investigate their full potential.