PRODUZIONE DI METABOLITI SECONDARI DI ORIGINE VEGETALE A PARTIRE DA COLTURE IN VITRO DI CALLI DALLA POLPA DI FRUTTA DI DIVERSE POMACEAE (VALUTAZIONE DELL’ATTIVITÀ BIOLOGICA DEI METABOLITI SECONDARI OTTENUTI DA COLTURE IN VITRO DI CELLULE DI POLPA DI CALLO)

This doctoral thesis focused on evaluating the biological activities of hydroalcoholic extracts derived from callus pulp cultures of three selected apples: Cydonia oblonga Mill., Mela Rosa Marchigiana, and Malus pumila cv Miller (Annurca Campana Apple). For each apple, the research followed the following workflow: (1) establishment of callus cultures and extract preparation, (2) qualitative-quantitative chemical characterization of SM extracts, and (3) evaluation of their biological activity. In Cydonia oblonga Mill., the hydroalcoholic extract, previously found to be rich in phenolic and triterpenic compounds, demonstrated a potent anti-inflammatory effect in in vitro models of LPS-stimulated RAW 264.7 macrophages and HaCaT keratinocytes. Results indicated significant reductions in NO production, and pro-inflammatory gene expression (iNOS, IL-6, IL-1β, ikBα, ICAM). Moreover, the extract reduced the radical oxygen species levels in the hydrogen peroxide (H2O2) and tert-butyl hydroperoxide- (TBHP) injured HaCaT keratinocytes, revealing an important antioxidant activity. For Mela Rosa Marchigiana, callus cultures from sub-cultures maintained high triterpenic acid content and bioactivity over the years. Comparative analysis with previous extracts confirmed consistent antioxidant, and anti-inflammatory effects, as demonstrated through cell-free (DPPH, ABTS, and ORAC) and cell-based assays (LPS-stimulated RAW 264.7 macrophages and H2O2 injured HaCaT keratinocytes). In Malus pumila cv Miller, two callus cultures were cultivated under darkness and photoperiod conditions. The selected conditions for generating the two types of calli facilitated the production of substantial amounts of triterpenic compounds. Both calli primarily exhibit anti-inflammatory and genoprotective effects (tested using the lipoxygenase inhibition activity assay and LPS-stimulated RAW 264.7 macrophages, and the nicking assay, respectively) while showing lower antioxidant activity (evaluated by DPPH, ABTS, and ORAC assays). The callus cultivated in light conditions was chosen for encapsulation in a nanocarrier, which allowed for the assessment of its anti-inflammatory activity in a cellular model of ferroptosis on HaCaT cells. The results highlight the promise of Annurca Apple Callus extract in addressing oxidative stress and inflammation linked to excess iron. The nanocarrier system greatly improves its therapeutic effectiveness, as evidenced by gene expression analysis showing that the encapsulated extract can modulate the expression of genes associated with the ferroptosis process (COX-2, GPX-4, NrF2, IL-6, and TNF-α). Overall, this study highlights the potential of callus culture technology for producing bioactive SMs with pharmaceutical, cosmetic, and nutraceutical applications. It also emphasizes the importance of optimizing culture conditions and leveraging advanced delivery systems to maximize the therapeutic potential of plant-derived compounds. These findings pave the way for scalable and sustainable production strategies in bioactive compound research and industry applications.