EVALUATION OF ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES OF TUSCANY CHERRY EXTRACT WITH DIFFERENT NANOSYSTEMS ON HUMAN ENDOTHELIAL CELLS

Aim. Cherry fruits have been widely described for their nutritional properties due to the high content in flavonoids and anthocyanins, which represent the most abundant protectors in the human diet against oxidative stress and inflammation. Their beneficial effects are mainly involved in the prevention of atherosclerosis, the major cause of all over the world cardiovascular diseases. The research has been addressed to cherry extracts (CEs) derived from Crognola, a particular tuscan sweet cherry variety, that have represented a good source of bioactives for the study, thanks to the high phenolic content. Since polyphenolic compounds are easily degraded in the gastrointestinal tract, CEs have been encapsulated in different types of nanosystems, based on mucoadhesive chitosan derivatives (Ch-der), i.e. quaternary ammonium chitosan (QA-Ch) and S-protected QA-Ch (QA-Ch-S-pro), or non mucoadhesive poly(lactic-co-glycolic acid) (PLGA). Therefore, the principle aim of this work has been to evaluate the ability of CEs and CEs-loaded nanoparticles (NPs), to protect human umbilical vein endothelial cells (HUVECs) -the in vitro study model for endothelial dysfunction- from both oxidative and inflammatory stress. Methods. HUVECs viability after CEs and NPs treatments, to different concentrations, has been measured by water-soluble tetrazolium salt (WST-1) assay. Reactive oxygen species (ROS) production after H2O2-induced oxidative stress has been detected by CM-H2DCFDA fluorescent probe. The secretion of cytokines (IL-6, IL10, TNF-α, PGE-2), nitric oxide and inflammasome NLRP3 has been quantified in supernatants by ELISA, after lipopolysaccharide-induced inflammatory stress on HUVECs. Moreover, the NPs cellular uptake has been studied by confocal laser scanning microscopy and by flow cytometry. The NPs ability to improve polyphenols permeability, by protecting them from degradation, was demonstrated in vitro by using a triple cell co-culture intestinal model (Caco-2/HT29-MTX/Raji B) and in ex vivo experiments on rat intestinal mucosa. Results. The results demonstrated that natural CEs from Tuscany cultivar protect endothelial cells from oxidative and inflammatory stress, i.e. by reducing ROS and inflammatory factors production. The results are particularly important since they are comparable with that of the synthetic corticosteroid dexamethasone. The encapsulation in Ch-der NPs or PLGA NPs improved the CEs positive effects. In particular, while QA-Ch-Spro and QA-Ch NPs enhance the antioxidant and the anti-inflammatory activity, respectively, of the lowest CEs polyphenolic concentrations tested, PLGA NPs maintain the activity of the highest ones. Ch-der NPs, thanks to their mucoadhesive properties and effective interaction with cells, can also promote CEs polyphenols intestinal permeation. In conclusion, although Ch-der NPs are promising delivery nanosystems, thanks to their physical properties, when we need high CEs concentrations, the use of low cytotoxicity PLGA NPs could be more convenient. Considering that fresh cherry fruit is only available a few days a year, the development of natural products delivery nanosystems could prolong and improve the beneficial effects deriving from cherry consumption, limited by the seasonality of the fruit.