BIOMOLECULES FROM OLIVE MILL WASTEWATER: COSMECEUTICAL AND NUTRACEUTICAL APPLICATIONS

Olive mill wastewater (OMWW), the main by-product of the olive oil industry, contain water, sugars, mineral salts and polyphenols. Hydroxytyrosol, the most abundant biophenol, has a lot of beneficial effects on human health, as anticancer activities. The aims of my doctorate project were to evaluate: 1) possible differences in the chemical composition of OMWW generated from two Sicilian olive cultivar (Cerasuola and Nocellara etnea); 2) safety and the moisturizing properties of a sugars and minerals enriched fraction (SMEF), obtained from Cerasuola-OMWW, by in vitro and in vivo studies; 3) the chemotherapeutic activities in vitro exhibited by hydroxytyrosol-enriched fraction (H-EF) obtained from Cerasuola-OMWW. As control, we used hydroxytyrosol commercial standard (HTyr). HPLC analysis evidenced a quantitative rather than a qualitative difference in the polyphenolic profiles of Cerasuola-OMWW and Nocellara etnea-OMWW. However, hydroxytyrosol and tyrosol were the most abundant biophenols present in both OMWW. Both waters were able to quench the DPPH radical and had a higher radical scavenging activity than the standard hydroxytyrosol, probably due to the synergistic effect of other phenolic compounds present in OMWW. Stability studies showed no significant changes in total phenolic, flavonoid and hydroxytyrosol content in both OMWW when stored at -20°C for 6 months. The lyophilized SMEF was rich in sugars (around 43%) and potassium. The in vitro study performed on MRC-5 cells has evidenced that: - the lowest concentrations of lyophilized SMEF (0.5% and 1% w/w) did not influence cell viability either after 6 or 24 hours of treatment. Conversely, the highest concentrations (2%, 5% and 10% w/w) determined a drastic decrease of cell viability in a dose- and time-dependent manner; - the degree of apoptosis and intracellular ROS levels increased, in treated fibroblasts compared to untreated controls, in a dose- and time-dependent way, in particular from 2% concentration to 5% w/w; - the DNA damage extent increased for the 2% and 5% SMEF treatment only in a concentration-dependent manner; - the treatment with the highest concentrations of SMEF for 6h induced early alterations of cell morphology as shrunken cytoplasm and numerous blebs and ruffles on the cell surface. Taking into account these data, 1% w/w of lyophilized SMEF was incorporated in an emulsion to formulate a body cream, able to increase skin hydration, as shown by in vivo study. H-EF added at different concentration in astrocytoma cells decreased cell viability, in a dose- and time-dependent manner, induced arrest in G1 phase and inhibited cell migration. These data are in accordance with the observed time-dependent increase in DNA damage and the decrease in p53 and p21 expression. An increase in ROS level with a parallel decrease in GSH level seemed to be involved in the genotoxic effect-H-EF- induced in astrocytoma cells. In addition, by investigating some possible epigenetic effects exerted by our extract, we observed a modulation of global DNA methylation expressed as hypomethylation at lowest concentration (50 µM) and hypermethylation when the cell treatment was performed at 100 µM. The additional epigenetic parameters we studied, SIRT1 expression and nuclear sirtuins activities, were both decreased. We observed different dose- and time-dependent effect for hydroxytyrosol (HTyr) 100 µM. The compound did not influence cell viability either after 24 or 48 hours of treatment but induced an arrest in S phase at 24 h and in G1 phase only at 48 h. In addition, it increased p53 expression without inducing any change in p21 expression and, finally, it hypomethylated DNA and increased both SIRT 1 expression and nuclear sirtuins activity. We demonstrated the potential use of SMEF and H-EF in cosmeceutical and nutraceutical field. However, the possible therapeutic use of H-EF in humans requires additional studies.