*Article* **Antioxidant Properties of Olive Mill Wastewater Polyphenolic Extracts on Human Endothelial and Vascular Smooth Muscle Cells**

**Anna Maria Posadino 1, Annalisa Cossu 1, Roberta Giordo 2, Amalia Piscopo 3, Wael M. Abdel-Rahman 2, Antonio Piga 4,\* and Gianfranco Pintus 1,2,\***


**Abstract:** This work aims to analyze the chemical and biological evaluation of two extracts obtained by olive mill wastewater (OMW), an olive oil processing byproduct. The exploitation of OMW is becoming an important aspect of development of the sustainable olive oil industry. Here we chemically and biologically evaluated one liquid (L) and one solid (S) extract obtained by liquid– liquid extraction followed by acidic hydrolysis (LLAC). Chemical characterization of the two extracts indicated that S has higher phenol content than L. Hydroxytyrosol and tyrosol were the more abundant phenols in both OMW extracts, with hydroxytyrosol significantly higher in S as compared to L. Both extracts failed to induce cell death when challenged with endothelial cells and vascular smooth muscle cells in cell viability experiments. On the contrary, the higher extract dosages employed significantly affected cell metabolic activity, as indicated by the MTT tests. Their ability to counteract H2O2-induced oxidative stress and cell death was assessed to investigate potential antioxidant activities of the extracts. Fluorescence measurements obtained with the reactive oxygen species (ROS) probe H2DCF-DA indicated strong antioxidant activity of the two OMW extracts in both cell models, as indicated by the inhibition of H2O2-induced ROS generation and the counteraction of the oxidative-induced cell death. Our results indicate LLAC-obtained OMW extracts as a safe and useful source of valuable compounds harboring antioxidant activity.

**Keywords:** antioxidant activity; olive mill wastewaters; phenolic extract; reactive oxygen species; vascular cells
