**1. Introduction**

The olive oil industry is a large productive sector globally, and three-quarters of world production takes place in Europe. Among different commercial categories, extra virgin olive oil (EVOO) is produced by the sole employment of mechanical processes, and its quality is tightly related to different parameters, such as agronomic practices, the olive cultivar, and the olive oil extraction technology used [1]. The final chemical composition of the EVOO is influenced by different factors, among which the olive variety is the first one to play a primary role across the supply chain. Indeed, the olive cultivars display a large range of genetic variability for several agronomic traits such as the fruit size, oil content, and the degree of adaptation to severe environmental stress [2,3]. Olive drupes possess various health-promoting bioactive substances primarily represented by biophenol

Giordo, R.; Piscopo, A.; Abdel-Rahman, W.M.; Piga, A.; Pintus, G. Antioxidant Properties of Olive Mill Wastewater Polyphenolic Extracts on Human Endothelial and Vascular Smooth Muscle Cells. *Foods* **2021**, *10*, 800. https://doi.org/10.3390/ foods10040800

**Citation:** Posadino, A.M.; Cossu, A.;

Academic Editors: Cristina Alamprese, Emma Chiavaro and Francesco Caponio

Received: 9 March 2021 Accepted: 6 April 2021 Published: 8 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

secoiridoids (oleuropein, ligstroside) and their hydrolytic derivatives [4]. Although olive fruit is rich in phenolic compounds, only 2% of the entire phenolic content transfers into the oil phase, while the rest goes into the olive mill wastewater (OMW) (approximately 53%), the pomace, the olive oil filtration residue, and olive leaves (approximately 45%) [5]. As a result, the high percentage of phenolic compounds in the olive industry's byproducts is attracting great interest as a potential source of such phenolic compounds, with special attention oriented to the olive mill, which is the primary potential source of such molecules. The major components in OMW include hydroxytyrosol, tyrosol, oleuropein, ligstroside and their secoiridoids derivatives, and a variety of hydroxycinnamic and hydroxybenzoic acids [6]. Given the potential environmental impact, active molecule extraction from olive oil byproducts should embrace methodologies that employ green technologies, considering their possible exploitation as food antioxidants or nutraceuticals [7]. In fact, phenol recovery from these byproducts should be organized in order to promote their reintroduction into the food chain and coincide with greater valorization and improved olive oil industry waste management. In addition to its main implications for the local and international economy, this could reduce the environmental impact of olive oil manufacturing and contribute to this valuable production chain's sustainability. However, the quality of OMW phenolic compounds differs according to several factors, including the olive oil production technological process employed, and for this reason, it is essential to evaluate different OMW technological processes to provide promising bioactive compounds. In this regard, OMW-derived products have been tested for certain biological effects and have showed an interesting bioactivity spectrum [8]. For instance, EVOO-containing phenolic compounds have shown In Vivo and In Vitro antioxidant activity, likely due to molecules such as hydroxytyrosol, tyrosol, and secoiridoid derivatives [9,10].

Reactive oxygen species (ROS) are aerobic metabolism products and exert a pivotal role in regulating cellular functions such as proliferation, differentiation, and migration [11]. Under physiological conditions, a series of cellular antioxidant mechanisms maintain vascular ROS levels in homeostatic conditions [12]. However, an aberrant modulation of the above-mentioned mechanisms leads to critical increases in ROS levels, thus promoting different vascular-associated pathological conditions, including cancer and cardiovascular diseases [13]. In this context, endogenous ROS, released by endothelial and vascular smooth muscle cells as a result of pro-inflammatory and pro-atherosclerotic stimuli, can trigger vascular injury and blood vessel restructuring by affecting diverse intracellular signaling pathways [14]. Indeed, ROS-activated molecular machinery can modulate both endothelial cells and vascular smooth muscle cells functions including proliferation, migration, and invasion, leading to vascular pathologies such as hypertension, atherosclerosis, and cancer [15], which may be counteracted by OMW-contained compounds [16].

In this light, the present work aims to investigate whether (i) the considered OMW samples can be a source of valuable antioxidants, and (ii) whether the obtained extracts can protect human vascular cells against oxidative cell death.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Unless stated in the text, all the reagents used were from Sigma (Sigma, St. Louis, MO, USA).

#### *2.2. Sample Collection*

Ottobratica olives were sampled in November 2019 at a ripening index of 4 according to Guzmán et al. [17]. Oil extraction was performed by means of a three-phase decanter system (Alfa Laval, Monza, Italy) at 25–26 ◦C and 20 min of malaxation parameters in an olive oil mill located in the Calabria region (Italy). The obtained OMWs were transferred to the Food Technologies laboratory of the Mediterranea University of Reggio Calabria for the experimental project.
