**1. Introduction**

Olive cultivation is typical of Mediterranean countries and it is developed in several regions of the world having similar climate features [1]. By-products and waste from olive production and the olive oil industry represent a relevant environmental issue because they contain several compounds that may also be phytotoxic [2]. Olive wastewater (OWW) contains a huge bulk of organic compounds, metals, minerals including potassium, calcium, and phosphorous. Furthermore, OWW has high biological and chemical oxygen demand (BOD and COD) and very low pH that make this olive waste highly pollutant [3]. In developed countries, the growing demand for food increases the mass of waste and by-products in an unsustainable manner. In the last few years, novel approaches, based on the new concept of the circular economy, lead to reduce waste biomass and valorize by-products. The improvement of extraction strategies provides a better exploit of olive by-products for functional food production, the development of pharmaceutical supplements and, cosmeceuticals [4]. Along the olive oil chain, olive mills produce tons of by-products including olive pomace (OP), olive mill wastewater (OWW), and olive leaves (OLs). These

**Citation:** Centrone, M.; D'Agostino, M.; Difonzo, G.; De Bruno, A.; Di Mise, A.; Ranieri, M.; Montemurro, C.; Valenti, G.; Poiana, M.; Caponio, F.; Tamma, G.et al. Antioxidant Efficacy of Olive By-Product Extracts in Human Colon HCT8 Cells. *Foods* **2021**, *10*, 11. http:// dx.doi.org/10.3390/foods10010011

Received: 12 November 2020 Accepted: 19 December 2020 Published: 23 December 2020

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latter are commonly used for animal feeding or direct combustion. Recent studies report that OLs contain certain bioactive compounds that may be useful for the preparation of nutraceuticals and supplements.

OP contains pulp, skin, stone and water. However, the chemical composition and the biological activity of OP may differ according to the oil extraction method. Pomace is mainly used to produce pomace olive oil and a more recent application includes the generation of fuel after microbiological fermentation processes [5]. In contrast, the disposal of the olive mill wastewater is still an expensive cost for the oil olive industry. Notably, recent reports propose new methods based on nanocentrifugation and ultrafiltration to revalorize the olive wastewater [6,7]. Chemical characterization studies revealed that some bioactive compounds are transferred from olives to oil and their by-products and waste during the oil production process [8]. Specifically, OP, OWW, and OLs contain bioactive compounds including secoiridoids, squalene, flavonoids, lignans, phytosterols, tocopherols, and phenols. Among them, phenols, which displayed a significant antioxidant capability, are extensively investigated in different fields of research. The highest bulk of phenols have been isolated in olive wastewater. By contrast, a low amount of phenols was detected in the oil [9]. Numerous studies investigated the functional involvement of phenols in human health and healthy aging. The biological actions of phenols may be due to their antioxidant and anti-inflammatory properties. Nevertheless, the molecular mechanism of action of these compounds, on different diseases, is still unclear because they may have multiple intracellular targets. In bronchial epithelial NCI-H292 cells, a green olive leaf extract (OLE), obtained using water as an extraction solvent, reduced the tBHP-induced reactive oxygen species (ROS) production. This extract indeed displayed a significant antioxidant capability in vegetable oil [10]. Furthermore, in renal collecting duct MCD4 cells, the green OLE counteracted the cytotoxic effects due to long exposure to low doses of cadmium [11]. Specifically, in cells exposed to cadmium, OLE reduced the frequency of micronuclei, DNA double-strand breaks, and anomalous alterations of the cytoskeleton by modulating the S-glutathionylation of actin [11]. Therefore, OLE may exert different cellular responses that may be only partially due to antioxidant features. The olive derived extracts and by-products contain numerous compounds such as hydroxytyrosol and oleuropein that can modulate different intracellular signal transduction pathways including the ones regulating autophagy and apoptosis [12,13]. Chronic and acute disturbances affecting the gastrointestinal tract such as inflammatory bowel diseases are associated with an abnormal intracellular level of reactive species due to a relevant unbalance between the ROS production systems and the antioxidant intestinal defenses [14]. Moreover, colonic mucosa of patients with chronic inflammatory diseases produced a high amount of ROS compared with normal mucosa [15]. Therefore, the administration of selective antioxidant phytocompounds may provide beneficial effects to counteract intracellular processes leading to abnormal production of ROS in the intestine. In the present study, the human colon HCT8 cells were used as an experimental model to assay the cell viability and the antioxidant activity of extracts deriving from olive by-products including OP, OWW, and OLs. HCT-8 cell line derived from the ileocecal colon displays a high proliferation and migration rate associated with high ROS production [16]. Moreover, compared with the fully differentiated HT-29 and Caco-2 colon cell lines, HCT-8 cells have poorly organized junctional complexes [16,17]. Interestingly, here, with the same phenolic concentration, the extract obtained from the olive wastewater displayed a higher antioxidant activity compared with the extracts derived from olive pomace and leaves.

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

#### *2.1. Chemicals and Reagents*

Cell culture media and FBS (fetal bovine serum) were from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Dihydrorhodamine-123 was obtained from Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). The tert-Butyl hydroperoxide (tBHP) was a kind gift from A. Signorile (University of Bari Aldo Moro, Bari, Italy).

#### *2.2. Extraction of Phenolic Compounds from By-Products*

#### 2.2.1. Olive Wastewater (OWW)

OWW was collected during the crop seasons 2019/2020 from Ottobratica olive cultivar and produced according to a three-phase centrifugation process. The phenolic extract was obtained following the method reported by Romeo et al. [18] with some modifications. Two liters of OWW was acidified to pH 2 with HCl and washed three times with hexane (1:1, *v*/*v*) to remove the lipid fraction. The mixture was vigorously shaken and centrifuged under 3000 rpm for 3 min at 10 ◦C. The extraction procedure was carried out using ethyl acetate three times and the solvent was recovered in a separating funnel (1:4, *v*/*v*). The twophases (water and ethyl acetate) were separated and both were evaporated using a rotary vacuum evaporator at 25 ◦C. Finally, the dry residues were again dissolved in 100 mL of water, filtered using PTFE 0.45 μm (diameter 15 mm) syringe filter, and stored at 4 ◦C until subsequent analyses.
