**1. Introduction**

Olive mill wastewaters (OMWWs) are byproducts of the olive oil production process, causing significant problems such as soil contamination and eutrophication, when they are discarded in the environment. Although OMWWs have been extensively considered as a byproduct, several studies have shown that they are rich in polyphenolic compounds (e.g., oleuropein, tyrosol, hydroxytyrosol, coumaric acid, ca ffeic acid, vanillic acid, ferulic acid, kaempferol, and quercetin) with important biological activities [1]. For example, phenolic compounds are widely known for their antioxidant properties [2]. Our research group has previously demonstrated that administration of feed supplemented with polyphenols from OMWW improves the redox status in farm animals [3–5].

Polyphenolic extracts from OMWW may be used as food supplements or preservatives, but their unpleasant bitter taste is a significant problem. Furthermore, the polyphenolic compounds can be chemically unstable under the influence of temperature, light, and oxygen [6]. Therefore, the encapsulation of polyphenolic extracts is an e ffective way to preserve their stability and bioactivity, while overcoming the problem of bad taste and simultaneously enhancing their bioavailability [7].

Spray drying encapsulation is a common and popular technique in terms of production cost, short process time, and low thermal stresses [8].

The entire vascular system consists of a monolayer of endothelial cells, the integrity of which is necessary to maintain a harmonic circulatory function. Apart from that, the endothelium regulates the homeostasis as well as the immune and inflammatory responses of the body [9,10]. Oxidative stress can severely damage the endothelium; thus, it constitutes one of the most important factors of pathological conditions of the vessels, along with atherosclerosis and thrombosis [10].

Thus, the aim of the present study was initially to evaluate the antioxidant potency of a polyphenolic extract from OMWW, encapsulated in three different carrier agents (i.e., maltodextrin, whey protein and gelatin) under different conditions, thus producing 17 samples. Then, the two samples exhibiting the greatest antioxidant activity (i.e., 3 and 9) were chosen in order to assess their antioxidant effects on the redox status of human endothelial cells.

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

#### *2.1. Preparation of Encapsulation Powders*

The liquid raw material that was used for the production of the OMWW originating antioxidant powders was supplied by Polyhealth S.A. (Larissa, Greece). This liquid raw material was a standard product of Polyhealth S.A., commercialized under the trademark 'MEDOLIVA® Liquid'. The polyphenolic composition of MEDOLIVA® liquid as assessed by HPLC analysis has been previously reported [11]. Thus, a mixed liquid material was prepared by mixing MEDOLIVA® liquid (an aqueous solution) with Twin 80 (liquid surfactant) and with either plain or mixed encapsulation agents (i.e., whey protein C80, maize maltodextrin DE18, and gelatin) according to the recipes given in detail in Table 1. Consequently, the mixture was thoroughly homogenized by ultrasonic energy. Finally, the homogenized aqueous solution was spray dried for water removal and production of 17 dry encapsulated OMWW polyphenol powders. A BUCHI Spray Dryer model B-290 connected with a B296 Dehumidifier was used to carry out the spray drying. The operating conditions used for each recipe are presented in Table 1. The conditions and the ingredient ratios given in Table 1 were selected after more than 100 trials (data not shown) and only 17 were found to be acceptable on the basis of giving good quality and stable powder at a yield of more than 75% of the initial aqueous solution dry matter. The initial temperature range tested was from 100 to 220 ◦C, while the gelatin percentage tested was from 5 to 30 *w*/*w*% of the maltodextrin or whey protein. In many cases, the tested conditions led to a failure to obtain powder and the finished product was slurry type or very sticky due to unfavorable glass transition conditions.



#### *2.2. Free-Radical Scavenging Activity*

The free-radical scavenging activity of the powders was evaluated using the 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•<sup>+</sup>) and 2,2-diphenyl-picrylhydrazyl (DPPH•) radical scavenging assays as previously described [12].

In the DPPH assay, 950 μL of 100 μM methanolic solution of DPPH• was mixed with 50 μL of the tested powder at different concentrations. Regarding the DPPH• assay, 1.0 mL of freshly made methanolic solution of DPPH• radical (100 μM) was mixed with the powder solution at different concentrations. The contents were vigorously mixed, incubated at room temperature in the dark for 20 min and the absorbance was measured at 517 nm. The measurement was conducted on a Hitachi U-1900 ratio beam spectrophotometer (Tokyo, Japan). In each experiment, the tested powder alone in methanol was used as blank and DPPH• alone in methanol was used as control.

In the ABTS•<sup>+</sup> assay, ABTS•<sup>+</sup> radical was produced by mixing 2 mM ABTS with 30 μM H2O2 and 6 μM horseradish peroxidase (HRP) enzyme in 1 mL of distilled water. The solution was vigorously mixed and incubated at room temperature in the dark for 45 min until ABTS•<sup>+</sup> radical formation. Then, 10 μL of different powder concentrations were added in the reaction mixture and the absorbance at 730 nm was read. The measurement was conducted on a Hitachi U-1900 ratio beam spectrophotometer (Tokyo, Japan). In each experiment, the tested powder in distilled water containing ABTS and H2O2 was used as blank, and the ABTS•<sup>+</sup> radical solution with 10 μL H2O was used as control.

The percentage of the radical scavenging capacity (RSC) of the tested powders for both assays was calculated according to the following equation:

$$\text{Radical scavenging capacity (\%)} = \text{[(A}\_{\text{control}} - \text{A}\_{\text{sample}}) / \text{A}\_{\text{control}}] \times 100 \tag{1}$$

where, Acontrol and Asample are the absorbance values of the control and the tested samples, respectively. Moreover, in order to compare the radical scavenging capacity of the samples, the IC50 value showing the concentration that induced 50% scavenging of DPPH• and ABTS•<sup>+</sup> was calculated. In both assays vitamin C was used as a positive control. All experiments were carried out in triplicate and at least on two separate occasions.

#### *2.3. Peroxyl-Radical-Induced Plasmid DNA Strand Cleavage*

The peroxyl-radical-induced DNA plasmid strand cleavage assay was performed as described previously [12]. In brief, peroxyl radicals (ROO) were produced from thermal decomposition of 2,2-azobis(2-amidinopropane hydrochloride) (AAPH). The reaction mixture (10 μL) containing 1 μg Bluescript-SK+ plasmid DNA, 2.5 mM AAPH in phosphate-buffered saline (PBS) and the tested powder at different concentrations was incubated in the dark for 45 min at 37 ◦C. Then, the reaction was stopped by the addition of 3 μL loading buffer (0.25% bromophenol blue and 30% glycerol). After analyzing the DNA samples by agarose gel electrophoresis, they were photographed and analyzed using the Alpha Innotech Multi Image (ProteinSimple, San Jose, CA, USA). In addition, plasmid DNA was treated with each powder alone at the highest concentration used in the assay in order to test their effects on plasmid DNA conformation. The percentage of the protective activity of the tested powders from ROO-induced DNA strand breakage was calculated using the following equation:

$$\% \text{ Inhibition} = \text{I} (\text{S} - \text{S}\_{\text{o}}) / (\text{S}\_{\text{control}} - \text{S}\_{\text{o}}) \text{I} \times 100 \tag{2}$$

where, Scontrol is the percentage of supercoiled DNA in the negative control (plasmid DNA alone), So is the percentage of supercoiled plasmid DNA in the positive control (without the tested powder but in the presence of the radical initiating factor), and S is the percentage of supercoiled plasmid DNA in the tested powder along with the radical initiating factor. Moreover, the IC50 values showing the concentration that inhibited the AAPH-induced DNA relaxation by 50% were calculated. Vitamin C was used as a positive control. At least three independent experiments were performed for each sample.
