**1. Introduction**

It is well known that within living organisms, the reactive oxygen species (ROS) are produced endogenously under physiological processes such as metabolism and inflammation [1,2]. Low ROS levels are needed for the progression of several basic biological processes including cellular proliferation and differentiation [2,3]. However, excessive intracellular concentration of ROS can lead to oxidative stress, a pathological condition associated with the development of various of diseases including

cancer, diabetes, rheumatoid arthritis and other degenerative diseases in humans [4,5]. Oxidative stress causes healthy cells of the body to lose their function and structure as a consequence of ROS interaction with proteins, lipids and DNA [6].

One of the tissues that is especially susceptible to oxidative stress is vascular endothelium, a critical regulator of vascular homeostasis. Endothelial cell injury or dysfunction by ROS can be both a cause and consequence of many vascular complications including atherosclerosis, thrombosis and cardiovascular diseases [7,8]. In particular, vascular endothelium cells are susceptible to ROS damage, since ROS derived from di fferent tissues circulate in the bloodstream and can interact directly with endothelial cells in the inner wall of blood vessels [9,10]. In addition, oxidative stress may cause damage to the endothelium through leukocyte adhesion [11].

However, human organism possesses several antioxidant mechanisms to counteract the overproduction and harmful e ffects of ROS [12]. The antioxidant protective mechanisms act in order to keep a balance between free radical production and scavenging. These antioxidants are either produced endogenously or obtained through diet, especially from plant foods [13]. The antioxidant proprieties of plant foods are mainly attributed to polyphenols, a large group of secondary metabolites, that possess antiradical activities due to their phenolic hydroxyls acting as reducing agents, metal ion chelators, antioxidant enzymes activators, and oxidases inhibitors [14,15]. Thus, there is currently a grea<sup>t</sup> interest in natural sources of antioxidants in order to improve the redox status and protect the organism from the detrimental e ffects of oxidative stress.

Many plant species of the *Rosaceae* family are considered to be of high importance because of their use in various food preparations as jam, tea and beverages. The *Rosa* genus of *Rosaceae* family consists of approximately 200 species located mainly in the Northern hemisphere in rainy areas and deserts [16]. *Rosa* species produce rose hips, a pseudocarp or a fruit. A number of studies have shown that rose hips demonstrate a grea<sup>t</sup> variety of bioactivities such as antioxidant, anticancer, anti-inflammatory and anti-obesity activities [17–20]. Wild fruits of *Rosa canina* are rich in vitamin C and are used for the prevention and/or treatment of many diseases including diabetes, flu, arthritis, inflammations, pain and diarrhea [17,18,21]. *Rosa sempervirens,* the evergreen rose, is a representative member of the *Rosa* species. It is a thorny, climbing rose characterized by long branches with few or no prickles. Moreover, it is an important source of vitamin C, carotenoids, polyphenols, organic acids and tocopherols [22]. *Pyracantha coccinea* of the genus *Pyracantha* of the *Rosaceae* family is a plant growing from South Europe to South-East Asia. Its fruits are known for their rich content in fatty acids, polyphenolic compounds, phytosterols and vitamins. Thus, they have been used in traditional medicines for cardiac and diuretic properties [23].

The aim of the present study was to investigate the antioxidant properties of polyphenolic extracts derived from the following three wild *Rosaceae* species of Greece; *Pyracantha coccinea* (fruit extract), *Rosa sempervirens* (fruit extract) and *Rosa canina* (fruit extract). The extracts were initially examined for their free radical scavenging activity against DPPH• and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS)•<sup>+</sup> radicals and for their protective e ffects against peroxyl (ROO•) radical-induced DNA damage. Moreover, for the first time, the ability of these extracts to enhance the antioxidant defense in human endothelial cells was assessed. As mentioned above, the oxidative stress-induced endothelium damage is a crucial etiological factor for cardiovascular diseases, and so the identification of compounds that could protect from such damage is important.

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

#### *2.1. Plant Material and Preparation of the Extracts*

Fresh plant material of the investigated *Rosaceae* family plants *Rosa sempervirens* (fruit), *Rosa canina* (fruit) and *Pyracantha coccinea* (fruit) were collected during early summer of 2014, from respective wild plants colonies that grow on Parnitha mountain (Attica Region, Greece). A voucher specimen for each sampling material has been deposited in the herbarium of the Agricultural University of Athens (Athens, Greece).

After air-drying, 500 g of each plant material were crushed, homogenized with a blender and lyophilized to provide a powder, which was stirred in darkness with 1 L of methanol (HPLC-analytical grade) for 48 h. Then, the solution was filtered and the solid was re-extracted twice following the same procedure. The combined methanolic extracts (3 L in total) were evaporated to dryness under reduced pressure to provide crude extracts, which were further elaborated for the assessment of their chemical content and bioactivities.

#### *2.2. Determination of Extracts' Polyphenolic Content Using HPLC and Ultra Performance Liquid Chromatography–Tandem Mass Spectrometer (UPLC-MS-MS) Analysis*

The chemical composition of the extracts was determined using HPLC analysis performed on a Hewlett Packard HP1100 (Hewlett Packard, Palo Alto, CA, USA) equipped with an Agilent 1100 diode-array detector (Agilent Technologies, Santa Clara, CA, USA) (measuring absorbance over the full wavelength range during the entire run), a quaternary pump, degasser and coupled to HP ChemStation utilizing the manufacturer's 5.01 software package system (Hewlett Packard, Palo Alto, CA, USA).

The column used was a Zorbax Eclipse Plus C18, 5 μm, 150 × 4.6 mm i.d. chromatographic column (Agilent Technologies, Santa Clara, CA, USA), connected with a guard column of the same material (8 × 4 mm). Injection was by means of a Rheodyne injection valve (model 7725I) with a 20 μL fixed loop. For the chromatographic analyses HPLC-grade water was prepared using a Milli-Q system (Merck Millipore, Burlington, MA, USA), whereas all HPLC solvents (except acetonitrile) were filtered prior to use through cellulose acetate membranes of 0.45 μm pore size.

The mobile phase was composed of a gradient system of 0.3% acetic acid in water (A) and acetonitrile (B). The flow rate was maintained at 1 mL/min and the column gradient elution program consisted of: 25% B (0 min), 25% B (5 min), 30% B (10 min), 40% B (15 min), 50% B (20 min) and 70% B (30 min) where it remained for additional 5 min, and returned during 2 min to initial conditions, where it stayed for additional 2 min. This routine was followed by a 15 min equilibration period with the zero-time mixture prior to injection of the next sample. Prolonged runtimes (extended until 100 min) were also applied to determine constituents that elute after the 35 min (betulinic and ursolic acids). Peaks were identified by comparing their retention times and UV–vis spectra with the reference compounds, and data were quantitated using the corresponding curves of the reference compounds as standards (Extrasynthese, Genay Cedec, France; Sigma-Aldrich, St. Louis, MO, USA; Alfa Aesar, Haverhill, MA, USA; Fluka, Buchs, Switzerland). Confirmatory UPLC-MS-MS analysis was carried out on a Thermo Scientific Ultra High Performance Liquid Chromatography system (Waltham, MA, USA) coupled to a TSQ Quantum Vantage (Thermo Fischer Scientific, San Jose, CA, USA) triple quadrupole mass spectrometer. Mass spectrometric analysis was conducted using a heated electrospray ionization (HESI) operating in two complementary modes (positive and negative mode). Selected ion monitoring (SIM) mode was primarily used to confirm the presence of analytes. In selected cases of compounds, tandem mass spectrometry (MS/MS) utilizing the multiple reaction monitoring mode (MRM) was employed for additional confirmation [quinic acid: parent ion, *m*/*z* 191 ( *m*/*z* 85, 93 product ions), rutin: parent ion, *m*/*z* 609 ( *m*/*z* 300, 271 product ions), quercetin: parent ion, *m*/*z* 300.9 ( *m*/*z* 179, 151 product ions), quercitrin: parent ion, *m*/*z* 447.1 ( *m*/*z* 301, 300, 271 product ions)]. The working conditions were the following: spray voltage 4.2 kV; vaporizer and capillary temperatures 280 and 260 ◦C respectively, while sheath and auxiliary gas at 60 and 40 arbitrary units respectively. The LC separation was achieved on a Hypersil Gold. 3 μm. 150 × 3 mm i.d. chromatographic column (Thermo Fischer Scientific, San Jose, CA, USA). The mobile phase and the gradient system were identical to the above mentioned for the HPLC-UV analysis, using a flow rate of 0.3 mL/min. Water, acetonitrile, and acetic acid were purchased from Merck (Darmstadt, Germany) and all were LC-MS grade. PTFE filters (0.45 μm) were obtained from Macherey-Nagel, Duren, Germany. All measurements were repeated three times.

#### *2.3. Analytical Method Validation*

With respect to the analytical method validation part, the linearity for all analytes was determined within the ranges of 10–1000 ng/mL (using matrix matched calibration standards), demonstrating acceptable correlation coefficient values (*r2* ≥ 0.99). Recovery of the investigated compounds (as a criterion of the trueness of the method) was evaluated at two concentration levels (40 and 200 ng/mL) by the addition of mixed solutions of the standards into the respective extract and fell within the acceptable range of 70%−120%. Precision values were always acceptable with percent Relative Standard Deviation (RSD%) < 15%.

#### *2.4. Assessment of the Total Polyphenolic Content of the Extracts*

The total polyphenolic content (TPC) of the extracts was evaluated by the Folin-Ciocalteu method as described previously [24]. Briefly, 20 μL of the extract were added to a tube containing 1 mL of deionized water. 100 μL of Folin-Ciocalteu reagen<sup>t</sup> was added to the reaction mixture, followed by incubation for 3 min at room temperature. Afterwards, 280 μL of 25% *w*/*v* sodium carbonate solution and 600 μL of deionized water were added to the mixture. Following 1 h incubation at room temperature in the dark, the absorbance was measured at 765 nm versus a blank containing Folin-Ciocalteu reagen<sup>t</sup> and distilled water without the extract. The measurement of absorbance was conducted on a Hitachi U-1900 ratio beam spectrophotometer (Tokyo, Japan). The optical density of the sample (20 μL) in 25% *w*/*v* solution of sodium carbonate (280 μL) and distilled water (1.7 mL) at 765 nm was also measured. TPC was determined by a standard curve of absorbance values in correlation with standard concentrations (50–1500 μg/mL) of gallic acid. The total polyphenolic content was expressed as mg of gallic acid equivalents (GAE) per gram of dried weight (dw) of extract.

#### *2.5. Total Flavonoid Content of the Extracts*

The total flavonoid content (TFC) of the extracts was evaluated as described previously with minor changes [25]. In particular, 1 mL of the methanolic extract was added into a 10 mL flask containing 4 mL of deionized water. Then, 0.3 mL of sodium nitrite (5%) were added to this mixture and allowed to stand for 5 min at room temperature. Then, 0.3 mL of AlCl3·6H2O (10% ethanolic) was added, the mixture was allowed to stand for 1 min at room temperature and 2 mL of potassium hydroxide (1 M) was added. The solution was diluted to 10 mL with the addition of deionized water and the absorbance of the solution versus a blank at 510 nm was measured immediately. Flavonoid content was expressed as mg of catechin equivalents (CE) per gram of dry weight of extract by using a standard curve (absorbance versus concentration) prepared from authentic catechin sample.

#### *2.6. Free Radical Scavenging Activity*

Free radical scavenging activity of the extracts was evaluated using the 2,2-diphenyl-picrylhydrazyl (DPPH•) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•<sup>+</sup>) radical scavenging assays [26,27]. Regarding the DPPH• assay, 1.0 mL of freshly-made methanolic solution of DPPH• radical (100 μM) was mixed with the tested extract 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 sample alone in methanol was used as blank and DPPH• alone in methanol was used as control. ABTS•<sup>+</sup> radical scavenging activity of the extracts was determined as described previously [27] with slight modifications. Briefly, 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, incubated at room temperature in the dark for 45 min until ABTS•<sup>+</sup> radical formation. Then, 10 μL of different extract 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 sample 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 radical scavenging capacity (RSC) of the tested extracts, for both assays was calculated according to the following equation:

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

where Acontrol and Asample are the absorbance values of the control and the tested sample respectively. Moreover, in order to compare the radical scavenging e fficiency of the extracts, the IC50 value showing the concentration that caused 50% scavenging of DPPH• and ABTS•<sup>+</sup> radical was calculated from the graph plotted RSC percentage against extract concentration. All experiments were carried out in triplicate and at least on two separate occasions.

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

The peroxyl radical-induced DNA plasmid strand cleavage assay was performed as described previously [28]. 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-bu ffered saline (PBS) and the tested extract at di fferent concentrations was incubated in the dark for 45 min at 37 ◦C. Then, the reaction was stopped by the addition of 3 μL loading bu ffer (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 extract alone at the highest concentration used in the assay in order to test their e ffects on plasmid DNA conformation. The percentage of the protective activity of the tested extracts from ROO•-induced DNA strand breakage was calculated using the following formula:

$$\text{\textbullet\text{\textbullet Inhibition}} = \text{[(S - S\_o) / (S\_{\text{control}} - S\_o)]} \times 100$$

where Scontrol is the percentage of supercoiled DNA in the negative control sample (plasmid DNA alone), So is the percentage of supercoiled plasmid DNA in the positive control sample (without tested extracts but in the presence of the radical initiating factor) and S is the percentage of supercoiled plasmid DNA in the sample with the tested extracts and the radical initiating factor. Moreover, IC50 values showing the concentration that inhibited the AAPH-induced relaxation by 50% were calculated from the graph plotted percentage inhibition against extract concentration. At least two independent experiments in triplicate were performed for each tested compound.

#### *2.8. Cell Culture Conditions*

As previously described [29], human endothelial EA.hy926 cells were cultured in normal Dulbecco's modified Eagle's medium (DMEM) in plastic disposable tissue culture flasks at 37 ◦C in 5% carbon dioxide.

#### *2.9. XTT Cell Viability Assay*

For examining the extracts' antioxidant activity in endothelial cells, non-cytotoxic concentrations were used. For selecting these concentrations, extracts' cytotoxicity in endothelial cells was checked using the XTT cell viability assay kit (Roche, Switzerland) as previously described [30]. Briefly, EA.hy926 cells were seeded into a 96-well plate with 1 × 10<sup>4</sup> cells per well in DMEM medium. After 24 h incubation, the cells were treated with di fferent concentrations of the extracts in serum-free DMEM medium for 24 h. Then, 50 μL of XTT test solution was added to each well. After 4 h of incubation, absorbance was measured at 450 nm and also at 630 nm as a reference wavelength in a Bio-Tek ELx800 microplate reader (Winooski, VT, USA). The negative control was DMEM serum-free

medium. The absorbance values of the control and samples were used for calculating the percentage inhibition of cell growth caused by the extract treatment. All experiments were carried out in triplicate and on two separate occasions.

#### *2.10. Treatment of EA.hy926 Cells with the Extracts*

The extracts from *R. sempervirens*, *R. canina* and *P. coccinea* were examined for their antioxidant capacity in endothelial EA.hy926 cells. The cells were cultured in flasks for 24 h. Afterwards the medium was replaced with a serum-free medium containing the tested extracts at non-cytotoxic concentrations. The cells were treated with the extracts for 24 h, and then they were trypsinized, collected and centrifuged twice at 300× *g* for 10 min at 5 ◦C. At the end of the first centrifugation, the supernatant fluid was discarded and the cellular pellet was resuspended in PBS. After the second centrifugation, the cell pellet was collected and used for measuring the glutathione (GSH) and ROS levels.

#### *2.11. Assessment of GSH and ROS Levels by Flow Cytometry Analysis in Endothelial Cells*

The GSH and ROS levels in EA.hy926 cells were assessed using mercury orange and DCF-DA, respectively, as described previously [31,32]. In brief, the cells were resuspended in PBS at 1 × 10<sup>6</sup> cells/mL and incubated in the presence of mercury orange (10 μM) or 2,7-Dichlorofluorescin diacetate (DCF-DA) (40 μM) respectively, in the dark at 37 ◦C for 30 min. Then, the cells were washed, resuspended in PBS, and submitted to flow cytometric analysis using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with excitation and emission wavelengths at 488 and 530 nm for ROS and at 488 and 580 nm for GSH. Data were analyzed using 'BD Cell Quest' software (Becton Dickinson, Franklin Lakes, NJ, USA). Each experiment was repeated at least three times.
