**3. Results**

#### *3.1. Extract Composition in Bioactive Compounds*

The TPC values of the extracts were 267.67, 290.00 and 226.93 mg GAE/g dw, while the TFC values were 65.78, 118.56 and 99.16 mg CE/gr dw for *R. sempervivens*, *R. canina* and *P. coccinea*, respectively (Table 1).


**Table 1.** Bioactive compounds of extracts from dried fruits *R. canina*, *R. semprevirens* and *P. coccinea*.


**Table 1.** *Cont.*

ND: not detected. a Values are expressed as μg/g of dried weight of extract and are the mean ± SD from three measurements. b TPC: Total Polyphenolic Content, expressed as mg of gallic acid equivalent/g dried weight extract. c TFC: Total Flavonoid Content, expressed as mg of catechin equivalent/g dried weight extract. d,e,f Values with different superscript letters are significantly different between them (*p* < 0.05). g,h,i Values with different superscript letters are significantly different between them (*p* < 0.05).

The results from the qualitative and quantitative assessment of the chemical composition of the extracts as assessed by using HPLC with Diode-Array Detection complemented by UPLC-MS-MS (especially for the quantitative analysis of non-UV sensistive compounds) analysis are depicted in Table 1. The HPLC analysis of the extracts identified polyphenols belonging to different subclasses of flavonoids such as flavanols (e.g., (+)-catechin and (−)-epicatechin), flavonols (e.g., hyperoside, rutin, astragalin, quercitrin, quercetin and kaempferol), flavanonols (e.g., taxifolin), flavanones (e.g., eriodictyol and isosakuranetin), isoflavones (e.g., genistein). In this respect, various polyphenolic acids were also detected such as hydroxybenzoic acids (e.g., gallic acid, syringic acid and protocatechuic acid), hydroxycinnamic acids (e.g., caffeic acid and *p*-coumaric acid) and the chlorogenic acid. Finally, a series of additional molecules were identified such as phloridzin and vanillin polyphenols, quinic acid and the terpenoids betulinic acid and ursolic acid. The latter compounds were detected only in *R. canina* extract.

Specifically, the chemical analysis showed that the *R. canina* extract is particularly rich in polyphenols hyperoside (308.11 μg/g dw), astragalin (172.48 μg/g dw), (+)-catechin (134.75 μg/g dw) and (−)-epicatechin (120.99 μg/g dw) (Table 1). Moreover, *R. canina* extract contained significant concentration of quinic acid (1102.59 μg/g dw) and the terpenoid, ursolic acid (138.23 μg/g dw) (Table 1). In *R. sempervirens* extract, the compounds identified at higher concentrations were quinic acid (389.95 μg/g dw), and the polyphenols (−)-epicatechin (34.01 μg/g dw), (+)-catechin (25.48 μg/g dw), astragalin (9.16 μg/g dw), and hyperoside (8.31 μg/g dw) (Table 1). Finally, *P. coccinea* extract exhibited higher concentrations of hyperoside (170.72 μg/g dw), rutin (25.82 μg/g dw), (−)-epicatechin (10.23 μg/g dw), astragalin (9.13 μg/g dw), (+)-catechin (7.93 μg/g dw), vanillin (7.89 μg/g dw) and syringic acid (6.23 μg/g dw) (Table 1).

#### *3.2. Free Radical Scavenging Activity of the Extracts*

All three of the extracts exhibited strong free radical scavenging activity. As known, the lower the IC50 value, the higher the antioxidant activity. Thus, in the DPPH assay the potency order and the IC50 values were: *R. canina* (100 μg/mL) > *R. sempervivens* (130 μg/mL) > *P. coccinea* (500 μg/mL) (Table 2). Similar order of portency was observed in ABTS•<sup>+</sup> assay; *R. canina* (60 μg/mL) > *R. sempervivens* (85 μg/mL) > *P. coccinea* (140 μg/mL) (Table 2).


**Table 2.** Free radical scavenging activity against DPPH and ABTS radicals, protective activity against peroxyl radical (ROO•)-induced DNA damage of the extracts.

a Values are the mean ± SD of at least two separate triplicate experiments. b Values are the mean ± SD from three independent experiments. \* *p* < 0.05, indicates significant difference from the control values. a,b,c Values with different superscript letters are significantly different between them (*p* < 0.05). d,e,f Values with different superscript letters are significantly different between them (*p* < 0.05). g,h Values with different superscript letters are significantly different between them (*p* < 0.05).

Finally, all three extracts exhibited protective activity against ROO•-induced DNA plasmid breakage with IC50 values and potency order *R. canina* (530 μg/mL) > *R. sempervivens* (570 μg/mL) > *P. coccinea* (950 μg/mL) (Table 2 and Figure 1).

**Figure 1.** Protective activity of polyphenolic extracts from ( **A**) *Pyracantha coccinea*, (**B**) *Rosa sempervivens* and ( **C**) *Rosa canina* species against ROO• radical: Lane 1, pBluescript-SK+ plasmid DNA without any treatment; lane 2, plasmid DNA exposed to ROO• radical alone; lanes 3–8 plasmid DNA exposed to ROO• radical in the presence of different concentrations of extracts (*P. coccinea*: 0.063, 0.125, 0.250, 0.500, 1.0 and 1.5 mg/mL; *R. sempervivens*: 2.0, 1.5, 1.0, 0.500, 0.250, 0.125 mg/mL; *R. canina*: 0.063, 0.125, 0.250, 0.500, 1.0 and 1.5 mg/mL); lane 8, plasmid DNA exposed to the maximum tested concentration of each extract alone. OC: open circular; SC: supercoiled.

#### *3.3. E*ff*ects of Extracts on the Antioxidant Status of Endothelial Cells*

To examine the extracts' antioxidant activity in endothelial cells, flow cytometry analysis was performed. At first, the extract's e ffect on cell viability was assessed using the XTT assay, in order to use non-cytotoxic concentrations. The cell viability assay showed that significant cytotoxicity was exhibited at concentrations above 2.5 mg/mL for *R. sempervivens* and 2.0 mg/mL for *R. canina* (Figure 2B,C). None of the concentrations used for *P. coccinea* had cytotoxicity (Figure 2A). Thus, the selected non-cytotoxic concentrations of the extracts in the following assays were up to 1.00 mg/mL.

**Figure 2.** Cell viability following the treatment with polyphenolic extracts from (**A**) *Pyracantha coccinea*, (**B**) *Rosa sempervivens* and (**C**) *Rosa canina* species. The results are presented as the means ± SD of three independent experiments carried out in triplicate. \* *p* < 0.05 indicates significant difference from the control value.

The assessment of the extracts' effects on the antioxidant capacity of endothelial cells was based on the measurement of GSH and ROS levels by flow cytometry analysis. The results demonstrated that *R. canina extract* significantly increased GSH levels by 15.0, 10.4, 28.4 and 43.1% at 0.13, 0.25, 0.50 and 1.00 mg/mL, respectively compared to control (Figure 3C). *P. coccinea* extract also significantly increased GSH levels by 29.2 and 32.3% at 0.50 and 1.00 mg/mL, respectively, compared to control (Figure 3A). However, *R. sempervirens* extract did not affect GSH levels at any of the examined concentrations (Figure 3B).

**Figure 3.** Effects on GSH levels after treatment with *P. coccinea*, *R. sempervivens* and *R. canina* extracts at different concentrations for 24 h in EA.hy926 cells. The histograms of cell counts versus fluorescence of 10,000 cells analyzed by flow cytometry for the detection of GSH levels after treatment with (**A**) *P. coccinea*, (**B**) *R. sempervivens* and (**C**) *R. canina*. FL-2 represents the detection of fluorescence in the FL-2 channel using 488 and 580 nm as the excitation and emission wavelength, respectively. Bar charts indicate the GSH levels as % of control as estimated by the histograms in EA.hy926 cells after treatment with (**D**) *P. coccinea*, (**E**) *R. sempervivens* and (**F**) *R. canina* extracts. All values of bar charts are presented as the mean ± SD of three independent experiments. \* *p* < 0.05 indicates significant difference from the control.

The results from the assessment of extracts' effects on ROS levels are shown in Figure 4. According to the results, only one of the three extracts affected ROS levels. In particular, *R. canina extract* significantly reduced ROS by 9.73 and 13.37% at 0.50 and 1.00 mg/mL, respectively, compared to control

(Figure 4C). However, *P. coccinea* and *R. sempervivens* extracts did not significantly affect ROS levels, compared to control (Figure 4A,B).

**Figure 4.** The diagrams show the changes in ROS levels after treatment with *P. coccinea*, *R. sempervivens* and *R. canina* extracts in EA.hy926 cells. The histograms demonstrate the cell counts versus fluorescence of 10,000 cells analyzed by flow cytometry for the detection of ROS levels after treatment with (**A**) *P. coccinea*, (**B**) *R. sempervivens* and (**C**) *R. canina*. FL-1 represents the detection of fluorescence in the FL-1 channel using 488 and 530 nm as the excitation and emission wavelength, respectively. Bar charts indicate the ROS levels as % of control as estimated by the histograms in EA.hy926 cells after treatment with (**D**) *P. coccinea*, (**E**) *R. sempervivens* and (**F**) *R. canina* extracts. All values of bar charts are presented as the mean ± SD of three independent experiments. \* *p* < 0.05 indicates significant difference from the control.
