Antioxidant Activity and Phytochemical Characterization of Senecio clivicolus Wedd.

Antioxidant phytochemicals play a key role in oxidative stress control and in the prevention of related disorders, such as premature aging, degenerative diseases, diabetes, and cancer. The aim of this study was to investigate the potential antioxidant activity and the phytochemical profile of Senecio clivicolus Wedd., a perennial shrub, belonging to the Asteraceae family. Despite the wide interest of this family, this specie has not been investigated yet. S. clivicolus aerial parts were extracted with 96% ethanol. Then, the ethanol extract was fractionated by liquid/liquid extraction using an increasing solvents polarity. Total polyphenol and terpenoid contents were measured. Moreover, the antioxidant activity was evaluated by six different complementary in vitro assays. The Relative Antioxidant Capacity Index (RACI) was used to compare data obtained by different tests. The sample showing the highest RACI was subjected to characterization and quantitation of its phenolic composition using LC-MS/MS analysis. The ethyl acetate fraction, investigated by LC-MS/MS analysis, showed 30 compounds, most of them are chlorogenic acid and flavonoid derivatives. To the best of our knowledge, this is the first report about the evaluation of antioxidant activity and phytochemical profile of S. clivicolus, underlying the importance of this species as a source of health-promoting phytochemicals.


Introduction
Plants are an important source of bioactive compounds with antioxidant capacity and during the last years, the interest for the use of natural products has significantly increased. The protective effects of plant secondary metabolites can be attributed to direct scavenging activities against reactive oxygen species (ROS), as well as to the induction of intracellular antioxidant effect [1,2]. In fact, different epidemiological studies have shown as the decrease of premature death and mortality from cancer or other chronic diseases are associated with antioxidant-rich diet including fruit, vegetables, and other botanicals [3,4]. Reactive oxygen and nitrogen species (RNS) are physiologically produced during metabolic processes and especially during electron transport chain reactions [4], low concentration of these species is essential for several biochemical processes. An important endogenous antioxidant system compensates the production of ROS, RNS, and other free radicals, but an overall increase highest extraction yield (63.39 ± 5.23%); instead, the water and ethyl acetate fractions demonstrated the lower extraction yields, 7.60 ± 0.42 and 6.87 ± 0.39%, respectively. Total phenolic content (TPC) of Sc EtOH and its fractions was carried out based on the reaction of the samples with Folin-Ciocalteu reagent, which results in a blue colored solution whose intensity was directly proportional to the amount of phenolic compounds present. Gallic acid was used as standard and results were expressed as milligram of gallic acid equivalents per gram of dry extract (mg GAE/g) and shown in Figure 2. TPC ranged from 24.32 ± 0.43 to 170.11 ± 1.49 mg GAE/g in ScW and ScEA fractions, respectively. Out of all samples, ScEA had the highest value, followed by ScB and Sc EtOH (82.50 ± 1.94 and 48.31 ± 1.32 mg GAE/g, respectively).

Figure 2.
Total polyphenolic content (TPC) and total terpenoids content (TTeC). Results were expressed as mean ± standard deviation in mg of gallic acid equivalents per gram of dried sample (mg GAE/g), and in mg of linalool equivalents per gram of dried sample (mg LE/g). In each tests, the values with the same letter are not significant different at the p < 0.05 level, according to oneway analysis of variance (ANOVA). Samples are crude ethanol extract (Sc EtOH), n-hexane fraction Total phenolic content (TPC) of Sc EtOH and its fractions was carried out based on the reaction of the samples with Folin-Ciocalteu reagent, which results in a blue colored solution whose intensity was directly proportional to the amount of phenolic compounds present. Gallic acid was used as standard and results were expressed as milligram of gallic acid equivalents per gram of dry extract (mg GAE/g) and shown in Figure 2. TPC ranged from 24.32 ± 0.43 to 170.11 ± 1.49 mg GAE/g in ScW and ScEA fractions, respectively. Out of all samples, ScEA had the highest value, followed by ScB and Sc EtOH (82.50 ± 1.94 and 48.31 ± 1.32 mg GAE/g, respectively). Total phenolic content (TPC) of Sc EtOH and its fractions was carried out based on the reaction of the samples with Folin-Ciocalteu reagent, which results in a blue colored solution whose intensity was directly proportional to the amount of phenolic compounds present. Gallic acid was used as standard and results were expressed as milligram of gallic acid equivalents per gram of dry extract (mg GAE/g) and shown in Figure 2. TPC ranged from 24.32 ± 0.43 to 170.11 ± 1.49 mg GAE/g in ScW and ScEA fractions, respectively. Out of all samples, ScEA had the highest value, followed by ScB and Sc EtOH (82.50 ± 1.94 and 48.31 ± 1.32 mg GAE/g, respectively). Total polyphenolic content (TPC) and total terpenoids content (TTeC). Results were expressed as mean ± standard deviation in mg of gallic acid equivalents per gram of dried sample (mg GAE/g), and in mg of linalool equivalents per gram of dried sample (mg LE/g). In each tests, the values with the same letter are not significant different at the p < 0.05 level, according to oneway analysis of variance (ANOVA). Samples are crude ethanol extract (Sc EtOH), n-hexane fraction Total polyphenolic content (TPC) and total terpenoids content (TTeC). Results were expressed as mean ± standard deviation in mg of gallic acid equivalents per gram of dried sample (mg GAE/g), and in mg of linalool equivalents per gram of dried sample (mg LE/g). In each tests, the values with the same letter are not significant different at the p < 0.05 level, according to one-way analysis of variance (ANOVA). Samples are crude ethanol extract (Sc EtOH), n-hexane fraction (ScH), chloroform fraction (ScC), ethyl acetate fraction (ScEA), n-butanol fraction (ScB), and water fraction (ScW). Moreover, total terpenoids content (TTeC) was evaluated for their characteristic reddish brown color at 538 nm. The linalool, a monoterpene, was used as standard reagent and results ( Figure 2) were expressed as mg of linalool equivalents per gram of dry sample (mg LE/g). TTeC ranged from 470.77 ± 3.61 to 2074.24 ± 12.76 mg LE/g in ScEA and ScC fractions, respectively. ScC, followed by ScB, ScW, and Sc EtOH, exhibited higher values.
Results demonstrated as the yield of phenolic and terpenoid clearly differs on the basis of solvent polarity. Fractions obtained by using more polar solvents reported the highest total polyphenols, indicating that the majority of polyphenolic compounds in the aerial parts of S. clivicolus could be of polar nature, data congruent with what reported previously in literature [32].

Antioxidant Activity
Low concentrations of biological radicals are important in the human body; in fact, they are involved in biological functions as vascular homeostasis, neurotransmission, antimicrobial, antioxidant and antitumor activities. In contrast, the overproduction of biological radicals is associated with several diseases [1]. Phenolic compounds are gaining attention due to their significant antioxidant activities. Different in vitro assays were reported for the measurement of antioxidant activity on foods and plants and it has been demonstrated as more than one method is necessary to elucidate the antioxidant capacity of samples because these assays differ in the principles and experimental conditions. In this study, the antioxidant activity of the Sc EtOH extract and its fractions (ScH, ScC, ScEA, ScB, and ScW) were tested using six different complementary in vitro antioxidant assays.
In particular, the radical scavenging activity was evaluated by using synthetic cationic and neutral (ABTS and DPPH) and physiological (superoxide anion and nitric oxide) radicals. The ScEA showed the highest radical scavenging-activity in all investigated tests (Table 1). In particular, the ScEA reported 409.53 ± 9.53 mg TE/g and 317.53 ± 5.81 mg TE/g values in ABTS and DPPH assays respectively, followed by ScB and Sc EtOH. Instead, in both assays, the lowest activity was found in ScH and ScC fractions. Samples are crude ethanol extract (Sc EtOH), n-hexane fraction (ScH), chloroform fraction (ScC), ethyl acetate fraction (ScEA), n-butanol fraction (ScB) and water fraction (ScW). Data are expressed as means ± standard deviation from three experiments; mg GAE/g = mg of gallic acid equivalents per gram of dried sample; mg TE/g = mg of Trolox equivalents per gram of dried sample; IC 25 mg/mL = concentration of the sample required to inhibit the activity of the radical by 25%; %AA = percentage of antioxidant activity at initial sample concentration of 1 mg/mL; different superscripts in the same row indicate significant difference (p < 0.05); nc = not calculable.
The ability of samples to scavenge superoxide anion and nitric oxide was expressed as IC 25 , as it was not possible to reach 50% inhibition at tested concentrations, and the results were compared with ascorbic acid. The S. clivicolus extract and fractions caused a dose-dependent inhibition in the superoxide anion assay (SO) and ScEA fraction showed the highest inhibition, that means the lowest IC 25 value (IC 25 of 0.08 ± 0.00 mg/mL), followed by ScC, ScH, and ScB (Table 1). These samples showed a higher activity than that measured for ascorbic acid (IC 25 of 0.26 ± 0.02 mg/mL).
The scavenging ability against the biological nitric oxide ( · NO) was not detectable in any fraction, but ScEA showed a dose-dependent inhibition ability (IC 25 of 1.11 ± 0.05 mg/mL); its value was four times lower than ascorbic acid (IC 25 4.78 ± 0.09 mg/mL), demonstrating its effectiveness.
The reducing ability of samples in a redox reaction was assessed using the FRAP test. ScEA fraction also in this case showed the highest activity (507.66 ± 5.26 mg TE/g) followed by ScB fraction (184.18 ± 4.59 mg TE/g). ScC and ScH were again the less active (Table 1).
To get a broader overview of the antioxidant potential of the plant complex of S. clivicolus samples, the β-carotene bleaching test (BCB) was conducted. The phenolic compounds compete with β-carotene for binding to the radical derived from linoleic acid and prevent the destruction of the conjugated system of the molecule responsible for coloring, for which, the higher is the antioxidant activity of the extract, the greater is the concentration of β-carotene in solution. Results were expressed as percentage of antioxidant activity (%AA) at the initial sample concentration of 1 mg/mL ( Table 1). The analysis evidenced that the most active sample in BCB test was the ScEA, followed by Sc EtOH and ScB (55.82 ± 2.22, 53.11 ± 0.45 and 51.70 ± 1.97 %AA, respectively). While, there was a low activity for the apolar ScH fraction.
Moreover, correlations were calculated based on the average of the results by the Pearson test. This test calculates the linear correlation coefficient (r), a dimensionless number between -1 and 1, inclusive, reflecting the extent of a linear relationship between two datasets. The more the value of the coefficient comes close to the extremes, the more the correlation is present, in a positive or negative manner. The correlation value of 0 indicates no linear correlation. High correlation was found between the total polyphenol content and radical-scavenging activity (r TPC/ABTS = 0.98; r TPC/DPPH = 0.98; r TPC/NO = 0.92; r TPC/SO = 0.77) and reducing power (r TPC/FRAP = 0.99) demonstrating as the polyphenols are the compounds mostly involved in the antioxidant activity [33]. The highest correlation was observed between the reducing power of the samples and the radical-scavenging activity by ABTS and DPPH methods (r FRAP/ABTS = 0.99 and r FRAP/DPPH = 1.00). Polyphenols are less involved in the inhibition of lipid peroxidation (r TPC/BCB = 0.61). The antioxidant activity determined by the BCB method, a lipophilic system constituted by the emulsion β-carotene-linoleic acid, generally has a low correlation with the phenolic compounds and the other methods tested because of the effectiveness of lipophilic compounds [34]. Instead, terpenoids are clearly less involved in tested activities ( Table 2). Total phenolic content (TPC); total terpenoids content (TTeC); ABTS assay; DPPH assay; Super oxide anion scavenging activity (SO); nitric oxide radical scavenging activity (NO); Ferric reducing antioxidant power assay (FRAP); β-carotene bleaching assay (BCB).
The results of the antioxidant activity obtained by the above-described chemical methods have been integrated by calculating the Relative Antioxidant Capacity Index (RACI). The RACI index allows the comparison of phytocomplex antioxidant capacity derived from different chemical methods (ABTS, DPPH, SO, NO, FRAP, and BCB) even the TPC. It has recently been shown, that the results obtained with the method of Folin-Ciocalteu reagent can be interpreted to determine the total content of polyphenols, and as an alternative way to measure the total reducing capacity of the samples since the Folin reagent reacts with any reducing substance present in solution [22]. This index is dimensionless and measures the distance between the average of the results obtained and the raw data expressed in standard deviation units. RACI values obtained agrees with the results obtained so far ( Figure 3) evidencing as ScEA fraction presented the highest value (1.73), followed by the ScB (0.16). The ScH fraction presented the lowest index (−0.73) and, therefore, a relative lack of antioxidant activity. In particular, the RACI values may be related to the high total polyphenol content of the ethyl acetate fraction.
Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules total content of polyphenols, and as an alternative way to measure the total reducing capacity of the samples since the Folin reagent reacts with any reducing substance present in solution [22]. This index is dimensionless and measures the distance between the average of the results obtained and the raw data expressed in standard deviation units. RACI values obtained agrees with the results obtained so far ( Figure 3) evidencing as ScEA fraction presented the highest value (1.73), followed by the ScB (0.16). The ScH fraction presented the lowest index (−0.73) and, therefore, a relative lack of antioxidant activity. In particular, the RACI values may be related to the high total polyphenol content of the ethyl acetate fraction. To date, no study reported the antioxidant activity of S. clivicolus extracts, but Conforti et al. [28] evaluated the antioxidant activity of Senecio gibbosus aerial parts. This study reported as the methanol extract and ethyl acetate fraction had potent antioxidant property on DPPH radical as well as on lipid peroxidation. In particular, the paper reported an IC50 of 0.01 mg/mL for the ethyl acetate fraction of S. gibbosus on DPPH and showed higher inhibition than that ScEA (IC50 of 0.10 ± 0.00 mg/mL) obtained in present study. It is possible to explain these results with the different methods used to obtain the ethyl acetate fraction. In fact, Conforti et al. extracted the plant materials with methanol; then, the methanolic extract was acidified with 2.50% H2SO4 and partitioned with nhexane, dichloromethane, and ethyl acetate. Moreover, in this paper the n-hexane fraction tested by DPPH assay was inactive, such as ScH.
Similarly, the in vitro antioxidant activity of Senecio inaequidens and Senecio vulgaris by the DPPH assay showed that the ethyl acetate fraction gave the 61.60% and 44.57% of inhibition, respectively, at a concentration of 0.31 mg/mL [35]. In present study, ScEA showed a stronger inhibition compared to both species tested by Conforti et al. (90.55 ± 0.61% of inhibition at 0.31 mg/mL).

Identification and Quantification of Polyphenols by Mass Spectrometry
The compounds previously identified in the aerial parts of S. clivicolus belong mainly to the class of alkaloids, eremophilanes and furanoeremophilanes [17,25]. To evaluate the compounds responsible of the measured antioxidant activity, ScEA fraction partitioned from ethanol extract of S. clivicolus aerial parts was subject to mass spectrometry analysis in negative ionization [36]. The LC-MS profile of ScEA is shown in Figure 4. To date, no study reported the antioxidant activity of S. clivicolus extracts, but Conforti et al. [28] evaluated the antioxidant activity of Senecio gibbosus aerial parts. This study reported as the methanol extract and ethyl acetate fraction had potent antioxidant property on DPPH radical as well as on lipid peroxidation. In particular, the paper reported an IC 50 of 0.01 mg/mL for the ethyl acetate fraction of S. gibbosus on DPPH and showed higher inhibition than that ScEA (IC 50 of 0.10 ± 0.00 mg/mL) obtained in present study. It is possible to explain these results with the different methods used to obtain the ethyl acetate fraction. In fact, Conforti et al. extracted the plant materials with methanol; then, the methanolic extract was acidified with 2.50% H 2 SO 4 and partitioned with n-hexane, dichloromethane, and ethyl acetate. Moreover, in this paper the n-hexane fraction tested by DPPH assay was inactive, such as ScH.
Similarly, the in vitro antioxidant activity of Senecio inaequidens and Senecio vulgaris by the DPPH assay showed that the ethyl acetate fraction gave the 61.60% and 44.57% of inhibition, respectively, at a concentration of 0.31 mg/mL [35]. In present study, ScEA showed a stronger inhibition compared to both species tested by Conforti et al. (90.55 ± 0.61% of inhibition at 0.31 mg/mL).

Identification and Quantification of Polyphenols by Mass Spectrometry
The compounds previously identified in the aerial parts of S. clivicolus belong mainly to the class of alkaloids, eremophilanes and furanoeremophilanes [17,25]. To evaluate the compounds responsible of the measured antioxidant activity, ScEA fraction partitioned from ethanol extract of S. clivicolus aerial parts was subject to mass spectrometry analysis in negative ionization [36]. The LC-MS profile of ScEA is shown in Figure 4.  Several compounds (>26) were detected and tentative identification of most of them could be reached through accurate masses and fragmentation pattern and aided by the existing literature. As many as 19 of the compounds were of chlorogenic acid derivatives. Only eight phenolic compounds could be identified in the ethyl acetate fraction by comparing their retention times with those of the available commercial standards. In particular, these compounds were phenolic derivatives of benzoic acid (4-hydroxybenzoic acid (1)), phenylpropanoids, and in particular cinnamic acid derivatives (chlorogenic acid methyl ester (3), 3,5-di-O-caffeoyl quinic acid (7), 3,4-di-O-caffeoyl quinic acid (8), caffeic acid (10) and chlorogenic acid (15)), and flavonols (rutin (4) and quercetin-3-O-glucoside (5)).
Previous reports evidenced as identified compounds are able to exert a strong antioxidant activity.
Also, flavonoid derivative identified compounds in S. clivicolus EA fraction, showed to be effective when tested for their antioxidant activity. For example, rutin exhibited a strong DPPH radical scavenging activity and at 0.05 mg/mL showed 90.40% inhibition compared with BHT (58.80%) as reported from Yang et al. [40]. Moreover, rutin showed to be also effective in preventing lipid peroxidation. Razivi et al. [41] instead showed the cytotoxic, antimicrobial, and antioxidant activities of flavonol quercetin 3-O-glucoside.
The antioxidant activity analysis was effective in identifying the important polyphenolic contributors to the antioxidant capacity of S. clivicolus aerial parts, as assessed by TPC, TTeC, and in vitro antioxidant assays. The ethyl acetate fraction demonstrated the highest relative antioxidant capacity index and was subjected to mass spectrometry analysis. In the present study, the known antioxidant compounds such as phenolic derivatives of benzoic acid, cinnamic acid derivatives, and flavonoids were reported for the first time in Senecio genus. The most abundant of them were the known antioxidant 3,5-di-O-caffeoyl quinic acid and 3,4-di-O-caffeoyl quinic acid.
Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA, USA). All spectrophotometric measurements were done in 96-well microplates or cuvettes on a UV-VIS spectrophotometer (SPECTROstar Nano BMG Labtech, Ortenberg, Germany).
LC-MS/MS analysis were performed on a Q-Tof Premier mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA). Mass spectrometry quantification of the polyphenols was performed using multiple reaction monitoring (MRM) experiments using Waters Acquity (Waters Corporation, Milford, MA, USA) ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS).

Plant Material and Sample Preparation
The aerial parts of S. clivicolus (Asteraceae family) were collected in the 2014, near the Aymaya population/community (18.45 • S to 66.46 • W; 3750 msnm), Bustillo province, Potosí department, Bolivia (Provincia di Nor Yungas, GPS coordinates −16.190040, −67.884402) dried at room temperature and crushed. A voucher specimen has been stored at the University of La Paz (number SC108-2014). Dried plant material (110.00 g) have been extracted by maceration in a darkness shaker set at 25 • C using 96% ethanol for 24 h at a solid to solvent ratio of 1:10 (w/v) per extraction (3 times). The extracts were filtered through a Buchner funnel (0.45 µm) and combined; subsequently supernantants have been dried using a rotary evaporator with a water bath set at 37 • C.
Twenty grams of ethanol extract (Sc EtOH) were solubilized in water (400.00 mL) and subjected to liquid/liquid partitioning in triplicate using an equal volume of n-hexane, chloroform, ethyl acetate and n-butanol in order to separate the compounds on the basis of increasing solvent polarity. Then the n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions (ScH, ScC, ScEA, ScB and ScW, respectively) were dried and stored in darkness at room temperature until further use.
Sc EtOH and all the above-mentioned fractions were analyzed for their polyphenolic and terpenoids contents and their antioxidant activities.

Total Phenolic Content (TPC)
Total phenolic content (TPC) of S. clivicolus samples was determined by Folin-Ciocalteu assay as reported by Todaro et al. [52] with slight modification. The results were expressed as milligrams of gallic acid equivalents per gram of dried extract (mg GAE/g).

Total Terpenoid Content (TTeC)
The total terpenoid content (TTeC) was determined using linalool as standard reagent [53]. The absorbance was measured at 538 nm and the results were expressed as milligrams of linalool equivalents per gram of dried sample (mg LE/g).

Radical-Scavenging Activity
The radical scavenging capacity of S. clivicolus was evaluated by four different complementary in vitro assays. In particular, the synthetic coloured ABTS + and DPPH · radicals, and the biological super oxide anion (O 2 − ) and nitric oxide (NO) radicals. The electron transfer involves reduction of a colored oxidant and the capacity of the samples to scavenge the radicals was monitored by spectrophotometer and quantified in Trolox equivalents, used as standard [15].

ABTS Assay
The free radical-scavenging capacity of all S. clivicolus samples was studied using the 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical assay, by the procedure described by Armentano et al. [54] with slight modification. The reaction between ABTS salt and potassium persulfate generates the ABTS + radical after 16 h of incubation at room temperature. The reaction for scavenging the cationic radicals from the samples was monitored at 734 nm and the results were expressed as milligrams of Trolox equivalents per gram of dried extract (mg TE/g) by the Trolox standard curve.

DPPH Assay
The radical-scavenging ability of samples was also evaluated by in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) neutral radical and, also in this case, Trolox was used as standard. The reaction was monitored at 515 nm and the data were expressed as milligrams of Trolox equivalents per gram of dried extract (mg TE/g) [22].

Super Oxide Anion Scavenging Activity (SO)
Superoxide anion radicals (O 2 − ) were generated in vitro by the NADH/PMS system as described by Russo et al. [55]. The scavenging activity of samples on the inhibition of formazan formation was monitored at 560 nm by spectrophotometer and ascorbic acid was used as positive control. The results were expressed as the concentration inhibiting 25% of radical inhibition in mg/mL (IC 25 ).

Nitric Oxide Radical Scavenging Activity (NO)
The nitric oxide radical (NO) was spontaneously generated at physiological pH from nitroprusside. Then, the nitric oxide interacts with oxygen to give nitrite ions that can be determined spectrophotometrically by Griess reagent [55]. Results were expressed as IC 25 of radical inhibition in mg/mL and ascorbic acid was used as positive control.

Ferric Reducing Antioxidant Power Assay (FRAP)
FRAP reagent was prepared fresh before the experiment by mixing 300 mM sodium acetate buffer at pH 3.60, 20 mM FeCl 3 ·6H 2 O in distilled water and 10 mM TPTZ in 40 mM HCl in a ratio of 10:1:1. The reaction was monitored at 593 nm and Trolox was used as reference antioxidant standard. FRAP values were expressed as milligrams of Trolox equivalents per gram of dried extract (mg TE/g) [56].

β-Carotene Bleaching Assay (BCB)
Lipid peroxidation inhibition was evaluated by the β-carotene bleaching method (BCB) [57]. The β-Carotene emulsion (950.00 µL) was mixed with 50.00 µL of sample at 1.00 mg/mL and BHT was used as positive control, then the mixture (250.00 µL) was transferred into a 96-well plate. Outer wells were filled with 250.00 µL of water to provide a large thermal mass because the reaction was temperature-sensitive and close temperature control throughout the plate was essential in this assay [58]. The microplate was placed at 50 • C for 3 h and the absorbance was measured at 470 nm at 0 , 30 , 60 , 90 , 120 , 150 , and 180 . The results were expressed as percentage of β-carotene bleaching inhibition (% Antioxidant Activity, %AA).

Identification and Quantification by Liquid Chromatography Mass Spectrometry
LC-MS characterization was carried out on a Q-Tof Premier mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA) as described previously [59,60]. Accurate mass measurements of the analytes were achieved through the use of an internal reference compound (leucine-enkephalin). The separation of the compounds was performed on an Atlantis T3 C18 column (Waters Corporation, Milford, USA, 100.00 mm × 2.10 mm; 3.00 µm particle size) maintained at 40 • C and using 0.10% aqueous formic acid (solvent A) and 0.10% formic acid in acetonitrile (solvent B). A stepwise gradient from 10% to 90% solvent B was applied at a flow rate of 0.30 mL/min for 25 min. Electrospray mass spectra data were acquired on a negative ionization mode for a mass range m/z 100 to m/z 1000. Cone voltage and capillary voltage were set at 30 V and 3 kV, respectively. Collision induced fragmentation (CID) of the analytes was achieved using 12 eV to 30 eV energy with argon as the collision gas.
For the quantification of the polyphenols in the ethyl acetate fraction of S. clivicolus, Waters Acquity (Waters Corporation, Milford, MA, USA) ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was used. The compounds were separated on a Waters Acquity HSS T3 C18 column (2.10 × 100.00 mm; 1.80 µm particle size) using a binary solvent system consisting of water containing 0.10% formic acid (mobile phase A) and acetonitrile containing 0.10% formic acid (mobile phase B). The following gradient program was carried out: 0-2.50 min 2% B, 2.50-3.00 min 10% B, 3.00-7.50 min 15% B, 7.50-8.50 min 35% B, 8.50-9.50 min 98% B and 9.50-10.00 min 2% B at a flow rate of 0.50 mL/min. The injection volume for all the samples and the standards was 3.00 µL. All the standards in the concentration ranging from 0.01 to 50.00 µg/mL were dissolved in 80% methanol. Multiple reaction monitoring (MRM) quantitative method was developed for each of the standard compound using the Waters Intellistart software, and the quantifications of the data were carried out using the Waters TargetLynx™ software. The ionization source conditions were as follows: capillary voltage 3 kV, cone voltage 35 V, source temperature 150 • C, desolvation temperature 350 • C, desolvation gas flow 200 L/h, cone gas flow 50 L/h, and collision gas flow 0.10 mL/min. The column temperature was maintained at 40 • C.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) of three independent experiments performed in triplicate. To verify the correlation among used methods, the p value of 0.05 or less was considered significant and calculated by one-way analysis of variance (ANOVA). Pearson coefficient and all other statistical calculations were determined using GraphPad Prism 5 Software (San Diego, CA, USA).

Conclusions
In this report the ethanol extract of S. clivicolus aerial parts was subjected to liquid/liquid fractionation using solvent with increasing polarity. The antioxidant activity, determined by six complementary in vitro assays, showed different values among fractions. Relative Antioxidant Capacity Index (RACI), evidenced S. clivicolus ethyl acetate fraction as the most active. More in detail, fraction reported activity was: n-hexane < water < chloroform < n-butanol < ethyl acetate. Moreover, this study reports the profile of the phenolic compounds in S. clivicolus ethyl acetate fraction. The phytochemical investigation allowed the identification or tentative identification of 30 compounds and confirms that the LC-MS-based profiling is a powerful technique for the phenolic characterization. This first report on S. clivicolus phenolics demonstrated as it could be considered a rich source of health promoting compounds, particularly chlorogenic acid, and flavonoid derivatives. Hung et al. (2006) demonstrated as this compounds are able to exert a protective role against oxidative stress. In fact chlorogenic acid, the most abundant isomer among caffeoylquinic acid isomers, is a biologically active polyphenol, which play several biological activities such as antioxidant [42].
In conclusion, based on the observed results in conjunction with the existing literature, it is anticipated that S. clivicolus, especially its polar extracts, might be used for mitigating human and animal diseases. The presence of potential nutraceuticals is suitable and promising for the development of safe food products, natural additives and cosmetics. Its compounds, previously tested for their biological activity, seems to explain the most of the measured antioxidant activity. This will further trigger extensive research for better understanding of the impact of S. clivicolus phenolic compounds on health.