Leafy Stems of Phagnalon saxatile subsp. saxatile from Algeriaas a Source of Chlorogenic Acids and Flavonoids with Antioxidant Activity: Characterization and Quantification Using UPLC-DAD-ESI-MSn

Phagnalon saxatile subsp. saxatile is a wild species widespread in Algeria which is utilized for medicinal purposes as analgesic and anticholesterolemic. However, information is still scarce regarding its phytochemical content. The objective of this study was to identify and quantify the phenolic compounds from different extracts of its leafy stems. For this purpose, the effects of four extracting solvents were investigated on the content of phenolic compounds and the antioxidant activity of this plant. The extracts prepared with polar solvents (methanol and water) contained higher amounts of phenolic compounds and showed better antioxidant activity than the extracts with apolar solvents (hexane, dichloromethane). The methanolic extract, richest in total phenolic and total flavonoid, had significant antioxidant activity as regarded by DPPH° scavenging capacity (IC50 of 5.5 µg/mL), ABTS+° scavenging capacity (IC50 of 63.8 µg/mL) and inhibition of oxidation of linoleic acid (IC50 of 22.7 µg/mL), when compared to synthetic antioxidants. Chlorogenic acids and several flavonoids were identified and quantified by UPLC-DAD-MSn. The di-O-caffeoylquinic acids isomers were the most concentrated phenolics (25.4 mg/g DW) in the methanolic extract.

Phagnalon saxatile subsp. saxatile (Phagnalon saxatile (L.) Cass., immortal rocks) is a very common plant, endemic to the Mediterranean region and found in sunny and dry rocky places [12]. Its aerial parts and flowers are traditionally used as an analgesic and to lower blood cholesterol [13]. Moreover, this species has been proposed as a potentially helpful plant to protect against Alzheimer's disease (AD). This pathology is characterized by oxidative and inflammatory processes leading to neuronal death [14]. The methanolic extract of the flowering aerial parts of P. saxatile were shown to exhibit anticholinesterase activity, cholinesterases constituting a master target for treating AD [9]. Interestingly, this property of the P. saxatile methanol extract was associated with the anticholinesterase activity of some of its phenolic components, notably caffeic acid, dicaffeoylquinic acid, apigenin and luteolin [6]. The neuroprotective property of P. saxatile extract could also be explained by its anti-inflammatory, antioxidant and hypocholesterolemic activities [11]. It was shown that the antioxidant therapy is essential to reduce ROS, which has an impact on neuronal degeneration and that cholesterol is strongly linked to the onset of this disease since high cholesterol increases the risk of AD by three times in old age [14]. From a phytochemical aspect, the aerial part of P. saxatile was shown to be a source of an essential oil rich in sesquiterpenoids [15]. In addition, it contains phenolic compounds, namely, 3,3-dimethylallyl-p-benzoquinone [16], 2-alkylhydroquinone glucoside [10], caffeic acid, methylchlorogenic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and flavonoids such as luteolin, apigenin and apigenin-7-glucoside [9]. Different biological properties were reported for these molecules such as radical-scavenging, antioxidant [17], anti-inflammatory and anti-carcinogenic activities [18].
Phenolic compounds, one of the main classes of secondary metabolites, can be ranged from simple molecules, such as phenolic acids, to highly polymerized compounds, such as tannins. They are phytochemicals including at least one aromatic ring and one or more hydroxyl groups [19]. They are antioxidants and are notably studied in order to find molecules that can protect the human body against the effects of reactive oxygen species (ROS) and replace synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tert-butylhydroquinone (TBHQ) used for many years in food, pharmaceutical and cosmetics industries and which may have a certain toxicity for consumers [20]. These phenolic antioxidants, mainly phenolic acids and flavonoids, have free radicals scavenging, metals chelating and reducing properties while they can also inhibit enzymatic systems responsible for the generation of ROS [21].
This study aimed to investigate the phenolic profile of P. saxatile leafy stems (LSPSs), which exhibit significant antioxidant activities (total antioxidant capacity, DPPH· and ABTS ·+ scavenging ability, reducing power, chelating capacity and β-carotene bleaching test), through LC-MS n analyses. Using extraction solvents with contrasting polarity was a very important step to reach this objective.

Extract Yields, Polyphenol Contents and Antioxidant Activities
The extract yields were affected by the solvent used, apolar solvents leading to lower values than methanol and water. The apolar solvents also led to poor total phenolic and total flavonoid contents, but revealed the presence of tannins. The contents of total phenolics and total flavonoids were higher for both extracts prepared with polar solvents. The methanolic extract had the highest phenolic (23.1 ± 0.1 mg GAE/g DW) and flavonoid (15.9 ± 0.1 mg CE/g DW) contents (Table 1). The results of the antioxidant assays for all extracts are summarized in Table 2. Because of their low activity, the hexane and dichloromethane extracts were assayed only for the total antioxidant capacity, DPPH radical scavenging and reducing power, and the values of IC 50 and EC 50 were not achieved. Polar solvents (methanol and water), showed impor-tant antioxidant capacity compared with standards. The total antioxidant capacity of the extracts, expressed in mg GAE/g DW, increased in proportion to the total phenol content. The DPPH • and ABTS +• scavenging capacities of the polar extracts were better than the BHT reference, with the lowest IC 50 values for the methanolic extract (5.5 ± 0.1 µg/mL and 63.8 ± 0.9 µg/mL, respectively). The methanol extract performed also better than the BHA reference for the β-carotene bleaching assay. However, the ability of the extracts to reduce the iron remained low compared to the BHT (IC 50 = 99.6 ± 1.8 µg/mL) and ferrous ion chelating activity was much lower than the positive control EDTA, with IC 50 values reached only for the aqueous extract (4450 ± 3 µg/mL).

Identification and Quantification of Phenolic Compounds Contained in the Polar Extracts of P. saxatile
The methanolic and aqueous extracts that showed the highest antioxidant activity were analyzed by UPLC/MS. Compounds found are given in Table 3, and were numbered by their order of elution. They were characterised based on their UPLC retention time, UV spectra and mostly on their MS n fragmentation. All of these identified compounds were quantified from the UPLC-DAD chromatographic peak areas. The concentrations of hydroxycinnamic acids and flavonoids are expressed in µg/g of the dried weight (DW).

•
Phenolic compounds identified in methanolic and aqueous extracts of P. saxatile.
The UPLC chromatograms at 320 nm of the methanolic and the aqueous extracts of LSPSs are presented in Figure 1A,B, respectively. The phenolic compounds detected were hydroxycinnamic acid esters and flavonoids (Table 3).

Flavonoids
In addition to the phenolic acid derivatives described above, some flavonoids were also identified in methanolic and aqueous LSPSs extracts. In the aqueous extract, all flavonoids were found in the form of derivatives generally glycosylated. Free aglycones were found in methanolic extract, in addition to the glycosylated forms.

Unknown Compound
Compound 4 was unknown and was eluted at 6.1 min in both extracts.
• UPLC quantification of phenolic acid derivatives and flavonoids The peak numbers follow their retention time (Rt) and are the same as the ones in Figure 1. Phenolics from the methanolic and aqueous extracts were quantified ( Table 3). The total amounts of compounds (mg/g DW) were calculated by summarizing individual amounts of all constituents (Table 4). Di-caffeoylquinic acid isomers were the most concentrated in the methanolic extract (25.37 mg/g DW), followed by caffeoylquinic acid isomers (12.56 mg/g DW), flavonoids (4.38 mg/g DW) and coumaroylquinic acid (0.07 mg/g DW) ( Table 4). The phenolic concentrations in the aqueous extract were 3 to 10-fold lower than in the methanolic extract for caffeoylquinic acids and dicaffeoylquinic acids, respectively. Interestingly, the aqueous extract was more concentrated in caffeoylquinic acids (3.90 mg/g DW) than the di-caffeoylquinic acids (2.80 mg/g DW).
Regarding flavonoids, the luteolin glucoside and quercetin glucoside were the most abundant in the extracts. The methanolic extract contained a higher diversity of flavonoids with a globally higher concentration, whereas only luteolin di-glucoside was more concentrated in the aqueous extract. Free aglycones were only found in the methanolic extract but at low concentration (luteolin at 0.13 mg/g DW and apigenin at 0.03 mg/g DW; Table 4).
The distribution between phenolic acids and flavonoids were 90% and 10%, respectively, in the methanolic extract and 76% and 24% in the aqueous extract (Table 4). The chromatographic profiles are followed at 320 nm which corresponds to a wavelength where both phenolic esters and flavonoids can be detected. The numbers on the peaks are related to their identification described in Table 3.

Discussion
The present work presented the phenolic characterization and antioxidant properties of different LSPSs extracts. The nature and polarity of the solvent used for the extraction impacted directly the yield and extractability of phenolic compounds. This is in agreement with previous studies that have demonstrated that the yields of phenolic compounds obtained with polar solvents were higher than those of less polarity [8,9,28,29]. Unexpectedly, the total tannin assay led to detect the presence of a small fraction of tannins in the apolar fractions only. Since hexane and dichloromethane extraction were previously shown to extract tannins with low efficiency [30,31] the fact that no trace of tannin was detected in the polar fraction is puzzling. UPLC analyses confirmed the absence of tannins in the polar extractions, however, since UPLC analyses were not conducted on the apolar fractions, we cannot strictly confirm the nature of the compound(s) that led to a positive result with the total tannin assay.
To our knowledge, a few studies have investigated the phenolic composition and antioxidant properties on Phagnalon species, including those carried out on P. saxatile from Italy [9] and Algeria [10,11], P. graecum Boiss. from Turkey [8] and P. lowei from Madeira archipelago [32]. The antioxidant activities measured in our methanolic extract were close to the Italian P. saxatile extract, regarding the DPPH and β-carotene bleaching test [9]. Accordingly to the study on P. graecum, the LSPSs aqueous extract appeared more active than the other solvent for the iron chelating activity [8].
In total, eight phenolic compounds were characterized and quantified in the LSPSs methanolic and aqueous extracts, including flavonoids and three hydroxycinnamic acid esters using UPLC-DAD-ESI-MS n analysis. Hydroxycinnamic acid esters, more precisely caffeoyl and dicaffeoylquinic acids were predominantly detected. These metabolites have already been detected in other Phagnalon species [9,10,33,34] and appeared as the main phytochemical components in a diversity of Asteraceae [32,35]. Quantitatively, caffeoyl and dicaffeoylquinic acids were the most abundant phenolic compounds in LSPSs extracts analyzed. Their total concentration in the methanolic extract reached 38 mg/g DW (i.e., 3.8 g/100 g) which ranks them in the upper part of the caffeoyl-quinic derivativeaccumulating plants, far below Ilex paraguariensis (herbal mate, 9.2 g/100 g) but in the same range as Arabica coffee beans (4.9 g/100 g) and above white and green tea (Camilla sinensis, 1.3-1.6 g/100 g) [36][37][38][39]. In addition, the concentrations in caffeoyl and dicaffeoyl-quinic acids measured in our P. saxatile samples are similar to the concentrations measured in the methanol extracts of P. lowei which was highlighted as the richest for these compounds on a panel of 10 Asteraceae [32]. Interestingly, in the same study, the authors identified that the P. lowei extract had the best inhibitory activities on a set of digestive enzymes (α-amylase, αand β-glucosidases, lipase) that are related to diabetes and obesity control, which appeared to be linked to the concentration of caffeoylquinic acid derivatives. Considering this study, a future study should be conducted on the beneficial impact of P. saxatile in the control of diabetes and obesity.
Flavonols and flavones were also identified in this plant, showing interesting, concentrations for luteolin glucosides and quercetin glucosides. The presence of aglycone flavonoids, only in the methanolic extract, can be due to their solubility, considered to be strongly affected by the nature of both the solvent and the flavonoid structure. There is, however, very limited data on direct measurement of solubility of differently substituted flavonoids, which are obtained by comparing extraction or chromatography data, such as, for example, comparisons of glycosylated and non-glycosylated compounds. The presence of a sugar moiety usually increases the solubility of the flavonoids in polar solutions. For example, glycosylated flavonols such as glycosylated quercetin have considerably better water solubility compared to the corresponding aglycone [40]. The position of the sugar moiety and the number of the bound sugars also play important roles. For example, the diglucoside quercetin is more stable than the monoglucoside [41].
From the results of quantification, we can notice that the amount of total phenolic compounds obtained by the UPLC analysis was different from that estimated by the Folin-Ciocalteu method, which can be explained by the limitation of the phenolic substances used as standard [42]. However, the absence of tannins by the assay was confirmed by UPLC.
The relationship between the antioxidant activity and phenolic composition of the extracts is a valid approach to understand the activity of the extracts of P. saxatile. It was previously reported that cinnamic acid derivatives display a high antioxidant capacity and that the caffeoyl group is essential to the radical scavenging activity and lipid antiperoxidation [7,43]. Several studies have shown that the isomers of caffeoylquinic and di-caffeoylquinic acids, the major components identified in LSPSs extracts, have antioxidant activity with a higher activity for the latter one [43][44][45]. This property is in accordance with the high antioxidant activity measured in our methanolic extracts. These natural antioxidants have commercial applications in medicine, food, and cosmetics [46]. Their medical interest lies in their various biological activities such as anti-oxidative ability, antiviral, antibacterial, anti-inflammatory, antispasmodic activities, reduction in the relative risk of cardiovascular disease and type 2 diabetes, inhibition of the mutagenicity of carcinogenic compounds [47].
Additionally, it was shown that these molecules, but not caffeine, confer a significant protection against oxidative stress, in vitro, and could be responsible for the beneficial health effects associated with the consumption of green coffee bean derived products, such as extracts commercialized as nutraceuticals or soluble green coffee either alone or blended with roasted coffee [48]. Therefore, regarding their high concentration in these molecules, the extracts of LSPSs could be used as a primary source in the supply chain of the cosmetic, pharmaceutical or health industries which uses chlorogenic acids as active principles. In addition, LSPS extracts could also be used to generate an innovative functional product, such as food supplements, as is the case for coffee-derived food supplements, which have attracted a lot of attention for their promise of quick weight loss [49].
Aglycones and glycosylated flavonoids present in the analysed extracts are of great general interest due to their antioxidant and chelating abilities. Their structure-activity relationships as antioxidants were comprehensively studied. It was previously shown that aglycones are more potent antioxidants than their corresponding glycosides and that flavonoids are able to reduce highly reactive free radicals through the conjugations and hydroxyl groups in particular in position 3 of the B ring and 3 of the C ring. They are able to inhibit a wide range of enzymes generating ROS, have a reducing power and can also chelate catalysts of oxidation reactions such as metal ions (iron and copper). Several studies have shown the antioxidant capacity of quercetin and luteolin [45,[50][51][52][53].

Plant Material and Extract Preparation
The flowering aerial parts of P. saxatile were collected in March 2011 from Tlemcen, in the west of Algeria. They were identified in the Laboratory of Natural Products, Department of Biology, University of Tlemcen, Algeria. Voucher specimens were deposited at the Herbarium of the Laboratory.
The plant parts were dried at room temperature for two weeks. Ten grams of fine powder of the leafy stems were extracted successively with hexane, dichloromethane, methanol and water, in a Soxhlet apparatus, following the protocol as already described by Chaouche et al. [29].

Determination of Total Polyphenol, Total Flavonoid, and Total Tannin Contents
To select the extracts rich in phenolic compounds, the contents of total polyphenols, flavonoids and tannins in the hexane, dichloromethane, methanol and water extracts, were determined. Estimation of different phenolic class contents was realized as already described in our previous paper [11]. The total phenolic content was determined using the Folin-Ciocalteu reagent and was expressed with reference to the gallic acid standard curve (Y = 0.00253 x + 0.01344, R 2 = 0.995). Total flavonoid content was determined using sodium nitrite and aluminum chloride and was expressed with reference to the catechin standard curve (Y = 0.00355 x + 0.09903, R 2 = 0.987). The total tannin content was determined using a vanillin-methanol solution and was expressed with reference to the catechin standard curve (Y = 0.00076 x − 0.0076, R 2 = 0.994).
For scavenging DPPH and ABTS radicals, chelating power and β-Carotene bleaching methods, lower IC 50 value (the concentration required to cause a 50% inhibition) indicates a higher antioxidant activity. For reducing power, lower EC 50 value (the effective concentration giving an absorbance of 0.5) indicates a higher antioxidant activity.

LC-DAD-ESI-MS n Analysis
The phenolic composition of the extracts (50 µL) was analyzed on an HPLC-DAD-ESI-MS n system composed by a binary solvent delivery pump (Ultimate 3000, Thermo Scientific-Dionex, Bremen, Germany) connected to a diode array detector (Surveyor PDA plus, Thermo-Finnigan, USA) and an LTQ Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) according to the method reported by Chougui et al. [54]. Briefly, the molecules were separated on a C18 LichroCART (250 mm × 4.6 mm) column (Merck, Darmstadt, Germany) using water formic acid 0.1% as solvent A and MeOH, acetonitrile, formic acid (50/50/0.1) as solvent B. The separation method consisted of an isocratic step at 1% of B for 2 min followed by a linear gradient from 1% to 40% of B in 100 min, then to 70% of B in 25 min and 90% of B in 55 min with a flow rate at 700 µL/min. The mass spectrometer conditions and data processing were the same as described by Chougui et al. [55]. Experimental exact masses and MS 2 fragmentation data were compared to metabolomics data banks (Pubchem Compound: https://pubchem.ncbi.nlm.nih.gov/, accessed on 21 February 2021; ReSpect: https://spectra.psc.riken.jp/, accessed on 21 February 2021; Mass Bank: https://www.massbank.jp, accessed on 21 February 2021) and available literature to identify the nature of the phenolic compounds.
The phenolic quantification was realized on a U-HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary solvent delivery pump connected to a diode array detector. Three to five microliters of the extracts were separated on a C18 Kinetex (100 mm × 2.1 mm) column (Phenomenex, Torrance, CA, USA) by using a gradient elution from 10 to 45% MetOH for 13 min, then 99% MetOH for 3 min with a flow rate of 300 µL min −1 . The column was rinsed for 1 min with 99% MetOH and re-equilibrated to 10% MetOH for 2 min prior to the next run. Compound quantification was based on measurement of area under each peak determined at 320 nm and 350 nm and expressed relative to calibration curves with chlorogenic acid (for caffeoyl and dicaffeoylquinic isomers), coumaric acid (for coumaroyl quinic acid), apigenin (for aglycon and mono-glycosylated flavonoids) and rutin (for diglycosylated flavonoids).

Statistical Analysis
All measurements were performed in triplicates and results are expressed as mean ± standard deviation (SD) and were subjected to analysis of variance (ANOVA). Differences were considered to be significant at the level of p < 0.05.

Conclusions
P. saxatile showed important antioxidant activity. The methanolic extract of leafy stems had the highest amounts of phenolic compounds and showed stronger antioxidant capacities regarding the DPPH, ABTS and β-carotene bleaching assays.
Phenolic compounds were identified and quantified in this plant from Algeria, for the first time, by the reliable and reproducible UPLC-DAD-ESI/MS n method. LSPSs constitutes an important and abundant source of caffeoylquinic and dicaffeoylquinic acids isomers which has demonstrated pharmacological properties.
Therefore, these results may provide useful information for researchers in phytopharmacy and phytotherapy because of the protective roles of these molecules in pathologies associated to oxidative stresses.
Additionally, this study revealed the presence of important metabolites that would allow emphasising unknown species such as P. saxatile under a nutritional aspect. It will be interesting to explore the potential of LSPSs extracts as a functional supplement in the food industry. This aspect deserves further studies to determine the maximum-tolerated dose and potential toxicity of this plant for human.