Chemical Composition and Antioxidant, Anti-Inflammatory, and Enzyme Inhibitory Activities of an Endemic Species from Southern Algeria: Warionia saharae

Warionia saharae Benth. & Coss. (Asteraceae) is an endemic species of North Africa naturally grown in the southwest of the Algerian Sahara. In the present study, this species’ hydromethanolic leaf extract was investigated for its phenolic profile characterized by ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MS). Additionally, the chemical composition of W. saharae was analyzed by gas chromatography–mass spectrometry, and its antioxidant potential was assessed through five in vitro tests: DPPH● scavenging activity, ABTS●+ scavenging assay, galvinoxyl scavenging activity, ferric reducing power (FRP), and cupric reducing antioxidant capacity. The UHPLC-DAD-ESI/MS analysis allowed the detection and quantification of 22 compounds, with taxifolin as the dominant compound. The GC–MS analysis allowed the identification of 37 compounds, and the antioxidant activity data indicate that W. saharae extract has a very high capacity to capture radicals due to its richness in compounds with antioxidant capacity. The extract also showed potent α-glucosidase inhibition as well as a good anti-inflammatory activity. However, weak anti-α-amylase and anticholinesterase activities were recorded. Moreover, an in silico docking study was performed to highlight possible interactions between three significant compounds identified in W. saharae extract and α-glucosidase enzyme.


Introduction
It is recognized that medicinal plants have been used since the dawn of civilization to treat a wide range of ailments [1,2]. Their therapeutic potential has been attributed to a large quantity and variety of substances called secondary metabolites belonging to several classes such as phenolic compounds (e.g., flavonoids, chlorogenic acids) [3], terpenes [4], and alkaloids [5]. In parallel with increasing knowledge about their biological properties, plant secondary metabolites are increasingly used as drugs [6] and pharmaceutical food additives for aromatic and culinary purposes [7]. Various biotic and abiotic (light, temperature, soil water, soil fertility, and salinity) elicitors can influence their biosynthesis and accumulation [8][9][10].
Asteraceae is one of the most prominent plant families. It covers 23,000 species distributed across more than 1,620 genera. Among them, Warionia saharae Benth. & Coss. is an endemic species grown in Beni Ounif Zousfana and Bechar (south-west of Algerian Sahara). It was also found in various regions in the south of Morocco [11,12]. The leaves of W. saharae are used in folk medicine to treat many health problems, among them inflammatory diseases, gastrointestinal disorders, and epileptic crises [13,14].
Warionia saharae, until now, did not arouse researchers' interest, and few works have been published about this species. These works were focused on different types of W. saharae extracts: aqueous extract [15], essential oil and ethyl acetate extract [16,17], and flavonoid-enriched extract [14]. Their antimicrobial [16,18], antioxidant [16,19], antidiabetic, and antihypertensive [14,20] activities were revealed. Moreover, only ten known compounds were isolated from W. saharae chloroform and ethyl acetate extracts and were identified using UV, IR, and NMR methods [19]. As far as we know, the UHPLC-ESI-MS and GC-MS compositions of W. saharae leaves have not been studied.
Furthermore, this species' extracts and secondary metabolites remain less explored regarding their anti-inflammatory, cytotoxic, and enzyme inhibition activities. Therefore, our study will be the pioneer in this field. Simultaneously, evaluating the extracts' antioxidant and anti-inflammatory potentials and their inhibition ability toward key enzymes involved in diabetes and neurodegenerative diseases (α-glucosidase, α-amylase, and acetylcholinesterase enzymes) was also an objective. Molecular docking experiments investigate the most energetically favorable interactions between bioactive compounds and enzymes [21]. In this context, molecular docking studies against α-glucosidase were conducted to unveil any possible interactions with the major phenolic compounds of W. saharae extract.
This research is expected to provide a better understanding of the chemical composition and the health benefits of this plant, which could contribute to new perspectives for developing new and potent pharmacological agents.

UHPLC-DAD-ESI-MS/MSn Characterization of W. saharae Extract
The UHPLC-DAD-ESI-MS/MS n analysis of W. saharae hydromethanolic (80/20, v/v) extract allowed the identification, for the first time, of 24 compounds. Figure 1 shows the chromatographic profile of this extract, and in Table 1, with respect to their elution order, the peak characteristics, the assigned identification, and the quantification are detailed. The compounds' assigned identification was enabled by comparing retention times and MS data from reference standards or comparing with previously reported MS and UV-Vis data.  Among the 24 metabolites, hydroxycinnamic acid derivatives and flavonoids were identified, which are secondary metabolites usually associated with anti-inflammatory properties. Actually, the quantification shows around 76 µg/mg extract of hydroxycinnamic acid derivatives and around 80 µg/mg extract of flavonoids (Table 1).
The flavonoid family is represented in the hydromethanolic extract of W. saharae by 10 derivatives, 7 aglycones, and 3 glycosides. These results are consistent with those previously reported because we also found luteolin, esculetin, taxifolin, and quercetin derivatives, although Mezhoud et al. [19] mainly reported the flavonoids in their aglycone form.
Among the identified flavonoids, taxifolin derivatives can be highlighted (peaks 14 and 15, Table 1) with a pseudomolecular ion [M-H] − at m/z 303, MS 2 fragment ion at m/z 285, and UV spectrum data also in agreement with a taxifolin derivative [25][26][27]. Other flavonoid derivatives, such as luteolin derivatives (peaks 20, 21and 22, Table 1) and quercetin derivatives (peaks 10 and 13, Table 1), were identified using a pure standard [23,28] and based on MS 2 fragmentation [29]. Isorhamnetin (peak 23) was identified using a standard and literature data, from which the MS 2 fragment at m/z 300 obtained by losing the methyl group can be highlighted [30,31]. Finally, peak 16, with a pseudomolecular ion [M-H] − at m/z 477 and MS 2 fragment at m/z 315, which corresponds to the loss of a glycosyl group, was assigned to isorhamnetin-O-hexoside (Table 1).
Other compounds were identified, for example, peak 2 with a pseudomolecular ion [M-H] − at m/z 137, characteristic of hydroxybenzoic acid [32], peak 3 with a pseudomolecular ion [M-H] − at m/z 339 and a fragment ion at m/z 177 characteristic of esculetin-6-O-glucoside [33], and peak 24 with a pseudomolecular ion [M-H] − at m/z 299 and predominant fragment ions at m/z 271 and 255 characteristic of naringenin derivatives (Table 1).

GC-MS Characterization of W. saharae Extract
GC-MS analysis is the most suitable technique to assess plant extracts lipophilic profile [34,35]. It requires, in some cases, a derivatization procedure to convert many secondary metabolites to volatile derivatives [36], and silylation is the most recent derivatization procedure [37,38]. The GC-MS analysis of the hydromethanolic extract of W. saharae allowed the identification of four families of compounds: sugars (214.735 µg/mg), alcohols (128.520 µg/mg), carboxylic acids and esters (124.6 µg/mg), and fatty acids (11.364 µg/mg). The peak characteristics and the identification of the compounds are presented in Table 2, respecting their elution order. The quantification data were expressed as µg compound per mg of extract. The chromatogram is shown in Figure 2.
As far as we know, no studies have been carried out on the lipophilic composition of W. saharae, so this research provides new knowledge about this plant s fatty acids and important alcohols ( Table 2). Since we analyzed the hydromethanolic extract and, before its analysis by GC-MS, the extract was silylated, important chemical compounds such as sugars were revealed by the GC-MS analysis since they became lipophilic upon the silyla-tion procedure. The sugars profile is, however, important to establish the plant nutritional value, and its richness in small carboxylic acids and alcohols also emphasizes the plant value. Moreover, they can be responsible for the observed activities discussed herein. Naturally, fatty acids represented the less significant group identified in the hydromethanolic extract of W. saharae because they are more lipophilic. Nevertheless, these compounds are known to benefit human health by decreasing cancer risk, cardiovascular and neurological disorders and through their antioxidant potential and anti-inflammatory activity [39,40]. file [34,35]. It requires, in some cases, a derivatization procedure to convert many secondary metabolites to volatile derivatives [36], and silylation is the most recent derivatization procedure [37,38]. The GC-MS analysis of the hydromethanolic extract of W. saharae allowed the identification of four families of compounds: sugars (214.735 µg/mg), alcohols (128.520 µg/mg), carboxylic acids and esters (124.6 µg/mg), and fatty acids (11.364 µg/mg). The peak characteristics and the identification of the compounds are presented in Table 2, respecting their elution order. The quantification data were expressed as µg compound per mg of extract. The chromatogram is shown in Figure 2.

Total Bioactive Content
Total phenolic and flavonoid contents in the hydromethanolic extract of W. saharae were found to be 119.8 ± 8.3 mg GAE/g of extract and 24.25 ± 0.070 mg QE/g extract, respectively. The most important properties of these compounds are their capacity to act as potent antioxidant [41], anti-inflammatory [42], anti-atherogenic [43], and anti-carcinogenic agents [44,45].

Antioxidant Activities
The antioxidant activity of W. saharae extract was evaluated through five in vitro tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) scavenging activity, 2,2 -azino-bis(3-ethylbenzoth iazoline-6-sulfonic acid (ABTS •+ ) scavenging assay, galvinoxyl • scavenging assay, ferric reducing power (FRP), and cupric reducing antioxidant capacity (CUPRAC). The results obtained by the different tests, expressed in IC 50 and A 0.50 , respectively, are shown in Table 3. The antioxidant reaction with free radicals involves different mechanisms: the hydrogen atom transfer (HAT) method, in which the free radical removes one hydrogen atom from the antioxidant, and single electron transfer (SET), in which the antioxidant provides an electron to reduce compounds such as metals, carbonyls, and radicals. An antioxidant reaction can also involve both methods (HAT and SET) [46]. The use of a single assay could not assess the total antioxidant potential because of the implication of different mechanisms in the neutralization of free radicals [47]. Hence, to better assess the overall antioxidant effect, the W. saharae extract was assessed through five different trials (see Table 3).
According to the HSD Tukey test following ANOVA (F4,12 = 88.01, p < 0.001), in the ABTS •+ scavenging assay, there is not a significant difference between Trolox and ascorbic acid and between BHT and BHA (q Tukey = 1.04 and 4.23, respectively). The studied extract exhibits high antioxidant activity (IC 50 = 4.19 ± 0.35 µg/mL), very close to those of the standards.
On the other hand, the reduction potential of the hydromethanolic extract of W. saharae was determined using FRP. In this assay, the antioxidant activity causes the reduction of the Fe 3+ /ferricyanid complex to the ferrous form. The FRP of extracts is linked with reductants, which exert antioxidant action by donating electrons and reacting with free radicals to convert them to more stable products [48,49]. A significant difference in the FRP assay was noted with ANOVA comparing all tests (F 4,12 = 1446.84, p < 0.001, q Tukey > 4.65). Data show that W. saharae extract activity (A0.5 = 31.21 ± 0.14 µg/mL) was weak compared to the used standards (5.25 ± 0.20, 7.99 ± 0.87, and 3.62 ± 0.29 µg/mL for Trolox, BHA, and ascorbic acid, respectively). Reducing capacity detected by cupric acid activity was significantly higher compared to that by the FRP assay.
Overall, present data indicate that W. saharae extract showed a greater capacity to capture radicals. The results of the DPPH • activity are in agreement with those reported in previous works on the ethyl acetate extract of the Algerian W. saharae (IC 50 = 6.98 µg/mL) [19], but are significantly higher than those reported in another study conducted on the same species grown in Morocco (IC 50 = 182 µg/mL) [16]. The nature of the extraction solvent, the plant growth area, and the collection period point out the discrepancy in antioxidant activity results between the referred studies. It should be mentioned that no previous studies were conducted on the antioxidant activity of W. saharae using the ABTS •+ scavenging assay, galvinoxyl assay, FRP, and cupric reducing antioxidant capacity.
The DPPH • , ABTS •+ , and galvinoxyl scavenging data suggest that the components present in W. saharae extract act as excellent scavenging free radicals through a mechanism of electron or hydrogen donation to stabilize the free radicals, and should be able to protect biological matrices from free radical-mediated oxidative degradation [51,52].
According to the CUPRAC and FRP data, W. saharae was as effective as the positive standards in reducing Cu 2+ to Cu + but had a moderate effect in reducing the Fe 3+ to Fe 2+ . The good reducing behavior of W. saharae extract suggests that its constituents, mainly chlorogenic acids, flavonoids, and sugars, are capable of terminating a chain reaction by eliminating free radical intermediates and hence are considered antioxidants [53]. A Molecules 2021, 26, 5257 8 of 20 significant positive correlation between total phenolic and reducing sugar contents with the potent antioxidant capacity was established for other species [54]. Therefore, it is important to emphasize the important role of reducing sugars in antioxidant activity, since the extract of W. saharae contains a significant amount (around 215 µg/mg).
Furthermore, phenolic acids' and flavonoids' antioxidant potentials are closely related to their chemical structure [55] with double bonds and hydroxyl groups [56,57], making them ideal for scavenging free radicals and chelating metal ions. These properties also conferred effective antioxidant potential in vivo [58]. In the present study, the chemical composition analysis of the hydromethanolic extract of W. saharae reveals that phenolic acids are the most dominant group identified in this species. In addition, many compounds detected in the W. saharae hydromethanolic extract are known to possess great antioxidant capacity. For example, simple hydroxycinnamic acids have been reported to possess potent antioxidant activity against DPPH • and ABTS •+ [59]. Chlorogenic acids are considered responsible for both green and medium roasted coffee's antioxidant capacity, with 5-Ocaffeoylquinic acid being the major contributor [60].
Li et al. [61] reported that di-O-caffeoylquinic acids exhibit high antioxidant activity using conventional antioxidant assays. The same authors proposed that their antioxidant mechanisms might include electron transfer, H + transfer, and Fe 2+ chelation.
Taxifolin (peak 14) is the major compound present in the hydromethanolic extract of W. saharae and may contribute to its strong antioxidant activity. A previous study reported that in antioxidant assays, taxifolin scavenges HO • , DPPH • , and ABTS •+ efficiently and enhances the relative Cu 2+ and Fe 3+ reducing levels [62]. Zu et al. [63] also mentioned that taxifolin is a free radical scavenger, and its antioxidant capacity is superior to other flavonoids. The results obtained by Topal et al. [64] from an in vitro study showed that taxifolin possesses marked antioxidant, reducing ability, radical scavenging, and metalchelating activities.
Although it is present in lower amounts, luteolin has received increasing attention due to its various biological activities and pharmacological effects, including antioxidant activity [65], and has been widely applied in medicine and functional foods [66,67].
The high antioxidant potential recorded in this study may be linked to the presence of these compounds that could act individually or combined to provide the hydromethanolic extract of W. saharae with its strong antioxidant power.

Anti-Inflammatory Activity
The anti-inflammatory effect of W. saharae was evaluated by the NO • scavenging method. NO • is one of the main chemical mediators involved in inflammatory events. Regarding its ability to scavenge NO • , the relevant anti-inflammatory activity of W. saharae extract (IC 50 = 76.66 ± 4.16 µg/mL) is better than that of ascorbic acid (IC 50 = 144.380 ± 7.035 µg/mL) but lower than that of curcumin (IC 50 = 7.49 ± 0.58 µg/mL). To the best of our knowledge, no anti-inflammatory effect had been previously described for W. saharae extract. Thus, this is the first report describing the anti-inflammatory potential of this species.
The phenolic compounds highly present in W. saharae extract are probably responsible for the anti-inflammatory property observed in this study, and flavonoids can be highlighted [42,68]. Taxifolin, the main phenolic compound present in W. saharae extract, was previously found to possess significant anti-inflammatory activity against carrageenan-induced edema involving the exudative phase of inflammation and also against formaldehyde-induced arthritis [69]. These results corroborate the traditional use of this species in folk medicine.

Enzyme Inhibitory Activities
The enzyme inhibitory activities of the W. saharae extract and the standard inhibitors tested toward α-glucosidase, α-amylase, and acetylcholinesterase expressed in IC 50 or the percentage of inhibition are shown in Table 4. The α-glucosidase and α-amylase inhibitors are used to treat diabetes mellitus as they slow down the digestion of carbohydrates in the small intestine and thus reduce the rapid rise in blood sugar after eating [70,71]. The inhibitory effect of the W. saharae species toward these two enzymes was evaluated in this study.
Several earlier studies have demonstrated the role of medicinal plants' phenolic compounds as inhibitors of α-glucosidase. Among those flavonoids such as taxifolin and luteolin, and hydroxycinnamic acids, major compounds of W. saharae extract exhibited strong inhibitory activity against α-glucosidase in in vitro studies [72][73][74]. In a recent report, it was found that taxifolin s pre-administration can significantly improve postprandial hyperglycemia in rats [74].
Furthermore, the obtained results with the α-amylase inhibition assay agree with those of Nyambe-Silavwe and Williamson [75] and Proença et al. [76], suggesting that chlorogenic and other phenolic acids, major compounds in W. saharae extract, weakly inhibit human α-amylase activity despite publications that claim otherwise [74].
The UHPLC-DAD-ESI/MS analysis shows that W. saharae extract contains these phenolic compounds abundantly. Consequently, it can be said that the synergistic effect could explain the high inhibitory effect recorded in the W. saharae extract among these compounds.
Apostolidis et al. [77] reported that medicinal plants that selectively inhibit α-glucosidase are better for controlling glucose absorption. Indeed, both enzymes' inhibition leads to abnormal bacterial fermentation in the colon resulting from the undigested carbohydrates. Thus, the inhibition of α-glucosidase by the W. saharae extract could be considered a promising way to control diabetes mellitus.
Acetylcholinesterase inhibitors prevent the breakdown of acetylcholine in the body by blocking the action of the enzyme acetylcholinesterase responsible for its separation, which leads to an increase in its level in the space between nerve endings and thus gives it more opportunity to cross into another cell and carry out its functions. This mechanism is important for people with Alzheimer s disease, who already suffer from a decrease in acetylcholine level since these inhibitors temporarily improve the symptoms of the disease and its stability [78].
We also evaluated the inhibitory effect of the acetylcholinesterase enzyme here. At the highest dose of the extract (1 mg/mL in this assay), the enzyme s inhibition was observed (28.578 ± 2.979%). The inhibitory action on AChE in the in vitro assay proved to be lower than the donepezil used as a positive control (IC 50 = 13.32 ± 1.34 µM). Thus, the weak inhibitory activity of W. saharae extract toward AChE activity could be related to the chemical composition of this species since we considered that the major substance in the plant extract is usually responsible for its biological activity.
Taxifolin, highly present in the W. saharae extract, has been shown to have relatively weak AChE inhibitory properties (IC 50 = 133.1 µg/mL) [79] despite studies that claim otherwise [80]. Although chlorogenic acids are mainly responsible for inhibiting the AChE enzyme in green coffee extracts [81], their presence in W. saharae extract could not be sufficient to inhibit this enzyme strongly. Some of them, like 4,5-di-O-caffeoylquinic, one of the major compounds in W. saharae extract, showed AChE inhibition under 50% [81].
In addition, some phenolic compounds may act as activators of AChE. Earlier studies demonstrated that caffeic acid was shown to increase AChE activity in in vitro studies [82]. Some authors have hypothesized that caffeic acid (or its derivatives) could trigger the AChE catalytic site s activation in a reaction at a second site [83]. Hence, the presence of caffeic acid in W. saharae extract could, in part, explain these results.

Molecular Docking
An in silico study was performed to further screen the mechanisms of interaction between α-glucosidase and the three major compounds in W. saharae extract. Figure 3 shows the 2D and 3D structures of the docked compounds at the active site of the α-glucosidase enzyme and the types of interactions involved.
Based on the docking test's interaction data, the major constituents of W. saharae extract interact with α-glucosidase, engaging different amino acids (Table 5). Taxifolin showed a higher binding affinity, as indicated by its lower binding energy of -5.89 (kcal/mol), followed by 4,5-O-dicaffeoylquinic acid and 1,3-O-dicaffeoylquinic acid with a binding energy of -4.17 and -4.02 (kcal/mol), respectively.
Herein, it can be observed that the best docking pose obtained was for taxifolin docked to α-glucosidase. Different interactions, such as three hydrogen bonds with Asn715, Ala687, and Gly690, π-cation, and π-alkyl with Ile691 to the aromatic cycle and many other Van Der Waals interactions can be observed. Taxifolin, which has shown the best binding affinity within the major compounds, was previously reported as a competitive inhibitor of the α-glucosidase enzyme with an IC 50 of 0.038 mg/mL [74].
Several types of interactions were found between both compounds, 4,5-O-dicaffeoylquinic acid and 1,3-O-dicaffeoylquinic acid, and the α-glucosidase active site (Figure 3), most of which are hydrogen bonds, π interactions, van der Waals, and carbon-hydrogen bonds.
These findings further support our in vitro assay results. However, in vivo studies are still necessary to better confirm these results. Acetylcholinesterase inhibitors prevent the breakdown of acetylcholine in the body by blocking the action of the enzyme acetylcholinesterase responsible for its separation, which leads to an increase in its level in the space between nerve endings and thus gives it more opportunity to cross into another cell and carry out its functions. This mechanism is important for people with Alzheimer′s disease, who already suffer from a decrease in acetylcholine level since these inhibitors temporarily improve the symptoms of the disease and its stability [78].
We also evaluated the inhibitory effect of the acetylcholinesterase enzyme here. At the highest dose of the extract (1 mg/mL in this assay), the enzyme′s inhibition was observed (28.578 ± 2.979%). The inhibitory action on AChE in the in vitro assay proved to be lower than the donepezil used as a positive control (IC50 = 13.32 ± 1.34 µM). Thus, the weak inhibitory activity of W. saharae extract toward AChE activity could be related to the chemical composition of this species since we considered that the major substance in the plant extract is usually responsible for its biological activity.
Taxifolin, highly present in the W. saharae extract, has been shown to have relatively weak AChE inhibitory properties (IC50 = 133.1 µg/mL) [79] despite studies that claim otherwise [80]. Although chlorogenic acids are mainly responsible for inhibiting the AChE enzyme in green coffee extracts [81], their presence in W. saharae extract could not be sufficient to inhibit this enzyme strongly. Some of them, like 4,5-di-O-caffeoylquinic, one of the major compounds in W. saharae extract, showed AChE inhibition under 50% [81].
In addition, some phenolic compounds may act as activators of AChE. Earlier studies demonstrated that caffeic acid was shown to increase AChE activity in in vitro studies [82]. Some authors have hypothesized that caffeic acid (or its derivatives) could trigger the AChE catalytic site′s activation in a reaction at a second site [83]. Hence, the presence of caffeic acid in W. saharae extract could, in part, explain these results.

Molecular Docking
An in silico study was performed to further screen the mechanisms of interaction between α-glucosidase and the three major compounds in W. saharae extract. Figure 3 shows the 2D and 3D structures of the docked compounds at the active site of the α-glucosidase enzyme and the types of interactions involved.
(a) Based on the docking test's interaction data, the major constituents of W. saharae extract interact with α-glucosidase, engaging different amino acids (Table 5). Taxifolin showed a higher binding affinity, as indicated by its lower binding energy of -5.89 (kcal/mol), followed by 4,5-O-dicaffeoylquinic acid and 1,3-O-dicaffeoylquinic acid with a binding energy of -4.17 and -4.02 (kcal/mol), respectively.
Compounds used as standards to elucidate the identification of the phenolic constituents and to elaborate the calibration curves were obtained from EXTRASYNTHESE (Genay CEDEX, France).
Formic acid and acetonitrile HPLC-grade solvents were purchased from Panreac (Barcelona, Spain). Ultrapure water was obtained by a Direct-Q ® water purification system (Merck Life Science, Darmstadt, Germany). All other chemicals were of analytical grade.

Extract Preparation
Aerial parts of W. saharae were collected from Djebel Antar (Bechar, Southwest Algeria; 31 • 56 34 N, 1 • 55 52 W). A voucher specimen was deposited in the Herbarium of the VAREN laboratory of Mentouri University of Constantine, Algeria, under the reference number VAREN/CWS04/05. The leaves were shade air-dried at room temperature (25 • C) before they were ground to a fine powder. The powder was then macerated at room temperature with a 80:20 mixture of methanol:water (v/v) for 72 h three times. The filtrates were concentrated under reduced pressure using a rotary evaporator at 30 • C. The residual water was freeze-dried in a freeze dryer and then stored at 4 • C until future use.

Total Phenolic Content
Total phenolic content was determined using the Folin-Ciocalteu method [84]. An aliquot of 15 µL of plant extract (1 mg/mL) was mixed with 15 µL of Folin's reagent and 60 µL of water. After 5 min of incubation, 150 µL of Na 2 CO 3 solution (20% w/v) was added. The mixture was then incubated in the darkness for 60 min, and absorbance was read at 760 nm. The standard curve of gallic acid was obtained under the same conditions as above, and the total phenolic content was measured as µg equivalents of gallic acid per mg of extract (µg GAE mg of extract).

Total Flavonoid Content
The total flavonoid content of the plant's crude extract was determined according to the method described by Türkoglu et al. [85] with a slight modification. A total of 100 µL of plant extract was mixed with 100 mL of AlCl 3 (2%) solution. The mixture was incubated at room temperature for 10 min, and then the absorbance was measured at 415 nm. The standard curve of quercetin was obtained under the same conditions as above, and the total phenolic content was measured as µg equivalents of quercetin per mg of extract (µg GAE/mg of extract).

UHPLC-DAD-ESI-MS/MSn Characterization of W. saharae Extract
Phenolic composition of hydromethanolic extract of W. saharae was determined using an Ultimate 3000 (Dionex Co., San Jose, CA, USA) apparatus equipped with a binary pump, an automatic sampler, and a diode array detector (Dionex Co., San Jose, CA, USA). The MS analysis was performed using a Thermo LTQ XL mass spectrometer (Thermo Scientific, San Jose, CA, USA), which was equipped with an electrospray ionization interface (ESI). The separation was carried out with a Hypersil Gold (Thermo Scientific, USA) C18 column (100 mm length; 2.1 mm i.d.; 1.9 µm particle diameter, end-capped) at room temperature 25 • C. The extract was prepared at a 1 mg/mL concentration and the injection volume was 10 µL. The solvents used were formic acid in water (A) and acetonitrile (B). The flow rate was set at 2 mL/min. Spectra were recorded in negative mode. MS and MS/MS data were processed using the Thermo Xcalibur Qual Browser data system (Thermo Scientific, USA).

GC-MS Characterization of W. saharae Extract
The lipophilic composition of the hydromethanolic extract of W. saharae was analyzed by GC-MS using a Shimadzu Gas Chromatograph QP2010 Ultra. Three aliquots of the W. saharae extract (20 mg) were submitted to silylation procedure according to the method described by Freire et al. [86]. Silylation is the substitution of the active hydrogen atoms by a trimethylsilyl group (TMS), so that all the compounds with hydroxy groups will be transformed into the correspondent TMS derivatives. The sample was dissolved in 250 µL of dichloromethane. A total of 200 µL of hexatriacontane as internal standard (IS), 250 µL of pyridine, 250 µL of BSTFA, and 50 µL of TMSCl were then added to the sample. The mixtures were incubated for 30 min at 70 • C and then injected in triplicate into the GC-MS apparatus. Compound separation was carried out in J&W DB-5 capillary column (30 m × 0.25 mm inner diameter, 0.25 µm film thickness) using helium as the carrier gas (35 cm s −1 ). The following standards: eicontane, palmitic acid, tetradecanol, D-(+)-mannose, mannitol, and cholestanol, were used to elaborate calibration curves. Compounds were identified by comparing their mass spectra with mass spectra in the Wiley and NIST libraries and those available in the literature.
3.6. Antioxidant Activities 3.6.1. Determination of 1,1-Diphenyl-2-Picrylhydrazyl Radical Scavenging Activity The 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) free radical-scavenging activity was evaluated by a slightly modified method [87,88]. Briefly, the solution of 0.1 mM of DPPH • was prepared in ethanol, and 160 µL of this solution was added to 40 µL of sample solutions diluted in methanol at different concentrations. Twenty-five minutes later, the absorbance was measured at 517 nm. The scavenging capability of DPPH • was calculated using the following equation. The results were given as IC 50 (mg/mL) corresponding to the concentration of 50% inhibition.
where Ac refers to the absorbance of control; A S refers to the absorbance of sample.
The sample concentration providing 50% free radical scavenging activity (IC 50 ) was calculated from the graph of DPPH • scavenging effect percentage against sample concentration. BHA, BHT, Trolox, and ascorbic acid were used as antioxidant standards for comparison of the activity.

Determination of 2,2 -Azinobis(3-Ethylbenzothiazoline-6-Sulfonic Acid) Scavenging Activity
The 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free cation-radical (ABTS •+ ) scavenging activity was obtained by spectrophotometric analysis according to the slightly modified method proposed by Re et al. [89]. The ABTS •+ was regenerated by adding 2.45 mM potassium persulfate to an aqueous solution of 7 mM ABTS •+ , which was stored in the dark at room temperature for 12 h. Before use, the ABTS •+ solution was diluted until an absorbance of 0.700 ± 0.025 at 734 nm with ethanol was obtained. Then, to each well, in a 96-well plate, 160 µL of this solution and 40 µL of sample solutions diluted in methanol at different concentrations were added. After 10 min, the percentage inhibition at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). The scavenging capability of ABTS •+ was calculated using the following equation: where Ac refers to the absorbance of control; As refers to the absorbance of sample.
The sample concentration providing 50% cation radical scavenging activity (IC 50 ) was calculated from the graph of ABTS •+ scavenging effect percentage against sample concentration. BHA, BHT, Trolox, and ascorbic acid were used as antioxidant standards for comparison of the activity.

Galvinoxyl Radical Scavenging Activity
The trapping activity of the galvinoxyl radical was determined according to the method described by Shi et al. [90] in 96-well microplates. A total of 40 µL of the extract to be tested at different concentrations was placed in the wells of the microplate. A total of 160 µL of methanolic galvinoxyl solution (4 mg of galvinoxyl in 100 mL of methanol) was added to each well. The plate was then incubated in the dark for 2 h. Absorbance was measured at the wavelength of 428 nm.
A blank was prepared by mixing 160 µL of methanol with 40 µL of each extract. All tests were performed in triplicate. The percentage of inhibition of the galvinoxyl • is expressed by the following formula: Galvinoxyl • Scavenging effect (%) = (Ac − As)/Ac × 100 where Ac refers to the absorbance of control; As refers to the absorbance of sample.
The concentration of the sample providing 50% inhibition (IC 50 ) was calculated by relating the inhibition percentages to the concentrations of the extracts. The antioxidant capacity of the extract was compared with ascorbic acid, BHT, BHA, and Trolox used as antioxidant standards and prepared under the same conditions.

Ferric Reducing Power Assay
The FRP assay was carried out according to the method of Oyaizu [91] with slight modification. Briefly, 40 µL of phosphate buffer (pH 6.6) and 50 µL of potassium ferricyanide (1%) (1 g of K 3 Fe(CN) 6 in 100 mL H 2 O) were mixed with 10 µL plant extract at different concentrations and then incubated 20 min at 50 • C. A mixture of 50 mL trichloroacetic acid (TCA) (10%), 40 mL H 2 O, and 10 µL FeCl 3 (0.1%) was then added. The absorbance of the reaction mixture was then recorded at 700 nm. Increased absorbance of the reaction mixture indicated increased FRP. The results were given as A 0.50 , which corresponds to the concentration producing 0.500 absorbance.

Cupric Ion Reducing Antioxidant Capacity Assay
The cupric ion reducing antioxidant capacity (CUPRAC) assay was determined by the method described by Apak et al. [92]. A total of 40 µL of W. saharae extract at different concentrations was added to a solution composed of 50 µL Cu Cl 2 , 2H 2 O (10 mM), 50 µL neocuproine (7.5 mM), and 60 µL ammonium acetate buffer solution (1 M, pH 7.0). The reaction mixtures were incubated in the dark for 1 h. The absorbance was then measured at 450 nm.
The results were calculated as A 0.50 (µg/mL) corresponding to the concentration indicating 0.500 of absorbance. The reduction capacity of the extracts was compared with that of BHA, BHT, Trolox, and ascorbic acid prepared under the same conditions.

Anti-Inflammatory Activity
The in vitro anti-inflammatory activity was determined by the ability of the extract to scavenge NO • following the method described by Afonso et al. [93]. A total of 100 µL of each extract concentration was mixed with 100 µL of sodium nitroprusside (3.33 mM) solution prepared in PBS 100 mM (pH 7.4). After incubation for 10 min at room temperature under light irradiation, 100 µL of Griess reagent (equal volume of 1% of sulfanilamide and 0.1% of naphthyl ethylenediamine dihydrochloride in 2.5% of phosphoric acid) was then added to the mixture. The absorbance was measured at 562 nm in spectrophotometer. Curcumin was used as the reference compound.
The scavenging ability of NO • was calculated using the following equation: NO • Scavenging effect (%) = (Ac − As)/Ac × 100 where Ac refers to the absorbance of control; As refers to the absorbance of sample. The sample concentration providing 50% NO • scavenging activity (IC 50 ) was calculated from the graph of NO • scavenging effect percentage against sample concentration.
3.8. Inhibition of Enzymatic Activities 3.8.1. Inhibition of α-Glucosidase Activity α-Glucosidase inhibition activity was measured by the method described by Neto et al. [94] with brief modifications. A total of 50 µL of each extract concentration was mixed with 50 µL of 4-nitrophenyl α-D-glucopyranoside (PNPG) solution used as substrate. A total of 100 µL of the α-glucosidase enzyme solution was then added to the mixture. The absorbance was measured at 405 nm at 37 • C, for 20 min every minute. Acarbose was used as reference.
The α-glucosidase inhibitory activity was expressed as percentage inhibition and was determined according to the following formula: Inhibition % = (Ac − As)/Ac × 100 where Ac refers to the absorbance of control (enzyme and buffer); As refers to the absorbance of sample (enzyme and inhibitor).
IC values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined.
3.8.2. Inhibition of α-Amylase Activity α-Amylase inhibition activity was determined according to the method previously described by Wickramaratne et al. [95] with slight modifications. Briefly, 200 µL of each extract concentration was added to 400 µL of a 0.8% (w/v) starch solution dissolved in 20 mM phosphate buffer (pH 6.9, containing 6 mM of NaCl). After incubation at 37 • C for 5 min, the reaction was started with the addition of 200 µL of α-amylase solution. A total of 200 µL of the mixture was then mixed with 600 µL of 3,5-dinitrosalicylic acid (DNS) reagent.
A second aliquot of 200 µL of the first mixture was also collected 15 min later and mixed with DNS reagent. The mixtures were boiled for 10 min. After cooling, 250 µL was transferred to each well in a 96-well microplate and the absorbance was read at 450 nm.
The α-amylase inhibitory effect was calculated by the following equation: Inhibition % = (Ac − As)/Ac × 100 where Ac refers to the absorbance of control (enzyme and buffer); As refers to the absorbance of sample (enzyme and inhibitor). IC values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined. Acarbose was used as reference compound.

Inhibition of Acetylcholinesterase Activity
Inhibition of acetylcholinesterase (AChE) activity was determined as described by Ellman et al. [96] with slight modifications. A total of 150 µL of 0.025 U/mL solution of AChE was added to 50 µL of each extract at different concentrations. After incubating at 37 • C for 5 min, 50 µL of DTNB (0.5 mM) and 50 µL of acetylthiocholine iodide (2.5 mM) were added to the mixture. The absorbance was read at 415 nm each 150 s for 7.5 min. Donepezil was used as a reference. The percentage inhibition of AChE enzyme was determined according to the following formula: Percentage of inhibition = (E − S)/E × 100.
where E is the activity of the enzyme without extract; S is the activity of the enzyme with the extract.
IC 50 values (concentration of inhibitor required to inhibit 50% of enzyme activity) were determined.

Molecular Docking Study
To support our findings from in vitro inhibition of α-glucosidase activity by the W. saharae extract, three compounds were docked to the α-glucosidase enzymatic activity. The selected compounds were taxifolin, 1,3-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, which are the most abundant in the W. saharae extract. The 3D structures of the considered molecules were downloaded from PubChem database [97]. All three structures were performed by AM1 [H2] semi empirical method by using Orca-4.2.1 program package [98]. The crystal structure of the α-glucosidase (PDB code 3W37) was retrieved from the Protein Data Bank [99]. Water molecules were removed from the protein structure. The docking software AutoGrid and AutoDock (version 4.2.6) implemented in AutoDock Tools (ADT 1.5.6) program were used for the docking experiments. The most stable receptor-ligand complexes were generated using Chimera [100] and BIOVIA Discovery studio visualizer (version 1.10.2) [101].

Statistical Analysis
All of the data were analyzed with MINITAB software (version 16, State College, PA, USA) using one-way ANOVA followed by a post hoc honestly significant difference (HSD) Tukey's test at p ≤ 0.05. Data were recorded as the mean ± standard deviation (m ± SD) from three independent assays.

Conclusions
Our results provided new information and allowed the characterization of the chemical profile of the hydromethanolic extract of W. saharae. The extract showed potent antioxidant and α-glucosidase inhibition properties, highlighting that this species provides a promising source for obtaining natural compounds that could be used to develop food additives or pharmaceutical products. Anti-inflammatory properties were also observed with the hydromethanolic extract of W. saharae. Finally, further toxicological and pharmacological studies must be performed for pharmaceutical purposes.