Polyphenol composition and antioxidant activity of Searsia tripartita and Limoniastrum guyonianum growing in Southeastern Algeria

Due to the notable medicinal value of Searsia tripartita and Limoniastrum guyonianum, it was of great interest to conduct phytochemical and antioxidant investigations. Polyphenol extracts of the dried powder of plants were prepared by maceration in 70% of methanol. The concentrated extracts were successively fractioned with distilled water, ethyl acetate and n-butanol. Obtained extracts were analysed for their phenol compositions by colorimetric processes and were evaluated for their total antiradical capacity by ABTS, DPPH and ORAC assays. Additionally, the phytochemical components were characterized in the methanolic extracts by HPLC–DAD–ESI-MS/MS in the negative ionisation mode. S. tripartita exhibited the higher quantity of phenolic compounds. The ethyl acetate extract of S. tripartita had the greatest quantities of polyphenols (55.5 ± 4.9 mg gallic acid equivalent per gram of dry residue (GAE/g DR)), flavonoids (44.2 ± 0.8 mg rutin equivalent per gram of dry residue (RE/g DR)), and tannins (12.5 ± 2.5 mg GAE/g DR). In L. guyonianum, the aqueous fraction had the highest quantity of polyphenols and tannins (10.2 ± 1.4 and 3.2 ± 1.9 mg GAE/g DR, respectively), whereas flavonoids (1.8 ± 0.2 mg RE/g DR) were higher in the ethyl acetate portion. The antioxidant capacity of the hydromethanolic extract of S. tripartita was found to be 3–16 times more effective than that of L. guyonianum using ORAC, DPPH, ABTS tests. HPLC analysis of hydromethanolic extracts provided tentative identification of four flavonoid glycosides in S. tripartita (myricetin-3-O-glucoside, myricetin-3-O-rhamnoside, quercetin-3-O-glucoside, and quercetin-3-O-rhamnoside) and five phenolic metabolites of the flavonoid class in L. guyonianum (myricetin-3-O-rhamnosylglucoside, myricetin-3-O-glucuronide, myricetin-3-O-pentoside, quercetin-3-O-glucuronide, and eriodictyol-7-O-rutinoside). Our findings revealed that these plants could be used as a potent source of health molecules.


a b s t r a c t
Due to the notable medicinal value of Searsia tripartita and Limoniastrum guyonianum , it was of great interest to conduct phytochemical and antioxidant investigations. Polyphenol extracts of the dried powder of plants were prepared by maceration in 70% of methanol. The concentrated extracts were successively fractioned with distilled water, ethyl acetate and n -butanol. Obtained extracts were analysed for their phenol compositions by colorimetric processes and were evaluated for their total antiradical capacity by ABTS, DPPH and ORAC assays. Additionally, the phytochemical components were characterized in the methanolic extracts by HPLC-DAD-ESI-MS/MS in the negative ionisation mode. S. tripartita exhibited the higher quantity of phenolic compounds. The ethyl acetate extract of S. tripartita had the greatest quantities of polyphenols (55.5 ± 4.9 mg gallic acid equivalent per gram of dry residue (GAE/g DR)), flavonoids (44.2 ± 0.8 mg rutin equivalent per gram of dry residue (RE/g DR)), and tannins (12.5 ± 2.5 mg GAE/g DR). In L. guyonianum , the aqueous fraction had the highest quantity of polyphenols and tannins (10.2 ± 1.4 and 3.2 ± 1.9 mg GAE/g DR, respectively), whereas flavonoids (1.8 ± 0.2 mg RE/g DR) were higher in the ethyl acetate portion. The antioxidant capacity of the hydromethanolic extract of S. tripartita was found to be 3-16 times more effective than that of L. guyonianum using ORAC, DPPH, ABTS tests. HPLC analysis of hydromethanolic extracts provided tentative identification of four flavonoid glycosides in S. tripartita

Background
The potential detrimental effects of molecular oxygen are believed to be due to the synthesis of partially reduced and highly harmful species, called free radicals such as reactive oxygen and/ or nitrogen species (ROS/RNS) [ 1 , 2 ]. These species are generated from normal physiological or biochemical processes in all living organisms. The overproduction and disruption of homeostasis between these toxic species and the overcapacity of detoxification systems lead to the appearance of oxidative stress phenomenon [ 3 , 4 ]. This overconcentration of ROS/RNS is able to oxidize biomolecules like, unsaturated lipids in cellular membrane, structural and functional proteins, carbohydrates and deoxyribonucleic acids, causing tissue destruction and cell injury that may be responsible for various chronic and degenerative illnesses, such as tumour, cardiovascular disease, neural diseases, skin irritation and inflammation [ 5 , 6 ]. Fortunately, there are well-designed protective systems in place, with functions based on enzymes (mainly superoxide dismutase, glutathione and ascorbate peroxidase, and catalase), primary antioxidant compounds (e.g., ascorbic and caffeic acids, kaempferol, glutathione, anthocyanins and polyphenols), and certain macromolecules (such as albumin, ceruloplasmin, and ferritin) [3] , although these systems may become overwhelmed.
Currently, there is an increase in the number of studies aiming to identify new sources of natural antioxidants that are important for health care benefits. Various classes of secondary antioxidants generally occur in plants. Among these phytochemical substances, polyphenols have been largely described for their ability to act as antioxidant agents. Polyphenols are of vast importance owing to their multiple biological activities, such as antimicrobial, anticoagulant, anticancer, hepatoprotector, cardioprotector and vasodilatory effects [7] . These actions have been attributed to their reducing power that relates to the mobility of phenolic hydrogen atoms, which allows human cells to defend against oxygen-induced injury [8] .
Algeria, with its geographical position and extensive area, enjoys a very diverse range of ecosystems and soils. This promotes the development of a rich and diverse flora, represented by 30 0 0 plant species, of which approximately 10 0 0 are considered to be medicinally important [ 9 , 10 ]. These phytoresources constitutes an inestimable treasure that should be valorised and utilised as a potential source of natural biological compounds. Therefore, to appreciate the natural plant resources in Algeria, we undertook to study two local Saharan species, Searsia tripartita (Ucria) Moffett (synonym: Rhus tripartita ) and Limoniastrum guyonianum Durieu ex Boiss., which grow especially well in North Africa. The leaves of both species are commonly used in Algeria for various medicinal purposes, such as for treating various digestive, skin, circulatory, metabolic, and respiratory diseases [ 11 , 12 ]. Leaves and galls of L. guyonianum are also traditionally used for their hypoglycaemic activity, although no study has been undertaken to explore this aspect [13] .
Our study was focused to characterize and quantify the phenolics, flavonoids, and tannins in the aerial parts of S. tripartita and L. guyonianum and to evaluate whether these phytomolecules have free radical quenching potential. Furthermore, the main polyphenols were characterized by reverse-phase high-performance liquid chromatography coupled to UV-visible spectrometry and ion-trap mass spectrometry used in the MS and MS/MS modes, in order to examine their therapeutic potential, as suggested by their use in traditional medicine.

Plant materials collection
The plant material consisting of the aerial part of S. tripartita (Anacardiaceae) and L. guyonianum (Plumbaginaceae), were harvested during the autumn season from their natural biotopes, Ghardaïa (32 °13 09.1 N, 0 03 °30 0 0.1 E, altitude 519 m) and Ouargla (33 °12 30.9 N, 006 °07 59.6 E, altitude 299 m), that are regions located in the north-eastern Algerian Sahara. The botanical identification of species was carried out according to the Flora of Sahara [14] .
To dehydrate the plant material by a similar method to that employed by traditional medicine, the fresh aerial parts were left to dry in air and in darkness at 25-30 °C room temperature for two weeks. The dried samples were powdered into a fine powder using an electric mortar and were put in glass vials maintained at a low temperature ( −20 °C) until phytochemical and biological analysis were begun.

Extraction of phenolic metabolites
A sample of 100 g of the dried and powdered aerial parts of each plant was extracted by immersion in 500 mL of hydroalcoholic mixtures (methanol/ water, 70/30: V/ V) under magnetic stirring for 24 h at laboratory temperature and then filtered through filter paper (Whatman No. 1). Then, the residue was exhausted successively by maceration twice using the same volume of the initial solvent.
The filtrates were combined and evaporated to a dry residue under reduced pressure at 40 °C using a rotary evaporator until dryness was achieved. The obtained dry residue constituted the "crude extract". A part of the dried crude extract was solubilized in absolute methanol (1 mg/mL) to measure their ability to scavenge free radicals and for quantifying the total extractable polyphenols. The remaining crude extract was suspended in 100 mL distilled water. Next, the extract was extracted three times with ethyl acetate (3 × 100 mL) and then with n -butanol (3 × 100 mL). The aqueous, ethyl acetate, and butanolic phases were evaporated at reduced pressure at 40 °C using a rotary evaporator until dryness was achieved [15] . Residues corresponding to the ethyl acetate, butanolic, and aqueous fractions were solubilized in absolute methanol to make a final concentration of 1 mg/mL for subsequent analysis.

Total phenolic content
The concentration of total phenols was estimated by the Folin-Ciocalteu method [16] . A volume of 100 μL of the sample was combined with 500 μL of the Folin-Ciocalteu reagent (diluted 10-fold) and 10 0 0 μL of distilled water. After vigorous shaking and incubation for 5 min in the dark, 1500 μl of an aqueous solution of sodium carbonate (200 g/L) were added.
The reaction mixture was left for 2 h in the dark to develop blue colour. Finally, the optical density was measured at 765 nm using a SHIMADZU UV mini-1240 UV-Vis spectrophotometer (Shimadzu, Japan). The total phenolic amount was estimated by referring to a standard curve made with gallic acid and was expressed as mg gallic acid equivalent per gram of dry residue (mg GAE/g DR).

Total flavonoid content
The flavonoid content was determined according to the colorimetric method of Lamaison and Carnatusing [17] , based on the formation of a yellow complex, due to the interaction between the flavonoid and aluminium and which absorbent at 430 nm [18] . An aliquot of 1 mL of each extract was mixed with 1 mL of a 2% aluminium chloride solution (2% in methanol). After incubation at room temperature for 10 min, the optical density was measured with a SHIMADZU UV mini-1240 UV-Vis spectrophotometer. The flavonoid content was determined by using a standard curve made with rutin and was expressed as mg rutin equivalent per gram of dry residue (mg RE/g DR).

Total tannin content
The total tannin level was measured according to the protocol developed by Makkar et al. [19] with some modifications. 100 mg of gelatin protein was mixed with 1 mL of distilled water and 1 mL of each sample. After incubation for 15 min at 4 °C, the mixture was centrifuged for 10 min at a centrifugal force of 30 0 0 g. 20 0 μL of supernatant were used to measure the non-tannin phenolics using the Folin-Ciocalteu method. Total tannin amount was calculated by subtracting non-tannin phenolic from total phenolic amount.

Measurements of total antioxidant capacity (TAC)
Antioxidant activities were evaluated by three complementary radical scavenging assays involving different mechanisms because multiple reactions are involved in natural extracts [20] . The antioxidant activity measurements were performed with respect to the regression equation of the calibration curve of trolox and expressed as trolox equivalent in micromoles per gram of dry residue (μmol TE/g DR).

ABTS (2,2 ʼ-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay
The antiradical activity against the ABTS radical of each sample was made following the procedure reported by Re et al. [21] . The stock solutions contain 7 mM ABTS and 2.45 mM potassium persulfate. The ABTS radical cation (ABTS . + ) was prepared by mixing 5 mL of the ABTS solution with 88 μL of potassium persulfate, the mixture left in the dark at room temperature for 12-16 h before use. The solution was diluted daily with distilled water to obtain an absorbance of 0.7 ± 0.02 at 734 nm. The assay was adapted to 96-well Microplates, and 10 μL of extracts or standard was added to 290 μL of freshly diluted ABTS . + solution and incubated for 5 min in the dark. The absorbance was measured at 734 nm using a VERSAmax Microplate Reader (Molecular Devices, CA, USA) with Softmax Pro-software

DPPH (2,2-diphenyl-1-picrylhydrazyl) assay
The DPPH assay is based on measurement of the loss of the DPPH °purple colour to yellow at 515 nm after reaction with antioxidants and carried out according to the procedure described by Brand-Williams et al. [ 22 ]. A total of 10 mL of each extract or standard was mixed with 290 μL of DPPH °methanolic solution (60 μM) in a 96-well Microplate. The decrease in absorbance was monitored at 515 nm every minute on the VERSAmax Microplate Reader until the reaction reached a plateau.

ORAC (oxygen radical absorbance capacity) assay
The ORAC test evaluates the antiradical ability against peroxyl radicals. This test was performed according to the method described by Ou et al. [23] . Analyses were conducted in a 75 mM phosphate buffer solution with pH 7.4 at 37 °C. The peroxyl radical was generated using AAPH that was freshly prepared for each run. Fluorescein was used as the substrate. Fluorescence was recorded using a SAFAS (Monaco) FLX-Xenius spectrofluorometer. ORAC analyses were performed in 96well black opaque Microplates. The excitation wavelength was 485 nm and emission wavelength was 520 nm. 20 μL of antioxidant solution was mixed with 180 μL of 120 nM fluorescein and incubated for 15 min at 37 °C. The reaction was initiated with 100 μL of 12 mM AAPH solution. The fluorescence was monitored every minute for 2 h. Fluorescence data were plotted and the area under the curve (AUC) was calculated. The net AUC was calculated by subtracting the AUC of the blank from the AUC obtained in the presence of an antioxidant (AUC extracts -AUC blank).

Analysis of individual phenolic compounds by analytical RP-HPLC-IT-MS
The detection and identification of phenolic compounds were performed on the aerial part of the plants. To that end, 1 g of dried material was prepared by dissolving in 5 mL of 70% (v/v) aqueous methanol containing 1% v/v formic acid for 30 min in an ultrasonic bath (Brasson 2200, USA). The procedure was repeated three times and the combined extracts were filtered through a polytetrafluoroethylene (PTFE) membrane (0.45 μm, Uptidisc Interchim, France) and injected into an RP-HPLC-UV-visible-MS system. Analyses were performed using an LC-DAD-ESI-MS (liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry) system composed of an SCMA10 0 0 degasification system (ThermoQuest, San Jose, CA, USA), an automatic injection system (ThermoFinnigan, San Jose, CA, USA), an 1100 series binary pump (Agilent Technologies, Palo Alto, CA, USA), and a Spectra photodiode array detector system UV60 0 0LP (Ther-moFinnigan, San Jose, CA, USA). The mass spectrometer was an ion trap LCQ Deca (ThermoFinnigan, San Jose, CA, USA) equipped with an electrospray ionization source. A sample volume of 2 μL was injected onto a Zorbax Eclipse XDB-C18 column (2.1 mm × 150 mm, 3.5 μm; Agilent Technologies) and maintained at 30 °C. The mobile phase consisted of solvent A (aqueous formic acid, 0.1% v/v) and solvent B (acetonitrile containing 0.1% formic acid v/v). The solvents were filtered through a 0.45 μm PTFE membrane. The following linear gradient elution was applied at a constant flow rate of 0.2 mL/min: 97%-91% A, 0-5 min; 91%-84% A, 5-15 min; 84%-50% A, 15-45 min; 50%-10% A, 45-48 min; and 48-51 min isocratic and reconditioning the column for 15 min.

Statistical analysis
The data were expressed as means ± standard deviation (SD) and statistically analysed using a Tukey's HSD test, differences were considered statistically significant at the 5% level. Linear regressions were used to examine the relationships between antioxidant activity and phenolic component content according to Bravais-Person correlation test. All statistical analyses were performed using XLSTAT statistical software version 2009.6.01. All experiments were executed in triplicate.

Phenolic fractionation in the plant species
Solvent-directed extraction is an essential step for the elucidation of bioactive components from medicinal plants. However, the nature of the extracting solvent is the most controversial parameter that can influence the quantity and the quality of molecules isolated from the plant extracts, namely, phenolics [24] . In our work, four solvents with increasing polarity were used to evaluate the polyphenol content in the extracts. S. tripartita possesses significantly higher content ( p < 0.001) of total phenolic compounds, flavonoids, and tannins than that of L. guyonianum except for the content of total phenolics ( p = 0.102) and tannins ( p = 0.248) in the aqueous fraction ( Table 1 ). Together, these compounds represent 16.3% of the dried S. tripartita plant versus 3.4% in L. guyonianum. For both species, phenolics were quantitatively the most abundant. Based on the solvent fraction, the flavonoid content ranged from 0.3 ± 0.1 to 53.6 ± 1.8 mg RE/g DR for S. tripartita and from 0.2 ± 0.0 to 5.7 ± 0.1 mg RE/g DR for L. guyonianum . The tannin content ranged from 7.9 ± 1.9 to 28.5 ± 5.2 mg GAE/g DR in S. tripartita and from 0.3 ± 0.1 to 5.4 ± 1.7 mg GAE/g DR in L. guyonianum . The ethyl acetate fraction was the richest in S. tripartita for the three classes of compounds (57.1% for phenolics, 75.8% for flavonoids, and 40.6% for tannins). The residual aqueous fraction had the lowest content, whereas L. guyonianum exhibited higher content of polyphenols (70.3%) and tannins (80%). The opposite was true for flavonoids, as the ethyl acetate extract was the richest in flavonoids (51.4%).

Total antioxidant capacity
Due to the complexity of the oxidation processes and the chemical diversity of antioxidants, with both hydrophilic and hydrophobic components, the total antioxidative ability cannot be fully evaluated by one single chemical reaction. Therefore, more than one type of radical system is required to investigate the total antioxidant activity of natural extracts and pure isolated substances [25] . In the current study, total antiradical capacity was measured using the ABTS, DPPH and ORAC The total phenolic and tannins content values were expressed as mg gallic acid equivalent/ g of dry residue. The flavonoids content was expressed as mg rutin equivalent /g of dry residue.
Values with different letters in the same row represent significant differences between the similar fractions of the two species ( p < 0.05). The differences were tested using Tukey's test.  assays ( Table 2 ). The ORAC technique measures the ability of an antioxidant to quenching free radicals (peroxyl radicals) by hydrogen atom transfer [26] , whereas the other two radicals may be neutralized either via electron transfer or via hydrogen donation [27] . Overall, the total antioxidant properties of the two studied species were strongly different. The effectiveness differed between and within the two species, depending on the scavenging assay used and on the tested fraction. S. tripartita with high levels of phenolic compounds displayed very high significant antioxidant capacity ( p < 0.001) in all assays (except for the aqueous fraction in the ORAC assay, p = 0.080). The crude extract possessed the highest antioxidant activity as determined by the ORAC (1243.1 ± 46.4 μmol TE/g DR), ABTS (214.7 ± 5.2 μmol TE/g DR) and DPPH (551.9 ± 3.9 μmol TE/g DR) assays. The results were 3 to 16-fold higher than those in L. guyonianum (369.5 ± 85.7, 14.2 ± 1.0 and 33.0 ± 0.9 μmol TE/g DR, respectively). Antioxidant capacity in S. tripartita was highest in the ethyl acetate extract, followed by the butanolic and aqueous portions, whereas in L. guyonianum the organic fractions exhibited the lowest antioxidant capacity, with the butanolic fraction always being the lowest. The antiradical activity of plant extracts increased proportional to the phenolic content. The total phenolic, flavonoid, and tannin content all correlated strongly and positively with antioxidative potential ( Table 3 ). The highly significant correlation coefficients were 0.96 ≤ R 2 ≥ 0.98 ( p < 0.0 0 01) 0.93 ≤ R 2 ≥ 0.99 ( p < 0.0 0 01), and 0.87 ≤ R 2 ≥ 0.93 ( p < 0.0 0 01), respectively.

Phenolic structure elucidation by LC-ESI-MS/MS
A qualitative composition of phenolic compounds in the aqueous-methanol fractions was carried out by HPLC -DAD -ESI-MS/MS in negative ionization mode. This mode was selected owing to the greater sensitivity and selectivity for phenolics in comparison with the positive mode. Polyphenols are weakly acidic compounds, indicating that dissociation is easier than protonation [ 28 , 29 ]. Data acquisition and analysis were performed using the Xcalibur software version 2.1 ( Table 4 ,  ( Fig. 3 D).
Compound 5 exhibited a deprotonated molecule at m/z 595 (aglycone + disaccharides) and a product ion at m/z 287 characteristic of the aglycone eriodictyol, which may be attributed to the loss of a rutinoside moiety. This compound has been assigned as eriodictyol −7-Orutinoside ( Fig. 3 E).

Discussion
Polyphenols are among the important group of secondary metabolites in plants and they are detected at high concentrations in many medicinal plants [ 34 , 35 ]. Undoubtedly, structure-related and dose-related mechanistic investigations are required for understanding the basis of phenolic compound pharmaceutical activity. S. tripartita and L. guyonianum are two halophyte species widely used as medicinal plants that possess several pharmacological effects. The root cortex extract of R. oxyacantha has been indicated to contain proanthocyanidic oligomers and polymers with high antioxidant property and the ability to prevent thymocyte death in rats [36] . Stems of R. tripartita are essentially rich in phenolic compounds and show great cardioprotective potential in human cells and rats [37] . Leaf and gall infusions of L. guyonianum have been utilized by the Tunisian traditional healers for the treatment of diarrhoea and dysentery [38] . Recently, Krifa et al. [39] stated that an aqueous gall extract of this plant exhibited antioxidant activities, as well as immunomodulation actions on splenocytes, natural killer cells, and macrophages.
Since the two species in the present study may be characterized by diversified classes of phenolic compounds, we first explored the qualitative and quantitative extraction performance of different solvent systems. The present study showed a great variation of phenolic content as a function of species and solvent polarity. S. tripartita exhibited higher quantities of phenolic compounds than that of L. guyonianum. Together, these compounds represented 16.3% of the dried extract for the S. tripartita plant versus 3.4% for L. guyonianum. The high level of phenolic compounds from S. tripartita could be related to intrinsic (genetic) factors also extrinsic (environmental and extraction conditions) factors [40] . It has been demonstrated that their concentrations are higher when the living environment of the plant is not adequate, whereby the plant promotes the synthesis of secondary metabolites to adapt and survive [41] . In the region of Ouargla and Ghardaïa, the environmental conditions are drastic. For example, the average temperature reached respectively 36.7 °C and 35.25 °C in summer (2010- 2015), and the pluviometry was at the most 7 mm and 11.6 mm a month. These conditions probably contributed to favour metabolic pathways producing metabolites that allow plants to survive.
For both species, high variation in polyphenol content was found between the different fractions depending on the solvent polarity. The content of all phenolic classes was in the following order from high to low: ethyl acetate fraction > butanolic fraction > aqueous fraction in S. tripartita and aqueous fraction > ethyl acetate fraction > butanolic fraction in L. guyonianum (except flavonoids). The richness of the ethyl acetate portion of the crude extract of S. tripartita in phenolic compounds might be linked to the richness of this species in moderately polar compounds (e.g., aglycones and monoglycosylates). However, the presence of higher amounts of polyphenols and tannins in the aqueous fraction of the crude extract of L. guyonianum than that in the organic fractions suggests the richness of this species in hydrolysable glycosylated polyphenols. The solubility of phenolic compounds is particularly dependent to the polarity of solvent used, the molecular weight and length of constituent hydrocarbon chains, the presence and position of hydroxyl groups, and the degree of polymerization of phenolics [42] . Many previous studies support the observation that phenol content can vary widely as a function of the polarity of the solvent in liquid-liquid extraction and fractionation systems. Kosar et al. [43] proved that the ethyl acetate fraction had the greatest phenolic content in Rhus coriaria L. (540 mg GAE/g extract), which is an Anacardiaceae species as well as S. tripartita , followed by the water fraction (5.1 mg GAE/g extract). Kim et al. [44] reported in R. vernici- flua , another Anacardiaceae species, that the total phenolic content in the ethyl acetate fraction (723 mg GAE/g) and in 80% ethanolic extract (597 mg GAE/g) was higher than that in other fractions, whereas the 80% ethanolic extract was the highest in flavonoids, equalling 201 mg QE/g (quercetine equivalent).
In a study dedicated to L. guyonianum , Belfar et al. [45] first extracted the polyphenols with 60% ethanol before partitioning. These authors found high quantities of both total phenolic and total flavonoid content, 75.81 mg GAE/g and 13.44 mg QE/g (quercetin equivalent) in a butanolic fraction, and 15.51 mg GAE/g and 3.56 mg QE/g in an ethyl acetate fraction, respectively. In comparison, our results in L. guyonianum for total phenolic and total flavonoid content were 1.2 mg GAE/g DR and 0.2 mg RE/g DR in the butanolic fraction, and 3.1 mg GAE/g DR and 1.8 mg RE/g DR in the ethyl acetate fraction, respectively. These differences might be related to the fact that in the present study the reference was rutin, whereas it was probably (although not indicated) quercetin for the study by Belfar et al. [45] . Furthermore, they did not analyse the aqueous fraction, which was demonstrated to be important for the total phenolic content (10.2 mg GAE/g DR in our analysis). For S. tripartita , the total phenolic content in the aerial parts was 80.7 mg GAE/g DR in our study. In Tunisia, Itidel et al. [46] found levels of phenolics of 45-102.1 mg GAE/g in leaves, 77-219 mg in the stem cortex, and 39-121 mg in fruits of the same species (under the name R. tripartita (Ucria) Grande). For flavonoids, we found 53.6 mg RE/g DR and Itidel et al. [ 46 ] reported quantities of 50.7-74 mg RE/g in leaves, 5.6-12.7 in the stem cortex, and 5-11.9 in fruits.
Currently, great importance is given to the indication of total antioxidant capacity as a tool for identifying natural products that may have beneficial effects against degenerative diseases [47] . TAC values of plant extracts were largely different depending on the polarity of the solvent. Thus, effective antioxidant activity was found in S. tripartita . Our findings are in concord with those from a previous study by Mahjoub et al. [48] in Tunisian R. tripartitum ( = R. tripartita ) aerial parts after methanolic extraction. The ABTS assay showed TAC values of 1.20 mM TE/mg extract. Our results were 214.7 μmol TE/g DR. Ben Miled et al. [49] reported that polar extracts of R. tripartitum root (water and methanol) have a same and better ORAC activity with respective values of 8.95 and 8.55 μmol TE/ mg than non-polar ones (hexane and dichloromethane). On the other hand, Wu et al. [50] indicated effective antioxidant activity of 1544 μmol TE/g in the ORAC assay for an ethanolic extract of R. hirta L. fruits that was higher than that of our methanolic extract (1243.1 μmol TE/g DR). For L. guyonianum extract, Trabelsi et al. [51] found a powerful ability to quench DPPH radical with an IC50 of 120 μg/mL, which is difficult to compare to our extract with a TAC in the DPPH assay of 33 μmol TE/g DR. Furthermore, Souid et al. [52] demonstrated that the hydromethanolic extracts obtained from leaves of eight Limonium species have a highly ORAC levels, varied between 2631.50 μM TE/G DW for L. spathulatum and 5108 for L. virgatum .
The difference in the activity between the different fractions can be connected to the nature of the phenolic compounds in each fraction. Based on a study by Cowan [53] , during liquid-liquid extraction, the phytomolecules are distributed between the solvents based on their polarity and solubility. Consequently, a change in solvent nature alters its efficacy to extract a specific group of antioxidant compounds and influence the antioxidant properties of the extract [54] . Kratchanova et al. [55] mentioned the significant effect of solvent extraction on the assessment of antioxidant activity and polyphenol extraction. It is consequently advocated to utilize more than one extraction system to improve the investigation of the antioxidant activity of natural products.
The values of TAC estimated by the ORAC assay were notably higher than those obtained by the ABTS and DPPH assay methods. The diversity in radical scavenging between the diverse systems shown in these experiments may be ascribed to the difference in the reaction stoichiometry between the natural antioxidants in the extracts and various radicals. Free radical scavenging by antioxidants is dependent upon two types of mechanisms as follows: via release of a hydrogen atom from hydroxyl groups (i.e., fast kinetics of some phenolic acids and derivatives) or via the release of an electron (i.e., slow kinetics derived glycosylated and anthocyanins) [20] .
Similar to other studies, we established a positive linear relationship between the content of the phenolic compounds and the antioxidant power, suggesting that phenolic components present in these extracts are the principal contributors to the antioxidant action of the selected plants [ 43 , 50 , 56 , 57 ]. Indeed, phenolic compounds encompass a range of structures that are widely distributed in plants and they have been informed to have various biological effects, including antiradical capacity. The antioxidant activities of the compound are primarily due to their redox properties, hydrogen donors, singlet oxygen quenchers, and metal chelators, which can act essential roles in trapping free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [58] .
Earlier studies showed that related polyphenolic compounds were encountered in different Limonium species [ 52 , 61 ].
In contrast to our results, Trabelsi et al. [62] , demonstrated that different fractions from an ethyl acetate extract of L. guyonianum leaves contained variable and powerful antioxidants, such as a phenolic acid ( p -coumaric acid) and two flavonoids (catechin and epigallocatechin-3-O-gallate) in their fraction 3, four phenolic compounds (gallo-catechin, sinapic acid) and two amides of phenolic acids (N-E-caffeoyl tyramine (1) and N-E-feruloyl tyramine) in fraction 4 and a new dimer of phenolic acid amide (Limoniastramide) in fraction 5. In addition, Trabelsi et al. [51] demonstrated that phenolic compound composition can vary, both qualitatively and quantitatively, within the same species depending on the provenance. Thus, six phenolic compounds were identified from the L. guyonianum collected in El Akarit (Tunisia) with three phenolic acids (gallic, 4-hydroxybenzoic and 3,4-dimethoxybenzoic acids) and three flavonoids (gallocatechin, catechin and epigallocatechin-3-O-gallate). However, only four compounds were identified from this medicinal halophyte native to the Oued Ran station, namely, gallocatechin, catechin 3,4-dimethoxybenzoic acid and vanillic acid.

Conclusions
Based on the present results, extracts and fractions of the two medicinal plants L. guyonianum and S. tripartita exhibited high quantities of bioactive substances and powerful antioxidant activities. The HPLC-DAD-ESI-MS/MS analysis of the crude extracts provided a tentative identification of major phytocompounds. The identified compounds belong to the flavonoid glycoside class, particularly the flavonol and flavanone classes (derived from aglycones; quercetin, myricetin, and eriodictyol). The sugar moieties consist of hexosides, disaccharides, deoxyhexosides, and pentosides. The presence of this group of secondary metabolites is probably the main contributor to the antiradical capacity of these plants, as well as supporting their uses in folk medicine to treat certain illness in relation to oxidative stress. Consequently, we propose a detailed study of these species to isolate the individual phenolic compounds to extensively elucidate their antioxidant capacity, which may be further exploited in herbal formulations.