Six species were selected based on their traditional curative traits, their abundance in nature, and their sustainable utilization. For each plant, scientific and common name, family, used organs, original habitat location and climatic characteristics, and the sampling date are given in Table 1. Shoots of M. crystallinum and M. edule were sampled from the sandy coasts of Jerba, in March 2006. Leaves, stems and roots of S. kali L. were collected from Soliman seashore, successively at the vegetative (May 2006) and reproductive (July 2006) stage. L. monopetalum leaves were sampled from Enfidha saline land in May 2006. T. gallica leaves and flowers were harvested from Enfidha and Takelsa localities in May 2006. Finally, leaves of C. maritima were sampled (June 2006) in two Tunisian littoral sites: Tabarka and Jerba.

Plant parts of all species were air dried at room temperature and in the dark for two weeks. Sample extracts were obtained by magnetic stirring of 2.5 g of dry matter powder with 25 mL of pure methanol for 30 min [24]. In the case of L. monopetalum leaves, five solvent extracts with increased polarity were used: hexane, ethanol, acetone, methanol and deionizer water. All extracts were kept for 24 h at 4 °C, filtered through a Whatman N°4 filter paper, and evaporated under vacuum to dryness. They were stored at 4 °C until analysis began.

Colorimetric quantification of total phenolics was determined, as described by [25]. Briefly, 125 μL of suitable diluted sample extract was dissolved in 500 μL of distilled water and 125 μL of the Folin–Ciocalteu reagent. The mixture was shaken, before adding 1250 μL Na₂CO₃ (70 g/L), adjusting with distilled water to a final volume of 3 ml, and mixed thoroughly. After incubation for 90 min at 23 °C in darkness, the absorbance versus a prepared blank was read at 760 nm. A standard curve of gallic acid was used. Total phenolic content of plant parts was expressed as mg gallic acid equivalents per gram of dry weight (mg GAE/g DW) through the calibration curve with gallic acid. The calibration curve range was 0–400 μg/mL (R2=0.99). All samples were analyzed in three replications.

Total flavonoids were measured by a colorimetric assay according to Dewanto et al. [25]. An aliquot of diluted sample or standard solution of (+)-catechin was added to a 75 μL of NaNO₂ solution, and mixed for 6 min, before adding 0.15 mL AlCl₃ (100 g/L). After 5 min, 0.5 mL of NaOH was added. The final volume was adjusted to 2.5 mL with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm against the same mixture, without the sample, as a blank. Total flavonoid content was expressed as mg catechin/g dry weight (mg CE/g DW), through the calibration curve of (+)-catechin. The calibration curve range was 0–400 μg/mL (R2=0.99). All samples were analyzed in three replications.

Proanthocyanidins were measured using the modified vanillin assay described by Sun et al. [26]. To 50 μL of properly diluted sample, 3 ml of methanol vanillin solution and 2.5 mL of H₂SO₄ were added. The absorption was measured at 500 nm against extract solvent as a blank. The amount of total condensed tannins is expressed as mg (+)-catechin/g DW. The calibration curve range was 0–400 μg/mL (R2=0.99). All samples were analyzed in three replications.

The DPPH^⋅ quenching ability of plant extracts was measured according to Hanato et al. [27]. One ml of the extract at different concentrations was added to 0.5 mL of a DPPH^⋅ methanolic solution. The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark. The absorbance of the resulting solution was then measured at 517 nm. The antiradical activity was expressed as IC₅₀ (μg/mL), the antiradical dose required to cause a 50% inhibition. A lower IC₅₀ value corresponds to a higher antioxidant activity of plant extract. The ability to scavenge the DPPH radical was calculated using the following equation:

Superoxide scavenging capacity was assessed using the method of Duh et al. [28]. The reaction mixture contained phosphate buffer, 200 μL of halophyte extracts, 200 μL of PMS solution, 200 μL of NADH, and 200 μL of NBT. After incubation at ambient temperature, the absorbance was read at 560 nm against blank. Evaluating the antioxidant activity in organ extract was based on IC₅₀. The IC₅₀ values were expressed as μg/ml. As for DPPH, lower IC₅₀ value corresponds to a higher antioxidant activity of plant extract. The inhibition percentage of superoxide anion generation was calculated using the following formula:

The chelating of ferrous ions by plant extracts was estimated as described by Dinis et al. [29], moderately modified by Zhao et al. [14]. Briefly, different concentrations of plant part extracts were added to a 0.05 mL FeCl₂, 4H₂O solution (2 mmol/L) and left for incubation at room temperature for 5 min. After the reaction was initiated by adding 0.1 mL of ferrozine (5 mmol/L), the mixture was adjusted to 3 mL with deionised water, shaken vigorously, and left standing at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm (Anthelie Advanced 2, SECOMAN). Analyses were run in triplicates. The percentage of inhibition of ferrozine–Fe²⁺ complex formation was calculated using the formula given bellow:

The capacity of plant extracts to reduce Fe³⁺ was assessed by the method of Oyaizu [30]. Each extract was mixed with 2.5 mL of sodium phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of potassium ferricyanide (10 g/L), and the mixture was incubated at 50 °C for 20 min. 2.5 mL of trichloroacetic acid (100 g/L) were then added, and the mixture was centrifuged at 650g for 10 min. The upper layer (2.5 mL) was mixed with 2.5 mL of deionised water and 0.5 mL of ferric chloride (0.01 g/L) and thoroughly mixed. The absorbance was measured at 700 nm against a blank in a spectrophotometer. A higher absorbance indicates a higher reducing power. EC₅₀ value (mg/ml) is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained from linear regression analysis. Ascorbic acid was used as control.

Means were statistically compared using the STATI-CF program with Student's t-test at the P<0.05 significance level. A one-way analysis of variance (ANOVA) and Newman–Keuls multiple range test were carried out to test any significant differences between solvents used at P<0.05.

The antioxidant capacity within the genus Mesembryanthemum was found to be significantly variable, despite the both investigated species (M. edule and M. crystallinum) were harvested from the same region. Shoot phenolic content was significantly higher in M. edule (70.07 mg GAE/g DW) as compared to M. crystallinum (1.43 mg GAE/g DW) (Table 2). Similarly, total flavonoïd and condensed tannin contents were considerably higher in the former species (respectively 200-fold and 119-fold of M. crystallinum values). Both the antioxidant activity against DPPH radical and the iron reducing power were significantly lower in shoot methanolic extracts of M. edule, IC₅₀ and EC₅₀, being respectively 5 and 8.5-fold lower than M. crystallinum, hence indicating a notably higher efficiency in M. edule shoots. These findings may be related to the higher polyphenol contents in M. edule, as compared to M. crystallinum. Indeed, several authors have reported a positive and significant relationship between the antioxidant components including phenols, polyphenols and tannins, respectively with the reducing power and DPPH radical scavenging capacity [31,32]. Comparing three Artemisia species, Djeridane et al. [33] found a significant difference in their antioxidant capacities. For example, total phenolic content varied from 3.42 (Artemisia arboresens) to 20.38 mg GAE/g DW (Artemisia campestris), while the antioxidant activity ranged from 11.6 to 25 mmol TEAC/g DW. These data were corroborated by Oszmianski et al. [34], who found large inter-species variations of antioxidant capacities between plants from Rosaceae family. A small difference was however observed in the antioxidant capacity of four varieties of Chrysanthemum morifolium Ramat [28]. Overall, the literature describes that antioxidant capacities are more variable in plants of different species (inter-specific) than within the same species (intra-specific).

In T. gallica, the comparison between leaves and flowers showed that both phenolic content and antioxidant activities were organ-dependent (Table 3). Flower methanolic extracts were characterized by higher polyphenol contents (70.56 mg of GAE/g DW), as compared to the leaf extracts (20.69 mg of GAE/g DW). These findings agree with previous ones indicating that secondary metabolites distribution may fluctuate between different plant organs [13,35,36]. As found for total phenolic content, antioxidant activities of flower were 2 to 8-fold higher than those of leaf extracts. Concerning DPPH scavenging activity, a considerable antiradical ability was found especially in flower methanolic extracts (IC₅₀ value > 1 μg/ml). Similarly, the highest activities with respect to chelating and reducing powers were registered in flower extracts (EC₅₀: 5.3 mg/ml and 84.3 μg/ml, respectively). Such a result may be likely ascribed to the higher polyphenol content in T. gallica flowers as compared to the leaves, as found for M. edule when compared to M. crystallinum.

In contrast to T. gallica, polyphenol content and antioxidant capacities were lower in S. kali flower than in leaf extracts. The higher phenolic content in leaves (ca. 5-time higher than that of flowers) reflected the better antiradical activity and reducing power with the lowest IC₅₀ and EC₅₀ (respectively, 10.33 and 165 μg/ml). Considering the fact that polyphenol compounds contribute directly to the antioxidant activities [7], the correlation level between total phenolic content and antioxidant activities organs seems to be an interesting aspect to explore. In fact, previous reports showed a significant correlation between the antioxidant activity and total phenolic content of Algerian and Chinese medicinal plants [33,37].

Leaf and stem extracts showed a significant decrease of their phenolic contents and consequently their antiradical activities at the reproductive stage, as compared to the vegetative one, while root extract showed the opposite tendency (Table 4). For instance, the total polyphenol contents of both leaves and stems were 3 times lower at the reproductive stage. Similarly, a 5-fold reduction was observed for total flavonoid and tannin contents, with a more pronounced effect in stem extracts. Our results corroborate previous reports on tomato and Anethum graveolens cultivars [13,38], concluding that phenolic content varied as a function of plant growth. With respect to DPPH scavenging activity, data showed that this antiradical activity was significantly different in the same organ at the two developmental stages. As for phenolic contents, this capacity to quench free radical seemed to be related to the physiological stage too, as IC₅₀ values largely differed between the two periods. For instance, IC₅₀ values of leaves and stems ranged from 11 and 13.5 to 14 and 46 μg/ml, respectively at the vegetative and reproductive stage. On the other hand, phenolic content and antiradical activities seemed also to be related, since they varied in the same way in all studied organs as function of the developmental stage. These results are partially in agreement with those of Zainol et al. [39] who showed a significant correlation between antioxidant activity and phenolic compounds in Centella asiatica.

Both phenolic contents and antioxidant activities of C. maritima were influenced by the harvest site (Table 5). The comparison between the two provenances showed that phenolic content was 1.4 fold higher in Jerba leaves as compared to Tabarka. The same trend was observed for antioxidant activities against DPPH radical and superoxide anion: their IC₅₀ values (respectively 610 and 1.7 μg/ml) were significantly lower, indicating a better activity in Jerba provenance than in Tabarka. Thus, these two parameters were stimulated in the plants growing in the arid zone (Jerba) as compared to those originating from the humid zone (Tabarka). The extreme climatic conditions in terms of salinity, low rainfall, and high radiation, characterising Jerba, are likely related to the increase of C. maritima antioxidant potentialities. Previous studies suggested that abiotic stresses (salinity, luminosity, water deficit, etc.) widely present in the arid zone may enhance phenolic compound synthesis as a response to the oxidative stress generated by the formation of reactive oxygen species in these hostile environments [11,40,41].

In order to further assess this assumption, two closer provenances of T. gallica originating from two arid regions (superior and inferior bioclimatic stages), differing by edaphic factors especially soil salinity, were compared (Table 1). As expected, total polyphenols content and antioxidant activities against DPPH and superoxide anion in the two provenances were significantly different (Table 5), with values ca. twice higher in T. gallica harvested from Enfidha salty soil than that originating from Takelsa (woodland). For instance, phenolic contents were 79.24 and 34.44 mg GAE/g DW, respectively in Enfidha and Takelsa plants. Considering that soil salinity is the major different parameter between Enfidha and Takelsa provenances, one may attribute to this factor a major influence on phenolic biosynthesis, and consequently a better antioxidant activity. In agreement with our findings, Parida et al. [42] showed that polyphenol content increased significantly in Aegiceras corniculatum plants challenged with 250 mM NaCl. Other authors confirmed this relationship too [11,12].

Among the several parameters that influence antioxidant capacities in plant analysis, solvent nature is the most controversial one [10,43]. In our study, five solvent kinds with different polarity were used to evaluate the antioxidant potential of L. monopetalum leaves and revealed a wide range of leaf polyphenols contents as function of the used solvent, closely dependent on the solvent polarity (Table 6). The extraction with pure methanol showed the highest leaf polyphenol content (15.85 mg GAE/g DW), followed by acetone extract (9.47 mg GAE/g DW). The last group included water, ethanol and hexane extracts which exhibited the lowest amount (1 to 2.6 mg GAE/g DW). As for total phenolics, flavonoid and condensed tannin contents also varied depending on the solvent extraction with maximal values of 4.2 and 3.9 mg EC/g DW, respectively (Table 6). The effect of solvent in flavonoid solubility showed the same classification as phenolics, while differing for tannins. Leaf extract had better tannin content (3.91 mg EC/g DW) in pure acetone, followed by pure methanol (1.47 mg EC/g DW). In the same way, L. monopetalum extracts exhibited a variable activity to quench DPPH radical as a function of the solvent type. The IC₅₀ values of these extracts ranged from 45 (methanol) to 175 μg/ml (water). Leaf extracts with pure methanol showed the highest ability to reduce DPPH, with an IC₅₀ value about 45 μg/ml, followed by acetone (76 μg/ml), ethanol, water and hexane (IC₅₀ values over 100 μg/ml for the three last solvents). As discussed above, the significant differences in antioxidant potential between the five solvents used in this experiment was essentially due to the difference in polarity, and thus different extractability, of the antioxidative compounds [7]. Thus, the difference in DPPH scavenging activity of plant extracts might be due to the difference in solvent selectivity for extracting certain phenolic groups [33]. Several studies showed that solvent natures, notably polarity, have significantly different extraction capacities for phenolic compounds in plants [42,44]. Therefore, there is no uniform or completely satisfactory procedure that is suitable for extraction of all phenolics or a specific class of phenolic substances in plant materials. Methanol and acetone, and to a lesser extent water and ethanol, and their mixture are frequently used for phenolic extraction [45]. In recent studies, numerous others factors like chemical treatment and agronomical crop management practices have been demonstrated to have a great influence on plants antioxidant pool under abiotic stresses. For instance, exogenous application of triazole derivatives can ameliorates the tolerance to these environmental constraints by enhancing the activities of several enzymes, especially those related to detoxification of active oxygen species and antioxidant metabolism in medicinal plants such as Catharanthus roseus [46,47] and Withania somnifera [48,49].