Characterization of Silybum marianum and Silybum eburneum seed oils: Phytochemical profiles and antioxidant properties supporting important nutritional interests.

Milk thistle seed oil is still not a well-known edible oil. Silybum marianum (milk thistle), is present in several countries and is the only known representative of the genus Silybum. However, Silybum eburneum, which is an endemic plant in Spain, Kenya, Morocco, Algeria, and Tunisia, is considered a marginalized species. The present work is the first report that gives information on the lipid and phenolic profiles of Tunisian S. eburneum seed oil compared to those of Tunisian S. marianum seed oil. In addition, the antioxidant properties of these oils were determined with DPPH, FRAP, and KRL assays, and their ability to prevent oxidative stress was determined on human monocytic THP-1 cells. These oils are characterized by high amounts of unsaturated fatty acids; linoleic acid and oleic acid are the most abundant. Campesterol, sitosterol, stigmasterol, and ß-amyrin were the major phytosterols identified. α-tocopherol was the predominant tocopherol found. These oils also contain significant amounts of phenolic compounds. The diversity and richness of Silybum marianum and Silybum eburneum seed oils in unsaturated fatty acids, phenolic compounds, and tocopherols are associated with high antioxidant activities revealed by the DPPH, FRAP, and KRL assays. In addition, on THP-1 cells, these oils powerfully reduced the oxidative stress induced by 7-ketocholesterol and 7ß-hydroxycholesterol, two strongly pro-oxidant oxysterols often present at increased levels in patients with age-related diseases. Silybum marianum and Silybum eburneum seed oils are therefore important sources of bioactive molecules with nutritional interest that prevent age-related diseases, the frequency of which is increasing in all countries due to the length of life expectancy.

Tunisian Silybum Species: Important Sources of Polyphenols, Organic Acids, Minerals, and Proteins across Various Plant Organs.

Silybum marianum and Silybum eburneum are wild edible Mediterranean plants used in the human diet. This study presents the initial findings on the phytochemical characterization of Tunisian S. marianum and S. eburneum organs. It examined their mineral, sugar, organic acid, polyphenolic, and seed storage protein contents, as well as their antioxidant potential. In S. marianum, stems had high sodium and potassium contents, while the immature and mature seeds were rich in calcium and magnesium. However, S. eburneum had high potassium levels in stems and high sodium and calcium levels in the flowers. S. marianum showed substantial fructose variation among its organs. Conversely, S. eburneum exhibited significant heterogeneity in glucose, sucrose, and maltose levels across its organs, with maltose exclusively detected in the immature seeds. A notable organ-dependent distribution of organic acids was observed among the two species. Higher levels of phenolic contents were detected in both mature and immature seeds in both species compared to the other plant parts. The seeds possessed higher antioxidant activities than other plant organs. In both S. marianum and S. eburneum seeds, albumins and globulins were the predominant protein fractions. This study brings evidence supporting the important potential of Silybum organs as sources of nutrients with antioxidant properties for producing functional food.

Phytochemical Screening, Antioxidant Potential, and LC-ESI-MS Profiling of Ephedra alata and Ephedra altissima Seeds Naturally Growing in Tunisia.

Most Ephedra species are adapted to arid and desert conditions and are widely used in folk medicine to treat several disorders. The design of the current study was to determine the functional properties of seeds of two Ephedra species (E. alata and E. altissima) naturally growing in Tunisian arid zones by evaluating their mineral contents and bioactive compounds. The flame atomic absorption spectrometry revealed that seeds contained remarkable amounts of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), and iron (Fe). The colorimetric investigation revealed high total polyphenol, flavonoid, and condensed tannin contents. Furthermore, by utilizing high-performance liquid chromatography-electrospray ionization-mass spectrometry method (HPLC-ESI/MS), a total of 11 phenolics were identified and quantified including 7 flavonoid compounds and 4 phenolic acids that were mostly predominated by gallic acid and quercetrin. Results so far have been very encouraging and proved that Ephedra seeds are a valuable source of natural bioactive compounds and minerals which could potentially be used for industrial and pharmaceutical purposes.

Minerals, fatty acids, and antioxidant activity in sea knotgrass (Polygonum maritimum L.) seeds.

The design of the current study was to determine the functional properties of P. maritimum seeds by evaluating their mineral content, fatty acid composition, and biactive compounds. Results showed that seeds contained remarkable amounts of Na, K, Ca, Mg, Cu, Zn, and Fe. The oil yield was found to be 4.58% and contained higher unsaturated fatty acids predominated by linoleic and oleic acids. The colorimetric investigation revealed high total polyphenol (33.56 mg gallic acid equivalent/g), flavonoid (46.3 mg quercetin equivalent/g), and condensed tannin (22.3 mg catechin equivalent/g) contents. Furthermore, the LC-ESI/MS analysis revealed the presence of 13 phenolics mostly predominated by (+) catechin, quercetin-3-O-galactoside, gallic acid, and quinic acid. The seeds possessed strong antioxidant potential evidenced by ABTS and DPPH radical scavenging activities, total antioxidant capacity, and reducing power. The present findings suggest that P. maritimum seeds may be used as a potential source of biologically active natural products.

Limoniastrum guyonianum behavior under seasonal conditions fluctuations of Sabkha Aïn Maïder (Tunisia).

In Sabkha biotope, several environmental factors (i.e., salinity, drought, temperature, etc.) especially during dry season affect halophytes developments. To cope with these harmful conditions, halophytes use multiple mechanisms of adaptations. In this study, we focused on the effect of environmental condition changes over a year in the Sabkha of Aïn Maïder (Medenine - Tunisia) on the physiological and biochemical behavior of Limoniastrum guyonianum using a modeling approach. Our study showed that the model depicted well (R2 > 0.75) the monthly fluctuations of the studied parameters in this habitat. During the dry period (June to September), the salinity of the soil increased remarkably (high level of EC and Na+ content), resulting in high Na+ content in the aerial parts followed by a nutrient deficiency in K+, Ca2+, and Mg2+. As a result of this disruption, L. guyonianum decreased its water potential to more negative values to maintain osmotic potential using inorganic osmolytes (i.e., Na+) and organic osmolytes (i.e., sugars: sucrose, fructose, glucose, and xylitol, and organic acids: citric and malic acids). In addition, CO2 assimilation rate, stomatal conductance, transpiration rate, and photosynthetic pigments decreased significantly with increasing salinity. The phenolic compounds contents and the antioxidant activity increased significantly in the dry period as a result of increased levels of H2O2 and lipid peroxidation. This increase was highly correlated with soil salinity and air temperature. The maintenance of tissue hydration (i.e., moderate decrease of relative water content), the accumulation of sugars and organic acids, the enhancement of phenolic compounds amounts, and the increase of antioxidant activity during the dry period suggest that L. guyonianum possesses an efficient tolerance mechanism that allows the plant to withstand the seasonal fluctuations of climatic conditions in its natural biotope.

Seasonal environmental changes affect differently the physiological and biochemical responses of two Limonium species in Sabkha biotope.

To cope with abiotic stresses, the halophytes use various tolerance mechanisms that are not all known for some species of halophytes. This work aimed to model the responses of two halophytes, Limonium pruinosum and Limonium tunetanum, to changing environmental conditions over a year in Sabkha biotope, Tunisia. Our findings indicated that the model could describe well the seasonal variations of all studied traits over a year in this region (R2  > 0.80). The pH, electrical conductivity (EC), and mineral composition (i.e., Sodium [Na+ ], potassium [K+ ], calcium [Ca2+ ], and magnesium [Mg2+ ] concentrations] in the soil significantly varied during the year (P < 0.01). The highest soil Na+ concentration was found during the dry period (June-August), which matched with a high Na+ content in the aerial parts (i.e., 2455.1 and 3366.0 µmol g-1 SDM in August for L. pruinosum and L. tunetanum, respectively). This accumulation is concomitant with a deficit in nutrients, particularly K+ and Mg2+ and, to a lesser extent, Ca2+ . Following these disturbances, both species decreased their photosynthetic activity, water potential as well as the relative water content to a lesser extent, especially in August. To counteract these harmful effects, these species have accumulated organic substances, but in a different way. Indeed, L. pruinosum accumulated sucrose, fructose, and citrate, while L. tunetanum accumulated sucrose, xylitol, citrate, and malate, indicating a major role of these osmolytes in the stress tolerance mechanisms. In both species, the results also showed that all traits were highly correlated with the edaphic variables (i.e., pH, EC, Na+ , K+ , Ca2+ , and Mg2+ concentrations) and with temperature as the climatic variable.

Characterization of lipids, proteins, and bioactive compounds in the seeds of three Astragalus species.

Despite the interest of legumes for food and feed purposes, the phytochemicals of Astragalus armatus (AA), A. caprinus (AC), and A. gombiformis (AG) have not been reported in-depth yet. Thus, the lipid contents, fatty acids composition, proteins, and bioactive compounds in the seeds of these species were investigated. Total lipids were ranged from 12.2 (AG) to 36.5 g 100 g-1 DW (AC) and consisted of oleic acid, linoleic acid, α-linolenic acid, and palmitic acid. The crude protein contents were in the range from 52.2 (AG) to 54.7 g 100 g-1 DW (AA). Globulin and albumin were the predominant soluble protein fractions. The seeds consisted significantly of different contents of total polyphenols (3.4-6.5 mg GAEg-1 DW), total flavonoids (1.24-5.15 mg QEg-1 DW), and total condensed tannins (12-23.2 mg CEg-1 DW). The extracts mainly consisted of quinic acid, p-coumaric acid, and cirsiliol. All these findings can be assets for functional foods and/or food ingredients.

Flower, seed, and fruit development in three Tunisian species of Polygonum: Implications for their taxonomy and evolution of distyly in Polygonaceae.

Polygonum is the largest genus of Polygonaceae and 5 species are reported in Tunisia. In order to characterized flower, seed, and fruit development in Polygonum, flower and fruit of Polygonium equisetiforme (var. graecum and peyerinhoffi), P. aviculare and P. maritimum, collected from Tunisia, were examined. Flowers are composed of five oblong tepals. P. equisetiforme and P. aviculare have whitish-pink distylous flowers with dimorphism of style, filament and anther height, pollen diameter and stigma size. In contrast, P. maritimum shows white homostylous flowers. The floral vasculature showed that the tepals are inserted in one whorl and their traces arise independently in 3+2 manner. The eight stamens are arranged in a 5+3 manner and the staminal bundles arise independently in the two whorls. The epidermis and endothecium cells width were higher in P. maritimum and the lowest endothecium width was observed in P. aviculare. Polygonum aviculare and P. equisetiforme showed circular pollen with shallow colpi and trilobite pollen shape with deep colpi, while P. maritimum rarely showed shallow colpi. The ovule is anatropous with basal placentation in P. equisetiforme and P. aviculare and apical placentation in P. maritimum. The young seed coat was formed by an endotesta with thick-walled cells, a mesotesta and exotesta with thin-walled cells and a tegmen composed of radially elongated cells. The fruits of the studied species are trigonous with ovate-lanceolate shape. In P. aviculare, the exocarp is thicker compared to the two other species, in P. equisetiforme, the mature exocarp consists of smaller rectangular cells with narrow cavities, and in P. maritimum showed a thinner exocarpIn conclusion, P. equisetiforme and P. aviculare are a typically distylous species from the morphological point of view and we discussed the significance of heterostyly in Polygonaceae. From this first morpho-anatomical study of Polygonum species in North Africa, we can conclude mainly that there is no significant difference between P. equisetiforme var. graecum and var. peyerinhoffi supporting a taxonomic grouping of these two varieties.

Effect of long-term storage on phenolic composition, antioxidant capacity, and protein profiles of Calicotome villosa subsp. intermedia seeds.

Phytochemicals from Calicotome villosa seeds were evaluated during long-term storage. Total phenols were affected during storage, ranging from 34.6 (5-years-aged seeds) to 45.1 mg GAE/g DW (16-years-aged seeds). Flavonoids increased with the storage period varying from 21.4 (seeds collected at 2013) to 34.1 mg QRE/g DW (seeds collected at 2002). The total condensed tannins were low for all storage periods and the highest value was registered for 16-years-aged seeds (0.15 ± 0.01CTE/g DW). LC-ESI/MS analysis showed five phenolic acids and 11 flavonoids, which remained steady during the long-term storage. High-maintained storage protein content (50.2% DW) occurred in C. villosa seeds even under long-term storage (up to 16 years). Globulins were the major proteins (47.6% of total proteins). All these findings made C. villosa a source of natural antioxidants with high industrial value and could be used as a potential food source. PRACTICAL APPLICATIONS: In this research paper, we have evaluated and discussed the effect of long storage duration (5, 10, 13, and 16 years) on phenolic contents, antioxidant potential, and protein profiles of C. villosa seeds. The total phenolic content determined was not affected by storage duration, while the values of flavonoid content were enhanced. The storage length significantly increased the total condensed tannin content. However, the phenolic acids and flavonoid compounds remained stable. Moreover, high-maintained storage protein content (globulins, albumins, glutelins, and globulin) occurs in C. villosa seeds even under long-term storage. The obtained results showed that the quality of the seeds was not negatively affected by long storage duration. C. villosa seeds show the prospective potential applications in the food industry.

Population structure and genetic diversity of a medicinal plant species Retama raetam in southern Tunisia.

Retama raetam is a stem-assimilating, C3, evergreen, medicinal plant species, desert legume common to arid ecosystems around the Mediterranean basin. This study addresses the genetic diversity and relationship among and within three populations collected from different habitats in southern Tunisia by Random Amplified Polymorphic DNA (RAPD). Estimates of the percentage of polymorphic bands, Shannon's diversity information index and Nei's gene diversity index were determined. Results showed that population from the Island Djerba has the lowest Nei's gene diversity; this also was for Shannon diversity index. An analysis of molecular variance indicated that the majority of variation existed within populations (68%) and that there was significant differentiation among populations (phiPT = 0.316, p < 0.001). Genetic distance (phiPT based values) between pairwise populations ranged from 0.098 to 0.505 and the differentiation between pair-wise populations was significant when individual pairs of populations were compared. Based on the coefficient of gene differentiation (Gst), gene flow (Nm) was estimated and was found to vary from 0.490 to 4.609 between pair-wise populations and 1.42 among populations. The results of UPGMA cluster analysis and PCoA analysis indicated that most variation occurred within populations and that genetic differentiation had happened between populations. These findings are important for a better understanding of the adaptive strategy of R. raetam in southern Tunisia and will be useful for conservation managers to work out an effective strategy to protect this important species.

An efficient DNA extraction method for desert Calligonum species.

Genetic conservation programs in arid environments rely on molecular methods for diversity assessments. DNA-based molecular profiling will aid in conservation and protection of species from genetic erosion. Obtaining intact genomic DNA from Calligonum species, of sufficiently high-quality that is readily amplifiable using PCR, is challenging because of the presence of the exceptionally large amount of oxidized polyphenolic compounds, polysaccharides, and other secondary metabolites. The present method involves a modification of the available CTAB method employing higher concentrations of NaCl and CTAB, and incorporating PEG 6000 (1%) and glucose. The yield of DNA was 60-670 µg g(-1) of fresh tissue. The protocol has been tested with two species from the arid region. The DNA isolated was successfully amplified by two ITS primer pairs. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region among and within Calligonum species followed by sequencing is under way.