Afef Béjaoui

Variation of the Chemical Composition of Essential Oils in Tunisian Populations of Thymus algeriensisBoiss. et Reut. (Lamiaceae) and Implication for Conservation

The variation of the essential‐oil composition among 14 Tunisian natural populations of Thymus algeriensis Boiss. et Reut. (=Thymus hirtus Willd. ssp. algeriensis Boiss. et Reut.) was assessed by GC (RI) and GC/MS. The populations were collected from different geographical regions belonging to the sub‐humid, upper semi‐arid, mean semi‐arid, lower semi‐arid, and upper‐arid bioclimates. A total of 47 constituents, representing 81.0 to 96.5% of the total oil, were identified. The main volatiles at the species level were 1,8‐cineole (17.7%), α‐pinene (15.5%), and camphor (8.2%). A high variation among populations for the majority of the compounds was shown. Camphor (0.2–14.0%), linalool (0.2–22.4%), borneol (<0.01–24.3%), caryophyllene oxide (<0.01–18.8%), thymol (<0.01–54.9%), γ‐terpinene (0.4–6.5%), α‐copaene (0.4–7.6%), linalyl acetate (<0.01–6.4%), and methyl eugenol (<0.01–6.9%) were the main constituents differentiating the populations. The chemical differentiation among populations, assessed by principal component analysis (PCA) and a UPGMA (unweighted pair‐group method with averaging) cluster analysis performed on all populations and compounds, was high. Six chemotypes according to the main compounds have been distinguished, i.e., caryophyllene oxide/1,8‐cineole/α‐pinene, 1,8‐cineole/α‐pinene, 1,8‐cineole/α‐pinene/camphor, borneol/1,8‐cineole/α‐pinene, linalool, and thymol chemotypes. The spatial chemotype distribution was linked to the geographic distance among populations rather than to bioclimates, indicating that local selective environmental factors act on the chemotype diversity.

Essential oil composition and antibacterial activity of Origanum vulgare subsp. glandulosum Desf. at different phenological stages.

Variation in the quantity and quality of the essential oil (EO) of wild population of Origanum vulgare at different phenological stages, including vegetative, late vegetative, and flowering set, is reported. The oils of air-dried samples were obtained by hydrodistillation. The yield of oils (w/w%) at different stages were in the order of late vegetative (2.0%), early vegetative (1.7%), and flowering (0.6%) set. The oils were analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). In total, 36, 33, and 16 components were identified and quantified in vegetative, late vegetative, and flowering set, representing 94.47%, 95.91%, and 99.62% of the oil, respectively. Carvacrol was the major compound in all samples. The ranges of major constituents were as follows: carvacrol (61.08-83.37%), p-cymene (3.02-9.87%), and γ-terpinene (4.13-6.34%). Antibacterial activity of the oils was tested against three Gram-positive and two Gram-negative bacteria by the disc diffusion method and determining their diameter of inhibition and the minimum inhibitory concentration (MIC) values. The inhibition zones and MIC values for bacterial strains, which were sensitive to the EO of O. vulgare subsp. glandulosum, were in the range of 9-36 mm and 125-600 μg/mL, respectively. The oils of various phenological stages showed high activity against all tested bacteria, of which Bacillus subtilis was the most sensitive and resistant strain, respectively. Thus, they represent an inexpensive source of natural antibacterial substances that exhibited potential for use in pathogenic systems.

Genetic Diversity and Population Structure of Teucrium polium (Lamiaceae) in Tunisia

Random amplified polymorphic DNA markers were used to assess the genetic diversity within and among seven Tunisian diploid and polyploid populations of Teucriumpolium L. from five bioclimatic areas. Out of the 141 bands generated from eight selected primers, 124 were polymorphic. The genetic diversity within a population (Shannon’s index) was high and varied according both the ploidal levels and bioclimatic zones. The genetic differentiation among populations assessed by GST and ΦST statistics was high, suggesting a low level of gene flow among them. The major proportion of the variation was attributable to individual differences within populations. The UPGMA analysis based on Nei and Li’s coefficient showed that individuals from each population clustered together. In a dendrogram using the ΦST distance matrix, population grouping is concordant with bioclimates and cytotypes. Conservation strategies should take into account the level of the genetic diversity of the populations according to their bioclimate and ploidal levels.

Variation of volatiles in Tunisian populations of Teucrium polium L. (Lamiaceae).

Volatiles from wild Tunisian diploid (2n=2x=26) and tetraploid (2n=4x=52) Teucrium polium L. populations from five bioclimatic zones were assessed by GC and GC/MS. Thirty‐eight compounds were identified. Main volatiles at the species level were myrcene (6; 15.3%), germacrene D (27; 9.0%), α‐pinene (6.6%), β‐pinene (3; 5.8%), and α‐cadinol (38; 5.1%). A significant variation among populations was observed. The tetraploid populations belonging to the sub humid and lower semi arid bioclimates showed a high amount of myrcene (6; 24.2–43.8%), those from the upper arid contain high percentages of α‐cadinol (38; 50.6%). The dominant compounds in diploid populations, mainly located in the semi arid zone, are α‐pinene (1) and germacrene D (27). Population chemical structure, assessed by a principal component analysis (PCA) using all identified compounds, is concordant with bioclimate and ploidy level. In situ conservation of populations should be made appropriately according to these two factors.

Bioactive compounds from Hypericum humifusum and Hypericum perfoliatum: inhibition potential of polyphenols with acetylcholinesterase and key enzymes linked to type-2 diabetes

Abstract Context: Natural products are reported to have a wide spectrum of pharmacological properties such as antimicrobial, anti-inflammatory and anti-cholinesterase. The genus Hypericum (Hypericaceae) is a source of a variety of molecules with different biological activities, notably hypericin and various phenolics. Objectives: The goals of the present work were the determination of total phenolic and flavonoid content, hypericin and hyperforin concentration as well as the evaluation of biological of Hypericum humifusum L. (Hhu) and Hypericum perfoliatum L. (Hper). Materials and methods: The various extracts of aerial parts were powdered, and then extracted with methanol. Antibacterial activity was performed according to minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) methods against four Gram-positive bacteria, four Gram-negative bacteria and yeast. Results: The results revealed that H. humifusum, bear the highest total phenolic and flavonoid content (48–113 mg GAE/g and 8–41 mg RE/g, respectively) as well as hypericin (60–90 mg/g) and hyperforin (8–30 mg/g) concentration. Both species showed significant antioxidant activity as revealed by DPPH, FRAP, ABTS, and metal chelating assays. H. humifusum exhibited a strong acetylcholinesterase (3.86–4.57 mg GALAEs/g), α-glucosidase (0.73–2.55 mmol ACEs/g) and α-amylase (3–8 mmol ACEs/g) inhibitory activity. The extract of H. humifusum exhibited strong antibacterial activity mainly against Staphylococcus epidermidis, Staphylococus aureus, and Enterococcus faecium (MIC values ranging from 200 to 250 μg/mL). The highest antifungal activity was showed for H. perfoliatum extract (MIC value = 250 μg/mL). Conclusion: The data suggest that H. humifusum could be used as valuable new natural agents with functional properties for pharmacology industries.

Genetic diversity and population structure of Hypericum humifusum L. (Hypericacae) in Tunisia: Implications for conservation

Abstract Sixteen Tunisian natural populations of Hypericum humifusum from different bioclimates (sub‐humid, upper semi‐arid and lower semi‐arid) were assessed for their genetic diversity using nine isozymes. For all populations and all enzymes, 11 polymorphic loci were detected. The allelic frequencies varied according to the populations. A high genetic diversity and an excess of heterozygosity were observed within populations (A p = 2.02; P% = 64.29; H o = 0.321). The outbreeding mating system and the high number of individuals in the starting populations may contribute to this high variation. A relatively high level of differentiation (F ST = 0.142) and a restricted gene flow among populations (N m =1.34) were revealed. The lower semi‐arid populations showed the highest differentiation (F ST = 0.205). The relationship between F ST and geographic distance matrices was not significant, indicating that the genetic structure among populations is more closely linked to habitat fragmentation and probably to founder events. Nei’s unbiased genetic distances among pairs of populations were low (0.012–0.119). The unweighted pair group method with arithmetic averaging algorithm cluster based on these distances showed four sub‐clusters. Population groupings had no evident relationship with bioclimates or geographic regions. The substantial differentiation between populations and their high genetic similarity suggest their recent divergence as a result of habitat fragmentation. Most of the total variation was found within populations, thus ex‐situ conservation should be based on sampling seeds within, rather than among, populations. Populations harbouring rare alleles and a high genetic diversity level should be collected first.

Genetic diversity in Tunisian Crataegus azarolus L. var. aronia L. populations assessed using RAPD markers

Abstract• The genetic diversity of nine wild Tunisian Crataegus azarolus var. aronia L. populations from different bioclimates was assessed using RAPD markers.• Eight selected primers generated a total of 105 bands, 81 of which were polymorphic. Shannon’s index (H′) ranged from 0.222 to 0.278 according to a population with an average of 0.245. The genetic variation within the species (HSP = 0.423) was relatively low. A high differentiation (GST = 0.421) among populations coupled with a low level of gene flow (Nm = 0.472) were observed. The analysis of molecular variance (AMOVA) revealed also significant differentiation among populations (ΦST = 0.371), even at a low scale space. The majority of variation occurred within populations (63.31%). The Mantel test performed on genetic (ΦST) and geographic distance matrices among population pairs did not reveal an isolation by distance.• Interpretation of Neighbour-joining tree based on Nei’s and Li’s genetic distance among individuals showed distinct population groupings. The UPGMA dendrogram based on ΦST values revealed two population sub-clusters, each including populations from different bioclimates and/or geographic regions.• The low level of genetic diversity and the high genetic structure of populations resulted from genetic drift caused both by habitat fragmentation and the low size of populations.• The high differentiation among populations and the similar low level of diversity within populations suggest that in situ conservation should interest all populations. The ex situ conservation should be based on the collection of seeds rather within than among populations because of the maximum of variation was revealed within populations.Résumé• La diversité génétique de neuf populations naturelles de Crataegus azarolus var. aronia L. en Tunisie, appartenant à différents étages bioclimatiques, a été analysée par des marqueurs RAPD.• Huit amorces retenues ont généré 105 bandes dont 81 sont polymorphes. L’indice de Shannon (H′) varie de 0,222 à 0,278 selon les populations avec une moyenne de 0,245. La variabilité génétique au sein de l’espèce est relativement faible (HSP = 0,423). Une différenciation importante entre les populations (GST = 0,421) et un faible flux de gènes entre elles (Nm = 0,472) ont été observés. L’analyse de la variance moléculaire (AMOVA) a révélé, elle aussi, une différenciation significative entre les populations considérées ensemble (ΦST = 0,371) ou regroupées selon leur localisation bioclimatique. La majeure proportion de la variabilité réside à l’intérieur des populations (63,31 %). Le test de Mantel, effectué sur les matrices des distances génétiques (ΦST) et géographiques entre les paires des populations, n’a pas révélé une isolation par distance.• Le dendrogramme, établi à partir des distances génétiques de Nei et Li, a montré des regroupements des individus dans leurs populations respectives. Le dendrogramme UPGMA, construit à partir des valeurs de ΦST entre les paires des populations, révèle deux groupes de populations. L’agrégation des populations au sein des groupes ne s’opère pas toujours selon leur proximité géographique et/ou leur localisation bioclimatique.• La faible diversité génétique intrapopulation et le niveau élevé de différenciation des populations résulteraient de la dérive génétique due à la fragmentation des habitats et à la taille réduite des populations.• Toutes les populations devraient bénéficier d’une priorité pour leur conservation in situ, vu le niveau élevé de leur différenciation et leur faible diversité génétique. La conservation ex situ devrait s’appuyer sur le prélèvement d’un maximum de semences plutôt au sein qu’entre les populations étant donné que la majeure proportion de la variabilité réside à l’intérieur des populations.

Population genetic structure of Tunisian Hypericum humifusum assessed by RAPD markers

The genetic variation within and among seven Tunisian natural Hypericum humifusum L. populations belonging to three bioclimatic zones (sub-humid, upper semi-arid, and lower semi-arid) was assessed using random amplified polymorphic DNA markers. Eight selected primers produced a total of 166 bands, of which 153 were polymorphic. The genetic diversity within a population, based on Shannon’s index and percentage of polymorphic loci, was relatively high. The level of variation among populations did not differ significantly. However, the variation among populations grouped according to their bioclimates was significant.A high differentiation and a low gene flow were observed at all spatial scales among all populations. The upper semiarid populations exhibited the highest differentiation. The relationship between genetic and geographic distances was not significant indicating that structuring occurred due to founding events. The UPGMA analysis based on Nei & Li’s coefficients showed that individuals from each population clustered together. The cluster analysis based on genetic distances among populations did not show clear groupings relevant to geographical distances or bioclimates.The high differentiation among populations even through a small geographic range implies the collection of seeds from all populations to preserve, ex-situ, extant variation in the species. Populations from the upper semi-arid zone showing the highest genetic diversity should be first prospected.

Chemical Variation and in vitro Antibacterial Properties of Volatiles from Tunisian Marrubium vulgare L. (Lamiaceae)

Abstract In this paper, we have studied the chemical composition of the volatile fraction from six wild Tunisian populations of Marrubium vulgare L. growing wild in four bioclimatic zones were assessed by GC-FID and GC/MS analysis. Volatiles were individually tested against a large panel of microorganisms. In total, 37 compounds were identified with a predominance of diterpenes and alcanes. The main compounds were marrubiin (9-34 %), doctriacontane (13-25 %), and squalene (6-47 %). The variance analysis showed that amounts of the identified compounds varied significantly across the populations. Antibacterial activity of the volatiles was tested against three Gram-positive, three Gram-negative bacteria, and one pathogenic yeast. The volatiles exhibited interesting antibacterial activity. The inhibition zones and MIC values for bacterial strains, which were sensitive to the volatiles of M. vulgare, were in the range of 7–15 mm and 300-850 μg/mL, respectively. Greater interest and increased knowledge of the variation of the volatile composition of M. vulgare may be of significant value in the quest for new antimicrobial agents.

Chemical composition and bioactivities of the polyphenolic-rich extract of Ormenis africana Jord. and Fourr

ABSTRACT Ormenis africana is an endemic North African species used in folk medicine because of its hypoglycemic property. In this study, the α-amylase and α-glucosidase inhibition and antioxidant activities of the polyphenolic-rich extract from O. africana were determined. The chemical composition was made using liquid chromatography with photodiode array and electrospray ionization mass spectrometry method and the identification of phenolics was assessed by comparing their retention times and ultraviolet and mass spectra with those of the standards and/or reported in the literature. The total phenolic content was estimated by the Folin–Ciocalteu method. The antidiabetic potential was estimated by the determination of α-amylase and α-glucosidase inhibition in vitro. Four assays were used for the evaluation of antioxidant activity of the extracts. Seventeen phenolic compounds were detected. The major peaks are chlorogenic acid, 5-O-di-caffeoylquinic acid, and apigenin and luteolin derivatives. The polyphenolic-rich extract showed remarkable α-amylase and α-glucosidase inhibition activity in a concentration dependent manner. Furthermore, the extract also demonstrated high antioxidant activities. O. africana can serve as a potential natural source for the development of a novel α-amylase and α-glucosidase inhibitory agents against diabetic complications.