Mohamed Larbi Khouja

Chemical composition of 8 eucalyptus species' essential oils and the evaluation of their antibacterial, antifungal and antiviral activities

BackgroundIn 1957, Tunisia introduced 117 species of Eucalyptus; they have been used as fire wood, for the production of mine wood and to fight erosion. Actually, Eucalyptus essential oil is traditionally used to treat respiratory tract disorders such as pharyngitis, bronchitis, and sinusitis. A few investigations were reported on the biological activities of Eucalyptus oils worldwide. In Tunisia, our previous works conducted in 2010 and 2011 had been the first reports to study the antibacterial activities against reference strains. At that time it was not possible to evaluate their antimicrobial activities against clinical bacterial strains and other pathogens such as virus and fungi.MethodsThe essential oils of eight Eucalyptus species harvested from the Jbel Abderrahman, Korbous (North East Tunisia) and Souinet arboreta (North of Tunisia) were evaluated for their antimicrobial activities by disc diffusion and microbroth dilution methods against seven bacterial isolates: Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes. In addition, the bactericidal, fungicidal and the antiviral activities of the tested oils were carried out.ResultsTwenty five components were identified by GC/FID and GC/MS. These components were used to correlate with the biological activities of the tested oils. The chemical principal component analysis identified three groups, each of them constituted a chemotype. According to the values of zone diameter and percentage of the inhibition (zdi, % I, respectively), four groups and subgroups of bacterial strains and three groups of fungal strains were characterized by their sensitivity levels to Eucalyptus oils. The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.ConclusionsE. odorata showed the strongest activity against S. aureus, H. influenzae, S. agalactiae, S. pyogenes, S. pneumoniae and against all the tested fungal strains. In addition, E. odorata oil showed the most cytotoxic effect. However, the best antiviral activity appeared with E. bicostata. Virus pretreatment with E. bicostata essential oil showed better antiviral activity (IC50 = 0.7 mg/ml, SI = 22.8) than cell-pretreatment (IC50 = 4.8 mg/ml, SI = 3.33). The essential oil of E. astringens showed antiviral activity only when incubated with virus prior to cell infection. This activity was dose-dependent and the antiviral activity diminished with the decreasing essential oil concentration.

Intraspecific Variation of Capparis spinosa L. in Tunisia

An analysis of caper (Capparis spinosa L.) variability in Tunisia using morphological descriptors revealed intraspecific diversity. This diversity was relatively high in the prickly caper but relatively low for inermis and shaggy types that formed one homogenous group. All studied descriptors showed discriminative power and enabled classification of the studied accessions in two morphotypes: one prickly and one that included the inermis and downy caper. The later morphotype is characterized by creeping shoots and relatively large leaves, low stomatal density, and high numbers of stamens. The heterogeneous prickly group is characterized by erect shoots and relatively small leaves, high stomatal density, and a small numbers of stamens.

Evaluation of Pistacia lentiscus seed oil and phenolic compounds for in vitro antiproliferative effects against BHK21 cells

Abstract Context: Within the global context of increasing cancer diseases, natural products are important in devising new drugs and providing unique ideas in cancer therapy. In Tunisian folk medicine, Pistacia lentiscus L. (Anacardiaceae) fixed oil is used for cancer treatment. Objective: This investigation studied, for the first time, the antiproliferative effect of Pistacia lentiscus fixed oil and its phenolic extract on BHK21 cancer cells. Materials and methods: Oil was extracted from fruits harvested in northwest Tunisia and the phenolic fraction was obtained by mixing with methanol. The anti-proliferative activity of the two tested substances on BHK 21 cells were investigated in vitro using trypan blue assays. Cells were treated with different concentrations of P. lentiscus oil (0.009, 0.018, 0.036, and 0.09 g/mL) and the phenolic extract (0.007, 0.014, 0.03, and 0.07 g/mL) for 24, 48, and 72 h. Results: The inhibitory effect of Pistacia lentiscus fixed oil increases with the increase in dose. The IC50 value was estimated at 0.029 g/mL. The percentage of cell viability was 42.46 ± 3.4% at a dose of 0.09 g/mL and was significantly lower than that of the untreated control (96.24 ± 2.5%, p<0.01). The phenolic extract demonstrated a dose- and time-dependent inhibitory effect on BHK21 cell growth. After 48 h of incubation, the IC50 value was estimated at 0.15 g/mL. Discussion and conclusion: The results demonstrated the potential of Pistacia lentiscus fixed oil in treating cancer, as it is used in traditional medicine.

Breeding Improvement of Laurus nobilis L. by Conventional and In Vitro Propagation Techniques

SUMMARY Originating from Asia Minor and later transmitted through Greece, Laurus nobilis L. (Lauraceae) has overrun Europe and the Mediterranean area. The problems associated with the great variability of this dioïceous species, the difficulties in sexual reproduction and seed germination, could be overcome, using micropropagation and in vitro culture techniques, by mass clonal production, as was demonstrated in our laboratory with other species. Here we present preliminary results of the breeding improvement and micropropagation by axillary buds of selected mature individuals. An analysis of variance of the results showed significant differences in rooting due to the type of cutting. The buds also have an effect in this morphogenetic process, especially in the induction of the first root. In micropropagation trials, we could control the delicate stage of sterilization. Aseptic and vigorous cultures were obtained after treatment by calcium hypochlorite. Shoot multiplication and elongation were obtained by addition of benzyl-aminopurine combined with gibberellic acid. Root induction was obtained in MS medium salts at ⅓ strength with naphtalene-acetic acid.

Diversity of Sterol Composition in Tunisian Pistacia lentiscus Seed Oil.

Pistacia lentiscus L. seed oil is used in some Mediterranean forest area for culinary and medicinal purposes. In this study, we aim to examine, for the first time, the effect of growing area on sterol content of Pistacia lentiscus seed oil. Fruits were harvested from 13 different sites located in northern and central Tunisia. Gas chromatography‐flame‐ionization detection (GC‐FID) was used to quantify sterols and gas chromatography/mass spectrometry (GC/MS) was used to identify them. The major sterol identified was β‐sitosterol with a value ranging from 854.12 to 1224.09 mg/kg of oil, thus making up more than 54% of the total sterols. The other two main sterols were cycloartenol (11%) and 24‐methylene‐cycloartenol (5%). Statistical results revealed that growing location significantly (P < 0.001) affected phytosterol levels in these oils.

Essential-oil composition of the Tunisian endemic cypress (Cupressus sempervirens L. var. numidica TRAB.).

The essential oils isolated from leaves, wood, and cones of the Tunisian endemic cypress Cupressus sempervirens L. var. numidica Trab. collected from three natural populations were characterized by GC‐FID and GC/MS analyses. In the wood, leaf, and cone oils, 38, 35, and 26 constituents, representing 94.4, 97.8, and 98.5% of the total oil composition, respectively, were identified. Monoterpenes constituted the major fraction of the oils from all organs and for all populations. The oils were found to be of an α‐pinene (64.2%)/δ‐car‐3‐ene (11.1%) chemotype with considerable contents of α‐humulene (3.4%) in the leaf oil, cedrol (2.8%) in the wood oil, and sabinene (3.2%) in the cone oil, respectively. α‐Pinene, δ‐car‐3‐ene, limonene, carvacrol methyl ether, α‐humulene, and α‐amorphene were the main components that differentiated the oils of the three organs in the cypress of Makthar.

Résultats d’essais de comparaison de provenances du Pin pignon (Pinus pinea L.) en Tunisie septentrionale

Stone pine (Pinus pinea L.) is widely used for reforestation in northern Tunisia to produce both fruit and timber. A genetic improvement programme for this species has begun, the first stage of which is a geographic exploration of its genetic variability. Experimental plots have been established in several countries bordering the Mediterranean Sea. This article reports the results of 10 years of monitoring the two plots in northern Tunisia, at Jbel Essmaa and Oued El Bir. The survival and vigour data recorded for seedlings of various provenances are presented and analysed. Survival and growth of the provenances differ significantly between the two sites whereas differences are not significant within each site taken individually.

Fatty Acid Composition, Essential Oil and Antibacterial Activity of Berries of Laurus nobilis L.

Abstract Fruits of Laurus nobilis L. growing wild in different stations of Tunisia and Algeria were analysed for their fixed oil and essential oil composition. Fixed oil was extracted in Soxhlet apparatus by using hexane as solvent. The yield values were found approximatively the same for the fruit of the different stations. Fatty acids composition of the fixed oil was determined by using combined techniques of HPLC and GC. Fatty acids composition of L. nobilis were found as 54 % for satured fatty acids and 46 %for unsatured acids. Essential oils, isolated by hydrodistillation, from different parts of the berries (seeds and pericarp) of L. nobilis were analyzed by GC-MS and the main components were: 1,8-cineole, (E) β-ocimene, α-terpinyl acetate, α-pinene, β-pinene and β-longipinene. The yields in essential oil range between 0.6 - 1.2 % by weight for seeds and 0.8 -1 .1 % for pericarps. Study of the chemical composition indicate a small variability of the volatile oil and fixed oil between the different stations whereas big differences in the composition was observed for the seeds and pericarp.

Phenolic profile and effect of growing area on Pistacia lentiscus seed oil

In this investigation, we aimed to study, for the first time, the phenolic composition of Pistacia lentiscus seed oils from different growing areas. Extraction of the phenolic fraction from oils was done by methanol/water. Phenolic profiles were determined using chromatographic analysis by High Performance Liquid Chromatography (HPLC-DAD/MSD) and its quantification was done using an internal standard which is unidentified in the studied oil (syringic acid). Forty phenolic compounds were quantified and only eighteen of them were identified. The eight studied oils showed different phenolic profiles. The total phenols amount varied from 538.03 mg/kg oil in Jbel Masour oils to 4260.57 mg/kg oil in oils from Kef Erraai. The highest amount of secoiridoids was reached by Bouchoucha oil containing 366.71 mg/kg oil of Oleuropein aglycon. Oils from Kef Erraai locality contained the highest concentrations in flavonols (377.44 mg/kg oil) and in phenolic acids (2762.67 mg/kg oil).

An overview of adaptative responses to drought stress in Eucalyptus spp.

Abstract Eucalyptus is a plant frequently present and cultivated in arid regions because of its high adaptation to drought. Furthermore, it is known by its numerous species which represents a great opportunity to choose several tolerant species with variable uses. Therefore, the choice of species, hybrids or genotypes resistant to aridity becomes a necessity before any plantation in regions and countries characterized by semi-arid, arid and Saharan climates. Our review shows an important effect of drought on anatomical, physiological and biochemical parameters of Eucalyptus spp. Indeed, water stress acts directly on growth, yield and response to pests. However, the response to drought varies significantly among species, genotypes, hybrids and clones. This result represents a field for the selection of eucalypts tolerant and adapted to water deficit or climate change.