Mondher Mejri

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp.

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC50 of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC90 for 24h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.

Comparison of the effects of major fatty acids present in the Mediterranean diet (oleic acid, docosahexaenoic acid) and in hydrogenated oils (elaidic acid) on 7-ketocholesterol-induced oxiapoptophagy in microglial BV-2 cells

Increased levels of 7-ketocholesterol (7KC), which results mainly from cholesterol auto-oxidation, are often found in the plasma and/or cerebrospinal fluid of patients with neurodegenerative diseases and might contribute to activation of microglial cells involved in neurodegeneration. As major cellular dysfunctions are induced by 7KC, it is important to identify molecules able to impair its side effects. Since consumption of olive and argan oils, and fish is important in the Mediterranean diet, the aim of the study was to determine the ability of oleic acid (OA), a major compound of olive and argan oil, and docosahexaenoic acid (DHA) present in fatty fishes, such as sardines, to attenuate 7KC-induced cytotoxic effects. Since elaidic acid (EA), the trans isomer of OA, can be found in hydrogenated cooking oils and fried foods, its effects on 7KC-induced cytotoxicity were also determined. In murine microglial BV-2 cells, 7KC induces cell growth inhibition, mitochondrial dysfunctions, reactive oxygen species overproduction and lipid peroxidation, increased plasma membrane permeability and fluidity, nuclei condensation and/or fragmentation and caspase-3 activation, which are apoptotic characteristics, and an increased LC3-II/LC3-I ratio, which is a criterion of autophagy. 7KC is therefore a potent inducer of oxiapoptophagy (OXIdation+APOPTOsis+autoPHAGY) on BV-2 cells. OA and EA, but not DHA, also favor the accumulation of lipid droplets revealed with Massons trichrome, Oil Red O, and Nile Red staining. The cytotoxicity of 7KC was strongly attenuated by OA and DHA. Protective effects were also observed with EA. However, 7KC-induced caspase-3 activation was less attenuated with EA. Different effects of OA and EA on autophagy were also observed. In addition, EA (but not OA) increased plasma membrane fluidity, and only OA (but not EA) was able to prevent the 7KC-induced increase in plasma membrane fluidity. Thus, in BV-2 microglial cells, the principal fatty acids of the Mediterranean diet (OA, DHA) were able to attenuate the major toxic effects of 7KC, thus reinforcing the interest of natural compounds present in the Mediterranean diet to prevent the development of neurodegenerative diseases.

Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts

Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl acetate extract of olive leaf was the most active showing an IC50 of 5.11±0.71μg/ml of dry extract. Bio-guided fractionation of this extract was conducted and led to the identification of three active compounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of novel therapeutic approaches against Acanthamoeba infections.

Activity of olive leaf extracts against the promastigote stage of Leishmania species and their correlation with the antioxidant activity.

Leishmaniasis is one of the neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic and to present resistance issues. As consequent, the need for novel leishmanicidal molecules has notably increased in the recent years. In the present work an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of compounds in leaf extracts of 5 different Tunisian olive tree varieties, used as traditional medicine in this country. The concentration of extracts needed to inhibit 50% of the parasitic growth (IC50) was estimated using different Leishmania strains. All tested extracts showed an inhibitory effect on the parasite growth with IC50 ranging from 2.130±0.023 to 71.570±4.324μg/ml, respectively for the methanolic extracts of Limouni and Zarrazi against Leishmania donovani. In fact, this activity was significantly affected by the olive cultivar and the tested Leishmania strain. Furthermore, the activities against both Leishmania tropica and major species were correlated to the total phenolic compounds. These results could suggest that olive leaf extract could carry potential new compounds for the development of novel drugs against Leishmaniasis.

Programmed cell death in Acanthamoeba castellanii Neff induced by several molecules present in olive leaf extracts

Therapy against Acanthamoeba infections such as Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK), remains as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. Recently, the activity of Olive Leaf Extracts (OLE) was demonstrated against Acanthamoeba species. However, the molecules involved in this activity were not identified and/or evaluated. Therefore, the aim of this study was to evaluate the activity of the main molecules which are present in OLE and secondly to study their mechanism of action in Acanthamoeba. Among the tested molecules, the observed activities ranged from an IC50 of 6.59 in the case of apigenine to an IC50 > 100 μg/ml for other molecules. After that, elucidation of the mechanism of action of these molecules was evaluated by the detection of changes in the phosphatidylserine (PS) exposure, the permeability of the plasma membrane, the mitochondrial membrane potential and the ATP levels in the treated cells. Vanillic, syringic and ursolic acids induced the higher permeabilization of the plasma membrane. Nevertheless, the mitochondrial membrane was altered by all tested molecules which were also able to decrease the ATP levels to less than 50% in IC90 treated cells after 24 h. Therefore, all the molecules tested in this study could be considered as a future therapeutic alternative against Acanthamoeba spp. Further studies are needed in order to establish the true potential of these molecules against these emerging opportunistic pathogenic protozoa.

Supercritical CO2 Extract and Essential Oil of Ruta chalepensis L. Growing in Tunisia: A Natural Source of Undecan-2-one

Abstract The extraction of essential oil was carried out using two processes: hydrodistillation and supercritical CO2 extraction. The chemical composition of extracted essential oil was determined. The yield of extraction by supercritical CO2 was about 7.8 %. This yield is more important than that obtained by hydrodistillation process (5.51 %). The chemical composition of the supercritical CO2 extract indicates the presence of only two compounds. The major one is undecan-2-one (100 %). The second one is limonene which is present as trace. However, 23 compounds were identified in the essential oil of the aerial part of Ruta chalepensis extracted by hydrodistillation process. The major compound is also undecan-2-one (77.7 %).

Tunisian Milk Thistle: An Investigation of the Chemical Composition and the Characterization of Its Cold-Pressed Seed Oils

In this study, milk thistle seeds growing in different areas in Tunisia were cold pressed and the extracted oils were examined for their chemical and antioxidant properties. The major fatty acids were linoleic acid (C18:2) (57.0%, 60.0%, and 60.3% for the milk thistle seed oils native to Bizerte, Zaghouan and Sousse, respectively) and oleic acid (C18:1) (15.5%, 21.5%, and 22.4% for the milk thistle seed oils originating from Bizerte, Zaghouan and Sousse, respectively). High performance liquid chromatography (HPLC) analysis showed the richness of the milk thistle seed oils (MTSO) in α-tocopherol. The highest content was recorded for that of the region of Zaghouan (286.22 mg/kg). The total phenolic contents (TPC) of Zaghouan, Bizerte, and Sousse were 1.59, 8.12, and 4.73 Gallic Acid Equivalent (GAE) mg/g, respectively. Three phenolic acids were also identified (vanillic, p-coumaric, and silybine), with a predominance of the vanillic acid. The highest value was recorded for the Zaghouan milk thistle seed oil (83 mg/100 g). Differences in outcomes between regions may be due to climatic differences in areas. Zaghouan’s cold-pressed milk thistle seed oil had a better quality than those of Bizerte and Sousse, and can be considered as a valuable source for new multi-purpose products or by-products for industrial, cosmetic, and pharmaceutical utilization.

Optimized Extraction of Antioxidants from Olive Leaves Using Augmented Simplex Centroid Design

ABSTRACT The olive tree historically provided economic benefits to the countries in Mediterranean basin as a source of products contributing to health promotion. Some of the health benefits attributed to olive tree leaves are related to phenolic composition. In this study, a 10-point augmented simplex-centroid design was used to formulate a three-component mixture system using water, ethanol, and methanol, adequate for the extraction of phenolics from olive leaves. Phenolic and flavonoid content, antioxidant activity with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), oleuropein, and luteolin7-O-glucoside concentrations were selected as responses. The optimal extraction mixture was: 12.7% water, 14.8% ethanol, and 72.5% methanol. The yields of total phenolic and flavonoid compounds, oleuropein, and luteolin-7-O-glucoside were 163.69 ± 6.35 mg gallic acid g−1, 114.69 ± 3.56 mg rutin g−1, 858.29 µg g−1, and 21.04 µg g−1 dry weight of olive leaves. The antioxidant capacity was 238.71 ± 4.35 mM equivalent Trolox per g−1 dry weight of olive leaves.

Ruta chalepensis L. Essential Oil: Chemical Composition and Phytotoxic Activity

Abstract The aim of this work is to study the use of Ruta chalepensis essential oil as a bioactive substance in order to propose ways of valuation and rational exploitation of the plant. In this context, the extraction of essential oil was carried out using hydrodistillation process. 23 compounds were identified in the aerial part of Ruta chalepensis essential oil. The major compound is undecan-2-one (77.7 %). In addition, the phytotoxic activity of Ruta chalepensis essential oil was investigated. An important toxicity on the germination of the hard wheat was observed. Indeed, the rate of hard wheat germination passes from 65.55 % at 0.5 μ1.ml−1 essential oil concentration to 31.11% at 1 μ1.ml−1.

Investigation and Modeling of Extraction Parameters of Ruta chalepensis L. Essential Oil

Abstract To evaluate the effects of hydrodistillation parameters and the vegetative cycle of the plant (Ruta chalepensis L.) on the global essential oil yield the response surface methodology was used. The parameters tested are the volume of water in the suspension to be distilled and the time of distillation. The third parameter is the vegetative cycle of the plant. The responses were the global extraction yield and the extraction yield of following essential oil compounds: 2-undecanone, 2-decanone, 2-dodecanone and 2-tridecanone. The optimal conditions selected for the total output of the extraction obtained from the analysis of the response surfaces are: time of distillation: 120 minutes; volume of water in the suspension to be distilled: 500 ml; stage of vegetation: January (young growths). The results show that the time of distillation and the stage of vegetation are the parameters more meaning on the global essential oil yield.