Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis

The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in new drug developments from traditionally used medicinal plants which offers unprecedented diversity in structures and bioactivity. With this rationale, ethanolic extract of Tinospora sinensis Linn and its four fractions were tested in vitro against promastigotes and intracellular amastigotes and in vivo in Leishmania donovani infected hamsters. Ethanolic extract exhibited an appreciable activity against promastigotes (IC₅₀ 37.6 ± 6.2 μg/ml) and intracellular amastigotes (IC₅₀ 29.8 ± 3.4 μg/ml). In hamsters, it resulted in 76.2 ± 9.2% inhibition at 500 mg/kg/day × 5 oral dose level. Among fractions, n-butanol imparted highest in vitro and in vivo activities. Ethanolic extract and butanol fraction also enhances reactive oxygen species (ROS) and nitric oxide (NO) release. The results indicate that T. sinensis may provide new lead molecules for the development of alternative drugs against VL.     

In vitro and in vivo activity of <Emphasis Type="Italic">Aloe vera</Emphasis> leaf exudate in experimental visceral leishmaniasis

The leishmanicidal activity of Aloe vera leaf exudate (AVL) has been demonstrated in promastigotes and axenic amastigotes, but its effectiveness in animal models has not been evaluated. The presence of alkaloids, triterpenes, cyanidines, proanthocyanidines, tannins, and saponins in AVL was identified. Its effectiveness in four Leishmania donovani strains was studied both in promastigotes (IC₅₀ ranged from 70–115 μg/ml) and amastigotes (IC₅₀ ranged from 3.1–11.4 μg/ml). In amastigotes, the killing by AVL was facilitated through its induction of nitric oxide in leishmania-infected macrophages. The safety index was good as AVL up to 300 μg/ml remained non-toxic to monocytes and macrophages. In a L. donovani BALB/c mouse model, oral or subcutaneous administration of AVL (15 mg/kg body weight × 5 days) reduced parasitemia by >90% in the liver, spleen, and bone marrow without impairment of hepatic and renal functions. Collectively, we conclude that AVL shows promising antileishmanial activity and may provide a new lead agent in the treatment of Leishmaniasis. Chitra Mandal and Mitali Chatterjee should be considered as joint senior authors.     

In vitro evaluation of the effectiveness and cytotoxicity of meglumine antimoniate microspheres produced by spray drying against Leishmania infantum

The aim of the present study was to evaluate the in vitro activity and cytotoxicity of meglumine antimoniate microspheres produced by spray drying on Leishmania infantum and the effect of the excipients used in them. The parasite strain shows sensitivity to the meglumine antimoniate microspheres prepared. All the antimony IC₅₀ values from encapsulated meglumine antimoniate (3.80 ± 0.34 to 9.53 ± 0.70 μg SbV/ml for promastigotes assay) are considerably lower compared to the mean value of IC₅₀ in Glucantime solution (112 ± 12.74 μg SbV/ml). Interesting IC₅₀ values for the excipient chitosan (112.64 ± 0.53 mg/ml for promastigotes and 100.81 ± 26.45 mg/ml for amastigotes) were obtained (without cytotoxic activity), whereas the rest of the excipients did not show any activity. This new delivery system could offer a new pharmacological tool for the treatment of leishmaniosis that reduces the doses required, lowering toxic side effects because of meglumine antimoniate.     

Comparative studies of the anti-leishmanial activity of three <Emphasis Type="Italic">Crotalus durissus</Emphasis> ssp. venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus–Cdt, Crotalus durissus cascavella–Cdca, and Crotalus durissus collilineatus–Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 ± 1.72 μg/ml) in comparison with that of Cdca venom (IC50 value of 9.41 ± 1.21 μg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 ± 2.18 μg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 ± 9.50 μg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 ± 0.52 μg/ml and 19.95 ± 4.21 μg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 ± 2.21 μg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.     

Antiprotozoan activity of Brazilian marine cnidarian extracts and of a modified steroid from the octocoral <Emphasis Type="Italic">Carijoa riisei</Emphasis>

In the present investigation, we have evaluated the antileishmanial and antitrypanosomal activity of methanolic crude extracts obtained from eight species of cnidarians and of a modified steroid isolated from the octocoral Carijoa riisei. The antileishmanial activity of cnidarians crude extracts showed 50% inhibitory concentration (IC₅₀) values in the concentration range between 2.8 and 93.3 μg/mL. Trypomastigotes of Trypanosoma cruzi were less susceptible to the crude extracts, with IC₅₀ values in the concentration range between 40.9 and 117.9 μg/mL. The steroid (18-acetoxipregna-1,4,20-trien-3-one) displayed a strong antileishmanial activity, with an IC₅₀ value of 5.5 μg/mL against promastigotes and 16.88 μg/mL against intracellular amastigotes. The steroid also displayed mammalian cytotoxicity (IC₅₀ of 10.6 μg/mL), but no hemolytic activity was observed at the highest concentration of 12.5 μg/mL. The antileishmanial effect of the steroid in macrophages suggested other mechanism than macrophage activation, as no upregulation of nitric oxide was observed. The antitrypanosomal activity of the steroid resulted in an IC₅₀ value of 50.5 μg/mL. These results indicate the potential of cnidarian natural compounds as antileishmanial drug candidates.     

Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 μg/ml; mf = 13.17 μg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 μg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 μg/ml, CC50 = 212.5 μg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 μg/ml, CC50 = 262.3 μg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.     

In vitro activity of the essential oil of <Emphasis Type="Italic">Cymbopogon citratus</Emphasis> and its major component (citral) on <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Leishmaniasis causes considerable mortality throughout the world, affecting more than 12 million people. Cymbopogon citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries and is also known as a source of ethnomedicines. In this study, the inhibitory effect and the morphological and ultrastructural alterations on Leishmania amazonensis by the essential oil (EO) of C. citratus and its main constituent, citral, were evaluated. The results showed that the antiproliferative activity of EO on promastigotes and axenic amastigotes, and intracellular amastigote forms of L. amazonensis was significantly better than citral, and indicated a dose-dependent effect. Neither compound showed a cytotoxic effect on macrophage strain J774G8. The promastigote forms of L. amazonensis underwent remarkable morphological and ultrastructural alterations compared with untreated cultures. These alterations were visible by light, scanning, and transmission electron microscopy of promastigotes treated with EO and citral at concentrations corresponding to the IC₅₀ (1.7 and 8.0 μg/ml) and IC₉₀ (3.2 and 25 μg/ml), respectively, after 72 h of incubation. This study revealed that citral-rich essential oil from C. citratus has promising antileishmanial properties, and is a good candidate for further research to develop a new anti-protozoan drug.     

Antileishmanial activity of imidothiocarbamates and imidoselenocarbamates

In the present study, a family of 15 imidothio- and imidoselenocarbamates (1–15) analogs have been synthesized and screened for their in vitro antileishmanial potential against Leishmania infantum promastigotes. The six most active ones (2, 4, 7, 13, 14, and 15) were also tested in an axenic amastigote model. In order to establish their selectivity indexes (SI) the cytotoxic effect of each compound was also assayed against Jurkat and THP-1 cell lines. Compounds 2 and 4, both with a pyridine moiety, showed a moderate antileishmanial activity with an IC₅₀ value of 4.68 ± 0.46 and 3.03 ± 0.24 μM, respectively, in the amastigote model. The activity was compared with that of standard drugs, edelfosine (IC₅₀ = 0.82 ± 0.13 μM) and miltefosine (IC₅₀ = 2.84 ± 0.10 μM). Related to selectivity, the SI of both compounds are similar to those of the standard drugs when compared against the THP-1 cell line. Moreover, compound 4 was able to reduce the number of amastigote-infected THP-1 cells to 40% of that observed in untreated controls after a 96-h period of treatment. These derivatives thus represent two new leads for further studies aimed at establishing their mechanism of action.     

In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani

Control of Leishmania infection relies primarily on chemotherapy, and the current drug available for treating leishmaniasis is limited. Nitazoxanide (NTZ) is a broad spectrum antiparasitic agent with activity against protozoa, nematodes, cestodes, and trematodes. In the present study, the in vitro antileishmanial efficacy of NTZ was evaluated by incubation of Leishmania donovani promastigotes with NTZ, indicating that NTZ can affect the ultrastructure of parasite promastigote and efficiently inhibit the parasite growth. Moreover, 200 μg/ml NTZ inhibited >90% of promastigotes growth, showing similar activity of the reference drug amphotericin B (P > 0.05). Therapeutic efficacy of NTZ against L. donovani-infected BALB/c mice demonstrated that oral NTZ produced a significant reduction of parasite burden in spleen and liver from L. donovani-infected mice, compared with the untreated mice (P < 0.05). These results indicated NTZ may be a novel therapeutic drug for leishmaniasis.     

Purification and characterization of hexokinase from <Emphasis Type="Italic">Leishmania mexicana</Emphasis>

Hexokinase from Leishmania mexicana was purified to homogeneity from a glycosome-enriched fraction obtained after a differential centrifugation of promastigote form. The kinetic properties of the pure enzyme were determined and the Km values for glucose (Km = 66 μM) and ATP (Km = 303 μM) were comparable to those from hexokinase of Trypanosoma cruzi. L. mexicana hexokinase was able to use fructose (Km = 142 μM), which reflects the condition found in the insect host. In contrast with hexokinases from other trypanosomatids, the enzyme exhibited a moderate sensitivity to inhibition by glucose 6-phosphate. This inhibition was competitive with respect to both ATP and glucose, indicating that an allosteric site for glucose 6-phosphate does not exist in this enzyme. The enzyme was also inhibited by inorganic pyrophosphate, the inhibition being higher than that observed for T. cruzi enzyme. As expected, the enzyme was localized, by immunofluorescence analysis, in glycosomes and is present in both promastigotes and true amastigotes obtained from hamster lesion. Hexokinase specific activity increased with the aging of promastigote culture, and this increment was related to glucose consumption. However, the level of the hexokinase protein remains constant as determined by Western blotting. Several hypotheses are discussed to explain this result.     

In vitro antileishmanial activity of Adana propolis samples on Leishmania tropica: a preliminary study

Propolis (bee glue) is a natural resinous hive product, collected from various plant sources. It has attracted much attention as a useful substance applied in medicine due to its pharmacological activities. It was aimed to investigate the in vitro effects of an ethanolic extract of Adana propolis samples on the growth of Leishmania tropica. Parasite cells were treated with five concentrations (25, 50, 100, 50, 500, and 750 μg/ml) of the propolis. The number of promastigotes in each concentration was calculated using a hemocytometer slide at 24, 48, and 72 h after being harvested. In the experiments, it was determined that the concentrations up to 100 μg/ml of the propolis did not exhibit antileishmanial activity against the parasites cells. At these concentrations, there was no changes in terms of morphologically. In addition, there was no statistically significant difference in terms of cell count between control and these three groups (p > 0.05). However, in culture media containing the propolis samples at 250, 500, and 750-μg/ml concentrations, statistically significant differences in cell counts were observed, as compared to the control group (p < 0.05). Our results demonstrate that ethanolic extracts of Adana propolis samples reduce the proliferation of L. tropica parasites significantly.     

Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine

In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 μM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 μM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.     

An ethanolic extract of leaves of <Emphasis Type="Italic">Piper betle</Emphasis> (Paan) Linn mediates its antileishmanial activity via apoptosis

An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC₅₀ values of 9.8 and 5.45 μg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G₀/G₁ phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.     

GC-MS analysis and antileishmanial activities of two Turkish propolis types

Propolis is a honeybee product with a very complex chemical composition and various pharmacological properties. This study was aimed to investigate antileishmanial activities of “Bursa” and “Hatay” propolis samples against Leishmania infantum and Leishmania tropica strains. Propolis samples were analysed with the gas chromatography-mass spectrometry technique. Promastigotes were incubated in Roswell Park Memorial Institute culture medium in the absence and presence of several concentrations (50, 100, 250, 500, 750, and 1,000 μg/mL) of each propolis sample. The viability and cell morphology of promastigotes in each concentration were examined after 24, 48, 72, and 96 h of incubation. The growth of leishmania parasites was significantly suppressed in the presence of 500, 750, and 1,000 μg/mL of Hatay propolis. Bursa propolis was found to be efficient in inhibiting the growth of leishmania promastigotes in culture media at these concentrations, 250, 500, 750, and 1,000 μg/mL. Thus, the in vitro results showed that the Hatay and Bursa propolis samples decreased significantly the proliferation of L. infantum and L. tropica parasites (p < 0.001); however, Bursa propolis was found to be more effective than Hatay propolis against leishmania promastigotes. These two natural products may be useful agents in the prevention of leishmanial infections.     

Exenatide enhances kaliuresis under conditions of hyperkalemia

Experiments on Wistar rats showed that exenatide (0.015–0.5 nmol per 100 g body weight) somewhat increased renal excretion of potassium from 7 ± 1 to 16 ± 1 μmol/h/100 g body weight (p < 0.05) in animals with normal serum concentration of glucose (4.6 ± 0.4 mM) and potassium (4.3 ± 0.1 mM). Exenatide dramatically enhanced excretion of potassium under conditions of hyperkalemia (11.4 ± 0.4 mM) produced by intraperitoneal injection of 1.25% KCl solution (5 ml per 100 g body weight). During the fi rst postinjection hour, potassium excretion increased 2-fold and attained 97 ± 11 μmol/h/100 g body weight in comparison with potassium load alone (47 ± 9 μmol/h/100 g body weight, p < 0.05). The data attest to a possible role of peptide regulators in normalization of potassium balance via renal mechanisms.     

The effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of <Emphasis Type="Italic">Leishmania tropica</Emphasis> to meglumine antimoniate

Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL) with low efficacy and resistance is emerging. CL is increased significantly in respect to incidence rate and expanding to new foci. In the present study, the effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Leishmania tropica to meglumine antimoniate (MA, Glucantime) was evaluated using colorimetric assay (MTT) and in a macrophage model, respectively. Verapamil, as a calcium channel blocker, affects drug uptake by preventing of drug efflux from the cells. In promastigote form, several concentrations of MA with or without verapamil showed significant decrease (P < 0.05) in optical density. The overall mean IC₅₀ value with combination of MA plus verapamil (IC50 = 116.03 μg/ml) was significantly less than MA (IC50 = 225.14 μg/ml) alone (P < 0.05) for promastigote stage. Similarly, the amastigote stage was more susceptible to treatment with MA plus verapamil to that of MA alone (P < 0.05). Analysis of overall effect of different concentrations of MA alone, compared with combination of MA plus verapamil by mean infection rate of amastigotes in each macrophage showed a significant difference (P < 0.05).These findings indicated some degree of synergistic effects between MA and verapamil on in vitro susceptibility of L. tropica to MA. Further works are required to evaluate this synergistic effect on animal model or volunteer human subjects.     

A novel herbal formulation against dengue vector mosquitoes <Emphasis Type="Italic">Aedes aegypti</Emphasis> and <Emphasis Type="Italic">Aedes albopictus</Emphasis>

The objective of this study was to develop a herbal formulation to control dengue vector mosquitoes. PONNEEM, a novel herbal formulation prepared using the oils of neem (Azadirachta indica), karanj (Pongamia glabra) and their extracts, was tested for larvicidal, ovicidal and oviposition deterrent activities against Aedes aegypti and Aedes albopictus at 1, 0.5, 0.3 and 0.1 ppm concentrations. Cent percent larvicidal and ovicidal activities were observed at 0.1 ppm in the two mosquito species under laboratory and sunlight-exposed conditions up to 12 months from the date of manufacture. Oviposition deterrent activity of 69.97% and 71.05% was observed at 1 ppm concentration of PONNEEM against A. aegypti and A. albopictus, respectively. Reduction in enzyme levels for α-esterase was 0.089 ± 0.008 and 0.099 ± 0.140 μg napthol produced/min/mg larval protein; for β-esterase, it was 0.004 ± 0.009 and 0.001 ± 0.028 μg napthol produced/min/mg larval protein; for glutathione S-transferase, it was 10.4814 ± 0.23 and 11.4811 ± 0.21 μmol/min/mg larval protein and for total protein, it was 0.177 ± 0.010 and 0.008 ± 0.005 mg/individual larva in treated groups of A. aegypti and A. albopictus, respectively. The nontarget organisms such as Gambusia affinis and Diplonychus indicus were not affected. No mortality was observed in control. PONNEEM can be used effectively for the management of human vector mosquitoes.     

Development of a highly responsive needle-type glucose sensor using polyimide for a wearable artificial endocrine pancreas

To produce a long-life, stable, miniature glucose sensor for a wearable artificial endocrine pancreas (WAEP), we developed a novel microneedle-type glucose sensor using polyimide, designated the PI sensor (outer diameter, 0.3 mm; length, 16 mm), and investigated its characteristics in vitro and in vivo. In the in vitro study, we tested the sensor in 0.9% NaCl solution with varying glucose concentrations and observed an excellent linear relationship between the sensor output and glucose concentration (range: 0–500 mg/100 ml). In in vivo experiments, the PI sensor was inserted into the abdominal subcutaneous tissue of beagle dogs (n = 5), and interstitial fluid glucose concentrations were monitored after sensor calibration. Simultaneously, blood glucose concentrations were also monitored continuously with another PI sensor placed intravenously. The correlation and time delay between subcutaneous tissue glucose (Y) and blood glucose concentrations (X: 30–350 mg/100 ml) were Y = 1.03X + 7.98 (r = 0.969) and 6.6 ± 1.2 min, respectively. We applied the new WAEP system/PI sensor and an intravenous insulin infusion algorithm developed previously for glycemic control in diabetic dogs. The use of the WAEP system resulted in excellent glycemic control after an oral glucose challenge of 1.5 g/kg (post-challenge blood glucose levels: 176 ± 18 mg/100 ml at 65 min and 93 ± 23 mg/100 ml at 240 min), without any hypoglycemia. Thus, we confirmed that our new PI sensor has excellent sensor characteristics in vitro and in vivo. The new WAEP using this sensor is potentially suitable for clinical application.     

In vitro anthelmintic activity of the essential oils of <Emphasis Type="BoldItalic">Zanthoxylum zanthoxyloides</Emphasis> and <Emphasis Type="BoldItalic">Newbouldia laevis</Emphasis> against <Emphasis Type="BoldItalic">Strongyloides ratti</Emphasis>

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: β-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC₅₀ and IC₉₀) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC₅₀ values (19.5 and 18.2 μg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC₅₀ = 2.5 μg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC₅₀ = 46 μg/ml), N. laevis (IC₅₀ = 51 μg/ml), and the control [levamisole (IC₅₀ = 36 μg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC₅₀ values higher than 50 μg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.     

Screening of antileishmanial activity from marine sponge extracts collected off the Tunisian coast

The present study reports on the in vitro antileishmanial activity of two Ircinidae (Dictyoceratida, Demospongiae, Porifera) Ircinia spinosula and Sarcotragus sp. Sampled from the east coast of Tunisia. The ethyl acetate, dichloromethane, and aqueous extracts were tested against Leishmania major promastigotes. The anti-proliferative activity was checked using different extracts concentration during 72 h. We found that the IC50 (sub-inhibitory concentration) values ranged from 1.39 to 264.67 μg/ml. The most active extract was that from sarcotragus sp dichloromethane extract. Microscopic observations showed that the extracts promoted cellular alterations and induce enlargement of the nucleus and modification of the parasite shape. These promising results in relation with in vitro antileishmanial activity open the way for complementary investigation in order to purify and identify active molecules.