In vitro and ex vivo protoscolicidal effects of hydroalcoholic extracts of Eucalyptus microtheca on protoscoleces of Echinococcus granulosus sensu stricto: A light and scanning electron microscopy (SEM) study.

BACKGROUND: Cystic echinococcosis (CE) is one of the most widespread and important global helminth zoonoses. Treatment relies mainly on surgery and, or percutaneous interventions. However, spillage of live protoscoleces (PSCs) leading to recurrence is a problem during surgery. So, the application of protoscolicidal agents before surgery is required. This study aimed to investigate the activity and safety of hydroalcoholic extracts of E. microtheca against PSCs of Echinococcus granulosus sensu stricto (s.s.) both in vitro and also ex vivo, which is a simulation to Puncture, Aspiration, Injection, and Re-aspiration (PAIR) method. METHODS: Considering the effects of heat on the protoscolicidal effecacy of Eucalyptus leaves, hydroalcoholic extraction was performed by both soxhlet extraction at 80 °C and percolation at room temperature. The protoscolicidal action of hydroalcoholic extracts was assessed by in vitro and ex vivo assessments. Infected sheep livers were collected from the slaughterhouse. Then, the genotype of hydatid cysts (HCs) was confirmed by sequencing and, isolates were limited to E. granulosus s.s. In the next step, ultrastructural changes of Eucalyptus-exposed PSCs were studied by scanning electron microscopy (SEM). Finally, a cytotoxicity test was conducted by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the safety of E. microtheca. RESULTS: The prepared extracts by soxhlet extraction and percolation were, successfully exerted strong protoscolicidal effects in both in vitro and ex vivo tests. The results of in vitro assessment indicated that hydroalcoholic extract of E. microtheca prepared by percolation at room temperature (EMP) and hydroalcoholic extract of E. microtheca prepared by soxhlet extraction at 80 °C (EMS) killed all PSCs (100%) at concentrations of 10 and 12.5 mg/mL, respectively. Also, EMP showed 99% protoscolicidal action after 20 min in an ex vivo setting compared to EMS. SEM micrographs confirmed potent protoscolicidal and destructive effects of E. microtheca against PSCs. The cytotoxicity of EMP was tested on the HeLa cell line using MTT assay. The value of 50% cytotoxic concentration (CC50) was calculated at 46.5 µg/mL after 24h. CONCLUSION: Both hydroalcoholic extracts showed potent protoscolicidal activity and, especially EMP produced remarkable protoscolicidal effects compared to the control group.

Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to the fucose-mannose ligand of Leishmania infantum combined with glycyrrhizin.

BACKGROUND: The fucose-mannose ligand (FML) of Leishmania infantum is a complex glycoprotein which does not elicit adequate immunogenicity in humans. In recent years, adjuvant compounds derived from plants have been used for improving the immunogenicity of vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known immunomodulatory activities. In the present study, we investigated the effects of co-treatment with FML and GL on the production of cytokines and nitric oxide (NO) by macrophages, in vitro. METHODS: Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 µg/ml) of L. infantum and various concentrations of GL (1 µg/ml, 10 µg/ml and 20 µg/ml). After 48 h of treatment, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 were measured by sandwich ELISA and NO concentration by Griess reaction. RESULTS: Our results indicate that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α and IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone. CONCLUSIONS: Therefore, we concluded that GL can improve the immunostimulatory effect of FML on macrophages and leads to their polarization towards an M1-like phenotype.

In vitro efficacy of ethanolic extract of Artemisia absinthium (Asteraceae) against Leishmania major L. using cell sensitivity and flow cytometry assays.

Leishmaniasis is one of the most neglected human diseases with an estimated global burden ranking second in mortality and fourth in morbidity among the tropical infections. Chemotherapy involving the use of drugs like glucantime is the mainstay treatment in endemic areas of Iran. Drug resistance is increasingly prevalent, so search for alternative therapy is gathering pace. Medicinal herbs, like wormwood Artemisia, have chemical compounds effective against a number of pathogens. In this study, the efficacy of ethanol extract from Artemisia absinthium (Asteraceae) against Leishmania major L. was investigated in vitro. The outcome of different effective doses (1-40 mg/ml) of ethanol extracts from this medicinal herb, A. absinthium, on a standard Iranian parasite strain of L. major was examined. The L. major promastigote cell sensitivity and mortality or viability effects due to the addition of herbal extract were measured using the MTT assay and the flow cytometry technique, respectively. There was complete agreement between the two assays. The lethal concentration (LC50) was measured as 101 mg/ml. Some contrasting relationships between the medicinal herb concentrations and the viability of parasites were observed; so that there was an increased multiplication of the parasite at low concentrations of the drug, but an anti-parasitic apoptotic effect was seen at high concentrations of A. absinthium. It was concluded that there might be one or more chemical constituents within the herbal extract of wormwood which at high concentration controlled cell division and affected the relevant activity within the only one giant mitochondrion in this flagellate parasite. At low doses, however, it showed the opposite effect of leading to mitotic cell divisions.