Comparison of the Scolicidal Activity of Two Leaves Extracts of Myrtus communis from Algeria Against Echinococcus granulosus Sensu Lato Protoscoleces.

PURPOSE: During cystic echinococcosis surgery, the use of scolicidal agents such as hypertonic saline (20%) aims to reduce the risk of infection recurrence, but most of the used agents are associated with undesirable side effects. Therefore, the use of natural scolicidal agents such as medicinal plant extracts could reduce these medical issues. The present study aimed to compare in vitro the scolicidal activity between two extracts of the medicinal plant Myrtus communis from Algeria against Echinococcus granulosus sensu lato protoscoleces. METHODS: The ethanolic and aqueous extraction of plant leaves was performed. Phytochemical analysis by gas chromatography-tandem mass spectrometry (GC-MS/MS), determination of total phenolic and flavonoid contents, and in vitro antioxidant activity by DPPH were evaluated for both extracts. Finally, the in vitro scolicidal activity was tested by different concentrations. The viability was evaluated by the eosin exclusion test. RESULTS: The phytochemical analysis revealed 28 components for the ethanolic extract and 44 components for the aqueous extract. The major components were 2'-hydroxy-5'-methoxyacetophenone and 4-amino-2-methylphenol, respectively. The total phenolic and flavonoid contents were 45.9 ± 0.085 mg of gallic acid equivalent per g of extract (GAE/g E) and 16.5 ± 0.004 mg of quercetin equivalent per g (QE/g E) for the ethanolic extract, and 36.5 ± 0.016 mg GAE/g E and 18.2 ± 0.023 mg QE/g E for the aqueous extract, respectively. Furthermore, ethanolic and aqueous extracts of M. communis gave a value of IC50 = 0.009 ± 0.0004 mg/ml and IC50 = 0.012 ± 0.0003 mg/ml for the antioxidant activity, respectively. The in vitro scolicidal activity with concentrations of 50, 75, 100, and 150 mg/ml was tested for 5, 10, 15, and 30 min, and 5, 10, 15, 30, 60, 90, and 120 min for ethanolic and aqueous extracts, respectively. The mortality rate of protoscoleces at concentrations of 100 and 150 mg/ml was 98.8 and 100%, respectively, after 5 min of exposure to the ethanolic extract, while this rate was 100% at the same concentrations only after 60 min of exposure to the aqueous extract. CONCLUSIONS: The ethanolic extract showed a stronger scolicidal activity against E. granulosus s.l protoscoleces than the aqueous extract. In the future, other investigations are necessary to elucidate the mechanism of action and the possible toxicity on human cells. Moreover, experimental animal studies are required to investigate the efficacy of different extracts of this plant and its components as natural anti-parasitic alternatives for the treatment of human cystic echinococcosis.

Echinococcus granulosus tegumental enzymes as in vitro markers of pharmacological damage: a biochemical and molecular approach.

Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces.

Flubendazole and ivermectin in vitro combination therapy produces a marked effect on Echinococcus granulosus protoscoleces and metacestodes.

The aim of the present work was to evaluate the in vitro efficacy of the flubendazole (FLBZ) and ivermectin (IVM) combination against Echinococcus granulosus protoscoleces and metacestodes. Protoscoleces and groups of ten peritoneal cysts obtained from BALB/c mice were incubated with the two drugs, either separately or in combination, at the following final concentrations: 10 microg/mL FLBZ, 1 microg/mL FLBZ, 1 microg/mL IVM, 10 microg/mL FLBZ + 1 microg/mL IVM, and 1 microg/mL FLBZ + 1 microg/mL IVM. The maximum protoscolicidal effect was found with the combination 10 microg/mL FLBZ + 1 microg/mL IMV. After 1 day of incubation, the presence of numerous blebs in the tegument of protoscoleces was observed. Ultrastructural studies revealed that the primary site of damage was the tegument of the parasite. The effect of the two drugs on hydatid cysts obtained from mice was more rapidly detected in cysts treated with the combination of FLBZ + IVM than when drugs were used separately. Ultrastructural studies revealed that the germinal layer of treated cysts lost the multicellular structure feature and underwent considerable degenerative changes after in vitro treatment. The outcomes obtained demonstrated the favorable effect of the combination of FLBZ and IVM against E. granulosus.

Efficacy of thymol against Echinococcus granulosus protoscoleces.

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 mug/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 microg/ml, viability reduced to 53.5+/-11.9% after 12 days of incubation. At day 42, viability was 11.5+/-15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 microg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.