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.

Protoscolicidal activity of Atriplex halimus leaves extract against Echinococcus granulosus protoscoleces.

Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 µg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 µg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer.

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1ß, TGF-ß, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1ß that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-ß (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-ß expression and down-regulation of IFN-γ and IL-1ß expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.

Comparative study of nitric oxide (NO) production during human hydatidosis: relationship with cystic fluid fertility.

Human hydatidosis is characterized by a prolonged coexistence of Echinococcus granulosus and its host without effective rejection of the parasite. This parasitic infection constitutes a major health problem in Algeria. In this study, we investigated in vivo production of nitrite (NO(2)(-) + NO(3)(-)) in sera of Algerian patients carrying different cyst locations. Nitrite (NO(2)(-) + NO(3)(-)) levels were evaluated by the Griess method. Our results indicated that the levels of nitrite were significantly higher in the sera of hydatic patients than those of healthy controls supporting the involvement of nitric oxide (NO) in antihydatic action. The levels of nitrite in sera of the patients with hepatic hydatidosis were significantly higher than those with pulmonary infection. The lower serum (NO(2)(-) + NO(3)(-)) levels were observed in the relapsing cases. In addition, (NO(2)(-) + NO(3)(-)) levels of fertile hydatic fluids were significantly higher compared to infertile fluids. Our results suggest that the presence of NO products in hydatic fluids seems to be related to the location and the fertility of hydatic cysts. The assessment of protein concentration in hydatic fluids showed that the concentration of proteins was not exclusively dependent on the fertility but on the cyst locations. The assessment of (NO(2)(-) + NO(3)(-)) production in hydatic patients may be a useful tool to evaluate effector mechanisms of NO and clinical manifestations.

Mucosal intestinal alteration in experimental colitis correlates with nitric oxide production by peritoneal macrophages: effect of probiotics and prebiotics.

Inflammatory bowel disease (IBD) consists mainly of Ulcerative colitis (UC) and Crohn disease (CD). Although its aetiology is still not clearly established, it is thought to be due to overly aggressive immune response to enteric bacteria in genetically predisposed individuals. Manipulating the microbiota using probiotics or prebiotics is considered as a promising field of new therapeutic strategies used to attenuate immune disorders observed during IBD. The production of nitric oxide (NO) seems to be implicated in IBD pathogenesis. In our study, an acute UC was induced in Swiss mice using 3% Dextran Sulfate Sodium (DSS). The preventive effects of "Ultrabiotique®" (a probiotic) and inulin (a prebiotic) on the colitis were investigated. The production of NO was evaluated in the supernatants of peritoneal macrophages (pMφ) cultures. Colonic mucosa histology was subsequently examined. Results showed severe acute UC after administration of DSS. High levels of NO in pMφ cultures were also observed compared to control samples. These findings correlated with a significant destruction of the colonic mucosa. Oral administration of Ultrabiotique® or inulin decreased the severity of DSS-induced colitis. These treatments lead to a decrease in NO levels in pMφ cultures. A considerable reduction of colonic lesions was also noticed. Our findings suggest the involvement of NO in experimental UC pathogenesis. Pre- and pro-biotics, as discussed herein, seem to have an anti-inflammatory effect.

In Vitro Study of Nitric Oxide Metabolites Effects on Human Hydatid of Echinococcus granulosus.

Hydatidosis is characterized by the long-term coexistence of larva Echinococcus granulosus and its host without effective rejection. Previous studies demonstrated nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we investigated the direct in vitro effects of NO species: nitrite (NO(2) (-)), nitrate (NO(3) (-)) and peroxynitrite (ONOO(-)) on protoscolices (PSCs) viability and hydatid cyst layers integrity for 24 hours and 48 hours. Our results showed protoscolicidal activity of NO(2) (-) and ONOO(-) 24 hours and 3 hours after treatment with 320 muM and 80 muM respectively. Degenerative effects were observed on germinal and laminated layers. The comparison of the in vitro effects of NO species on the PSCs viability indicated that ONOO(-) is more cytotoxic than NO(2) (-). In contrast, NO(3) (-) has no effect. These results suggest possible involvement of NO(2) (-) and ONOO(-) in antihydatic action and point the efficacy of these metabolites as scolicidal agents.