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.

Effects of Zataria multiflora essential oil on the germinative cells of Echinococcus granulosus.

BACKGROUND: Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. This study was performed to establish an in vitro cell culture model for E. granulosus germinative cells and to evaluate the lethal effect of Zataria multiflora essential oil (ZMEO) on the cultured cells. METHODS: The inner surface of germinal layers of CE cysts was scraped, and the obtained materials were trypsinized to obtain a suspension of single germinative cells. Medium 199 was used as the basic culture medium and was supplemented with fetal bovine serum, 2-mercaptoethanol, L-cysteine, L-glutamine, glucose, sodium pyruvate, hydatid fluid, amphotericin B and antibiotics. The cells were cultured at a concentration of 104 cells/ml of culture medium and incubated at 37 °C. The culture medium was replaced every 7 days. Chemical composition of ZMEO was identified by GC-MS analysis. ZMEO was tested at concentrations of 0.5-8 mg/ml. Viability of the cells was assessed by trypan blue exclusion assay. RESULTS: A significant increase in the cell number was evident at 20, 30 and 45 days after cultivation. At 45 days of cultivation, the number of cells was approximately five-fold higher than on the first day. In GC-MC analysis, carvacrol, p-cymene, g-terpinene and thymol were found to be the main compounds of ZMEO. The lethal effect of ZMEO on the germinative cells at concentrations of 6, 7 and 8 mg/ml was 100% after 60, 25 and 7 min of exposure, respectively. CONCLUSIONS: At 45 days of cultivation, the cell concentration was suitable for the desired in vitro experiments. A high lethal effect of ZMEO on the germinative cells of E. granulosus may be considered an opportunity for the introduction of a novel, more effective and safer therapeutic agent for treatment of CE using an herbal product.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

Chemical Composition, Apoptotic Activity, and Antiparasitic Effects of Ferula macrecolea Essential Oil against Echinococcus granulosus Protoscoleces.

BACKGROUND: Today, the present protoscolicidals used to minimize the serious risks during hydatid cyst surgery are not completely safe and have various adverse side effects. The present study aimed to evaluate the chemical composition and apoptotic activity of Ferula macrecolea essential oil (FMEO) as well as its in vitro and ex vivo protoscolicidal effects against hydatid cyst protoscoleces. METHODS: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of FMEO. Protoscoleces of hydatid cysts were collected from liver fertile hydatid cysts of infected sheep and were then treated with various concentrations of the essential oil (75, 150, and 300 µL/mL) for 5-60 min in vitro and ex vivo. Then, by using the eosin exclusion test, the viability of the protoscoleces was studied. The caspase-3-like activity of the FMEO-treated protoscoleces was also evaluated through the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: According to GC/MS, the main constituents of the essential oil were terpinolene (77.72%), n-nonanal (4.47%), and linalool (4.35%), respectively. In vitro, the maximum protoscolicidal activity of FMEO was observed at the concentrations of 150 and 300 µL/mL, such that 100% of the protoscoleces were killed after 30 and 20 min of exposure, respectively. Based on the obtained findings, the results demonstrate that FMEO required a longer time to kill protoscoleces ex vivo; after 12 min of exposure to FMEO, only 13.4% of the protoscoleces remained alive. After 48 h of the treatment of protoscoleces, FMEO, in a dose-dependent manner and at doses of 75, 150, and 300 µL/mL, induced the activation of the caspase enzyme by 24.3, 35.3, and 48.3%, respectively. CONCLUSIONS: Our findings demonstrate the potent protoscolicidal effects of FMEO in vitro and ex vivo; however, further studies are required to assess the safety and the efficiency of FMEO as a promising scolicidal agent in a preclinical model and clinical setting.

Copper nanoparticles: Biosynthesis, characterization, and protoscolicidal effects alone and combined with albendazole against hydatid cyst protoscoleces.

BACKGROUND: Surgery remains the preferred treatment option for hydatid cyst (cystic echinococcosis); however, recent studies have demonstrated that the current protoscolicidal agents used during surgery are associated with some adverse side effects such as biliary fibrosis, hepatic necrosis, and cirrhosis. The present study aims to evaluate the in vitro and ex vivo anti-parasitic effects of copper nanoparticles (CuNPs) alone and combined with albendazole on hydatid cyst protoscoleces. METHODS: CuNPs was green synthesized using C. spinosa extract. Various concentrations of CuNPs (250, 500, and 750 mg/mL) alone and combined with albendazole (ALZ, 200 mg/mL) were exposed to protoscoleces collected from the liver fertile hydatid cysts of infected sheep for 5-60 min in vitro and ex vivo. Next, the eosin exclusion test was applied to determine the viability of protoscoleces. Caspase-3 like activity of CuNPs-treated protoscoleces was then evaluated using the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: Scanning electron microscopy (SEM) results showed that the particle size of CuNPs was 17 and 41 nm with the maximum peak at the wavelength of 414 nm. The maximum protoscolicidal activity of CuNPs was observed at the concentration of 750 mg/mL in vitro, so that 73.3 % of protoscoleces were killed after 60 min of exposure. Meanwhile, the mortality of protoscoleces was 100 % after 10 min of exposure to 750 mg/mL of CuNPs along with ALZ (200 mg/mL). Nevertheless, the findings proved that CuNPs even in combination with ALZ required a longer time to kill protoscoleces ex vivo. After 48 h of treating protoscoleces, CuNPs in a dose-dependent manner and at doses of 250, 500, and 750 mg/mL induced the caspase enzyme activation by 20.5 %, 32.3 %, and 36.1 %, respectively. CONCLUSION: The findings of the present investigation showed potent protoscolicidal effects of CuNPs, especially combined with albendazole, which entirely eliminated the parasite after 10-20 min of exposure. The results also showed that although the possible protoscolicidal mechanisms of CuNPs are not clearly understood, the inducing apoptosis through caspases is one of the main protoscolicidal mechanisms of CuNPs. However, supplementary studies, especially in animal models and clinical settings, are needed to approve these results.

In vitro potential effect of Pipper longum methanolic extract against protoscolices of hydatid cysts.

This study aimed to evaluate the efficacy of methanolic extract of P. longum (PLM) against protoscolices of hydatid cyst in vitro. Four different concentrations of PLM extract (25, 50, 100 and 150 mg/ml) were used for the experiments. The metabolites in the PLM extract were characterized by Gas chromatography-mass spectrometry (GC-MS). The results showed the highest lethality of PLM extract in 50 mg/ml for 60 min exposure. The IC50 value obtained about 20 mg/ml for 60 min of PLM extract exposure. In this study, valuable findings were obtained for the first time about the scolicidal activity of P. longum, which is expected to conduct further studies in this field in the future.

Effects of Allium sativum on IFN-γ and IL4 concentrations in mice with cystic echinococcosis.

The aim of the current investigation was to assess the impacts of methanolic extract of Allium sativum (MEAS) on IL-4 (a cytokine derived from Th2 cells) and IFN-É£ (a cytokine derived from Th1 cells) levels in mice infected with Echinococcus granulosus. Sixty healthy BALB/c female mice were used in this study. Each animal was intraperitoneally injected with 1500 protoscoleces. The infected animals were randomly divided into six groups: albendazole (100 mg/kg), MEAS 10 (10 mg/kg), MEAS 20 (20 mg/kg), MEAS 40 (40 mg/kg), MEAS 80 (80 mg/kg) and control group with no treatment. The studied animals received albendazole and/or MEAS through drinking water for 30 days. Serum IFN-γ concentration significantly increased in the MEAS 20 and 80 groups in comparison to the control, albendazole and MEAS 10 groups (P < 0.05). The serum IL-4 level showed no significant difference between the trial groups. The findings of this study showed that MEAS at 20 and 80 mg/kg concentrations enhanced Th1 cell response in mice with cystic echinococcosis.

A systematic review of medicinal plants used against Echinococcus granulosus.

Cystic echinococcosis (CE) is a zoonotic helminthiasis caused by different species of the genus Echinococcus, and is a major economic and public health concern worldwide. Synthetic anthelmintics are most commonly used to control CE, however, prolonged use of these drugs may result in many adverse effects. This study aims to discuss the in vitro/in vivo scolicidal efficacy of different medicinal plants and their components used against Echinococcus granulosus. Google Scholar, ScienceDirect, PubMed and Scopus were used to retrieve the published literature from 2000-2020. A total of 62 published articles met the eligibility criteria and were reviewed. A total of 52 plant species belonging to 22 families have been reported to be evaluated as scolicidal agents against E. granulosus worldwide. Most extensively used medicinal plants against E. granulosus belong to the family Lamiaceae (25.0%) followed by Apiaceae (11.3%). Among various plant parts, leaves (36.0%) were most commonly used. Essential oils of Zataria multiflora and Ferula asafetida at a concentration of 0.02, and 0.06 mg/ml showed 100% in vitro scolicidal activity after 10 min post application, respectively. Z. multiflora also depicted high in vivo efficacy by decreasing weight and size while also causing extensive damage to the germinal layer of the cysts. Plant-based compounds like berberine, thymol, and thymoquinone have shown high efficacy against E. granulosus. These plant species and compounds could be potentially used for the development of an effective drug against E. granulosus, if further investigated for in vivo efficacy, toxicity, and mechanism of drug action in future research.

Evaluation of the efficacy of albendazole sulfoxide (ABZ-SO)-loaded chitosan-PLGA nanoparticles in the treatment of cystic echinococcosis in laboratory mice.

Albendazole is known as the drug of choice for medical treatment of cystic echinococcosis (CE). Albendazole sulfoxide (ABZ-SO), as the main active metabolite of albendazole, has low efficacy in the disease due to low water solubility and poor absorptivity. PLGA nanoparticles (NPs) enhance the dissolution of poorly soluble drugs, and chitosan (CS) coating enhances oral drug delivery of NPs. In this study, the efficacy of ABZ-SO-loaded CS-PGLA NPs in the treatment of CE was evaluated in laboratory mice. ABZ-SO-loaded CS-PGLA NPs were prepared by nanoprecipitation and characterized by dynamic light scattering method and scanning electron microscopy. Thirty mice were intraperitoneally infected by 1000 protoscoleces of Echinococcus granulosus. Ten months later, the mice were allocated into 3 groups: groups 1 and 2 were treated with ABZ-SO and ABZ-SO-loaded CS-PGLA NPs, respectively, and the mice in group 3 remained untreated as the control group. The drugs were administered by gavage for 45 days at a daily dose of 10 mg/kg. Finally, all mice were opened and the cysts were collected, counted, weighed, and measured separately. The therapeutic effect of ABZ-SO in the number, weight, and volume of the cysts were not statistically significant compared with those in ABZ-SO-loaded CS-PGLA NPs and the control group. However, the therapeutic effect of ABZ-SO-loaded CS-PGLA NPs in the weight and volume of cysts were statistically significant when compared with that in the control group (p ˂ 0.05). In conclusions, this study revealed that ABZ-SO-loaded CS-PGLA NPs could enhance the therapeutic efficacy of ABZ-SO in the treatment of CE in laboratory mice.

The High Potential of Ozone Gas to Inactivate Echinococcus granulosus Protoscoleces During Hydatid Cyst Surgery.

BACKGROUND: In medicine, ozone therapy is effectively used in a broad spectrum of diseases. Reviews have shown that ozone gas demonstrates potent antimicrobial effects against a wide range of pathogenic microorganisms, such as oral bacteria, fungi, viruses, and parasite even in resistant strains. The present investigation was designed to assess the protoscolicidal effects of ozone gas on hydatid cysts protoscoleces in vitro and in vivo. METHODS: Hydatid cyst protoscoleces were acquired from sheep livers that were slaughtered at Kerman slaughterhouse, Iran. The viability of protoscoleces was assessed by the eosin exclusion examination after exposure with ozone gas for 1 to 14 min in vitro and ex vivo. RESULTS: In this study, in vitro assay showed that ozone gas at the concentration of 20 mg/L killed 85 and 100% of hydatid cyst protoscoleces after 4 and 6 min of treatment, respectively. However, in the ex vivo analysis, a longer time was needed to confirm a potent protoscolicidal activity such that ozone gas after an exposure time of 12 min, 100% of the protoscoleces were killed within the hydatid cyst. CONCLUSION: In conclusion, the findings of the present study showed that ozone gas at low concentrations (20 mg/L) and short times (4-6 min) might be used as a novel protoscolicidal drug for use in hydatid cyst surgery. However, more clinical surveys are required to discover the precise biological activity of ozone gas in animal and human subjects.

Zataria Multiflora bois as an auspicious therapeutic approach against Echinococcus granulosus: Current status and future perspectives.

Hydatidosis caused by Echinococcus granulosus is a major zoonotic diseases. In addition to imposing heavy economic losses, the disease is a public health problem worldwide. The larval stage of the parasite (hydatid cyst) is formed in a wide range of domestic, wild and human beings as intermediate hosts. On the other, its recurrence has been reported anywhere as a reemerging disease. Although the cysts have some evading mechanisms, both human TH1 and TH2 cells subsets are stimulated. Because of increasing resistance and adverse effects of medications such as abnormalities of liver and other organs functions and abdominal pain, seeking alternative therapeutic approaches to be inexpensive, easy available, with low side effects and toxicity seems essential. However, the lack of information on the social and economic welfares of herbal medicines for the industrial scale application is a limitation. Zataria Multiflora bois (ZMB) has exhibited huge advantages and tremendous protoscolicidal effects as demonstrated by numerous studies and its combination therapies with anti-parasitic drugs have exerted desirable outcomes in vitro and in vivo. Noticeably, the compound confers negligible side effects or toxicity even at high concentrations. ZMB has exhibited promising inhibitory effects against hydatid cyst, particularly when combined with chemical drugs and in formulations of nanoemulsions. Its immunomodulatory effects include increase of nitric oxide production (NO) and protection of hepatic cells (Kupffer cells, fat-storing cells, and endothelial cells), enhancement of macrophages and T cells and increase of cytokines production. This study aimed at assessment of ZMB traits for application against hydatid cyst protoscolices.

Scolicidal activity of Mesobuthus eupeus venom against the protoscolices of Echinococcus granulosus.

Hydatidosis is an important zoonosis caused by a parasitic tapeworm, namely Echinococcus granulosus. This infection is distributed worldwide and affects the health as well as economic loss in both humans and animals. In most cases, the disease needs chemotherapy with or without surgery. Conventional drugs have some major problems, including drug complications, harmful side effects, and also progressive resistance. According to the importance of biological productions as alternative medicine, a large number of studies confirmed that whole venom and many peptide ingredients of the scorpion venom have various different medical benefits, including antimicrobial properties, due to the mechanism of blocking gated ion channel. In this study, the venom peptides of Mesobuthus eupeus scorpionwere purified using gel filtration chromatography and subsequently ion exchange chromatography, followed by the determination of the molecular weights of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure. After collecting the hydatid cysts fluids from the liver of infected sheep, protoscolices were derived, washed, and encountered to the whole venom as well as eight different fractions of toxin 30, 60, 120, and 240 min after the exposure. In the next step, the viability of protoscolices was determined by eosin staining. The obtained results revealed that a venom fraction under 10 kDa killed all protoscolices after 30 min. Moreover, it was found that the scolicidal activity of fractions increases according to the time of exposure. As a result, it can be concluded that M. epeus venom peptides under its LD50 (1/2 LD50) can properly and quickly destroy the protoscolices of hydatid cysts at the level of applied concentrations and such components are good alternatives to treat hydatidosis.

In vitro evaluation of the protoscolicidal effect of Eucalyptus globulus essential oil on protoscolices of hydatid cyst compared with hypertonic saline, povidone iodine and silver nitrate.

OBJECTIVE: There are various protoscolicidal agents for inactivation of protoscoleces of hydatid cysts before and during surgical operation. The present study was aimed to evaluate the protoscolicidal effect of two concentrations of Eucalyptus globulus on protoscoleces of Echinococcus granulosus sensu lato under in vitro condition and to compare its efficacy with hypertonic saline, povidone iodine and silver nitrate. METHODS: Live protoscoleces obtained from the liver of naturally infected sheep were exposed to 0.5% and 1% of Eucalyptus globulus essential oil, 5% hypertonic saline, 10% povidone iodine and 0.5% silver nitrate for 1 and 3minutes. Phosphate buffered saline was used as a negative control. One percent eosin staining method was used to test the viability of protoscoleces in different groups. RESULTS: While the mean percentage of dead protoscoleces was 6.08% in the control group, the scolicidal power of 5% hypertonic saline was only 6.54% and 6.60% after 1 and 3min respectively. 0.5% E. globulus EO demonstrated 97.38% and 100% scolicidal activity after 1 and 3min respectively. The mean protoscolicidal power of 1% E. globulus EO, 10% povidone iodine and 0.5% silver nitrate was 100% after one minute. CONCLUSIONS: According to the results of this study, E. globulus EO demonstrated high scolicidal power in a short period of time. Hence, this herbal product could be considered as a potent natural scolicidal agent that could be used before and during surgery of hydatid disease.

In vitro evaluation on the scolicidal effect of Myrtus communis L. and Tripleurospermum disciforme L. methanolic extracts.

Hydatid disease, a zoonotic disease, is still endemic in many developing countries that is caused by the metacestode of Echinococcus (E.) granulosus. Surgical management is one of the best choices for the treatment of the hydatidosis and using effective scolicidal agents during hydatid surgery is essential to prevent the secondary infection. The aim of the present in vitro study was to evaluate the scolicidal effect of the methanolic extract of Myrtus communis and Tripleurospermum disciforme against protoscoleces of hydatid cyst. Protoscoleces of E. granulosus were aspirated aseptically from infected livers. Various concentrations of M. communis and T. disciforme extracts at different exposure times were examined for their scolicidal activity. Normal saline and silver nitrate were used as negative and positive groups, correspondingly. The viability of protoscoleces was evaluated by 0.1% eosin. The result of the current study indicated that the highest scolicidal effect (100%) of M. communis was obtained at 100 and 50 mg/ml concentrations and LC50 in 10, 20 and 30 min were 11.64 mg/ml, 7.62 mg/ml, and 6.47 mg/ml respectively. The scolicidal activity of T. disciforme was negligible even at high concentration. The findings have shown that the scolicidal activity of M. communis against echinococcosis protoscoleces was strong, while the T. disciforme extract showed fewer effects. However, further studies are required for identification of the active ingredients in the extract and its safety on cells in effective concentrations.

Towards green drugs against cestodes: Effectiveness of Pelargonium roseum and Ferula gummosa essential oils and their main component on Echinococcus granulosus protoscoleces.

Cystic echinococcosis is of great public health importance. Surgery is the efficient treatment for this infection. To minimize the risk of cyst intraoperative leakage, using scolicidals is crucial. To date, any efficacious scolicidal without side effect has not been introduced. Since essential oils of Pelargonium roseum and Ferula gummosa have shown several bioactivities, we evaluated their potential against protoscoleces of E. granulosus using albendazole as positive control. Furthermore, chemical composition of both essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS) analyses, and their main constituents were also evaluated for scolicidal activity. Different concentrations of essential oils and their two main constituents were tested for scolicidal activity. Mortality rate was measured by eosin staining. Results of GC-MS revealed citronellol and ß-pinene as the main constituents of P. roseum and F. gummosa essential oils, respectively. After 60 min of exposure to 50 µg/mL of P. roseum and F. gummosa, mean mortality rate of protoscoleces was 100%. However, ß-pinene and citronellol at the same time point with only 10 µg/mL concentrations resulted in approximately higher than 80% mortality. Essential oils of P. roseum and F. gummosa showed significant toxic effect on E. granulosus with 50% lethal concentration (LC50) values of 8.52 and 17.18 µg/mL, respectively. Based on the LC50 values, ß-pinene (2.20 µg/mL) was the most potent scolicidal agent in the present study. The overall toxicity of ß-pinene and citronellol was significantly higher than the whole essential oils of F. gummosa and P. roseum. Based on these results, ß-pinene and citronellol can be considered as candidate ingredients for the development of green scolicidals.

In Vitro Study on Protoscolicidal Effect of Methanolic Extract of Allium hirtifolium on Protoscoleces of Cystic Echinococcosis.

BACKGROUND: Cystic echinococcosis/ hydatidosis is one of the most important parasitic zoonoses worldwide. Usually, surgery is used for the treatment of active cysts. Due to the risk of cyst rupture during surgery, a protoscolicidal agent is usually injected into the cyst before surgery to prevent secondary cyst formation in case of cyst rupture. Due to the side effects of chemical protoscolicidal agents attempts have been focused on herbal medicine. In this regard, Allium sativum has already been used; however, the protoscolicidal effect of Allium hirtifolium from Allium family has not been investigated, so far. OBJECTIVE: The present study was conducted in order to assess the protoscolicidal effect of methanolic extract of Allium hirtifolium in different concentrations, temperature and exposure times. Cetrimide as a positive control and DMSO as a negative control were also used in this study. METHODS: The viability of protoscoleces was determined by 0.1% eosin staining method. RESULTS: The protoscolicidal effect of Allium hirtifolium on protoscoleces was as follows: The mean percent and standard deviation (SD) of protoscolicidal activity of 50 mg/ml concentration of Allium hirtifolium extract in room temperature at 10, 20, 30, 40 and 50 minutes of exposure was 5.88±2.39, 8.59±4.54, 11.82±5.81, 12.31±6.16and 16.96±8.42 respectively; while at 37°C it was 9.10±5.01, 11.52±7.36, 18.64±4.36, 27.07±6.57 and 38.15±12.80, respectively. CONCLUSION: In conclusion, although Allium hirtifolium from Allium family is similar to Allium sativum; the protoscolicidal effect of its methanolic extract is not similar and less than A. sativum extract.

In vitro effects of Artemisia sieberi on Echinococcus granulosus protoscolices.

Hydatidosis is a parasitic zoonotic disease. Surgery is one of its treatment modalities during which protoscolices are likely to be released into the peritoneal cavity and cause recurrence of the disease. Given the above problem and the complications associated with conventional anti-parasitic agents, it is imperative to find an effective and natural protoscolicidal agent. The present study was conducted to evaluate effects of Artemisia sieberi on Echinococcus granulosus protoscolices. Protoscolices were collected from slaughtered livestock in Kerman abattoir and the effect of three concentrations of aqueous extract of A. sieberi (25 mg ml-1, 50 mg ml-1 and 75 mg ml-1) was assessed over three different exposure periods. Results showed that scolicidal effect of this extract at exposure periods of 2, 5 and 10 min was 76 ±â€¯1.4, 76.8 ±â€¯1.41 and 85.7 ±â€¯3.29 percent at concentration of 25 mg ml-1 and 76.8 ±â€¯1.4, 78 ±â€¯3.18 and 86.4 ±â€¯24.9 percent at concentration of 50 mg ml-1 and finally 80 ±â€¯2.73, 90 ±â€¯0.79 and 92.6 ±â€¯1.27 percent at concentration of 75 mg ml-1, respectively. It can be concluded that the aqueous extract of A. sieberi has a protoscolicidal activity and can be considered a natural agent against hydatid cyst protoscolices.

Enhancement of the Therapeutic Effect of Albendazole on Cystic Echinococcosis using a Herbal Product.

PURPOSE: Albendazole is the drug of choice for chemotherapy of cystic echinococcosis (CE) but 40% of patients do not respond to it satisfactorily. With regard to the previously reported scolicidal activity of Zataria multiflora, this study was done to evaluate the efficacy of the co-administration of albendazole and Z. multiflora aromatic water (AW) on hydatid cysts in a murine model. MATERIALS AND METHODS: Laboratory mice were infected with 1,500 viable protoscoleces. After development of hydatid cysts, the infected mice were allocated into four treatment and one control group. The mice of groups 1, 2, 3, and 4 were treated orally with albendazole (100 mg/kg), Z. multiform AW (100 ml/l), albendazole (100 mg/kg) - Z. multiform AW and albendazole ((50 mg/kg) - Z. multiform AW respectively. Two months later, all the mice were euthanized and necropsied and the number, size and weight of hydatid cysts were compared between different groups. RESULTS: The size of the largest cysts, as well as the total weight of cysts, were significantly lower (p < .05) in the mice treated with albendazole (50 mg/kg) - Z. multiflora AW in comparison to those treated with albendazole (100 mg/kg) alone. CONCLUSIONS: The results of this study revealed the superiority of co-administration of albendazole and Z. multiflora AW to albendazole alone in the treatment of CE in laboratory mice. Since Z. multiflora AW is a safe and useful drink, it may be used successfully to enhance the therapeutic effect of albendazole in the patients infected with hydatid disease.

Allium Sativum Methanolic Extract (garlic) Improves Therapeutic Efficacy of Albendazole Against Hydatid Cyst: In Vivo Study.

After gas chromatography and mass spectrometry of prepared methanolic extract of Allium sativum, 40 laboratory BALB/c mice were infected intraperitoneally by injection of 1,500 viable protoscoleces. Five months after infection, the infected mice were allocated into four treatment groups, including 1- Albendazole (100 mg/kg); 2- Allium sativum methanolic extract (10 mL/L); 3- A. sativum methanolic extract (10 mL/L) + Albendazole (50 mg /kg); and 4- untreated control group. After 30 days of daily treatment, total number and weight of cysts and size of the largest cyst as well as blood serum bilirubin and liver enzymes were compared between the mice of different groups.The total number and weight of cysts and size of the largest cyst were significantly lower in treated groups A. sativum 10 mL/L + Albendazole 50 and Albendazole 100 in comparison to those of the control group (p < 0.05).The activity of alanine aminotransferase (ALT) enzyme and bilirubin concentration were significantly lower in the mice treated with A. sativum 10 mL/L and A. sativum 10 mL/L + Albendazole 50, when compared to the control group. In addition, bilirubin concentration revealed significant decrease in A. sativum 10 mL/L and A. sativum 10 mL/L + Albendazole 50 groups, when compared to the Albendazole group. In conclusion, administration of A. sativum 10 mL/L improved the anti-hydatidosis activity of Albendazole 50 mg /kg, due to parasitological effects similar to Albendazole 100 mg /kg but less hepatotoxic effects.