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.

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.

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.

A new method for laboratory rearing of Galba truncatula, the intermediate host of Fasciola hepatica.

In this study a relatively large and open top aquarium was designed, constructed and introduced as a suitable habitat for nutrition, growth and development as well as for egg laying and breeding of Galba truncatula under laboratory conditions. The soil and water used in the aquarium were collected from the locality in which the snails were collected. The aquarium was placed in a laboratory with temperature of 18-32 ºC and relative humidity of 22-37% respectively, according to the season. The artificial light was controlled by a light timer, giving 12 h of light and 12 h of darkness. The snails were fed with dried lettuce leaves, Cyperus alternifolius (aquatic plant), Spirulina (algae), Orthotrichum rupestre (moss) and cuttlebone (a supplementary source of calcium). Approximately five weeks after the start of study, there was evidence of reproduction and success in rearing of G. truncatula by the appearance of eggs and small snails (0.1-0.5 mm) in the aquarium. In conclusion, large scale laboratory rearing of G. truncatula is a feasible task. The method may be improved by balancing the temperature and relative humidity as well as by optimizing the soil type, the water quality and the type of food.

In vivo study of the efficacy of the aromatic water of Zataria multiflora on hydatid cysts.

Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were employed to determine the chemical composition of the essential oil (EO) from aromatic water (AW) of Zataria multiflora. Thymol (66.9%), carvacrol (15.2%), and carvone (7.3%) were found to be the major EO constituents. Eighty laboratory BALB/c mice were infected intraperitoneally by injection of 1,500 viable protoscolices and were divided into prevention (40 mice) and therapeutic (40 mice) groups. To prove the preventive effect of the Z. multiflora AW on development of hydatid cysts, the 40 infected mice were allocated into three treatment groups, including the albendazole group (10 mice that received 150 mg/kg body weight/day for 10 days), the Z. multiflora AW group (15 mice that received 20 ml/liter in drinking water for 8 months), and a control group (15 mice that received no treatment). To estimate the therapeutic effect of the Z. multiflora AW on the hydatid cyst, after 8 months of infection, the 15 remaining mice were allocated into three experimental treatment groups of five animals each, including the albendazole group (300 mg/kg/day for 20 days), Z. multiflora AW group (40 ml/liter in drinking water for 30 days), and control group (no treatment). All mice were then euthanized, and the sizes and weights of the cysts as well as their ultrastructural changes were investigated. The weights and sizes of the hydatid cysts significantly decreased upon treatment with the Z. multiflora AW in both the preventive and therapeutic groups (P < 0.05). The results of scanning electron microscopy also showed considerable damage in the germinal layer of the hydatid cysts recovered from the treated animals.

Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: an in vivo study.

The phenolic compounds of Zataria multiflora extract, were identified by HPLC analysis. Gallic acid, catechin, caffeic acid, and quercetin were found to be the major phenolic compounds. Eighty healthy laboratory Balb/C mice were infected intraperitoneally by injection of 1500 viable protoscoleces and were divided into prevention (40 mice) and therapeutic (40 mice) groups. To prove the preventive effect of Z. multiflora extract on development of hydatid cyst, the 40 infected animals were allocated into three treatment groups including Z. multiflora (4 g/l in drinking water for 8 months), albendazole (150 mg/kg BW/day for 10 days) and untreated (control) group. To estimate the therapeutic effect of Z. multiflora extract on the hydatid cyst, after 8 months of infection, the infected mice were allocated into three experimental treatment groups including Z. multiflora (8 g/l in drinking water for 30 days), albendazole (300 mg/kg BW/day for 20 days) and untreated (control) group. At the end of the treatment period, all mice were euthanized and necropsied, the hydatid cysts were carefully removed, weighed and their size were recorded. Weight and size of the hydatid cysts significantly decreased (p<0.05) upon the treatment with Z. multiflora extract in both prevention and therapeutic groups. The germinal layer of the hydatid cysts recovered from the treated mice, either from the prevention or therapeutic group, were completely damaged at ultrastructural level by scanning electron microscopy.

In vitro lethal effect of ajowan (Trachyspermum ammi L.) essential oil on hydatid cyst protoscoleces.

Various chemical scolicidal agents have been used for inactivation of hydatid cyst protoscolices, but most of them are associated with adverse side effects. Since ajowan (Trachyspermum ammi) has been shown to have a number of medicinal properties, in this study the scolicidal effect of the essential oil (EO) from the fruits of this herbal plant was investigated. Ajowan EO was obtained by hydrodistillation method. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were employed to determine the chemical composition of the EO. Protoscoleces were exposed to various concentrations of EO (3, 5 and 10mg/mL) for 10, 20, 30, and 60 min. Viability of protoscolices was confirmed by 0.1% eosin staining. A total of 18 compounds representing 99.54% of the total oil, were identified. Thymol (50.07%), γ-terpinene (23.92%), and p-cymene (22.9%) were found to be the major EO constituents. While the mortality rate of protoscolices in the control group was 6.67%, scolicidal power of ajowan EO at concentration of 3mg/mL was 31.34, 35.98, 45.17, and 51.58% after 10, 20, 30, and 60 min, respectively. The EO at concentration of 5mg/mL killed 51.89, 72.20, 88.64, and 100% of protoscolices after 10, 20, 30, and 60 min, respectively. One hundred percent scolicidal activity was observed with ajowan EO at concentration of 10mg/mL after 10 min of exposure. The results of this study revealed that the EO of ajowan is rich in thymol, γ-terpinene and p-cymene, has high scolicidal power and it may be used as a natural scolicidal agent.

In vitro effectiveness of garlic (Allium sativum) extract on scolices of hydatid cyst.

BACKGROUND: Surgery is still the main treatment for hydatid disease. Recurrence of the infection is one of the end points of surgery in treating the hydatid cyst which results from the dissemination of protoscolices-rich fluid. Installation of a scolicidal agent into the cyst is the most commonly employed measure to prevent recurrence. Many scolicidal agents have been used for inactivation of the cyst's content, but most of them are not safe due to their undesired side effects. In the present study, the scolicidal effect of methanolic extract of Allium sativum is investigated. METHODS: Protoscolices were aseptically collected from sheep livers containing hydatid cysts. Two concentrations (25 and 50 mg ml(-1)) of garlic extract were used for 10, 20, 30, 40, 50, and 60 min. Viability of protoscolices was confirmed by 0.1% eosin staining. RESULTS: Allium sativum extract at the concentration of 25 mg ml(-1) killed 87.9, 95.6, 96.8, 98.7, 99.6, and 100% of protoscolices following 10, 20, 30, 40, 50, and 60 min of application, respectively. Moreover, the scolicidal activity of Allium sativum extract at the concentration of 50 mg ml(-1) was 100% after 10 min of application. CONCLUSIONS: Methanolic extract of Allium sativum had a high scolicidal activity in vitro and thus might be used as a scolicidal agent in the surgical treatment of the hydatid cyst. However, further investigation on the in vivo efficacy of Allium sativum extract and its possible side effects is proposed.