Abstract
Various protoscolicidal agents are indicated for the prevention of recurrence of cystic echinococcosis caused by the larval stage of Echinococcus granulosus; however, most of these chemicals have adverse side effects. This study evaluated the effects of Zataria multiflora essential oil (ZEO) nano-emulsion and emulsion at the concentrations of 1, 2, 5, 10, 15, and 20 µl/ml on E. granulosus protoscoleces. Albendazole (5 mg/ml), normal saline, and nano-emulsion without ZEO served as control groups. Optimal concentrations of ZEO nano-emulsion and emulsion on the microcysts were also investigated. ZEO emulsion at a concentration of 20 µl/ml for the duration of 15 min and nano-emulsion for the duration of 10 min resulted in the death of 100% of the protoscoleces. Additionally, densely packed aggregates were formed inside the microcysts treated with ZEO nano-emulsion and emulsion at a concentration of 20 µl/ml after 40 min, but the complete destruction of laminated layers did not occur. The results indicate that ZEO nano-emulsions have a higher protoscolicidal effect than its emulsion, but these two compounds had similar effects on microcysts.
Keywords: Echinococcus granulosus, Protoscoleces, Microcyst, Nano-emulsion, Emulsion, Zataria multiflora, In vitro
Introduction
Hydatid disease, or cystic echinococcosis (CE), is a well-known parasitic zoonosis caused by the larval stage of Echinococcus granulosus tapeworm. The intermediate hosts of the parasite are herbivores, whereas definite hosts are canids, particularly dogs. Humans act as an accidental host and are infected by the ingestion of parasite eggs excreted into the environment through canine feces (Torgerson 2014). Hydatid cysts that may contain protoscoleces are most commonly found in the liver or lung, but other organs can also be affected. Human CE is an important health problem in endemic regions, including Iran (Shafiei et al. 2016).
The treatment options recommended by the WHO Informal Working Group on Echinococcosis include surgery, percutaneous management, anti-infective drug treatment, and observation for inactive and uncomplicated cysts (Brunetti et al. 2010). Spillage of infective cyst contents (protoscoleces) during surgery or percutaneous treatment enhances the risk of secondary cyst formation and subsequent recurrence of infection. To date, various protoscolicidal agents, including hypertonic saline, formalin, ethyl alcohol, hydrogen peroxide, and silver nitrate, have been employed during surgery to prevent recurrence of infection (Smego and Sebanego 2005). However, most chemical protoscolicidals have adverse side effects such as cholangitis and liver necrosis (Besim et al. 1998; Kahriman et al. 2017; Sharafi et al. 2017). A number of studies have identified the anti-infective properties of some herbal extracts such as Thymus vulgaris, Pistacia vera, and Olea europaea against various parasites and protoscoleces of Echinococcus granulosus (Kohansal et al. 2017; Mahmoudvand et al. 2016; Niazi et al. 2019; Yones et al. 2011).
Zataria multiflora is a medicinal plant that was used in Iranian traditional medicine, and its preventive and therapeutic effects of its hydro-alcoholic and methanolic extracts on E. granulosus protoscoleces and cysts have been studied in vitro (Moazeni et al. 2014a; Moazeni and Roozitalab 2012).
Nano-emulsions are thermodynamically stable systems in which two dissimilar liquids are mixed to form a single phase by means of an emulsifying agent in the range of 20–200 nm (Jaiswal et al.2015). Micro- and nano-emulsions serve as preservatives in food industries, and have potential for antimicrobial applications. It appears that the antimicrobial properties of nano-emulsions are due to their small particle size and high density. They can fuse with bacteria membranes, viruses, and fungi cells and eventually destroy them (Solorzano-Santos and Miranda-Novales 2012). Considering the satisfactory killing effect of Z. multiflora on hydatid cyst protoscoleces (Jahanbakhsh et al. 2016; Moazeni et al. 2014b, 2017), the effects of Z. multiflora nano-emulsion on protoscoleces and microcysts of E. granulosus were investigated.
Materials and methods
Preparation of Z. multiflora essential oil (ZEO) emulsion and nano-emulsion
Zataria multiflora Bioss essential oil was purchased from Barij Essence co., Kashan, Iran. ZEO emulsion, ZEO nano-emulsion, and nano-emulsion without ZEO were prepared in this investigation. ZEO emulsion (20 µl/ml) was prepared by mixing oleic acid and ZEO (1:1 v/v) before adding the required amount of normal saline solution containing tween 80 (16% w/v); then, the mixture was stirred for 15 min. The prepared ZEO emulsion was sonicated using an ultrasonicator (Hiescher 200 W, 24 KHZ, Germany) for 2 min to form nano-emulsion. The nano-emulsion formulation without ZEO was prepared as previously described (Ghosh et al. 2014).
Determination of droplet size and size distribution of ZEO nano-emulsions
The mean droplet size (Z-average), and size distribution (poly dispersity index (PDI)) of nano-emulsions were determined using a Malvern Zetasizer 2000 HS (Malvern, UK). Nano-emulsions were diluted 100 times with distilled water before measurements (Ghosh et al. 2014).
The droplet size and PDI of the ZEO emulsion were not determined. The appearance of the ZEO emulsion was examined visually, and its color and homogeneity were obsereved.
Short-term stability studies
The ZEO nano-emulsion was subjected to short-term storage stability tests at 5 ± 3 °C over a period of 0–3 days. Then, it was evaluated for droplet size and PDI as described previously, and samples were monitored for any phase separation or creaming.
The short-term stability of the ZEO emulsion and nano-emulsion without ZEO were followed visually over 0–3 days through the observation of the color and homogeneity of the samples and any signs of phase separation, creaming, and cracking.
The effects of ZEO emulsion and nano-emulsion on E. granulosus protoscoleces
Sheep livers and lungs infected with hydatid cysts were collected from a local slaughterhouse and transferred to the laboratory. Initially, the intact hydatid cysts were selected and protoscoleces were aspirated aseptically. The protoscoleces were washed three times with phosphate buffered saline (PBS) solution (pH 7.2) containing penicillin–streptomycin. Parasite viability was evaluated by a 0.1% eosin exclusion test. Samples of more than 95% living protoscoleces were used in this study. Approximately 2000 protoscoleces were incubated in 0.5 ml of each concentration (1, 2, 5, 10, 15, and 20 µl/ml) of nano-emulsions and emulsions of ZEO at 37 °C for 2, 5, 10, 15, and 20 min. Albendazole (5 mg/ml), normal saline, and nano-emulsion without ZEO served as the control groups.
After incubation periods, the supernatant was removed from the test tubes, and the protoscoleces were washed three times with normal saline. The samples were once again incubated with 0.1% eosin solution at 37 °C for 10 min. Finally, the rate of dead protoscoleces was estimated through an optical microscope (Jahanbakhsh et al. 2016).
In vitro development of microcysts and treatment with ZEO emulsion and nano-emulsion
Protoscoleces were cultured aseptically for 70–80 days in RPMI1640 supplemented with 20% (v/v) fetal bovine serum (FBS), and 4 mg/ml of glucose at 37 °C in 5% CO2. The culture medium was renewed every 5–6 days (Elissondo et al. 2004). Developed microcysts were selected using a sterile pipette under an inverted microscope. Each 15 healthy microcysts (1–4 mm diameter) were randomly allocated into final concentrations of 10, 15, and 20 µl/ml of ZEO emulsion and nano-emulsion, separately. Moreover, albendazole (5 mg/ml), normal saline, and nano-emulsion without ZEO were used as the control groups.
The flasks containing microcysts were incubated at 37 °C in 5% CO2. After 30, 40, 50, and 60 min, the degenerative changes of microcysts (formation of densely packed aggregate and destruction of laminated layer) were assessed using an inverted light microscope (Motic®, AE31, Spain).
Statistical analysis
Descriptive data, such as mean and standard deviation, was analyzed using SPSS software. A two-way ANOVA and a Tukey Multiple Comparison test were used to analyze the effect of ZEO nano-emulsion and emulsion concentrations and the duration of incubation on the mortality and degradation of the parasite.
Results
Droplet size and PDI of the nano-emulsions
Z-average sizes of different ZEO nano-emulsion and nano-emulsion without ZEO were in the range of 73–82 nm with a PDI lower than 0.3. Droplet size and PDI of ZEO nano-emulsion are shown in Fig. 1.
Fig. 1.
Droplet size and size distribution of ZEO nano-emulsion
The droplet size and PDI of ZEO emulsion were not determined. The ZEO emulsion appeared opaque and milky white without no phase separation, creaming, or cracking.
Short-term stability of nano-emulsions
The results on short-term stability of ZEO nano-emulsion at 5 ± 3 °C are shown in Table 1. The results showed that ZEO nano-emulsion was stable at 5 ± 3 °C for 3 days, and the Z-average and PDI did not change significantly.
Table 1.
Z-average and PDI of ZEO nano-emulsion during storage at 5 ± 3 °C for 3 days. Data are mean ± SD, (n = 3)
| Time (Day) | Mean droplet size (nm) | PDI |
|---|---|---|
| 0 | 77 ± 4 | 0.3 ± 0.1 |
| 1 | 80 ± 2 | 0.2 ± 0.1 |
| 2 | 82 ± 2 | 0.2 ± 0.1 |
| 3 | 87 ± 4 | 0.3 ± 0.1 |
No signs of phase separation, creaming, or cracking were observed during the 3 days of storage of the ZEO emulsion and nano-emulsion without ZEO. Therefore, all of the formulations in this study were considered as stable.
The effects of ZEO nano-emulsion and emulsion on E. granulosus protoscoleces
The maximum effect of ZEO nano-emulsion in the shortest possible time was obtained at a concentration of 20 µl/ml (Table 2). After 10 min of incubation, 100% of the protoscoleces died at this concentration. The protoscolicidal effect of ZEO nano-emulsion was significantly higher at a concentration of 20 µl/ml than at concentrations of 2, 5, or 10 µl/ml after 10 min of incubation (p < 0.05).
Table 2.
Mortality rates of E. granulosus protoscoleces (percent; mean ± SD) at the examined times with different concentrations of ZEO nano-emulsions
| Experiment groups | Exposure time (min) | ||||
|---|---|---|---|---|---|
| 2 | 5 | 10 | 15 | 20 | |
| Normal saline | 0 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.6 ± 0.5 | 1 ± 0 |
| Nano-emulsion without ZEO | 0 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.6 ± 0.5 | 1 ± 0 |
| Albendazole (5 mg/ml) | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
| ZEO nano-emulsions (µl/ml) | |||||
| 1 | 3 ± 1 | 3.6 ± 0.5 | 5.3 ± 0.5 | 16.6 ± 2.5 | 26.3 ± 1 |
| 2 | 10 ± 1 | 13.6 ± 1 | 27.6 ± 2 | 16.6 ± 2.5 | 80.6 ± 2 |
| 5 | 34.3 ± 5 | 70 ± 0 | 80 ± 0 | 84.6 ± 4 | 98.6 ± 0.5 |
| 10 | 76.6 ± 5 | 90 ± 0 | 93.9 ± 5 | 95 ± 0 | 99.6 ± 0.5 |
| 15 | 86.3 ± 2 | 91.3 ± 1 | 99 ± 0 | 100 ± 0 | 100 ± 0 |
| 20 | 93.6 ± 3 | 94.6 ± 0.5 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
The highest effect of ZEO emulsion in the least amount of time was obtained at a concentration of 20 µl/ml, but after 15 min of incubation in this form, 100% of the protoscoleces faced death (Table 3). The protoscolicidal effect of the ZEO emulsion was significantly higher in a concentration of 20 µl/ml than in concentrations of 2, 5, and 10 µl/ml at 15 min (p > 0.05).
Table 3.
Mortality rates of E. granulosus protoscoleces (percent; mean ± SD) at the examined times with different concentrations of ZEO emulsions
| Experiment groups | Exposure time (min) | ||||
|---|---|---|---|---|---|
| 2 | 5 | 10 | 15 | 20 | |
| Normal saline | 0 | 0 | 0.3 ± 0.5 | 0.3 ± 0.5 | 0.6 ± 0.5 |
| Albendazole (5 mg/ml) | 98 ± 0 | 98 ± 0 | 99 ± 0 | 99 ± 0 | 100 ± 0 |
| ZEO emulsions (µl/ml) | |||||
| 1 | 5.3 ± 1 | 6 ± 0 | 9 ± 1.5 | 14.6 ± 4 | 21.6 ± 5 |
| 2 | 8.6 ± 1 | 15 ± 2 | 28 ± 0 | 55 ± 5 | 74 ± 4.5 |
| 5 | 48.3 ± 2.5 | 77 ± 2.5 | 78.6 ± 0.5 | 81.6 ± 2.5 | 98 ± 1 |
| 10 | 77 ± 2.5 | 84.6 ± 4 | 95.6 ± 1 | 98.3 ± 0.5 | 100 ± 0 |
| 15 | 88 ± 1 | 92 ± 4 | 96.6 ± 1.5 | 99.6 ± 0.5 | 100 ± 0 |
| 20 | 91.6 ± 2.5 | 95.3 ± 2.5 | 98.3 ± 0.5 | 100 ± 0 | 100 ± 0 |
In general, nano-emulsions showed a more destructive effect in a faster time than emulsions of ZEO (Fig. 2). At a concentration of 20 µl/ml, ZEO emulsions destroyed all protoscoleces after 15 min, whereas it took nano-emulsions at the same concentration 10 min.
Fig. 2.
Mortality rates of E. granulosus protoscoleces (percent; mean ± SD) at different concentrations of ZEO nano-emulsions and emulsions (µl/ml)
The effects of ZEO emulsion and nano-emulsion on E. granulosus microcysts
ZEO emulsion and nano-emulsion caused a similar effect against E. granulosus microcysts. At 40 min post-incubation, all of the microcysts were affected by a concentration of 20 µl/ml of emulsion and nano-emulsion, and densely packed aggregates were formed inside the microcysts. In the altered group with concentrations of 10 and 15 µl/ml, aggregates were formed inside the microcysts in 50 and 60 min, respectively (Table 4 and Fig. 3).
Table 4.
Percentage of degenerated E. granulosus microcysts incubated with different concentrations of ZEO nano-emulsions and emulsions (mean ± SD)
| Experiment groups | Exposure time (min) | |||
|---|---|---|---|---|
| 30 | 40 | 50 | 60 | |
| Normal saline | 0 | 0 | 0 | 0 |
| Nano-emulsion without ZEO | 0 | 0 | 0 | 0 |
| Albendazole (5 mg/ml) | 0 | 0 | 0 | 0 |
| ZEO nano-emulsions (µl/ml) | ||||
| 10 | 0 | 0 | 78.8 ± 3.1 | 100 ± 0 |
| 15 | 0 | 84.9 ± 1.6 | 100 ± 0 | 100 ± 0 |
| 20 | 92.7 ± 2.5 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
| ZEO emulsions (µl/ml) | ||||
| 10 | 0 | 0 | 81.6 ± 2.5 | 100 ± 0 |
| 15 | 0 | 86 ± 0.8 | 100 ± 0 | 100 ± 0 |
| 20 | 94.1 ± 0.8 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
Fig. 3.
Light microscopy of E. granulosus microcysts (× 100). a Intact microcysts from control group. b Formation of aggregates inside microcyst incubated with ZEO nano-emulsion
Discussion
In this study, the effects of Z. multiflora nano-emulsion and emulsion on E. granulosus protoscoleces and microcysts were examined in vitro. The results showed that ZEO nano-emulsions at 20 µl/ml for 10 min and emulsions at the same concentration for 15 min destroyed 100% of the protoscoleces. Additionally, in the examination of ZEO nano-emulsions and emulsions, densely packed aggregates were observed to formed inside the microcysts with 20 µl/ml of ZEO at 40 min, 15 µl/ml at 50 min, and 10 µl/ml at 60 min, but no laminated layer destruction was observed.
Elissondo et al. studied the effect of thymol (a main component of Thymus vulgaris) at concentrations of 1, 5, and 10 µg/ml on protoscoleces which were kept in a glucose-enriched growth medium of M199 (Elissondo et al. 2008). The highest protoscolicidal effect was observed at a concentration of 10 μg/ml; after 12 days of incubation, about 50% of the protoscoleces were destroyed. In the current study, 20 µl/ml of ZEO nano-emulsion at 10 min resulted in the destruction of all protoscoleces. Although differences in the design of these two studies were present, such as the maintenance of the protoscoleces and the concentration levels, different results might have been possible if uniform conditions were fostered.
Moazeni et al. studied the protoscolicidal effect of aqueous aromatic extract of Z. multiflora at 1, 2, 3, 4, and 5 min under laboratory conditions and achieved protoscolicial effects of 93.78%, 99.16%, 99.48%, and 100%, respectively (Moazeni et al. 2014b).
There is a thin membrane around the protoscoleces which allows water flow inside organisms; thus, Z. multiflora aqueous extract penetrates the protoscoleces with ease and leads to their destruction because of the disruption of osmotic pressure. Yet, it should be noted that as the main goal of hydatid cysts treatment, the protoscoleces are within the cysts which have a thickened wall around them (laminated layer) that restricts the passage of aquatic compounds such as the aqueous extract of Z. multiflora.
In 2015, Jahanbakhsh et al. examined the lethal effects of Z. multiflora methanolic extract against E. granulosus protoscoleces. Their results showed a 100% destruction of protoscoleces in a concentration of 20 mg/ml in 10 min, the same rate achieved with ZEO nano-emulsions in the current study (Jahanbakhsh et al. 2016).
In 2016, Oryan et al. also examined the destructive effect of Z.multiflora methanolic extract on the germinal layer of hydatid cysts (Oryan et al. 2016). The results showed that the extract had its maximum destructive effect on the germinal layer at 30 mg/ml after 60 min. In this study, the cysts removed from infected sheep were cut and the effects of the drug were studied, while, in the current study, healthy microcysts achieved from culturing were used to study the effects of the drug on the laminated and germinal layers. ZEO nano-emulsion at 20 µl/ml in 40 min only caused microcyst collapse, which has a weaker effect than its methanolic extract due to the intact laminated layer in the microcysts and the inability of the nano-emulsions to penetrate this layer.
Mahmoudvand et al. demonstrated that concentrations of 12.5 and 25 µl/ml of Z. multiflora essential oil killed 100% of protoscoleces after 5 min of exposure. They reported that the composition of Z. multiflora essential oil and its biological activity were dependent on species, climate, and time of collection (Mahmoudvand et al. 2016).
Moazeni et al. showed that Z. multiflora nano-emulsion at a concentration of 2 mg/ml at 10 min had 100% scolicidal effect, while in the current study, the concentration of 20 µl/ml at 10 min resulted in the complete destruction of the protoscoleces (Moazeni et al. 2017).
The mentioned authors used water, oil extract and Tween 80 to synthesize Z. multiflora nano-emulsion, while normal saline was used instead of water in the current study. Moreover, the differences in drug efficacy may be due to the different strains of E. granulosus in these two regions (Farhadi et al. 2015; Hosseinzadeh et al. 2012).
Based on the results of these studies, it appears that Z. multiflora is a good candidate for the treatment of hydatid cysts. There are various formulations of this plant. Therefore, for a suitable formulation of this drug that is cost-effective, durable, and has a high effect, further studies are required.
Acknowledgements
This study is a result of an approved master’s thesis from Zanjan University of Medical Sciences (A-12-95-12; ZUMS.REC.1396.172). The authors of this article would like to sincerely thank colleagues in the Department of Parasitology and Mycology at Zanjan University of Medical Sciences for their assistance in this project.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Monire Karimi Yazdi, Email: m.karimi190@yahoo.com.
Ali Haniloo, Email: hani@zums.ac.ir.
Azadeh Ghaffari, Email: aghaffari@zums.ac.ir.
Negin Torabi, Email: torabin@zums.ac.ir.
References
- Besim H, Karayalcin K, Hamamci O, Gungor C, Korkmaz A. Scolicidal agents in hydatid cyst surgery. HPB Surg World J Hepat Pancreat Biliary Surg. 1998;10:347–351. doi: 10.1155/1998/78170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brunetti E, Kern P, Vuitton DA, WHO-IWGE WP Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop. 2010;114:1–16. doi: 10.1016/j.actatropica.2009.11.001. [DOI] [PubMed] [Google Scholar]
- Elissondo MC, Dopchiz MC, Brasesco M, Denegri G. Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique. Parasite (Paris, France) 2004;11:415–418. doi: 10.1051/parasite/2004114415. [DOI] [PubMed] [Google Scholar]
- Elissondo MC, Albani CM, Gende L, Eguaras M, Denegri G. Efficacy of thymol against Echinococcus granulosus protoscoleces. Parasitol Int. 2008;57:185–190. doi: 10.1016/j.paritit.2007.12.005. [DOI] [PubMed] [Google Scholar]
- Farhadi M, Fazaeli A, Haniloo A. Genetic characterization of livestock and human hydatid cyst isolates from northwest Iran, using the mitochondrial cox1 gene sequence. Parasitol Res. 2015;114:4363–4370. doi: 10.1007/s00436-015-4673-y. [DOI] [PubMed] [Google Scholar]
- Ghosh V, Mukherjee A, Chandrasekaran N. Eugenol-loaded antimicrobial nanoemulsion preserves fruit juice against, microbial spoilage. Colloids Surf B Biointerfaces. 2014;114:392–397. doi: 10.1016/j.colsurfb.2013.10.034. [DOI] [PubMed] [Google Scholar]
- Hosseinzadeh S, Fazeli M, Hosseini A, Shekarforoush SS. Molecular characterization of Echinococcus granulosus in south of Iran. Open J Vet Med. 2012;2:201–206. doi: 10.4236/ojvm.2012.24032. [DOI] [Google Scholar]
- Jahanbakhsh S, Azadpour M, Tavakoli Kareshk A, Keyhani A, Mahmoudvand H. Zataria multiflora Bioss: lethal effects of methanolic extract against protoscoleces of Echinococcus granulosus. J Parasit Dis. 2016;40:1289–1292. doi: 10.1007/s12639-015-0670-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jaiswal M, Dudhe R, Sharma PK. Nanoemulsion: an advanced mode of drug delivery system. 3 Biotech. 2015;5:123–127. doi: 10.1007/s13205-014-0214-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kahriman G, Ozcan N, Dogan S, Karaborklu O. Percutaneous treatment of liver hydatid cysts in 190 patients: a retrospective study. Acta Radiol (Stockh Swed 1987) 2017;58:676–684. doi: 10.1177/0284185116664226. [DOI] [PubMed] [Google Scholar]
- Kohansal MH, Nourian A, Rahimi MT, Daryani A, Spotin A, Ahmadpour E. Natural products applied against hydatid cyst protoscolices: a review of past to present. Acta Trop. 2017;176:385–394. doi: 10.1016/j.actatropica.2017.09.013. [DOI] [PubMed] [Google Scholar]
- Mahmoudvand H, Kheirandish F, Dezaki ES, Shamsaddini S, Harandi MF. Chemical composition, efficacy and safety of Pistacia vera (var. Fandoghi) to inactivate protoscoleces during hydatid cyst surgery. Biomed Pharmacother. 2016;82:393–398. doi: 10.1016/j.biopha.2016.05.012. [DOI] [PubMed] [Google Scholar]
- Moazeni M, Roozitalab A. High scolicidal effect of Zataria multiflora on protoccoleces of hydatid cyst: an in vitro study. Comp Clin Pathol. 2012;21:99–104. doi: 10.1007/s00580-010-1069-3. [DOI] [Google Scholar]
- Moazeni M, Larki S, Oryan A, Saharkhiz MJ. Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: an in vivo study. Vet Parasitol. 2014;205:107–112. doi: 10.1016/j.vetpar.2014.07.006. [DOI] [PubMed] [Google Scholar]
- Moazeni M, Larki S, Saharkhiz MJ, Oryan A, Lari MA, Alavi AM. In vivo study of the efficacy of the aromatic water of Zataria multiflora on hydatid cysts. Antimicrob Agents Chemother. 2014;58:6003–6008. doi: 10.1128/Aac.02963-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moazeni M, Borji H, Saboor Darbandi M, Saharkhiz MJ. In vitro and in vivo antihydatid activity of a nano emulsion of Zataria multiflora essential oil. Res Vet Sci. 2017;114:308–312. doi: 10.1016/j.rvsc.2017.06.003. [DOI] [PubMed] [Google Scholar]
- Niazi M, Saki M, Sepahvand M, Jahanbakhsh S, Khatami M, Beyranvand M. In vitro and ex vivo scolicidal effects of Olea europaea L. to inactivate the protoscolecs during hydatid cyst surgery. Ann Med Surg. 2019;42:7–10. doi: 10.1016/j.amsu.2019.04.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oryan A, Moazeni M, Zarei Kordshouli F. Administration of Zataria multiflora as a novel therapeutic strategy in destruction of the germinal layer of hydatid cyst. Res J Parasitol. 2016;11:41–47. doi: 10.3923/jp.2016.41.47. [DOI] [Google Scholar]
- Shafiei R, Teshnizi SH, Kalantar K, Gholami M, Mirzaee G, Mirzaee F. The seroprevalence of human cystic echinococcosis in Iran: a systematic review and meta-analysis study. J Parasitol Res. 2016;2016:1425147. doi: 10.1155/2016/1425147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharafi SM, Sefiddashti RR, Sanei B, Yousefi M, Darani HY. Scolicidal agents for protoscolices of Echinococcus granulosus hydatid cyst: review of literature. J Res Med Sci. 2017;22:92. doi: 10.4103/jrms.JRMS_1030_16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smego RA, Jr, Sebanego P. Treatment options for hepatic cystic echinococcosis. Int J Infect Dis. 2005;9:69–76. doi: 10.1016/j.ijid.2004.08.001. [DOI] [PubMed] [Google Scholar]
- Solorzano-Santos F, Miranda-Novales MG. Essential oils from aromatic herbs as antimicrobial agents. Curr Opin Biotechnol. 2012;23:136–141. doi: 10.1016/j.copbio.2011.08.005. [DOI] [PubMed] [Google Scholar]
- Torgerson PR. Helminth-Cestode: Echinococcus granulosus and Echinococcus multilocularis. In: Motarjemi Y, editor. Encyclopedia of food safety. Fribourg: Academic press; 2014. pp. 63–69. [Google Scholar]
- Yones DA, Taher GA, Ibraheim ZZ. In vitro effects of some herbs used in Egyptian traditional medicine on viability of protoscolices of hydatid cysts. Korean J Parasitol. 2011;49:255–263. doi: 10.3347/kjp.2011.49.3.255. [DOI] [PMC free article] [PubMed] [Google Scholar]