Insignificant activity of Allium paradoxium and Tanacetum parthenium on protoscoleces of Echinococcus granulosus: In vitro study

Medicinal plants are now recognized as most important alternative for chemical drugs resource, especially for anti-microbial targets. The current study aimed to determine the scolicidal effect of Allium paradoxium and Tanacetum parthenium as medicinal plants in vitro. Protoscoleces were exposed to different concentrations of chloroformic and hydroalcholic extracts (1, 10, 50 and 100 mg/mL) for 5, 10, 30, 60 and 120 min. Viability of protoscolices was confirmed by 0.1% Eosin staining. The results of this study showed that all extracts used were not significantly effective in comparison with the control group. Therefore, chloroformic and hydroalcholic extracts of A. paradoxium and T. parthenium could not be used during hydatid cyst treatment. However, there remains the need to perform advanced comparative clinical studies on the efficacy of A. paradoxium and T. parthenium and other scolicidal agents. Chloroformic and hydroalcoholic extract of A. paradoxium and T. parthenium, did not show any scolicidal activity at the concentration of 1, 10, 50 and 100 mg/ml.


INTRODUCTION
Hydatid disease due to Echinococcus granulosus remains an imperative, challenging medical problem (Brunetti et al., 2010). It is endemic in developing regions because of their poor socioeconomic conditions, but because of travelling and migration, it continues to have a global distribution. Hydatid cysts may be found anywhere in the body, but liver and lungs are the most infected organs (Pawlowski et al., 2001). Surgery is still the chosen method of treatment (Topcu et al., 2006). Repetition during the long-term follow-up period is the most usual problem with this therapeutic technique. For decrease in the recurrence rate of disease, it is necessary to avoid spilling of the cyst contents and the use of active scolicidal agents . The World Health Organization (WHO) regards the use of both effective and safe agents for intraoperative killing of infectious material, but unfortunately; there is no ideal agent up to now. According to WHO reports, therapeutic plants with a relatively low risk are the suggested for traditional treatment of disease (WHO, 1996).
Since A. paradoxium and T. partheniumon has been shown to have a number of medicinal and anti-parasitic properties and considering the origin of these plants to Iran, the present study aimed to assess the scolicidal effect of the chloroformic and hydroalcholic extracts at different concentrations and for various exposure times.

Parasite
Hydatid cysts of E. granulosus were detached from livers of naturally infected sheep slaughtered at Mazandaran abattoir, northern part of Iran under aseptic conditions. The fluid of hydatid cyst containing protoscoleces was removed by a 50 ml syringe and transferred into a 50 ml Falcon tube. After 30 min of immovability, settled protoscoleces were washed three times with normal saline (pH 7.2). The viability of the parasites was assessed with 0.1% eosin under a light microscope (400x) (Gholami et al., 2013).

Preparation of extracts
The sliced seeds of A. paradoxium and the aerial parts of T. parthenium were dried under shade and powdered mechanically using a commercial electric blender. Dried powder (100 g) were extracted by percolation method with hydroalchol and chloroform solution, separately for 72 h in room temperature. The obtained solution was passed through filter paper (Whatman No.3, Sigma, Germany) to remove plant debris. Finally, the extracts were concentrated using a rotary evaporator (Heidolph, Germany), placed in a sterile glass and stored at -4°C for later use (Shirzad et al., 2014).

Scolicidal tests
The scolicidal tests were done three times (triplicate) and based on Rahimi-Esboei et al. (2013). In the current study, four concentrations (1, 10, 50 and 100 mg/ml) of extracts were examined for 5, 10, 30, 60 and 180 min. 2 ml of each concentration was transferred to a test tube; 50 µl of sediment protoscoleces was added to the tube and mixed lightly. The tube was then incubated at 37°C for 5, 10, 30, 60 and 180 min. At the end of each incubation time, viability of the protoscoleces was evaluated by 0.1% eosin stain (Rahimi-Esboei et al., 2013).

Results of the effectiveness of different concentrations of
A. paradoxium and T. parthenium extract as a scolicidal agent are shown in Figures 1 to 4. While the viability rate in the control group was 88% after 180 min, scolicidal activity of chloroformic extract of A. paradoxium extract at concentration of 100 mg/mL was 90, 81, 78, 74 and 71% after 5, 10, 30, 60 and 180 min of application, respectively ( Figure 1). Scolicidal effect of hydroalcholic extract of A. paradoxium extract at concentration of 100 mg/mL was 88, 84, 79, 76 and 72% after 5, 10, 30, 60 and 120 min of exposure, respectively (Figure 2). While the viability rate in the control group was 85% after 180 min, scolicidal activity of chloroformic extract of T. parthenium extract at concentration of 100 mg/mL was 87, 85, 85, 81 and 78% after 5, 10, 30, 60 and 180 min of application, respectively (Figure 1).
Scolicidal effect of hydroalcholic extract of T. parthenium extract at concentration of 100 mg/mL was  In this study, the effectiveness of two plants were increased by increasing the time and concentration of plants, but there was no significant differences between times of exposure and concentration of extracts.

DISCUSSION
The current work examined the scolicidal activity of A. paradoxium and T. parthenium using the chloroformic and hydroalcholic extracts and an in vitro technique. Shirzad et al. (2014) showed high effectiveness of A. paradoxium and T. parthenium on Giardia lamblia in in vitro investigation with concentration of 100 mg/ml after 3 h. Also, this antigiardial activity of A. paradoxium was reproved by Elmi et al. (2014) in an in vivo study. Despite the anti-bacterial and anti-parasitic activity of these plants, the results of this study indicate insignificant efficacy against protoscoleces of hydatid cyst (Shirzad et al., 2014). It could be concluded that protoscoleces of hydatid cyst are more resistance than other used microorganisms and it depends on the physiological stricture of cells. In this study, the used plants were inactive against protoscoleces of hydatid cyst. Perhaps, a longer exposition time was needed while the same concentration and exposure time was effective against some microbial and parasitic infections (Sarkari et al., 2009;Mehrabi et al., 2011;Radwan et al., 2012).
This in vitro technique is simple but confirmed, trustworthy, sensitive and extensively used (Dey, 1980). Therefore, the results gained are permissible and reliable. As shown (Figures 1 to 4), the chloroformic and hydroalcholic extracts of A. paradoxium and T. parthenium did not exhibit any in vitro scolicidal activity, even at concentrations of 100 mg/ml after 180 min. From the extracts and plants, hydroalcholic extract of T. parthenium was the most effective extract in concentration of 100 mg/ml after 180 min.
This study strongly recommends evaluating the activity with a longer period and develops more research on the  phytochemistry of these plants.