In Vitro Assessment of the Protoscolicidal Activities of the Ephedra major Methanol Extracts

Copyright © 2017 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.


Background
Cystic echinococcosis is a widely zoonotic infection caused by larval stages (metacestodes) of the tapeworm Echinococcus granulosus.Hydatidosis affects mainly the intermediate hosts viscera, including the liver, lungs, and less frequently the muscle, kidney, bone, spleen, and other organs. 1 Hydatidosis is more prevalent in the countries in the Middle-East, Arabic, North Africa, and it is endemic in Iran. 2 Currently, the main methods for the treatment of hydatidosis are surgery (percutaneous aspiration, injection and reaspiration-PAIR) and chemotherapy (using benzimidazoles such as mebendazole and albendazole). 3,4he surgical treatment of hydatid diseases is still the most effective approach.It can be done successfully in a large number of patients if a cyst does not have a risky location.Injection of protoscolicidal agents into the cysts preoperatively is a traditional method. 5Spillage of protoscoleces can cause relapse or secondary infection that occurs in approximately 10% of postoperative cases. 6However, the absence of enough evidence about the efficacy and the presence of toxicity associated with the protoscolicidal agents led many surgeons to leave this routine stage in the operative management of hydatidosis. 7,8Therefore, the cystic fluid including many of protoscoleces has the potential to grow into new cystic echinococcosis. 9ecently, there has been a considerable attention in finding natural protoscolicidal agents from indigenous herbs to replace synthetic ones.1][12][13][14] Ephedra, a medicinal plant belonging to the Ephedraceae Dum.family, is genus of non-flowering seed plants belonging to the Gentlales, the closest living relative of the angiosperms. 15,16][19] There are several reports concerning in vitro antibacterial effect against different bacterial species, including Staphylococcus aureus, Bacillus antheracis, B. diphtheria, B. dysenteriae, B. typhosus and Pseudomonas aeruginosa. 20,21

Objective
The purpose of this study was to determine the in vitro activity of Ephedra major methanolic extracts against pro-toscoleces of hydatid cysts based on different concentrations and exposure times.

Plant Materials
The root, stem and leaf of Ephedra major were collected between 2014 and 2015 in their places of origin in Lorestan province in southwest of Iran.They were authenticated by Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Iran.

Extracts Preparation
Different parts of the plants were dried at room temperature and powdered.About 10 g of powdered subjects were extracted with 100 mL of absolute methanol (10% w/v) in a conical flask for maceration.The mixture was shaken for 3 days at room temperature.The suspension was filtered with a Whatman Filter Paper No.1. 22The procedure of extraction was operated via rotary evaporation at 37°C and the extracts were stored at 4°C until use.

Protoscoleces
Hepatic hydatid cysts from naturally infected sheep were obtained from an abattoir located in Haftjuy in the central district of Qods county, Tehran province, Iran.Protoscoleces were collected in aseptically conditions and washed at least 3 times with phosphate-buffered saline (PBS).The concentration of protoscoleces per milliliter of the hydatid fluid in normal saline solution (0.9% NaCl) containing 5000 protoscoleces in milliliter with more than 90% viability was used. 23ability After adding 10 μL eosin solution (0.1% w/v) to 10 μL, the viability of protoscoleces was reviewed microscopically for 15 minutes.Stained protoscoleces were considered as dead and unstained protoscoleces were recorded as alive.The control group included non-treated protoscoleces with the plant extracts.

Statistical Analysis
All experiments were done three times in each group.Findings are shown as means ± standard error of mean (SEM).Statistical differences were analyzed using Mann-Whitney non-parametric test.Probability (P) values of less than 0.05 were considered to be statistically significant.

Results
The mortality rates of protoscolices of hydatid cysts after ex posure to various concentrations of E. major extracts in different times are demonstrated in Table 1.Obtained findings showed that E. major stem extracts at the concentration of 0.1% after 60 minutes of exposure time killed 99.09% of protoscoleces.Similarly, the mean of mortality rate of protoscoleces after 60 minutes exposure with E. major leaves extract in the concentration of 0.1% was 90.25%.The protoscolicidal rate in the control group was 4.99% after in the some exposure times.Findings also revealed that E. major extracts at all concentrations had significant protoscolicidal activity (P = 0.001) compared with the control group (Table 2).

Discussion
There is a particular interest in studies about protoscolicidal activity in order to prevent the formation of secondary echinococcosis.An appropriate protoscolicidal effects demonstrated by its ability for rapid preparation, higher availability, stronger effects at low concentrations, high efficacy in shorter time after exposure, and less toxic effects. 24Recently, the scolicidal effects of several herbal extracts such as Olea europaea, Pistacia atlantica, Allium sativum, Zataria multiflora, and some chemicals including silver nitrate, cetrimide, ethyl alcohol, peroxide hydrogen, manithol, and hypertonic saline have been. 25We found that E. major stem extracts in various concentrations had potent protoscolicidal effects which are comparable with the protoscolicidal activity leaf and root extracts in different exposure times.It is noteworthy that increasing concentrations and exposure times showed more protoscolicidal efficacy.Findings indicated that stem extracts killed almost all of protoscoleces and its 0.1% concentration revealed strong scolicidal effects at 60 minutes, 0.001% concentration did not have enough lethality activity at the same time.In this in vitro experiment, we showed that the concentrations (0.1%-0.001%) of root extracts had less activity.Thus, the protoscoleces that were killed by root extracts (0.001% concentration) reduced the mortality rate to 9.54% at 60 minutes exposure time.
Although, to our knowledge, there is no report on antiparasitic activities of E. major methanolic extracts.Previous studies have proven antimicrobial properties of this plant.The Ephedra strobilacea showed activity against most of microorganisms.Based on the results, the methanolic extracts of Ephedra pachyclada significantly revealed antimicrobial effects against Klebsiella pneumonia and Bacillus subtilis as gram positive bacteria.Furthermore, the result revealed the methanolic extract of Ephedra procera wild plant significantly had highest antifungal activity against Candida albicans microorganisms.6][27][28][29] A survey which focused solely on the protoscolicidal activity of E. major leaves aquatic extracts demonstrated that the scolicidal effect against protoscoleces was not suggested and more research is also necessary to investigate by other extracts. 30inally, the present study demonstrated that the methanolic extracts of E. major might be a natural source and be used as new scolicidal agents during hydatid cysts surgery (PAIR) to reduce the risk of protoscoleces spillage.However, more researches will be needed to evaluate the in vivo effects of these extracts in a clinical setting on animals and humans.

Table 1 .
Scolicidal Activities of Different Concentrations of Ephedra major Root, Stem and Leaf Extracts in Different Exposure Time a a Concentrations of all of the plants extract perpetrated with one batch.b Root.c Stem. d Leaf.

Table 2 .
The Mean of Scolicidal Effects of Various Extracts of Ephedra major Root, Stem and Leaf Extracts in Exposure Times a a P value = 0.001