اثر اسکولکس کشی عصاره اتانولی زنجبیل (Zingiber officinale Roscoe) بر روی پروتواسکولکسهای کیست هیداتید

Treatment of hydatid cyst is often expensive and complicated and sometimes it requires extensive surgery and/or prolonged drug therapy. If surgery is carried out without complete and effective anti-infective treatment, frequent relapses will occur. Approximately 6.5% of cases experience relapse after an intervention; therefore, prolonged recovery time is required. Annual costs associated with cystic echinococcosis (CE) are estimated to be US$ 3 billion for treating cases and losses to the livestock industry can include liver condemnation, reduction in carcass weight, decrease of milk production and reduced fertility (WHO, 2018).

This multi-host disease is one of the most important public health infectious diseases in Iran (Sadjjadi, 2006; Rokni, 2008).  Sheep that present with a 35.21% infection rate and 88% fertilized cysts have been considered as the most important intermediate host in Iran (Fasihi Harandi, 2002; Rokni, 2009). Fasihi Harandi (2012) suggested that the overall annual cost of CE in Iran was estimated to be US$232.25 million, of which US$93.39 million and US$132.0 million were attributed to human CE and the total economic losses due to livestock CE in Iran, respectively.

Surgery is one of the best choices for the treatment of hydatidosis and to prevent relapse, effective scolicidal agents must be used after surgery (Brunetti, 2010). There are several agents, which have been used for inactivation of the cyst contents, for example, hypertonic saline, silver-nitrate, cetrimide, and ethanol, but various side effects such as sclerosane colangititis (biliary tract fibrosis), liver necrosis and methaemoglobinaemia have been reported following their use (Mahmoudvand, 2014a). At present, research is attempting to find a natural scolicidal agent with increased efficacy and low side effects. Herbs are very important and useful therapeutic agents against many pathological infections. Ginger (Zingiber officinale Roscoe) of the family Zingiberaceae, is one of these herbs whose rhizome has also been used in traditional herbal medicine (Park, 2002; Sivasothy, 2011). The characteristic fragrance and flavor of ginger result from volatile oils that compose 1-3% of the weight of fresh ginger, primarily consisting of zingerone, shagaols, and gingerols as the major pungent compound (An, 2016). Many studies have shown that ginger has antibacterial (Gao, 2010; Malu, 2009; Sebiomo, 2011), antifungal (Ahmed, 2012; Aghazadeh, 2016) and antiparasitic effects (Arbabi, 2016; Mekuriya, 2018; Wonhyung, 2013). The aim of this study was to evaluate the effect of ginger ethanolic extract against protoscolices of hydatid cyst, to explore the potential of ginger as natural scolicidal agent.

Materials and Methods

Ginger extraction: First, ginger rhizomes (Zingiber officinale) bought from the traditional Bazar of Rasht in the north of Iran, were washed with sterile water and then placed in the open air for   72 h to dry completely. The rhizomes were then cut into thin slabs and placed on clean gauze to dry completely. After drying the ginger, these sheets were milled to obtain an homogeneous ginger powder. For extraction (Baqer, 2014), 100 g of powder was dissolved in 500 ml of 70 % ethanol as much as possible under the action of a magnetic stirrer for two hours. The solution obtained was placed in an Erlenmeyer at room temperature for 48 h. After 48 h, the solution was filtered (Whatman paper No.1). The filtered solution was then placed in an oven for evaporation at 37 °C, and finally the dried extract was kept at 4°C until it was used in a sterile container.

Preparation of protoscolices: At this stage, ovine and caprine liver and lung samples contaminated with hydatid cysts were collected from Rasht slaughterhouse and immediately transferred to the laboratory. The fluid in the cysts containing protoscolices was drained using sterile syringes and placed in tubes in an incubator at 37 °C. After the protoscolices were deposited, the supernatant fluid was removed, and finally, a thick liquid containing protoscolices was obtained.

Scolicidal test: Approximately 20 μl of sediment containing about 500 protoscolices were treated with various concentrations of ginger extract (40, 50, 100, 150 and 200 mg/ml) and the effect of each concentration was evaluated across different specified time periods (15, 30, 45 and 60 min). Each tube was incubated at 37 °C and removed at the allocated time (15, 30, 45 and 60 min) for performing the subsequent tests. Each concentration/time was undertaken in triplicate and saturated saline and normal saline were used as positive and negative control, respectively in each stage (Mahmoudvand, 2014b).

Viability test:  At the end of each concentration/time, after removing the supernatant with sampler, one ml of 0.1% eosin solution was added to each microtube and mixed before being incubated for 15 min. The precipitates of each microtube were then expanded and protoscolices were examined under light microscopy (100x magnification) and the number of live and dead protoscolices were counted.

Statistical analysis: For each concentration/time, the numbers of live and dead protoscolices were counted to enable percentages for each sample and the mean percentage across the three samples to be calculated. SPSS software was used to analyze data and Tukey’s test was performed to compare the means of the different concentrations for each time period examined. Significance was set at P<0.05.

Results

In the viability test, the live protoscolices are not stained with eosin, while the dead protoscolices are stained (Fig. 1). Based on the results, as the concentration of the ginger extract increased, the viability of protoscolices decreased. The highest percentage of viability was observed at 40 mg/ml of Z.officinale extract and the lowest survival level was found at 200 mg/ml. As shown in Fig. 2, 100% mortality rate with ethanolic extract was observed at the concentrations of 200 mg/ml after 30 min of incubation. The mortality rate of the ethanolic extract of Z.officinale at concentration of 150 mg/ml was 72.7, 79.4, 88.4 and 93% after 15, 30, 45 and 60 min of incubation, respectively. These values for the concentration of 100 mg/ml were 62, 75, 81.4 and 92.4%, respectively. On the other hand, at the concentration of 50 and 40 mg/ml, after 60 min, 32 and 56.6% protoscolices were still alive.

Mean and standard deviation of protoscolicidal effects of ginger ethanolic extract and positive and negative controls in different times and concentrations are provided in Table 1. A significant difference in the percentage of live protoscolices was found between the different concentrations of ginger extract (P<0.05), with the exception of between two concentrations: 150 and 100 mg/ml (P>0.05). A significant difference in the percentage of live protoscolices also occurred between the different treatment times with ginger extract (P<0.05). At 200 mg/ml concentration of ginger extract, there was no significant difference in the percentage of protoscolices at different times (P<0.05). Moreover, the scolicidal power of saturated saline as the positive control was 100% after 15 min of application. Therefore, the scolicidal activity of ethanolic extract at the concentration of 200 mg/ml after 30 min was similar to the scolicidal activity of positive control. The scolicidal effect of various concentrations of the ginger ethanolic extract, except 40 mg/ml concentration was extremely significant (P<0.05) compared to the control group (normal saline) at all exposure times. In the 60th min, no significant difference was found between the three concentrations of 200, 150 and 100 mg/ml (P>0.05).

Table 1- Comparison of protoscolices viability in ethanolic extract of Ginger according to time and concentration by Tukey's multiple comparison test

Mean=M, Standard deviation=SD, Positive Control= C+, Negative Control= C-

(a, b, c, and d indicate no significant difference (p ≥0.05) in Tukey’s test)

Table 2- Summary of previous research examining the effect of herbs on protoscolices within hydatid cysts

In High Efficacy column, M= Mortality rate and everywhere that M hasn’t been written, it is 100%.

Discussion

Because of the low success rate of drug therapy, surgery is the best approach for treatment of hydatid cyst, but this method also has its own problems and to prevent surgery complications, the use of medication after surgery is common. Given the pathophysiological importance of hydatid cyst, researchers have always tried to find a suitable and effective way to control and treat the hydatid cyst. The use of medicinal herbs is one potential treatment option. Herbal extracts have fewer side effects than conventional treatments and might be more effective. Many medicinal herbs have been used to prevent and treat parasites in the world and the study of the effect of different herbs on protoscolices have also yielded satisfactory results. In this study, we investigated the effect of ginger root extract on the viability of hydatid cyst protoscolices.

In the current study research, at 40 mg/ml concentration of ginger extract, even after 60 min, 56.7% of the protoscolices were alive. As the concentration of the extract increased, the percentage of viable protoscolices decreased. At 50, 100 and 150 mg/ml concentrations of ginger extract, after 60 min, 32%, 7.7% and 7% protoscolices were alive, respectively. In contrast, ginger extract at the concentration of 200 mg/ml killed 100% of protoscolices in 30 min and only very few (4% average) survived at the 15 min. Accordingly, the concentration of 200 mg/ml had the highest effect, followed by 150, 100, 50 and 40 mg/ml, respectively and based on Tukey’s test, no significant difference was found between the two concentrations of 100 and 150 mg/ml. The greatest effect was observed in the 60 min and the lowest in 15 min.

The research on affective medicinal herbs on hydatid cyst has increased in recent decades, and most of these studies have been conducted in Iran (refer to Table 2). Numerous plants, such as extract of onion, Allium cepa and basil, Ocimum bacilicum (Haghani, 2014), Artemisia aucheri (Feizi, 2015), aqueous and hydro-alcoholic extracts of garlic (Sadjadi, 2009), squash seeds and hazel nut (Eskandarian, 2012) are reported as ineffective in the treatment and prevention of hydatid cysts. Herbs have also been investigated and can be effective on protoscolices, but after a several days, such as Dendrosicyos socotrana and Jatropha unicostata (Barzinji, 2009), Mentha piperita (Maggiore, 2012), Salvia officinalis, Origanum vulgare (Pensel, 2014) and Thymus vulgaris (Doaa, 2011; Pensel, 2014).

The most effective herb on protoscolices is Black Zira (Bunium persicum), only 25 μg/ml essential oil from its seeds can destroy one hundred percent of protoscolices in five min (Mahmoudvand, 2016a). The seeds of Green Zira and the leaves of Common Myrtle are also reported to be effective with a 100% mortality rate on protoscolices in dosage of 50 μg/ml essential oil at 10 min (Keyhani, 2017; Mahmoudvand, 2016b).

Heretofore, three studies have examined ginger, Zingiber officinale. Moazeni (2011) and Feizi (2015) found 100mg/ml methalonic extract of ginger destroyed one hundred percent of protoscolices in 30 and 40 min, respectively.

The only study on ethanolic extract of ginger was performed by Baqer (2014) who reported that in concentrations of 50 and 100 mg/ml ginger extract, 46% and 37.5% of protoscolices were alive after 30 min and 15.3% and 20% protoscolices were alive after 45 minutes. However, ginger extract in 50, 100 and 150 mg/ml concentrations, destroyed 100% of protoscolices in 120, 90, and 60 minutes, respectively. 

Although in this study the time was not checked after 60 min, 7% of the protoscolices were still alive in concentration of 150 mg/ml at 60 min and these conclusions are inconsistent with the result of the previous investigation (Baqer, 2014). This disparity may be due to the difference in the strains of the parasite in the two regions.

Based on Moazeni (2011) and Feizi (2015), 100 mg/ml methanolic extract of ginger destroys 100% of protoscolices at 30 and 40 min, respectively. Comparing these results with our results suggests that methanolic extract of ginger is stronger than its ethanolic extract.

Main mechanisms of scolicidal effects of ginger are not clear and further studies are needed to elucidate these mechanisms. Although Bahmani (2013) showed that ginger causes the paralysis and death of leeches and Lin (2014) suggested that Ginger has the cestocidal activity or ability to halt spontaneous parasite movement.

Ginger has few side effects (Marcello, 2001) and it has been introduced by the Food and Drug Administration (FDA) as a healthy substance. Based on the results of the present study, ethanolic extract of Zingiber officinale had a high scolicidal activity in vitro, However, further investigation on the in vivo efficacy of Zingiber officinale extract and its possible side effects is needed.

Acknowledgments

The budget of this research is provided by the scientific-literary associations of Rasht branch, Islamic Azad University. We would like to thank the chairman of this forum, Dr. Leila Asadpour and the vice President for Research and Technology in Rasht branch, Islamic Azad University.

Conflicts of interest

The author declared no conflict of interest.