Evaluation of the Effects of Bunium persicum and Albendazole on Killing Hydatid Cyst Protoscolices In Vitro

Objectives: Hydatidosis is a parasitic disease of zoonoses and the best treatment for this parasitic disease is cryosurgery. From time immemorial, many protoscolice-destroying chemicals have been used to inhibit the distribution of protoscolices within the cyst during surgery. However, the side effects of chemical compounds and medicinal plants should always be considered. Due to the antiparasitic properties of Bunium persicum (Boiss), this study was performed to evaluate and compare the effects of B. persicum (Boiss) essential oil and albendazole on protoscolices inside hydatid cyst in vitro. Materials and Methods: In this study, the essential oil used from the desired plant seeds was prepared. Gas chromatography-mass spectrometry was performed to determine its components. Hydatid cyst protoscolices were extracted from hydatid cyst-infected livers and exposed to B. persicum (Boiss) (2.5, 5, and 10 μg/mL) for 10, 30, 60, and 120 minutes, respectively. The viability of protoscolices was measured by the effect of plant essential oil by eosin staining. Albendazole was also used as an effective antiparasitic drug in this study. Results: The amount of essential oil protoscolices at 2.5 μg/mL in 10, 30, 60, and 120 minutes was 48.5%, 68.2%, 79.5%, and 96.1%, respectively. The antiparasitic drug albendazole showed less toxic effects at similar doses at the same intervals, but both treatment groups had a significant effect ( P <0.001). The highest toxic effects of essential oil were observed at a concentration of 10 μg/mL 30 minutes after treatment. For albendazole, this effect was achieved at a dose of 10 μg/mL after 2 hours. Conclusions: The results and statistical data of this study showed that the essential oil of B. persicum (Boiss) in comparison with the antiparasitic drug albendazole has an acceptable scolicide effects in hydatidosis and can be used as an alternative to scolicide compound.


Introduction
Hydatidosis is one of the most important and common parasitic diseases of zoonoses and livestock in all countries of the world and is caused by the larval stage of Echinococcus cestodes. This parasitic disease is more common in rural areas and livestock with a closer relationship between dogs, livestock and humans (1,2). Hydatidosis is considered a major public and economic health challenge by reducing livestock products, causing serious damage to human health and incurring medical costs, and the risk of death. In hydatic cyst surgery technique, if the necessary preventive measures are not observed, there is a risk of spreading of protoscolices and penetration into viscera and tissues during surgery, which is one of the main reasons for the recurrence of this parasitic disease and the formation of secondary cysts (3,4). In order to prevent widespread dissemination of cyst contents, effective scolicides is essential to reduce the rate of disease recurrence. So far, many chemical scolicides have been used, such as 2% formalin, 20%-30% hypertonic saline, silver nitrate, and betadine (5). In some cases, the use of these chemical compounds leads to serious side effects such as bile duct fibrinization, liver necrosis and healthy tissues adjacent to the cyst. The antiparasitic drugs benzimidazoles are used to treat human hydatid cysts with varying degrees of therapeutic success. Mebendazole was one of the first of these compounds, but due to the low absorption of the drug, its serum and intravesical levels were lower and not enough antiparasitic drug penetrated into the hydatid cysts (6)(7)(8)(9). Albendazole is a newer, broader spectrum antiparasitic drug that is more effective than mebendazole in treating human hydatid cysts. An alternative treatment for human hydatid cyst could be the formulation of new compounds that kill protoscolices using compounds derived from medicinal plants, including essential oils extracted from them (10,11). Very 90 few studies have been performed on the potential use of essential oils that kill hydatid cyst protoscolices (12,13). In this study, the seeds of Bunium persicum were collected in June 2019 from the wild plants, which found in the many regions of Iran. Numerous studies have also shown the antiparasitic activity of this aromatic plant against various species of parasites, for example; arthropods and protozoa (4,6). The present study investigated the scolicidal activity of essential oil of B. persicum oil on human hydatid cyst protoscolices in vitro conditions.

Plant Material
To start the practical work of this study, first the B. persicum plant was collected from different regions of Iran and after determining and confirming the plant species, the plant essential oil was extracted by water distillation. To prepare different dilutions of B. persicum, essential oils were dissolved as a solvent in olive oil (14,15). Essential oil components were identified using mass spectrum and gas chromatography-mass spectrometry system (16).

How to Prepare Protoscolices From Hydatid Cyst
For this purpose, a number of sheep liver infected with hydatid cyst were prepared from the slaughterhouse and transferred to a parasitology laboratory. After disinfecting the surface of the liver, the contents of the cysts, including the protoscolices of cestode Echinococcus granulosus and hydatid fluid, were drained into a sterile container. Then, we waited a few minutes for the protoscolices to settle.

Protoscolex Viability Test
To estimate the viability of protoscolices, some of the liquid containing protoscolices was poured into a glass slide by pasteurization pipette and the same volume of 0.1% eosin dye solution was placed next to the sample. Then gently place the lamellae on the prepared solution containing the dye. After 10 minutes, they were evaluated by counting the number of live protoscolices (Figures 1 and 2).

Investigation of Scolicidal Activity in Laboratory
In our study, the killing effects of 3 dilations of 25 and 50 and 100 mg/mL of essential oil of B. persicum plant at 10, 30, 60, and 120 minutes on hydatid cyst proto-scolexes was evaluated. To prepare the dilations of 25 and 50 and 100 mg/mL of essential oil of B. persicum in 9.7 mL of normal saline with 0.3 mL, Twin 80 was dissolved separately in each tube. After 10 minutes, the ratio of dead to live protoscolices was measured on the entire lamellar surface using a light microscope (4,6). The scolicidal effects of essential oil were studied and compared with albendazole at similar doses.

Results
The gas chromatography-mass spectrometry analysis results showed that in the first period of 10 minutes, the lowest dose of B. persicum essential oil (2.5 μg/mL) resulted in almost half (48.5%) of protoscolices. Albendazole with the same dose and in the same period showed a less lethal effect (22.3%) but compared to the control (0%), both treatments showed a significant effect (P=0.021). With increasing dose, the lethal activity increased so that at a dose of 5 mg/mL of essential oil of B. persicum killed 96.1% and albendazole killed 51.1% of protoscolices (P<0.001).

Discussion
The present study results showed that B. persicum essential oil has an acceptable scolicidal effect compared to the standard drug albendazole. Some studies showed the toxicity of essential Artemisia sieberi oil against protozoa Trichomonas gallinae in vitro and in vitro conditions. They reported a concentration of 10 μg/mL of essential oil of A. sieberi in the culture medium as the minimum inhibitory concentration for 24 hours (17). Other studies showed that the Artemisia plant at a concentration of 0.001 at the beginning of cultivation has a complete effect on Trichomonas vaginalis (18). In the present study, ► Hydatidosis is caused by the larval form of tapeworms of the genus Echinococcus. ► Bunium persicum (Boiss) essential oil has an acceptable scolicidal effect compared to albendazole.
Key Messages  the toxicity of B. persicum essential oil on hydatid cyst protoscolices was shown. The results of other researchers were positive for evaluating the protoscolicidal effect of thymol on hydatid cysts, and thymol had a lethal effect on Echinococcus granulosus protoscolices. Still, this killing took longer than usual (19). In another study the effect of B. persicum extract on hydatid cyst protoscolex during surgery showed that at a dose of 25 μg/mL in 100 minutes, 100% of protoscolices were killed (20). Some other researches showed that the methanolic extract of ginger has a very good effect on hydatid cyst protoscoleces. Still, methanolic extract of Artemisia aucheri did not have a good scolicidal effect on protoscoleces of hydatid cysts. However, the present study showed an acceptable effect of B. persicum essential oil on the killing property of scolex of hydatid cysts protoscoleces. The difference in the findings of another studies is probably due to the differences in the plant species studied and the constituent chemicals (21). researchers in their study of the simultaneous effect of albendazole and thymol against hydatid cyst protoscolex, showed a positive therapeutic effect and described the synergistic effect of this drug combination (22).
Our study showed that B. persicum essential oil could be considered as a suitable scolicide, and after passing hepatotoxicity and biliary system tests if it is safe to eliminate and inhibit protoscoleces be used. Further studies are needed to investigate the scolicide components of essential B. persicum oil, their mechanism of action, and the possible synergistic activity of essential B. persicum oil or its components with the standard drug albendazole (23). Due to the natural origin of this treatment and the extent of growth of this plant in Iran, further studies will provide the use of this substance during hydatid cyst surgery alone or with standard medicine.

Conclusion
The results of our study and those of other researchers showed that medicinal plants and their derivatives have many antiparasitic effects, and that these chemical compounds have the power to inactivate the protoscolices of cestode E. granulosus. It should be noted that there is still disagreement among various scientists about the mechanism of action of these plant extracts in killing protoscolices and more research is needed in this regard. Another very important issue about the extracts of medicinal plants is the degree of toxicity and their destructive effects on the cells of the human body, which is still a lot of research.