Effect of Saussurea costus extracts in the viability of Echinococcus granulosus protoscoleces of sheep origin In vitro

Cystic echinococcosis is one of the most prevalence and dangerous zoonotic parasitic disease in the world. Iraq is one of the most countries that affected by this disease. Surgery is usually the most effective therapy and can be used with injection of drugs in hydatid cysts before surgery to kill protoscoleces to avoid anaphylactic shock as a result of spilling of hydatid cyst fluid into peritoneal cavity, as well as this reduce the chance of secondary hydatid cysts production. Therefore, the current study aimed to evaluate the effectiveness of Saussurea costus extracts on protoscoleces viability of Echinococcus granulosus , where the protoscoleces of E. granulosus exposed to four different concentrations of Saussurea costus extracts at four different exposure times including; 15, 30, 45 and 60 min in vitro, The results of ethanolic extract showed highest efficacy at concentration 20,30mg/ml in 45,60min and 40mg/ml at all times. The highest scolicidal effect of petroleum ether extract was reported at 10 and 15mg/ml during 60min, while 20mg/ml reported similar effect at 45 and 60min. The data of cool aqueous extract showed reduction of protoscoleces viability to 0% at 200 and 250mg/ml in 45 and 60min, respectively, while 300mg/ml showed same reduction of viability at all experiment time periods. The data of hot aqueous extract showed 100% kill rate by using 350mg/ml at 60min and 400mg/ml at 45 and 60, as well as 450mg/ml at all experiment period times. Comparing to the control group. Generally, Saussurea costus extracts have concentration and exposure time-dependent effect on protoscoleces viability. Keyword: Echinococcus granulosus, Saussurea costus , protoscoleces جراخ مانغا لصا نم ةيبيبحلا ةكوشملل ةيلولأا تاسيؤرلا ةيويح يف يدنهلا طسقلا تاصلختسم ريثأت * يديبعلا لضاف ءامسأ 1 ليعامسا ملاس داؤف ، 2 1 ةعماج ،تانبلل ةيبرتلا ةيلك ،ةايحلا مولع مسق قارعلا ،لصوملا ،لصوملا 2 قارعلا ،لصوملا ، لصوملا ةعماج ،تانبلل ةيبرتلا ةيلك ،ةايحلا مولع مسق Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (72-82) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 74 ةصلاخلا ربتعيو .ملاعلا ءاحنا عيمج يف ةروطخو اً راشتنا ناويحلاو ناسنلاا نيب ةكرتشملا ةيليفطلا ضا رملاا رثكا نم يسيكلا تاكوشملا ءاد دعي يف ةيودلاا نقح عم اهمادختسا نكميو ضرملل ةيلاعف رثكلأا جلاعلا يه ةحا رجلا نوكت ام ةداع. ضرملا اذهب ةرثأتملا لودلا نم قا رعلا كلاا فيوجتلا يف يردعلا سيكلا لئاس راشتنأ ةجيتن )ةيقأتلا( ةمدصلا ثودح بنجتل ةيلولأا تاسيؤرلا لتقل ةحا رجلا ءا رجا لبق ةيردعلا ساي تابن تاصلختسم ةيلاعف مييقت ةيلاحلا ةسا ردلا تفده ،كلذل .ةيوناث ةيردع سايكأ نوكت ةصرف نم للقي اذه نا ىلا ةفاضلإاب ،ينوتيربلا Saussurea costus تاسيؤرلا تضرع ثيح .ةيبيبحلا ةكوشملل ةيلولأا تاسيؤرلا ةيويح ىلع يدنهلا طسقلا وا طسقلاب فورعملا يهو ضرعت تا رتف للاخ يدنهلا طسقلا تابن تاصلختسمل ةفلتخم زيكا رت ةعبرأ ىلا ةيبيبحلا ةكوشملا ليفطل ةيلولأا 45،30،15 و 60 لا جئاتن ترهظأ .يحلا مسجلا جراخ ةقيقد زيكرتلا دنع هل ةيلعاف ىلعا يلوناثيلاا صلختسم 20 و 30 يف لم/مغلم 45 و 60 ةقيقد ، 40 زيكرتلا دنع رثيأ مويلورتب صلختسمل لتاق ريثأت ىلعا ناكو .تاقولأا عيمج يف لم/مغلم 15 و 10 دعب لم/مغلم 60 و ةقيقد 20 لم/ما رغلم دعب 45 و 60 فخنا درابلا يئاملا صلختسملا جئاتن تققح دقو ةقيقد ىلا ةيويحلا ةبسن ضا 0 زيكا رت دنع % 200 و 250 يف لم/مغلم 60،45 و ةقيقد 300 تاقولأا يف لم/مغلم 15 ، 30 ، 45 و 60 لتق ةبسن تلجس دقف راحلا يئاملا صلختسملا جئاتن اما ةقيقد 100 دنع % زيكا رت 350 يف لم/مغلم 60 و ةقيقد 400 يف لم/مغلم 45 و 60 و ةقيقد 450 سملا تاقولأا عيمج يف لم/مغلم ةعومجم عم ةنراقملاب ةمدخت .ضيرعتلا ةدم ةدايزو زيكرتلا ةدايز عم اًيدرط اًبسانت يدنهلا طسقلا تاصلختسم ريثأت بسانت دقف ،مومعلا ىلع .ةرطيسلا :ةيحاتفملا تاملكلا ةيلولأا تاسيؤرلا ،يدنهلا طسقلا ،ةيبيبحلا ةكوشملا Introduction Echinococcosis or Hydatidosis is a zoonotic disease, caused by the metacestode or larval stage of a dog tapeworm Echinococcus granulosus [1,2]. E. granulosus spread in central Asia, China, North and East Africa, Australia and South America [3] . Life cycle of E. granulosus in definitive host, represented by the canids which harbors the adult worms in their intestines, and intermediate host includes livestock, as well as human which act as an accidental host. The intermediated host can be infected by orally ingestion of parasite eggs which develop into fluid-filled cysts in their liver, lungs, and other organs [4]. The disease causes expanded mortality and morbidity in human and livestock [5]. Echinococcosis disease is asymptomatic for several years, and the clinical symptoms can appear due to increase of cyst size which cause pressure on nearby tissues [6]. There are several methods of treatment echinococcosis including surgery, percutaneous technique, chemotherapy [7]. Surgery is the most effective therapy of the disease but with quite difficult for some cases because the cyst diffusion in to many organs or formed in risky location [8]. Chemotherapy by singing benzimidazole derivatives are necessary in cases of possible recurrence, injury to several organs in the body and addition to cases of the advanced stages [9]. However, it has side effects such as nausea, vomiting, diarrhea, abdominal pain, headache, it may cause gastro-intestinal and liver functions disturbances, hematuria and leukopenia [10]. In order to reduce the risk of hydatid fluid spillage of the cysts during surgery and to prevent reoccurrence of echinococcosis, which may occur in approximately 10% of postoperative cases[11]. Therefore, the use of more effective and less harmful agents to treat this disease is important [12], such as pistacia vera, zataria multiflora and other plants [13,14]. Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (72-82) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 75 Saussurea costus belongs to family of Asteraceae is one of the main species of the genus Saussurea [15] which mostly grow in the humid regions at altitude of 2600-4000m in Western Himalayan region of India and Pakistan, and it is also been started to cultivate in new areas for commercial purposes in 1920 [16]. The roots of S. costus are used for the treatment many diseases including chronic gastritis, stomach ulcer, asthma, bronchitis and rheumatoid [17], oil extracts from the roots of S. costus has been used in the treatment of leprosy [18]. Chemical studies of the S. costus plant have shown it contains many phytochemical compounds with biological activity, including alkaloids, flavonoids, sesquiterpene terpenes, anthraquiones and tannins [19,20]. This study aims to determinate the effect of S .costus extracts on viability of E. granulosus protoscoleces In vitro. Materials and methods Samples collection and protoscoleces isolation: The infected livers of sheep (figure1) were collected from butcher shops in Mosul city and protoscoleces of E. granulosus were isolated according to Smyth [21] by sterilizing the surface of hydatid cyst with 70% ethanol following by aspiration of hydatid cyst fluid using G21 needle. Protoscoleces were washed three times with phosphate buffered saline (PBS) using centrifuge at 3000rpm for 10minutes, with the addition of 1gm of streptomycin and 20000 UI of penicillin before starting a second wash. Estimation of protoscoleces viability: The assessment of the viability of protoscoleces was estimated by taking 20 ul of the hanging of protoscoleces was mixed with 20 ul of 0.1% eosin stain on the glass slide. Protoscoleces that appear by bright green in color considered alive ( figure 2) while protoscoleces red in color dead because stain entry inside it (figure 3) [22].The viability of the protoscoleces in this study was 100%. Figure1 : Hydatid cysts in the liver of sheep Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (72-82) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 76 Collection and preparation of the S. costus extracts S. costus plant was bought from perfumery shops in Mosul city. 25 gm from a root of plant powder were added to 500ml of petroleum ether for 72 hours on magnetic stirrer, then solution were filtered by Whatman No.1 filter paper, 500ml Ethanol 70% is added to the precipitant for 72 hours on magnetic stirrer [23], after filtering the solution, distilled water was added to precipitant on magnetic stirrer at medium speed and temperature 60C° to obtain a warm water extract, The solvent was removed by using rotary evaporator at 40C° [24] . Cool aqueous extract was prepared according to Rios et al. method [25], where 40g of root plant powder was added to 400 ml of distilled water following by mixing process using blender and then magnetic stirrer. The samples underwent to soak and mix for 24 hours at 4C°.The samples were filtered using Whatman filter paper No.1 following by store the crude extracts at 4C°, until been used. Effect of S. costus extracts on protoscoleces viability In vitro To evaluate the effect extracts on viability of the protoscoleces, experiments were designed that included four different concentrations at four different times, in addition to the control group. In each test tube 1ml of extract dissolved in PBS was added, with the addition of 2000 protoscoleces in the same tube and placed in a water bath at 37C⸰ according to the specified times washed with PBS solution to get rid of the effect of extracts and examined protoscoleces under a light microscope to count the living and dead ones. Statistical analysis Data analysis was carried out using the SPSS statistical analysis system, the means and standard error were used, in addition to using Duncon’s test to measure a significant difference between the means of experimental study at the level of significance p≤0.05. Results and Discussion The results of this study showed the effect of petroleum ether extract S. costus on protoscoleces viability in vitro using four concentrations 5,10,15 and 20 mg/ml at four different times 15,30,45 and Figure 2 : Live protoscoleces before treated with extracts after dye it with 0.1% eosin Figure 3 : Dead protoscoleces after treated with extract and dye it with 0.1% eosin Journal of Education and Science (ISSN 1812-125X), Vol: 30, No: 3, 2021 (72-82) Special Issue for Proceeding of 3 National (1 international conference of biology) (ICBSUM 2021) 5, 6 July College of Education for Pure Science, University of Mosul, Mosul, Iraq. Downloaded from https://edusj.mosuljournals.com/ 77 60minutes. The data of petroleum ether extract indicated 0% of viability rate at 10mg/ml and 15mg/ml during 60min, while 20mg/ml indicated 0% of viability at 45 and 60min. The highest percentage of viability (47.785%) reported at 5mg/ml during 15min (see table-1). Table 1. The effect of petroleum ether extract of S. costus on viability percentage of protoscoleces. Viability of protoscoleces (%) at different time Control Con. 60min. 45min. 30min. 15min. 100 2.500±1.443 a 19.440±0.329 d 25.850±2.626 e 47.785±1.278 f 5mg 0 a 13.330±0.408 c 18.220±0.408 d 42.565±1.405 e 10mg 0 a 1.495±0.863 a 9.830±1.056 b 17.140±0.790 d 15mg 0 a 0 a 1.515±0.874 a 10.800±0.408 bc 20mg Similar letters in the table above indicate there are no significant differences between values at p≤0.05, while different letters indicate there are significant differences between values at p≤0.05. The current study results showed the effect of Ethanolic extract S. costus on protoscoleces viability In vitro using four concentrations 10,20,30 and 40 mg/ml at four different times 15,30,45 and 60minutes. Lowest percentage of viability rate reported at 20mg/ml and 30mg/ml during 45 and 60min, while 40mg/ml indicated lowest viability percentage (0%) at all experimental time periods. The highest viability percentage (100%) was reported at 10mg/ml during 15min. (see table-2). Table 2 . The effect of ethanolic extract of S. costus on viability percentage of protoscoleces. Viability of protoscoleces (%) at different times Control Con. 60min 45min 30min 15min 100 23.640±0.871d 28.570±0.408e 69.7900±3.181f 100.000±0.000g 10mg 0a 0a 18.570±0.825c 27.470±1.749e 20mg 0a 0a 1.110±0.702a 7.566±1.261b 30mg


Introduction
Echinococcosis or Hydatidosis is a zoonotic disease, caused by the metacestode or larval stage of a dog tapeworm Echinococcus granulosus [1,2]. E. granulosus spread in central Asia, China, North and East Africa, Australia and South America [3] . Life cycle of E. granulosus in definitive host, represented by the canids which harbors the adult worms in their intestines, and intermediate host includes livestock, as well as human which act as an accidental host. The intermediated host can be infected by orally ingestion of parasite eggs which develop into fluid-filled cysts in their liver, lungs, and other organs [4]. The disease causes expanded mortality and morbidity in human and livestock [5]. Echinococcosis disease is asymptomatic for several years, and the clinical symptoms can appear due to increase of cyst size which cause pressure on nearby tissues [6].
There are several methods of treatment echinococcosis including surgery, percutaneous technique, chemotherapy [7]. Surgery is the most effective therapy of the disease but with quite difficult for some cases because the cyst diffusion in to many organs or formed in risky location [8]. Chemotherapy by singing benzimidazole derivatives are necessary in cases of possible recurrence, injury to several organs in the body and addition to cases of the advanced stages [9]. However, it has side effects such as nausea, vomiting, diarrhea, abdominal pain, headache, it may cause gastro-intestinal and liver functions disturbances, hematuria and leukopenia [10]. In order to reduce the risk of hydatid fluid spillage of the cysts during surgery and to prevent reoccurrence of echinococcosis, which may occur in approximately 10% of postoperative cases [11]. Therefore, the use of more effective and less harmful agents to treat this disease is important [12], such as pistacia vera, zataria multiflora and other plants [13,14].
Saussurea costus belongs to family of Asteraceae is one of the main species of the genus Saussurea [15] which mostly grow in the humid regions at altitude of 2600-4000m in Western Himalayan region of India and Pakistan, and it is also been started to cultivate in new areas for commercial purposes in 1920 [16]. The roots of S. costus are used for the treatment many diseases including chronic gastritis, stomach ulcer, asthma, bronchitis and rheumatoid [17], oil extracts from the roots of S. costus has been used in the treatment of leprosy [18]. Chemical studies of the S. costus plant have shown it contains many phytochemical compounds with biological activity, including alkaloids, flavonoids, sesquiterpene terpenes, anthraquiones and tannins [19,20].
This study aims to determinate the effect of S .costus extracts on viability of E. granulosus protoscoleces In vitro.

Samples collection and protoscoleces isolation:
The infected livers of sheep (figure1) were collected from butcher shops in Mosul city and protoscoleces of E. granulosus were isolated according to Smyth [21] by sterilizing the surface of hydatid cyst with 70% ethanol following by aspiration of hydatid cyst fluid using G21 needle. Protoscoleces were washed three times with phosphate buffered saline (PBS) using centrifuge at 3000rpm for 10minutes, with the addition of 1gm of streptomycin and 20000 UI of penicillin before starting a second wash.

Estimation of protoscoleces viability:
The assessment of the viability of protoscoleces was estimated by taking 20 ul of the hanging of protoscoleces was mixed with 20 ul of 0.1% eosin stain on the glass slide. Protoscoleces that appear by bright green in color considered alive ( figure 2) while protoscoleces red in color dead because stain entry inside it (figure 3) [22].The viability of the protoscoleces in this study was 100%.

Collection and preparation of the S. costus extracts
S. costus plant was bought from perfumery shops in Mosul city. 25 gm from a root of plant powder were added to 500ml of petroleum ether for 72 hours on magnetic stirrer, then solution were filtered by Whatman No.1 filter paper, 500ml Ethanol 70% is added to the precipitant for 72 hours on magnetic stirrer [23], after filtering the solution, distilled water was added to precipitant on magnetic stirrer at medium speed and temperature 60C° to obtain a warm water extract, The solvent was removed by using rotary evaporator at 40C° [24] . Cool aqueous extract was prepared according to Rios et al. method [25], where 40g of root plant powder was added to 400 ml of distilled water following by mixing process using blender and then magnetic stirrer. The samples underwent to soak and mix for 24 hours at 4C°.The samples were filtered using Whatman filter paper No.1 following by store the crude extracts at 4C°, until been used.

Effect of S. costus extracts on protoscoleces viability In vitro
To evaluate the effect extracts on viability of the protoscoleces, experiments were designed that included four different concentrations at four different times, in addition to the control group. In each test tube 1ml of extract dissolved in PBS was added, with the addition of 2000 protoscoleces in the same tube and placed in a water bath at 37C⸰ according to the specified times washed with PBS solution to get rid of the effect of extracts and examined protoscoleces under a light microscope to count the living and dead ones.

Statistical analysis
Data analysis was carried out using the SPSS statistical analysis system, the means and standard error were used, in addition to using Duncon's test to measure a significant difference between the means of experimental study at the level of significance p≤0.05.

Results and Discussion
The results of this study showed the effect of petroleum ether extract S. costus on protoscoleces viability in vitro using four concentrations 5,10,15 and 20 mg/ml at four different times 15,30,45 and 60minutes. The data of petroleum ether extract indicated 0% of viability rate at 10mg/ml and 15mg/ml during 60min, while 20mg/ml indicated 0% of viability at 45 and 60min. The highest percentage of viability (47.785%) reported at 5mg/ml during 15min (see table-1).  The current data showed the effect of hot aquatic extract of S. costus on protoscoleces viability in vitro using four concentrations 300,350,400 and450mg/ml at four different times 15,30,45 and 60min Lowest percentage of vitality rate (0%) Showed at 350mg/ml and 400mg/ml during 60 and 45min, respectively, while 450mg/ml indicated lowest viability percentage (0%) at all experimental time periods. The highest viability percentage (52.165%) was reported at 300mg/ml during 15min. (see table-3).  The viability rate of protoscoleces for petroleum ether extract decreased to zero at concentration 10 and 15mg/ml after 60min and 20mg/ml after exposure time of 45and60min, results are converged with a study of El-Bahy et al. [28] that achieved the maximum mortality rate among the protoscoleces 100% when used Nigella sativa oil at 100mg/ml concentration after 120min with the superiority of a current study in time and focus. The current study also outperformed in terms of achieving a high killing rate compared to the results of Hesari et al. [29] study , where they reported highest mortality rate 4% when used petroleum ether extract of Cucurbita moshata at concentration 10 mg/ml in 60min.
The present study showed that S.costus extracts inhibitive efficiency against protoscoleces of E. granulosus to different concentration at times different . The current data of ethanolic extract indicated 100% kill rate at 20 and 30 mg/ml during 45 and 60min, while the same kill rate was reported at 40mg/ml during all experiment time periods. This data is in an agreement with the data that obtained by previous study [26], where they reported 100% kill rate by using 50mg/ml of ethanolic extract of Salvadora persica at 20min.These results were superior in terms of concentration, time and killing rate on results Al-Aloosi et al. [27] when used alcoholic extract of viscum album showed high killing (80.7%) at 1500mg/ml in60 min.
The concentration 450mg/ml of hot aqueous extract showed highest kill rate (100%) at all experiment time periods. These results are in an agreement with previous data [30], where he reported reduction of protoscoleces viability from 87% to 0% by fourth day of experiment by using hot aqueous extract of ziziphus spina.