Abstract：
BACKGROUND: Cystic echinococcosis (CE) is one of the most widespread and important global helminth zoonoses. Treatment relies mainly on surgery and, or percutaneous interventions. However, spillage of live protoscoleces (PSCs) leading to recurrence is a problem during surgery. So, the application of protoscolicidal agents before surgery is required. This study aimed to investigate the activity and safety of hydroalcoholic extracts of E. microtheca against PSCs of Echinococcus granulosus sensu stricto (s.s.) both in vitro and also ex vivo, which is a simulation to Puncture, Aspiration, Injection, and Re-aspiration (PAIR) method.
METHODS: Considering the effects of heat on the protoscolicidal effecacy of Eucalyptus leaves, hydroalcoholic extraction was performed by both soxhlet extraction at 80 °C and percolation at room temperature. The protoscolicidal action of hydroalcoholic extracts was assessed by in vitro and ex vivo assessments. Infected sheep livers were collected from the slaughterhouse. Then, the genotype of hydatid cysts (HCs) was confirmed by sequencing and, isolates were limited to E. granulosus s.s. In the next step, ultrastructural changes of Eucalyptus-exposed PSCs were studied by scanning electron microscopy (SEM). Finally, a cytotoxicity test was conducted by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the safety of E. microtheca.
RESULTS: The prepared extracts by soxhlet extraction and percolation were, successfully exerted strong protoscolicidal effects in both in vitro and ex vivo tests. The results of in vitro assessment indicated that hydroalcoholic extract of E. microtheca prepared by percolation at room temperature (EMP) and hydroalcoholic extract of E. microtheca prepared by soxhlet extraction at 80 °C (EMS) killed all PSCs (100%) at concentrations of 10 and 12.5 mg/mL, respectively. Also, EMP showed 99% protoscolicidal action after 20 min in an ex vivo setting compared to EMS. SEM micrographs confirmed potent protoscolicidal and destructive effects of E. microtheca against PSCs. The cytotoxicity of EMP was tested on the HeLa cell line using MTT assay. The value of 50% cytotoxic concentration (CC50) was calculated at 46.5 μg/mL after 24h.
CONCLUSIONS: Both hydroalcoholic extracts showed potent protoscolicidal activity and, especially EMP produced remarkable protoscolicidal effects compared to the control group.
METHODS: Considering the effects of heat on the protoscolicidal effecacy of Eucalyptus leaves, hydroalcoholic extraction was performed by both soxhlet extraction at 80 °C and percolation at room temperature. The protoscolicidal action of hydroalcoholic extracts was assessed by in vitro and ex vivo assessments. Infected sheep livers were collected from the slaughterhouse. Then, the genotype of hydatid cysts (HCs) was confirmed by sequencing and, isolates were limited to E. granulosus s.s. In the next step, ultrastructural changes of Eucalyptus-exposed PSCs were studied by scanning electron microscopy (SEM). Finally, a cytotoxicity test was conducted by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the safety of E. microtheca.
RESULTS: The prepared extracts by soxhlet extraction and percolation were, successfully exerted strong protoscolicidal effects in both in vitro and ex vivo tests. The results of in vitro assessment indicated that hydroalcoholic extract of E. microtheca prepared by percolation at room temperature (EMP) and hydroalcoholic extract of E. microtheca prepared by soxhlet extraction at 80 °C (EMS) killed all PSCs (100%) at concentrations of 10 and 12.5 mg/mL, respectively. Also, EMP showed 99% protoscolicidal action after 20 min in an ex vivo setting compared to EMS. SEM micrographs confirmed potent protoscolicidal and destructive effects of E. microtheca against PSCs. The cytotoxicity of EMP was tested on the HeLa cell line using MTT assay. The value of 50% cytotoxic concentration (CC50) was calculated at 46.5 μg/mL after 24h.
CONCLUSIONS: Both hydroalcoholic extracts showed potent protoscolicidal activity and, especially EMP produced remarkable protoscolicidal effects compared to the control group.
摘要：
背景：囊性包虫病（CE）是全球最普遍，最重要的蠕虫人畜共患病之一。治疗主要依靠手术,或经皮干预。然而,导致复发的活的原头肌(PSC)的溢出是手术期间的问题。所以,手术前需要使用杀原剂。本研究的目的是研究微藻水醇提取物对细粒棘球蚴（s.s.）PSC的体外和离体的活性和安全性。这是对穿刺的模拟，吸气,注射,和再抽吸(PAIR)方法。
方法：考虑到热量对桉树叶的杀原骨作用的影响，通过在80°C下的索氏提取和在室温下的渗滤进行水醇提取。通过体外和离体评估评估水醇提取物的杀原骨作用。从屠宰场收集感染的绵羊肝脏。然后，通过测序证实了包虫囊肿(HCs)的基因型，分离株仅限于S.S.在下一步中，通过扫描电子显微镜（SEM）研究了暴露于桉树的PSCs的超微结构变化。最后,通过(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)法进行细胞毒性试验,以评价E.microtheca的安全性。
结果：通过索氏提取和渗滤制备的提取物为，在体外和离体测试中都成功地发挥了强大的杀原型作用。体外评估结果表明，通过室温渗滤（EMP）制备的小囊藻水醇提取物和通过索氏提取在80°C（EMS）制备的小囊藻水醇提取物杀死了所有PSC（100％）浓度为10和12.5mg/mL，分别。此外，与EMS相比，EMP在离体环境中20分钟后显示出99％的杀原骨作用。SEM显微照片证实了E.microtheca对PSC的有效的杀原骨和破坏作用。用MTT法测定EMP对HeLa细胞系的细胞毒性。在24小时后,50%细胞毒性浓度(CC50)的值计算为46.5μg/mL。
结论：两种水醇提取物均显示出有效的杀原骨活性，与对照组相比，尤其是EMP产生了显着的杀原骨作用。
方法：考虑到热量对桉树叶的杀原骨作用的影响，通过在80°C下的索氏提取和在室温下的渗滤进行水醇提取。通过体外和离体评估评估水醇提取物的杀原骨作用。从屠宰场收集感染的绵羊肝脏。然后，通过测序证实了包虫囊肿(HCs)的基因型，分离株仅限于S.S.在下一步中，通过扫描电子显微镜（SEM）研究了暴露于桉树的PSCs的超微结构变化。最后,通过(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)法进行细胞毒性试验,以评价E.microtheca的安全性。
结果：通过索氏提取和渗滤制备的提取物为，在体外和离体测试中都成功地发挥了强大的杀原型作用。体外评估结果表明，通过室温渗滤（EMP）制备的小囊藻水醇提取物和通过索氏提取在80°C（EMS）制备的小囊藻水醇提取物杀死了所有PSC（100％）浓度为10和12.5mg/mL，分别。此外，与EMS相比，EMP在离体环境中20分钟后显示出99％的杀原骨作用。SEM显微照片证实了E.microtheca对PSC的有效的杀原骨和破坏作用。用MTT法测定EMP对HeLa细胞系的细胞毒性。在24小时后,50%细胞毒性浓度(CC50)的值计算为46.5μg/mL。
结论：两种水醇提取物均显示出有效的杀原骨活性，与对照组相比，尤其是EMP产生了显着的杀原骨作用。
