Table1_Antiviral effects of the petroleum ether extract of Tournefortia sibirica L. against enterovirus 71 infection in vitro and in vivo.DOC

Enterovirus 71 (EV71) is the major cause of severe hand, foot, and mouth disease (HFMD). Compared to other HFMD pathogens, like coxsackievirus A16 (CVA16), EV71 can invade the central nervous system and cause permanent damage. At present, there are no available antivirals against EV71 for clinical treatment. Herein, multiple Chinese botanical drugs were collected, and 47 types of botanical extracts were extracted using aqueous solutions and organic solvents. Based on the cytopathic effect inhibition assay, petroleum ether extract of Tournefortia sibirica L. (PE-TS) demonstrated 97.25% and 94.75% inhibition rates for EV71 infection (at 250 μg/ml) and CVA16 infection (at 125 μg/ml), respectively, with low cytotoxicity. Preliminary mechanistic studies showed that PE-TS inhibits replication of EV71 genomic RNA and synthesis of the EV71 protein. The released extracellular EV71 progeny virus titer decreased by 3.75 lg under PE-TS treatment. Furthermore, using a newborn mouse model, PE-TS treatment protected 70% and 66.7% of mice from lethal dose EV71 intracranial challenge via administration of intraperitoneal injection at 0.4 mg/g and direct lavage at 0.8 mg/g, respectively. The chemical constituents of the PE-TS were analyzed by Gas Chromatography-Mass Spectrometer (GC-MS), and a total of 60 compounds were identified. Compound-target network analysis and molecular docking implied potential bioactive compounds and their protein targets against EV71 associated pathology. The present study identified antiviral effects of PE-TS against EV71/CVA16 infection in vitro and EV71 infection in vivo, providing a potential antiviral botanical drug extract candidate for HFMD drug development.

肠道病毒71型（EV71）是手足口病（HFMD）的主要致病因素。与其他手足口病病原体，如柯萨奇病毒A16（CVA16）相比，EV71能够侵袭中枢神经系统并造成永久性损伤。目前，针对EV71尚无有效的抗病毒药物用于临床治疗。本研究收集了多种中草药，采用水溶液和有机溶剂提取了47种草药提取物。基于细胞病变效应抑制实验，西伯利亚唐松草（Tournefortia sibirica L.）的石油醚提取物（PE-TS）对EV71感染（250 μg/ml）和CVA16感染（125 μg/ml）分别显示出97.25%和94.75%的抑制率，同时具有较低的细胞毒性。初步的机制研究表明，PE-TS能够抑制EV71基因组RNA的复制和EV71蛋白的合成。在PE-TS处理下，释放的细胞外EV71子代病毒滴度降低了3.75 lg。此外，通过新生小鼠模型，PE-TS以0.4 mg/g的剂量通过腹腔注射和0.8 mg/g的直接冲洗，分别保护了70%和66.7%的小鼠免受致死剂量EV71颅内挑战。PE-TS的化学成分通过气相色谱-质谱联用（GC-MS）进行分析，共鉴定出60种化合物。化合物-靶点网络分析和分子对接暗示了潜在的生物活性化合物及其针对EV71相关病理的蛋白靶点。本研究在体外和体内均证实了PE-TS对EV71/CVA16感染的抗病毒作用，为HFMD药物开发提供了潜在的抗病毒草药提取物候选物。