Graduate Institute of Biomedical Sciences, Chang Gung University, Kweishan, Taoyuan 333, Taiwan.
J Ethnopharmacol. 2012 Dec 18;144(3):533-44. doi: 10.1016/j.jep.2012.09.035. Epub 2012 Oct 3.
Ching-fang-pai-tu-san (CFPTS) is a Chinese herbal decoction that is used as a cure for the common cold, fever, headache, and poor circulation. However, no previous studies have investigated the mode of action of CFPTS against influenza virus infections. To investigate the antiviral mechanism of CFPTS, we examined viral entry, transcription, translation, viral glycoprotein hemagglutinin (HA) transport, and budding of the influenza virus.
The antiviral activity of nontoxic concentrations of CFPTS against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of CFPTS action was first examined with a time-of-addition assay of synchronized infections, followed by monitoring HA transport by immunofluorescence microscopy. Viral endocytosis was evaluated with attachment and penetration assays. The inhibition of viral replication was measured by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity and hemagglutinin activity assays.
Based on the inhibition of the virus-induced cytopathic effect in Madin-Darby canine kidney cells, the EC(50) of CFPTS was about 1.44 ± 0.22 mg/mL against influenza virus A/WSN/33. CFPTS displayed a broad spectrum of inhibitory activities against different strains of influenza A virus, as well as some enteroviruses. However, this extract proved less effective against clinical oseltamivir-resistant strains and influenza B viruses. CFPTS did not suppress viral RNA or protein synthesis. According to a time-of-addition assay, the antiviral mechanism of CFPTS may involve viral budding or intracellular viral glycoprotein transport. A plaque reduction assay showed that CFPTS reduced both the plaque size and plaque quantity. The intracellular transport of viral glycoprotein hemagglutinin was blocked by CFPTS by immunofluorescence microscopic analysis. Thus, it is possible that the antiviral mechanism of CFPTS might inhibit the assembly of progeny virions and/or their subsequent release.
Our results give scientific support to the use of CFPTS in the treatment of influenza virus infections. CFPTS has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.
清芳派土散（CFPTS）是一种中药方剂，用于治疗感冒、发热、头痛和血液循环不良。然而，以前没有研究调查 CFPTS 对流感病毒感染的作用模式。为了研究 CFPTS 抗病毒的机制，我们研究了流感病毒的病毒进入、转录、翻译、病毒糖蛋白血凝素（HA）转运和出芽。
通过检测（中和试验）其对病毒诱导的细胞病变效应的抑制作用，研究无毒浓度的 CFPTS 对流感病毒 A/WSN/33 的抗病毒活性。首先用同步感染的加药时间测定法检查 CFPTS 的作用方式，然后通过免疫荧光显微镜监测 HA 转运。用附着和渗透试验评估病毒内吞作用。通过定量实时 PCR、免疫印迹和免疫荧光显微镜检查来测量病毒复制的抑制作用。我们还进行了与病毒进入抑制相关的试验，如神经氨酸酶活性和血凝素活性试验。
根据在 Madin-Darby 犬肾细胞中抑制病毒诱导的细胞病变效应，CFPTS 对流感病毒 A/WSN/33 的 EC50 约为 1.44±0.22mg/ml。CFPTS 对不同株流感 A 病毒和一些肠道病毒表现出广谱的抑制活性。然而，该提取物对临床奥司他韦耐药株和 B 型流感病毒的效果较差。CFPTS 不能抑制病毒 RNA 或蛋白质的合成。根据加药时间测定法，CFPTS 的抗病毒机制可能涉及病毒出芽或细胞内病毒糖蛋白转运。噬斑减少试验表明，CFPTS 减少了噬斑大小和数量。免疫荧光显微镜分析显示，CFPTS 阻断了病毒糖蛋白血凝素的细胞内转运。因此，CFPTS 的抗病毒机制可能抑制了子代病毒颗粒的组装和/或随后的释放。
我们的结果为 CFPTS 在流感病毒感染治疗中的应用提供了科学依据。CFPTS 可能具有在季节性流感大流行中管理流感病毒感染的潜力，与其他临床可用药物一样。
