背景:已知Erigeron属植物具有抗病毒活性,包括针对呼吸道合胞病毒(RSV)的那些。在传统医学中,Erigeronannuus(L.)Pers(EA)已用于治疗肺部疾病和急性传染性肝炎。
目的:本研究的目的是确定最佳提取方法,以生产最有效的抗RSV提取物,在体外和体内模型中阐明其抗病毒活性的模式和机制,并确定生物活性化合物的化学结构。
方法:用乙酸乙酯提取整个EA,甲醇,乙醇,水,含水甲醇(60、80%和100%)和含水乙醇(50、75%和95%)使用浸渍,反流,和超声辅助提取方法。体外测定提取物的抗病毒活性。使用Hep-2细胞测定提取物的体外抗病毒活性。进行了四个体外实验,以确定最具活性的提取物的抗病毒活性模式,用50%乙醇回流制备的灯盏花素全植物提取物的乙酸乙酯部分(EAE),通过检查它直接灭活病毒的能力,抑制病毒的吸附和渗透,抑制病毒复制和预防作用。还确定了温度和处理持续时间对这些作用模式的影响。还在小鼠模型中体内评估EAE的抗病毒活性。肺指数,病毒载量,和肺组织组织学测量。进行qRT-PCR和ELISA研究以确定关键基因(TLR-3和TLR-4)和蛋白质(IL-2,IFN-γ,和TNF-α)与RSV感染有关。使用色谱技术分离出最具活性的抗病毒化合物,并通过电喷雾三重四极杆质谱和核磁共振谱鉴定了其化学结构。
结果:EAE对RSV最活跃。体外实验表明,EAE的抗病毒活性是通过直接灭活,抑制进入,和抑制病毒的增殖。体内实验表明,EAE有效抑制RSV在肺部的增殖,减轻RSV感染小鼠的肺组织损伤。EAE的抗病毒活性是通过下调肺TLR3和TLR4的表达来介导的,上调IL-2和IFN-γ的表达,下调TNF-α的表达。发现芹菜素7-O-甲基葡糖苷酸是EAE中的主要生物活性化合物。
结论:这项研究的结果证实了通过灭活EA的抗病毒活性,禁止进入,并抑制RSV的增殖。该活性通过调节免疫和炎症介质来介导。芹菜素7-O-甲基葡糖苷酸是EA中存在的生物活性化合物。
BACKGROUND: The plants in the genus Erigeron are known to exhibit antiviral activities, including those against the respiratory syncytial virus (RSV). In traditional medicine Erigeron annuus (L.) Pers (EA) has been used in the treatment of pulmonary diseases and acute infectious hepatitis.
OBJECTIVE: The aim of this study is to determine the optimum extraction method to produce the most potent anti-RSV extract, elucidate its mode and mechanisms of antiviral activity in both in vitro and in vivo models, and identify the chemical structures of the bioactive compounds.
METHODS: The whole plant of EA was extracted with ethyl acetate, methanol, ethanol, water, aqueous methanol (60, 80% and 100%) and aqueous ethanol (50, 75% and 95%) using maceration, reflux, and ultrasound-assisted extraction methods. The antiviral activities of the extracts were determined in vitro. The in vitro antiviral activities of the extracts were determined using Hep-2 cells. Four in vitro experiments were performed to determine the mode of antiviral activity of the most active extract, ethyl acetate fraction (EAE) of Erigeron annuus whole plant extract prepared by refluxing with 50% ethanol, by examining its ability to inactivate the virus directly, inhibit viral adsorption and penetration, inhibit viral replication and preventive effect. The effect of temperature and duration of treatment on these modes of action was also determined. The antiviral activity of the EAE was also assessed in vivo in a mouse model. The lung index, viral load, and lung tissue histology were measured. qRT-PCR and ELISA studies were performed to determine the expression of key genes (TLR-3 and TLR-4) and proteins (IL-2, IFN-γ, and TNF-α) related to RSV infection. The most active antiviral compound was isolated using chromatography techniques, and its chemical structure was identified through electrospray triple quadrupole mass spectroscopy and nuclear magnetic resonance spectroscopy.
RESULTS: The EAE was the most active on RSV. In vitro experiments showed that the antiviral activity of EAE is via direct inactivation, inhibition of entry, and inhibition of the proliferation of the virus. In vivo experiments showed that the EAE effectively inhibited the proliferation of RSV in the lungs and alleviated the lung tissue lesions in RSV-infected mice. The antiviral activity of the EAE is mediated by downregulating the expression of TLR3 and TLR4 in the lung, upregulating the expression of IL-2 and IFN-γ, and downregulating the expression of TNF-α. Apigenin 7-O-methylglucuronide was found to be a major bioactive compound in EAE.
CONCLUSIONS: The results of this study confirmed the antiviral activity of EA by inactivating, inhibiting the entry, and inhibiting the proliferation of RSV. The activity is mediated by regulating the immunity and inflammatory mediators. Apigenin 7-O-methylglucuronide is the bioactive compound present in EA.