Table_3_Willow (Salix spp.) bark hot water extracts inhibit both enveloped and non-enveloped viruses: study on its anti-coronavirus and anti-enterovirus activities.xlsx

IntroductionRecurring viral outbreaks have a significant negative impact on society. This creates a need to develop novel strategies to complement the existing antiviral approaches. There is a need for safe and sustainable antiviral solutions derived from nature.
ObjectiveThis study aimed to investigate the antiviral potential of willow (Salix spp.) bark hot water extracts against coronaviruses and enteroviruses. Willow bark has long been recognized for its medicinal properties and has been used in traditional medicines. However, its potential as a broad-spectrum antiviral agent remains relatively unexplored.
MethodsCytopathic effect inhibition assay and virucidal and qPCR-based assays were used to evaluate the antiviral potential of the bark extracts. The mechanism of action was investigated using time-of-addition assay, confocal microscopy, TEM, thermal, and binding assays. Extracts were fractionated and screened for their chemical composition using high-resolution LC-MS.
ResultsThe native Salix samples demonstrated their excellent antiviral potential against the non-enveloped enteroviruses even at room temperature and after 45 s. They were equally effective against the seasonal and pandemic coronaviruses. Confocal microscopy verified the loss of infection capacity by negligible staining of the newly synthesized capsid or spike proteins. Time-of-addition studies demonstrated that Salix bark extract had a direct effect on the virus particles but not through cellular targets. Negative stain TEM and thermal assay showed that antiviral action on enteroviruses was based on the added stability of the virions. In contrast, Salix bark extract caused visible changes in the coronavirus structure, which was demonstrated by the negative stain TEM. However, the binding to the cells was not affected, as verified by the qPCR study. Furthermore, coronavirus accumulated in the cellular endosomes and did not proceed after this stage, based on the confocal studies. None of the tested commercial reference samples, such as salicin, salicylic acid, picein, and triandrin, had any antiviral activity. Fractionation of the extract and subsequent MS analysis revealed that most of the separated fractions were very effective against enteroviruses and contained several different chemical groups such as hydroxycinnamic acid derivatives, flavonoids, and procyanidins.
ConclusionSalix spp. bark extracts contain several virucidal agents that are likely to act synergistically and directly on the viruses.

引言 反复发作的病毒性疫情对社会造成了显著的负面影响，这促使我们亟需开发新型策略以补充现有抗病毒手段，同时也亟需获取源自自然的安全、可持续的抗病毒解决方案。
研究目的 本研究旨在探究柳属（Salix spp.）树皮热水提取物针对冠状病毒与肠道病毒的抗病毒活性。柳属树皮的药用价值早已得到公认，并被广泛应用于传统医药中，但其作为广谱抗病毒制剂的潜力却尚未得到充分探索。
研究方法 本研究采用细胞病变效应抑制试验、杀病毒试验及基于定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）的试验，以评估柳树皮提取物的抗病毒活性。通过时间加样试验、共聚焦显微镜、透射电子显微镜（Transmission Electron Microscopy, TEM）、热稳定性试验及结合试验，探究其作用机制。此外，通过分级分离提取组分，并采用高分辨率液相色谱-质谱联用（High-resolution LC-MS）对提取物的化学组成进行分析筛选。
研究结果 天然柳属树皮提取物即便在室温条件下作用45秒后，仍对无包膜肠道病毒展现出优异的抗病毒活性，同时对季节性冠状病毒与大流行冠状病毒亦具有同等的抑制效果。共聚焦显微镜观测结果显示，新合成的病毒衣壳或刺突蛋白染色极弱，证实病毒的感染能力已丧失。时间加样试验表明，柳属树皮提取物可直接作用于病毒颗粒，而非通过细胞靶点发挥作用。负染透射电子显微镜（TEM）及热稳定性试验结果显示，其对肠道病毒的抗病毒作用是通过增强病毒粒子的稳定性实现的。与之相对，负染TEM观测证实，柳属树皮提取物可使冠状病毒的结构发生肉眼可见的改变。但qPCR试验验证显示，提取物并未影响病毒与宿主细胞的结合能力。此外，共聚焦成像研究表明，冠状病毒会在细胞内体中积聚，无法完成后续感染周期。本次测试的商用对照样品，包括水杨苷（salicin）、水杨酸（salicylic acid）、皮西因（picein）以及三蕊草苷（triandrin），均未表现出任何抗病毒活性。对提取物进行分级分离并辅以质谱分析后发现，多数分离组分对肠道病毒均具有显著抑制效果，且包含羟基肉桂酸衍生物、黄酮类化合物以及原花青素等多种化学组分。
研究结论 柳属（Salix spp.）树皮提取物中含有多种杀病毒活性成分，这些成分可能通过协同作用直接靶向病毒颗粒。