乳酸桿菌Pg4與中草藥的抗病毒能力評估

Abstract

動物常因高密度飼養使其對疾病敏感性增加，導致消化道與呼吸道易受環境中病原菌或病毒感染，甚至死亡，造成重大經濟損失，田間普遍使用抗生素或接種疫苗以預防或治療疾病，然而疫苗未能提供針對異源亞型病毒（heterosubtypic viruses）的保護。此外由於病毒的高突變性及容易產生抗藥性，使得目前的抗病毒藥物已無法有效的抑制或滅殺病毒，因此開發新的抗病毒藥物是必要的。近年來研究微生物製劑和中草藥製劑作為飼料添加物，以預防或治療疾病，頗受重視。益生菌常應用於維持動物腸道菌相之平衡，而中草藥長期以來被用於多種類型的疾病治療，其具有天然生物活性，常以複方形式維持動物或人體健康。 本研究之目的為探討乳酸桿菌Lactobacillus reuteri Pg4和中草藥（茵陳蒿湯、梔子仁、黃芩、黃連、甘草、南板藍、北柴胡以及菊花）之抗病毒能力，透過兩者之加成作用，評估其有效成分之聯合作用是否具有更佳的抗病毒能力，並進一步分析抗病毒機制。實驗分成三部分，第一階段取上述中草藥之水萃物，評估其對細胞之細胞毒性以及抗病毒活性。第二階段探討抗病毒機制的部分，透過病毒感染的時間效應分析、平均螢光強度，以及第一型干擾素及其相關下游基因，如ISG15、ISG56、Mx1和PKR等表現量，分析其可能之抗病毒機制。最後，進一步探討何種中草藥活性成分具有抗病毒活性，利用高效液相層析法（high-performance liquid chromatography, HPLC）分析在細胞預處理的階段，具有抗病毒活性之中草藥之主要活性成分含量是否有差異，並評估這些活性成分之抗病毒能力。 實驗證實，梔子、甘草、菊花以及茵陳蒿湯皆可在BHK細胞和Caco-2細胞模型中觀察到抗Sindbis virus（SBV）活性，其中梔子和茵陳蒿湯具有較佳的應用潛力；L. reuteri Pg4的胞內萃取物以及調整pH值之胞外培養液在BHK細胞中也觀察到具有良好的抗病毒能力，然而在Caco-2細胞中卻沒有觀察到L. reuteri Pg4具有抗病毒能力。透過時間效應分析，發現在病毒感染前或與病毒同時添加上述中草藥，可觀察到抗病毒活性，推測其抗病毒機制分別為預防病毒附著、預防病毒進入宿主細胞以及直接殺死病毒；而菊花不僅具有預防病毒感染之功效，在感染初期，亦可抑制病毒轉譯和複製。實驗也證實L. reuteri Pg4胞內萃取物對BHK細胞具有預防感染SBV之作用，以及在感染初期，可以抑制病毒轉譯和複製。利用第一型干擾素下游基因表現量分析，L. reuteri Pg4胞內萃取物抗病毒效果之途徑也可能是透過第一型干擾素下游之抗病毒基因Mx1所提供。梔子、柴胡以及菊花則可調控宿主細胞免疫，誘發第一型干擾素下游基因反應，如PKR、ISG15、ISG56等，以抑制病毒。而這些具有抗病毒活性的中草藥，其活性成分如菊花的綠原酸以及茵陳蒿湯的6,7-二甲氧基香豆精具有抗病毒活性。本研究更證實某些中草藥具有抗發炎及抗病毒活性，故飼料中添加中草藥不失為改善抗生素濫用問題和提升畜禽免疫力的方法之一，具有應用在經濟動物之潛力。

Due to high density populations of livestock and poultry in confinement, the infectious diseases spread rapidly and cause huge economic losses. The lack of vaccine availability and the existence of drug-resistant viruses further increase the impact of viral infectious diseases on animal industry. Thus, the development of novel antiviral drugs is necessary. The ban of antibiotic growth promoters in feed increase the studies of probiotics and Chinese herbal preparations as the alternative to antibiotics. Probiotics are used to maintain the balance of intestinal microflora, while Chinese herbal medicines are natural plants with particular bio-activities and have been used in the prevention and treatment of infectious diseases for a long history. The main purpose of this study is to investigate the antiviral activities of Lactobacillus reuteri Pg4 and the Chinese herbal medicines including Yin Chen Hao Tang, Gardeniae fructus, Scutellariae radix, Coptidis rhizome, Glycyrrhizae radix et rhizome, Baphicacanthi cusiae Rhizoma et radix, Bupleuri radix, and Chrysanthemi flos. The study was divided into three parts. First, the cytotoxicity and antiviral activity of the water extract of the Chinese herbal medicines and L. reuteri Pg4 were evaluated. Second, the antiviral mechanisms of the Chinese herbal medicines and L. reuteri Pg4 were evaluated by analysing time course, mean fluorescence intensity, and determining the transcription levels of the type I interferon related genes of the host cells by using quantitative real time-PCR (qRT-PCR）. Third, the contents of the major active ingredients of the Chinese herbal medicines were determined by high-performance liquid chromatography (HPLC) and their antiviral activities were evaluated. The results of cytotoxicity and antiviral activity assays for the Chinese herbal medicines revealed that the following herbs showed a low cytotoxicity and a high selectivity index (CC50/IC50）: Yin Chen Hao Tang, Gardeniae fructus, and Chrysanthemi flos. In the antiviral experiment, the intracellular extract and extracellular fluid (adjusted the pH value to 7.2） of the L. reuteri Pg4 could decrease the replication of SBV in the BHK cells, similar to the pure huIFN protein. According to the results of time course and qRT-PCR analyses, the antiviral activities of the Chinese herbal medicines and the intracellular extract of the L. reuteri Pg4 could be attributed to the inhibition of the viral invasion into the host cell, direct killing of the virus, or improving the expression of the type I interferon related genes such as PKR, ISG15, and ISG56 by the host cells. The active ingredients of these antiviral Chinese herbal medicines, such as chlorogenic acid in Chrysanthemi flos and 6,7-dimethylesculetin in Yin Chen Hao Tang, showed antiviral activity. In conclusion, this study demonstrated that L. reuteri Pg4 and the Chinese herbal medicines, including Yin Chen Hao Tang, Gardeniae fructus, and Chrysanthemi flos have the a potential to be used as feed additives with antiviral activity.