Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, Republic of Korea.
Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, Republic of Korea; College of Natural Sciences, Cantho University, Campus II, Cantho City, Viet Nam.
J Ethnopharmacol. 2020 Sep 15;259:112945. doi: 10.1016/j.jep.2020.112945. Epub 2020 May 7.
Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza.
Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation.
Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence.
The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC (μM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 μM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax.
Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).
白芷（Hoffm.）Benth. 和 Hook.f. ex Franch. 和 Sav.（伞形科）是一种草本、多年生植物，原产于亚洲北部和东部。白芷的根传统上被称为“白芷”，用作感冒药、头晕、溃疡和风湿的药用植物。此外，它也是各种处方的重要成分，如用于感冒和流感的 Gumiganghwal-Tang。
尽管已经报道了白芷根的各种生物活性及其化学成分，但在化合物水平上其发挥抗流感活性的详细机制尚未得到研究。因此，我们研究了通过生物活性导向分离得到的呋喃香豆素的抗流感特性。
从白芷根的 70%乙醇提取物中进行生物活性导向分离，产生了四种活性呋喃香豆素。评估细胞病变效应（CPE）的抑制作用，以确定这些化合物对甲型流感（H1N1 和 H9N2）病毒的抗病毒活性。最有效的化合物进行了详细的机制研究，如病毒蛋白合成抑制、病毒复制周期不同阶段的 CPE 抑制、神经氨酸酶（NA）抑制、使用流式细胞术的抗凋亡活性以及免疫荧光。
生物活性导向分离从正丁醇部分产生了四种活性呋喃香豆素，即异茴芹素（1）、氧化前胡素（2）、氧化前胡素水合物（3）和茴芹素（4）。其中，化合物 2（其次是化合物 1、4 和 3）表现出显著的 CPE 抑制作用，对 H1N1 和 H9N2 的 EC（μM）分别为 5.98±0.71 和 4.52±0.39，比阳性对照利巴韦林更强。化合物 2 呈剂量依赖性抑制 NA 和核蛋白（NP）的合成。在时间过程测定中，在用化合物 2 处理后 1 和 2 小时感染流感 A 感染的 MDCK 细胞的细胞病变效应减少了 80-90%，并且在感染后 3 小时急剧下降。与未感染细胞相比，用化合物 2（20 μM）处理的细胞中，两种蛋白的病毒 NA 和 NP 产量均降低至低于 20%，在感染后 2 小时。在分子对接分析中，化合物 2 对聚合酶酸性蛋白（PAC；-36.28 kcal/mol）的 C 端表现出比其他两个聚合酶亚基更强的结合亲和力。化合物 2 还对病毒感染细胞发挥抗凋亡作用，并显著抑制病毒感染诱导的 caspase-3 和 Bax 的 mRNA 表达。
我们的结果表明，化合物 2 可能通过抑制病毒复制周期的早期阶段发挥抗甲型流感活性，而不是直接中和表面蛋白（如血凝素和 NA），以及病毒感染引起的异常凋亡。综上所述，这些发现表明，伞形科中占优势的呋喃香豆素在其抗病毒活性中发挥着关键作用。这些发现还可以解释为什么这种植物在民族药理学中被用作许多传统中药（TCM）抗病毒处方的重要成分。
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