Elsevier

Journal of Ethnopharmacology

Volume 259, 15 September 2020, 112945
Journal of Ethnopharmacology

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2

https://doi.org/10.1016/j.jep.2020.112945Get rights and content

Abstract

Ethnopharmacological relevance

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.

Aim of the study

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.

Materials and methods

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.

Results

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 EC50 (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.

Conclusion

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).

Introduction

The world health organization (WHO) stated that approximately three quarters of world's population depends on natural medicine, mainly medicinal plants in different civilizations (Gilani and Rahman, 2005). Many countries have utilized a variety of formulas composed of various medicinal plants for the treatment of viral infection, such as influenza. Though pure compounds and synthetic drugs, which represent conventional medicine today, largely replaced the use of herbal medicine, there is a growing recognition of the herbal medicine as its effectiveness have been investigated scientifically (Eng et al., 2019). Anti-influenza properties of some representative formulas in traditional Korean medicine, such as Ma-Huang-Tang, and Galgeun-Tang were previously reported. Ma-Huang-Tang, which is composed of four medicinal plants and commonly used for treating the acute upper respiratory infection, was shown to reduce cytopathic effect induced by H1N1 infection and significantly attenuate histopathological changes and excessive secretion of inflammatory cytokines in an ICR pneumonia mouse model (Wei et al., 2018). Also, Galgeun-Tang, which is used for the patients with symptoms of headache, stiffness of neck, and fever, was reported to exert anti-influenza activity by increasing IL-12 production in the early phase of viral cycle (Kurokawa et al., 2002). A clinical trial demonstrated that a time to fever resolution in H1N1-infected participants was reduced by 19% in the group of patients prescribed with a combination of oseltamivir and maxingshigan-yinqiaosan compared to the group with oseltamivir C. Wang et al., 2011). Gumiganghwal-Tang (jiuweiqianghuo tang) listed in Dong-Eu-Bo-Gam (Heo, Joon, AD 1713) of Korea ancient pharmacopoeias, has been used for common cold and influenza with headache, body aches and fever regardless of seasons (Moon et al., 1999; Jin et al., 2014). As main one of its constituting medicinal herb, Angelica dahurica (under the name “Bai Zhi” in China) contributes to its overall activity by exerting antipyretic, analgesic, and anti-inflammatory properties (Choi et al., 2008).

Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., a perennial plant of the Umbelliferae (alt. Apiaceae), is widely distributed in northeastern China and Korea. The root of A. dahurica is used as a traditional folk medicine in China and Korea for abscesses, headaches, the common cold, fever, inflammation, flu, etc (Sarker and Nahar, 2004). Various laboratory-identified biological activities have been reported, including anti-inflammatory (Kang et al., 2007), anti-influenza A (Park and Lee, 2005), antiobesity (Lu et al., 2016), wound healing (Zhang et al., 2017) and antioxidant (Pervin et al., 2014) activities at even crude extract levels. For more detail, active compounds with a myriad of activities, such as anticancer (Luo et al., 2011), antibacterial, antifungal (Walasek et al., 2015), antiallergy (Xian et al., 2019), antihypertension (Zhang et al., 2011), cholinesterase inhibition (Tun and Kang, 2017), anti-human immunodeficiency virus (HIV), and anti-herpes simplex virus (HSV) activity (Koziol and Skalicka-Woźniak, 2016), have been studies. In addition, A. dahurica contains coumarin as the major class of compounds in the Angelica genus (Shokoohinia, 2014), steroids (Li and Wu, 2017), and aromatic essential oil attributed to myriad of monoterpenes and sesquiterpenes (Kim and Chi, 1990). Although there have been many studies about the bioactivity of the constituents isolated from A. dahurica in terms of their traditional uses, such as sweat-inducing herbs to combat harmful external pathogens, few studies about the direct antiviral property of compounds against influenza A virus have been conducted. Therefore, based on our screening data combined with literature reviews, we performed bioactivity-guided isolation and examined the efficacies of the isolates along with performing detailed mechanistic studies of the most active compound by using CPE assays, Western blotting, immunofluorescence, flow cytometry, etc. Moreover, we determined the content of these isolated compounds in the 70% EtOH total extract to determine the activity of the extract and the history of ethnopharmacological uses.

Section snippets

General information

A JEOL 600 MHz spectrometer (JEOL, Tokyo, Japan) with deuterated solvents was used for nuclear magnetic resonance (NMR) spectroscopy. Column chromatography (CC) was performed using various resins, including silica gel (particle size: 63–200 μm, Zeochem AG, Rüti, Switzerland) and reversed-phase (RP) C18 (particle size: 40–63 μm, Nacalai Tesque, Kyoto, Japan). Silica gel 60 F254 and RP-18 F254S thin-layer chromatography (TLC) plates were purchased from Merck (Darmstadt, Germany). A Gilson

Quantitative analysis of four furanocoumarins isolated from A. dahurica using HPLC-DAD

To quantify the four active furanocoumarins (Fig. 1) in the 70% EtOH extract of A. dahurica, an appropriate validation method was established by using HPLC-DAD. Linearity data, including the correlation regression equation, coefficient (R), LOD and LOQ, were successfully obtained. The intra- and interday precision and accuracy values were also calculated, and the % relative standard deviation (RSD) values of these assays were less than 5%, indicating that this method was well validated (Table 2

Conclusion

In our continuous efforts to find promising anti-influenza agents, bioactivity-guided isolation was performed to isolate four representative active furanocoumarins from the active 70% EtOH extract of A. dahurica. These four isolates were found to have dose-dependent CPE inhibition activity against H1N1 and H9N2. Of the compounds, as compound 2 exhibited the strongest activity in both subtypes, it was subjected to detailed mechanistic studies. Compound 2 was shown to exert its anti-influenza

Author contributions

Ba Wool Lee ([email protected]).

- performed quantitative analysis, validation and molecular docking analysis of compounds and wrote the manuscript.

Thi Kim Quy Ha ([email protected]).

- designed and performed biological experiments.

Hyo Moon Cho ([email protected]) and Jin-Pyo An ([email protected]).

- aided fractionation, isolation and characterization of compounds from A. dahurica.

Seong Kyun Kim ([email protected]) and Choong-Sik Kim ([email protected]).

- purified and provided virus stocks of

Funding

This work was supported financially by grants from the Korea Bioactive Natural Material Bank (NRF-2017M3A9B8069409) of the National Research Foundation of Korea (NRF) which is funded by the Ministry of Science and ICT and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Animal Disease Management Technology Development Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (318031031SB010).

Declaration of competing interest

All authors declare that they have no conflict of interest regarding this study.

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