Abstract
Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3–23), and three flavonoids (24–26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher’s analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.
Highlights
According to data from the Centers for Disease Control and Prevention (CDC), it is estimated that 5–10% of the human population is infected by seasonal human influenza virus each year, and approximately 290,000 to 650,000 people globally die from influenza-related respiratory diseases [1,2]
Major events that occur in viral replication of influenza A virus (IAV) are as follows: (i) attachment of a virion by interaction between the sialic acid receptor of the host cell and hemagglutinin (HA) on the virion surface; (ii) fusion and endocytosis of the virion into the cytoplasm of the host cell; (iii) release of viral ribonucleoprotein (RNP) into the cytoplasm and nuclear import; (iv) transcription and replication of viral RNA; (v) translation and protein synthesis; (vi) assembly of virion composition; and (vii) budding and release of a newly assembled virion by neuraminidase [4]
The cells were inoculated with influenza viruses (H1N1 A/PR/8/34 virus or H9N2 A/chicken/Korea/01210/2001 virus) at 0.01 MOI using Dulbecco’s modified Eagle’s medium (DMEM) containing 0.15 μg/mL trypsin and 5 μg/mL bovine serum albumin (BSA) (Sigma, St Louis, MO, USA)
Summary
According to data from the Centers for Disease Control and Prevention (CDC), it is estimated that 5–10% of the human population is infected by seasonal human influenza virus each year, and approximately 290,000 to 650,000 people globally die from influenza-related respiratory diseases [1,2]. Pharmaceutical companies have endeavored to develop inhibitors that block any of these aforementioned steps Among these targets, blocking viral entry and fusion has been emerging as a favorable inhibition target because hemagglutinin, especially, HA2 subunit (stem region of HA) responsible for viral fusion is highly conserved and able to trigger an antibody-mediated immune response during infection [5,6], in which the antibodies induced by HA2 show high cross-reactivity, even toward various different subtypes of influenza virus [7]. Neuraminidase inhibitors, including zanamivir and oseltamivir, which are most commonly prescribed to treat flu, have been used globally Their use has been facing limitations due to the rapid emergence of resistant influenza strains and the risk of side effects [15]. Various assays including, Western blot, immunofluorescence assay, flow cytometry, and NO production assay, were performed to investigate the anti-influenza mode of action
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