Antiviral Activities of Compounds Isolated from Pinus densiflora (Pine Tree) against the Influenza A Virus

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.

Keywords: H1N1; Pinus densiflora; anti-inflammation; anti-influenza; cytopathic effect; neuraminidase.

Figure 1

(A) Key HMBC (arrows) and COSY (bold) correlations of compounds 1 and 2. (B) Data from the modified Mosher’s method for 2a.

Figure 2

Chemical structures of compounds isolated from Pinus densiflora.

Figure 3

Antiviral activities of isolated compounds against the H1N1 A/PR/8/34 virus in the cytopathic effect (CPE) inhibition and neuraminidase (NA) inhibition assays. (A) The percent cell survival was evaluated by the effects of all isolated compounds (1–26) and ribavirin (Rib), a positive control, at 10 µM with the use of the CPE inhibition assay. (B) The inhibitory effects of compounds 3–26 at 50 µM and oseltamivir (Oselt; 100 nM), a positive control, on NA from H1N1. (C) The influence of compounds 5 and 24 (5, 10, and 20 μM) on cell viability was investigated using the CPE inhibition assay. Values are expressed as the mean ± SD of three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the virus control group (one-way analysis of variance followed by a two-tailed Student’s t-test).

Figure 4

Relative mRNA and protein viral expression levels decreased after treatment with several compounds from P. densiflora. (A) Madin–Darby Canine Kidney (MDCK) cells were infected with the H1N1 virus for 2 h and then treated with compounds 5, 8, 10, 20, 23, and 24 at various concentrations. After 24 h of incubation, viral NA and HA were evaluated by Western blot analysis. Data are presented as the mean ± SD (n = 2–4), * p < 0.05, ** p < 0.01, and # p < 0.05, ## p < 0.01 compared to the NA and HA virus control groups, respectively. (B) Compound 5 reduced NA in the cytoplasm of viral-infected cells as measured by the immunofluorescence method. After infection with the H1N1 virus for 2 h, MDCK cells were incubated with compound 5 or ribavirin as a positive control for 24 h. The cells were then fixed, stained, and visualized using a fluorescence microscope. (C) Compound 5 decreased the mRNA levels of NA and HA induced by virus replication in MDCK cells as assessed by real-time PCR. Values are expressed as the mean ± SD (n = 3), *** p < 0.001, and # p < 0.05, ## p < 0.01 compared to the NA and HA virus control groups, respectively.

Figure 5

Inhibition mode of compound 24 on the NA H1N1/PR/8/34 and H9N2 A/Chicken/Korea/O1310/2001 enzymes. Lineweaver–Burk plots for the inhibition of compound 24 (A) at three concentrations (C1 = 10 μM, C2 = 20 μM, and C3 = 40 μM) on NA from the H1N1 virus and (B) at three other concentrations (C1 = 5 μM, C2 = 10 μM, and C3 = 20 μM) on NA from the H9N2 virus for hydrolysis of the substrate. The values were analyzed in three replicates at each substrate concentration.

Figure 6

Illustration of the molecular docking simulation and 2D diagram of the interactions between (A) NP and compound 5, (B) PA-PB1 and compound 5, (C) NA and compound 24, and (D) NA mutant and compound 24 using Discovery Studio Client v19.1.0.18287/CDOCKER software. The structures of the proteins were obtained from the Protein Data Bank (http://www.pdb.org) (PDB ID code: 3RO5, 2ZNL, 3TI4, and 3CL0 for A-D, respectively).

Figure 7

Effects of compounds 5 and 24 on NO production and iNOS expression in RAW 264.7 cells. Treatment of the test compounds at various concentrations to inhibit NO production in RAW 264.7 cells by either (A) 100 ng/mL LPS treatment or (B) H1N1 A/PR/8/34 viral infection. (C) Western blot analysis of iNOS expression in activated RAW 264.7 cells decreased by compounds 5 and 24 at 20 μM. Data are presented as the mean ± SD (n = 3), * p < 0.05, ** p < 0.01, compared to the positive control group.