Table 1.
1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data (in CD3OD) of compound 1.
Figure 1.
Chemical structures of compounds 1–3 and (+)-pinoresinol 4-O-β-d-glucopyranoside.
Figure 2.
Selected HMBC and 1H–1H COSY correlations of compound 1.
Arrows indicate the heteronuclear multiple-bond correlation of hydrogen atom to neighboring carbon. Bold line demonstrated the correlations between two nearby hydrogen atoms.
Figure 3.
Mild alkaline hydrolysis of compound 1.
Figure 4.
UHPLC-MS chromatograms and HRESIMS data of the products from alkaline hydrolysis of compound 1.
The methanolic solution of compound 1 was treated with sodium methoxide to furnish (+)-pinoresinol 4-O-β-d-glucopyranoside and 4-hydroxy-3-methoxy benzoic acid methyl ester.
Table 2.
1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data (in CD3OD) of compounds 2 and 3.
Figure 5.
Plaque reduction assay of compound 1 against influenza viruses.
MDCK cells were infected with influenza viruses, including A/FM/1/47 (H1N1) (Top row), A/PR8/8/34 (H1N1) (Middle row) and A/Aichi2/68 (H3N2) (Bottom row) at 0.01 MOI for 2 h at 34°C. After viral adsorption, cell monolayer was covered with overlay medium containing compound 1 and further cultured at 34°C under 5% CO2 for 48 h. Then, the overlay medium was removed, and the cell monolayer was fixed with 10% formalin, stained with 1% crystal violet, and plaques were counted.
Table 3.
Antiviral activity of compound 1 against influenza viruses.
Figure 6.
Time course assay of compound 1.
(A) An illustration scheme shows the time of addition of compound 1. (B–C) MDCK cells (2×104 cells/well) in 48-well plates were infected with influenza virus A/PR/8/34 (H1N1) (MOI = 0.02). At post-infection, the medium was discarded and cells were washed with PBS three times. Compound 1 (134.3 µM) or oseltamivir (6.4 µM) was added at 0 h, 2 h, 4 h, 6 h, 8 h and 10 h after infection. At 12 h post-infection, the supernatants were collected and infectious titers were determined by CPE assay (B) and real time PCR assay (C). Data represent mean ± SD of 3 biological samples. Statistical significance was assessed by comparison between compound 1-treated group and virus control group by using student's t-test analysis (* p-value <0.001, ** p-value <0.01 and *** p-value <0.05).
Figure 7.
Inhibition of influenza virus-induced NF-κB activation in A549 cells by compound 1.
Compound 1 inhibited the influenza virus-induced NF- κB activation (A–J) but did not affect virus-induced Raf/MEK/ERK pathway activation (K). (A) and (K): A549 cells were infected with A/PR/8/34 (H1N1) (MOI = 0.1) in the absence or presence of different concentrations of compound 1 or the specific NF-κB inhibitor Bay11-7085 (10 µM) alone. A549 cell lysates were subjected to Western blot with specific antibodies against phospho-NF-κB p65 (Ser536), total NF-κB p65, phosphorylated ERK1/2 and ERK1/2. Equal protein load was verified using pan-antisera to GADPH. (B–J): A549 cells were infected with A/PR/8/34 (H1N1) (MOI = 1) and stained for against phospho-NF-κB p65 (Ser536) at 10 h of post-infection (green). Cell nuclei were stained with DAPI (blue).
Figure 8.
Effect of compound 1 on influenza virus-induced export of viral ribonucleoprotein complexes.
Immunofluorescence confocal microscopy was used to show the nuclear RNP export in the absence or presence of compound 1. A549 cells were infected with A/PR/8/34 (H1N1) (MOI = 1) and stained for influenza viral nucleoprotein (NP) at 10 h of post-infection (green). Cell nuclei were stained with DAPI (blue).