Fig 1.
Chemical structures and synthetic schemes of cyclobakuchiols A, B, C, and D.
(A) Transformation of (+)-(S)-bakuchiol (1) to cyclobakuchiols A (2), B (3), and C (4) under acidic condition. (B) Transformation of 2 and 3 to 4 and cyclobakuchiol D (5) by acetylation, epoxidation, and reduction.
Fig 2.
Toxicity of cyclobakuchiols A–D against MDCK cells.
The indicated concentrations of cyclobakuchiols A (2), B (3), C (4), and D (5) or (+)-(S)-bakuchiol (1) in DMSO (concentrations of 100 μM, 1%; 50 μM, 0.5%; 25 μM, 0.25%; 12.5 μM, 0.125%; 6.3 μM, 0.063%; 3.1 μM, 0.031%; 1.6 μM, 0.016%; 0.8 μM, 0.008%) were added to the MDCK cells. Cell viabilities were determined by MTT assay after incubation for 24 h (n = 5 each) (A) or 72 h (n = 5 each) (B). Data represent the mean ± SEM and were representative of three independent experiments. ***p < 0.001, for the comparison with DMSO-treatment. The results were reproducible in this experiment.
Fig 3.
Cyclobakuchiols A–D promotes the viability of MDCK cells infected with influenza A viruses.
Effects of cyclobakuchiols A (2), B (3), C (4), and D (5) on the viability of MDCK cells infected with influenza A viruses. 2–5 (0.4–25 μM) were mixed with or without the A/PR/8/34 (A), A/CA/7/09 (B), or A/WSN/33 (C) viruses and subsequently added to MDCK cells. DMSO (0.004–0.25%) and (+)-(S)-bakuchiol (1) (0.4–25 μM) were used as negative and positive controls, respectively. Cell viability was determined via naphthol blue-black staining after incubation for three days. Data are representative of three independent experiments. The results were reproducible across all experiments.
Fig 4.
Image analysis of inhibitory effect of cyclobakuchiols A–D on influenza A viral infection.
The corresponding concentrations of cyclobakuchiols A (2), B (3), C (4), and D (5) (3.1–12.5 μM; n = 9 each), (+)-(S)-bakuchiol (1) (3.1–12.5 μM; n = 9 each), or DMSO (0.031–0.125%; n = 9 each) were mixed with A/PR/8/34 (A and B) or A/WSN/33 (C and D) viruses and added to MDCK cells for 24 h. The infected MDCK cells were visualized by immunofluorescence staining of influenza A viral NP and then photographed under a microscope (A and C). The percentages of influenza A viral NP-positive cells per DAPI-positive cells were calculated based on the counts of influenza A viral NP-positive and DAPI-positive cells (B and D). The white scale bar in each image represents 100 μm. Data are expressed as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to DMSO-treatment. ††p < 0.01 relative to cyclobakuchiol D-treatment. The results were reproducible in this experiment.
Table 1.
Anti-viral effects of cyclobakuchiols A–D against A/PR/8/34 virus.
Fig 5.
Cyclobakuchiols A–D inhibits influenza A viral growth.
MDCK cells were infected with A/PR/8/34 (A) or A/WSN/33 (B) viruses prior to the addition of cyclobakuchiol A (2), B (3), C (4), and D (5) (12.5 μM; n = 9), DMSO (0.125%; n = 9) or (+)-(S)-bakuchiol (1) (12.5 μM; n = 9). The conditioned culture media were collected at the indicated time-points and added to MDCK cells, and the treated cells were immunostained. The viral titers were calculated from the number of stained cells. Data represent the mean ± SEM and are representative of three independent experiments. **p < 0.01, ***p < 0.001 for the comparison of DMSO-treatment. Results were reproducible in this experiment.
Fig 6.
Cyclobakuchiols A–D inhibits the expression of influenza A viral mRNAs.
Cyclobakuchiols A (2), B (3), C (4), and D (5) (12.5 μM), DMSO (0.125%), or (+)-(S)-bakuchiol (1) (12.5 μM) were mixed with A/PR/8/34 virus (MOI 0.1) and incubated for 30 min prior to the addition to 1 × 105 MDCK cells. (A–F) Total RNA was extracted from cell lysates 24 h post-infection. The relative expression levels of viral mRNAs [NP (A), NS1 (B), PA (C), PB1 (D), PB2 (E) or M2 (F)] (n = 9 each) were determined by RT-qPCR. These mRNA levels were normalized to 18s ribosomal RNA and expressed in relation to the levels in the DMSO-treated cells (set as 1). Data represent the mean ± SEM and are representative of three independent experiments. UI; uninfected cells. *p < 0.05, ***p < 0.001 for the indicated comparisons. Results were reproducible in this experiment.
Fig 7.
Cyclobakuchiols A–D inhibits the expression of influenza A viral proteins.
Cyclobakuchiols A (2), B (3), C (4), and D (5) (12.5 μM), DMSO (0.125%), or (+)-(S)-bakuchiol (1) (12.5 μM) were mixed with A/PR/8/34 virus (MOI 0.1) and incubated for 30 min prior to the addition to 1 × 105 MDCK cells. (A–C) The levels of influenza A viral NP and NS1 proteins in cell lysates were analyzed by western blotting at 4–12 h (A) or 24 h (B) post-infection. β-actin protein was analyzed as an internal control. Signal intensities at 24 h post-infection were measured using ImageJ software, and the protein levels of NP/β-actin or NS1/β-actin were analyzed, while the protein levels of NP (C, left panel) (n = 3 each) and NS1 (C, right panel) (n = 3 each) were normalized to that of β-actin. Data represent the mean ± SEM and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. The results were reproducible in this experiment.
Fig 8.
Cyclobakuchiols A–D inhibit the upregulation of Ifn-β and Mx1 mRNAs in influenza A virus-infected cells.
Cyclobakuchiols A (2), B (3), C (4), and D (5) (12.5 μM), DMSO (0.125%), or (+)-(S)-bakuchiol (1) (12.5μM) were mixed with or without A/PR/8/34 virus (MOI 0.1) and incubated for 30 min prior to the addition to 1 × 105 MDCK cells. Total RNA was extracted from cell lysates 24 h post-infection. The relative levels of Ifn-β (n = 9 each) (A) or Mx1 (n = 9 each) (B) mRNA were determined by RT-qPCR, normalized to β-actin mRNA, and expressed relative to the levels in DMSO-treated non-infected cells (set as 1). Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, ***p < 0.001 for the indicated comparisons. The results were reproducible in this experiment.
Fig 9.
Cyclobakuchiols A–D increase the mRNA expression of NAD(P)H quinone oxidoreductase 1 in MDCK cells, and cyclobakuchiols A–C induce the nuclear factor erythroid 2-related factor 2 activation.
(A) Cyclobakuchiols A (2), B (3), C (4), and D (5) (12.5 μM), DMSO (0.125%), or (+)-(S)-bakuchiol (1) (12.5 μM) were mixed with or without A/PR/8/34 virus (MOI 0.1) and added to 1 × 105 MDCK cells for 24 h. Total RNA was extracted from the cell lysates, and the mRNA levels of NAD(P)H quinone oxidoreductase 1 (Nqo1), a nuclear factor erythroid 2-related factor 2 (Nrf2)-induced gene (n = 9 each), were determined by RT-qPCR, normalized to β-actin mRNA, and expressed relative to the DMSO-treated non-infected cells (set as 1). (B) A Nrf2 reporter assay based on the dual luciferase system was performed in MDCK cells. MDCK cells (1 × 105) were transfected with pNQO1-ARE-Fluc, expressing a Firefly luciferase gene driven by Nrf2 activation, and pRL-TK-Rluc, expressing Renilla luciferase driven by the herpes simplex viral thymidine kinase promoter. At 24 h post-transfection, the cells were treated with 2–5 (12.5 μM) (n = 6 each), DMSO (0.125%) (n = 6), or 1 (12.5μM) (n = 6). The levels of Firefly and Renilla luciferase mRNA were analyzed by RT-qPCR after 24 h and normalized to β-actin mRNA. The relative levels of Firefly per Renilla luciferase mRNAs were calculated and compared with that observed in the DMSO-treated cells (set as 1) and expressed relative to the DMSO-treated cells (set as 1). Data are presented as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. The results were reproducible across all experiments.