Enhanced anti-influenza activity of fermented yellow soybean extract and daidzein co-treatment on MDCK cells

Abstract

The study investigated the effectiveness of pre- and co-treatment with fermented yellow soybean extract (FYSE) against anti-influenza A virus (IAV) on MDCK cells. FYSE, fermented with Bacillus subtilis, was evaluated for its anti-IAV activity by inhibiting the IAV PA gene expression. Daidzein was identified as a significant contributor to FYSE's antiviral effects. Co-treatment with FYSE and daidzein during IAV infection demonstrated superior anti-IAV activity compared to their respective pre-treatment (IC₅₀: FYSE; 8.65 vs 3.77 µg/mL, and daidzein; 6.01 vs 5.20 µg/mL). Both pre- and co-treatment with FYSE demonstrated higher therapeutic potential than daidzein (Selective index: pre-treatment; > 115.58 vs. 72.32 and co-treatment; > 265.04 vs. 83.56). Despite daidzein showing lower anti-IAV activity in both treatment methods compared to oseltamivir phosphate, it exhibited lower cytotoxicity (CC₅₀: 434.50 vs. 395.20 µg/mL). In conclusion, co-treatment with FYSE and daidzein presents a promising anti-IAV strategy with minimal cytotoxicity in vitro, potentially offering a safer alternative for IAV treatment.

Supplementary information: The online version contains supplementary material available at 10.1007/s10068-024-01673-2.

Keywords: Antiviral; Daidzein; Influenza; Yellow soybean.

Fig. 1

Effects of fermented yellow soybean extract (FYSE) on cell viability and anti-IAV activity, (A) Cell viability of FYSE on MDCK cells. The extract was treated to MDCK cells at concentrations ranging from 12.5 to 1000 µg/mL. Cell viability was measured after 48 h using a CCK-8 kit and expressed as a percentage of the viability of untreated control, represented as bars. (B) Anti-IAV activity of pre-treatment and co-treatment with FYSE on MDCK cells. MDCK cells were either pre-treated with FYSE for 24 h prior to IAV infection or co-treated with IAV and FYSE, and then incubated for 48 h. Virus yield in the medium was collected, RNA was extracted, converted to cDNA, and analyzed by qRT-PCR. The anti-IAV activity is expressed as the average of IAV PA gene expression, represented as bars. An asterisk above the bars indicates a significant difference in the PA gene expression between FYSE-treated and untreated IAV-infected cells (p-value < 0.01; Dunnett’s test)

Fig. 2

Representative chromatogram and ion mass spectra of fermented yellow soybean extract (FYSE). (A) Representative chromatogram of chemical profiling of FYSE using positive ionization mode (B) Representative chromatogram of chemical profiling of FYSE using negative ionization mode. The x-axis represents retention time, and the y-axis represents the intensity (%) of identified chemicals or metabolites. The analysis was performed using Ultra-high-performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC–ESI–Q-TOFMS). The peak numbers corresponding to identified chemicals or metabolites are listed in Table 1. (C) Ion mass spectra of daidzein, a key component of FYSE. The spectra include [M + H − CO₂ − H₂O], [M + H − CO₂], [M + H − CO], and [M + H] ions with m/z values of 181.051, 191.0758, 227.099 and 255.065 respectively

Fig. 3

Effect of pre-treatment (A) and co-treatment (B) with fermented yellow soybean extract (FYSE), and pre-treatment (C) and co-treatment (D) with daidzein, on apoptosis in IAV-infected MDCK cells. The effect of FYSE or daidzein on apoptosis in IAV-infected cells was analyzed by the Muse Annexin V and Dead Cell Assay. Different cell samples were stained with 7-aminoactinomycin D. Live, early apoptotic (Early apo), late apoptotic (Late apo), and dead cells were quantified using the Muse Cell Analyzer. The bars represent the mean ± SD, and an asterisk above the bars indicates a significant difference from untreated IAV-infected cells (p-value < 0.01; Dunnett’s test)

Fig. 4

Cell viability of (A) daidzein and (B) oseltamivir phosphate on MDCK cells. MDCK cells were treated with daidzein or oseltamivir phosphate at concentrations ranging from 2.5 to 400 µg/mL. Cell viability was measured after 48 h using a CCK-8 kit and expressed as a percentage of the viability of untreated control. An asterisk above the bars indicates a significant difference from the untreated control (p-value < 0.01; Dunnett’s test)

Fig. 5

Anti-IAV activity of pre- and co-treatment with (A) daidzein and (B) oseltamivir phosphate on MDCK cells. MDCK cells were either pre-treated with daidzein or oseltamivir phosphate for 24 h prior to IAV infection or co-treated with IAV. After infection, the cells were incubated for 48 h and assessed for IAV PA gene expression. Virus yield in the medium was collected, RNA was extracted, converted to cDNA and analyzed by qRT-PCR. The anti-IAV activity is expressed as the average of IAV PA gene expression and is represented as bars. An asterisk above bars indicates a significant difference in PA gene expression in daidzein- or oseltamivir phosphate-treated IAV-infected cells compared to untreated IAV-infected cells (p-value < 0.01; Dunnett’s test)