The potential mechanisms and material basis of Fuzheng Jiedu decoction broad-spectrum inhibiting coronaviruses

Abstract

Traditional Chinese medicine has unique advantages in preventing and treating COVID-19, and Fuzheng Jiedu decoction (FZJDD) was reported to be effective against COVID-19 in clinical trials. To investigate the potential mechanisms and material basis of FZJDD against SARS-CoV-2, we performed SARS-CoV-2 target protein inhibition analyses and a metabolite full spectrum analysis of FZJDD. Interestingly, FZJDD was found to block the binding of SARS-CoV-2 Spike protein with the receptor ACE2 and inhibit the activity of SARS-CoV-2 3CLpro. Moreover, FZJDD can regulate the TNF and the MAPK signaling pathway to inhibit the inflammatory response and alleviate the "cytokine storm". A total of 298 compounds were identified in FZJDD, among them, caffeic acid and octyl gallate were found to be the potential therapeutic agents of FZJDD. Importantly, FZJDD can broadly inhibit coronavirus infection, including SADS-CoV and porcine epidemic diarrhea virus (PEDV) live viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2 mutant pseudotyped viruses, which might be ascribed to the broad-spectrum anti-coronavirus activity of caffeic acid and octyl gallate. In conclusion, this study reveals the mechanisms and material basis of FZJDD against SARS-CoV-2 and identifies the broad-spectrum anti-coronavirus activity of FZJDD for the first time. Our data provide empirical evidence for the development and application of FZJDD.

Keywords: Broad-spectrum anti-coronavirus; Caffeic acid; Fuzheng jiedu decoction; Octyl gallate; SARS-CoV-2; Traditional Chinese medicine.

Fig. 1

Antiviral effect and time-of-addition assay of FZJDD against SARS-CoV-2-related pangolin coronavirus GX_P2V and SARS-CoV-2 trVLP. A The antiviral activity of FZJDD against GX_P2V in Vero E6 cells. The left and right Y-axis of the graphs represent the mean percentage of inhibition of virus yield and cytotoxicity of FZJDD, respectively (n ​= ​3). B The antiviral activity of FZJDD against SARS-CoV-2 trVLP in Caco-2-N cells. C Determination of GX_P2V yield in the supernatants of infected cells and FZJDD treatment by RT-qPCR (n ​= ​3). D Immunofluorescence images of FZJDD treatment after GX_P2V infection in Vero E6 cells. Scale bar, 100 ​μm. E The GX_P2V titers in the supernatant of FZJDD treatments with infected GX_P2V Vero E6 cells were analyzed by plaque assay. F Time-of-addition experiments of FZJDD against GX_P2V. The viral protein level in Vero E6 cell lysates was determined by Western blot. G Time-of-addition experiments of FZJDD against GX_P2V on Vero E6 (left) and 293 ​T-ACE2 (right) cell lines. The GX_P2V RNA loads in the infected cells was quantified by RT-qPCR. H Time-of-addition experiments of FZJDD against SARS-CoV-2 trVLP. The SARS-CoV-2 trVLP RNA loads in the infected Caco-2-N cells was quantified by RT-qPCR (n ​= ​3). I Time-of-addition experiments of FZJDD against GX_P2V on Calu-3 (left) and Caco-2 (right) cell lines. The GX_P2V RNA loads in the infected cells were quantified by RT-qPCR. J The cells were treated with drugs on an hourly basis, and the GX_P2V RNA loads in infected cells was quantitated by qRT-PCR (n ​= ​3). The error bars represent ±SD by T-test analysis, “ns” showed no significant difference. ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001.

Fig. 2

FZJDD targets the binding of SARS-CoV-2 RBD with hACE2 and the 3CLpro activity. A Diagram of the mechanism of action of FZJDD against SARS-CoV-2. B Effect of FZJDD on the stage of attachment and internalization of GX_P2V infected cells (n ​= ​3). The error bars represent ±SD by T-test analysis, “ns” showed no significant difference. ∗∗∗, P ​< ​0.001. C Inhibitory effect of FZJDD with different concentrations on SARS-CoV-2 wild-type pseudovirus (n ​= ​3). D, E ELISA assay was used to evaluate the effect of FZJDD on the binding of SARS-CoV-2 RBD with hACE2 (n ​= ​3). F, G The dose-dependent inhibition of FZJDD against SARS-CoV-2 3CLpro activity. Ebselen is used as the positive control to inhibit 3CLpro activity (n ​= ​3). H–J The activity of SARS-CoV-2 nsp13 (H, I) and SARS-CoV-2 nsp14 (J) assays in vitro. Punicalagin is used as a positive control to inhibit nsp13 activity (n ​= ​3). Data represent mean ​± ​SD for n ​= ​3.

Fig. 3

The transcriptome analysis of GX_P2V-infected Vero E6 cells treated with FZJDD. A Schematic diagram of transcriptome sample processing procedures (n = 3). B Heatmap showing the transcription levels (rows) across four groups (columns). The genes were clustered by the K-means method. C Principal component analysis (PCA). D Volcano map showing the differentially expressed genes between “V” and “VP” (up), and between “F” and “FP” (down) (fold change > 1, padj ≤ 0.05). E Heatmap showing the differentially expressed genes between “F” and “FP”. F The differentially expressed genes across four groups enriched in MAPK signaling and TNF signaling were analyzed by heatmap of the genes (fold change > 1.5, FDR ≤ 0.05).

Fig. 5

Broad-spectrum anti-coronavirus activity of FZJDD. A Coronaviruses that can be inhibited by FZJDD. B, C Inhibitory effect of FZJDD against PEDV (B) and SADS-CoV (C) (n ​= ​3). D, E Time-of-addition experiment of FZJDD against PEDV in Vero E6 cells (D) and SADS-CoV in Huh7 cells (E) (n ​= ​3). ∗∗∗∗, P ​< ​0.0001. F–P Inhibitory effect of FZJDD with different concentrations against pseudovirus of SARS-CoV-2 mutants, including Alpha, Beta, Gamma, Delta, Lambda, Aiot, Mu, and Omicron (BA.4, BF.7, XBB) (n ​= ​3). Q, R Inhibitory effect of FZJDD with different concentrations against pseudovirus of SARS-CoV (Q) and MERS-CoV (R) (n ​= ​3).

Fig. 6

Inhibitory effect of compounds in FZJDD against SADS-CoV and PEDV. A Dose-dependent analysis of the effective components identified in the primary screening against PEDV. The left and right Y-axis of the graphs represent the mean percentage of inhibition of virus yield and cytotoxicity of compounds, respectively. Vero E6 cells infected with PEDV (MOI ​= ​0.01) were treated with kaempferol-7-O-glucoside, icaritin, kaempferol, and octyl gallate at different concentrations. After 48 ​h of culture, the virus yield in the cells was detected by RT-qPCR. The inhibition rate was calculated by the ratio of the experimental group to the control group. CC₅₀ was calculated by cell activity assay. The EC₅₀ and CC₅₀ curves were fitted by GraphPad Prism 9 (n ​= ​3). B Time-of-addition assays of kaempferol-7-O-glucoside (6.25 ​μM), icaritin (12.5 ​μM), kaempferol (12.5 ​μM), and octyl gallate (6.25 ​μM) against PEDV (MOI ​= ​0.01). The virus yield in cell lysate was quantitatively detected by RT-qPCR and analyzed by T-test (n ​= ​3). C Dose-dependent analysis of the effective components identified in the primary screening against SADS-CoV. Huh7 cells infected with SADS-CoV (MOI ​= ​0.01) were treated with nobiletin, arctigenin, caffeic acid, and echinatin at different concentrations. After 48 ​h of culture, the virus yield in the cells was detected by RT-qPCR. The inhibition rate was calculated by the ratio of the experimental group to the control group (n ​= ​3). D Time-of-addition assays of nobiletin (12.5 ​μM), arctigenin (25 ​μM), caffeic acid (12.5 ​μM), and echinatin (12.5 ​μM). The virus yield in cell lysate was quantitatively detected by RT-qPCR and analyzed by T-test (n ​= ​3). ∗, P ​< ​0.05; ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001.

Supplementary Figure 1

Direct inactivation effect of FZJDD, caffeic acid and octyl gallateon GX P2V. A FZJDD. B Caffeic acid. C Octyl gallate. After the drug was incubated with the virus for 2 hours, the cells were directly infected with the virus, and the titer of the virus was detected by plaque assay.

Supplementary Figure 2

Differentially expressed genes. A The counts of differentially expressed genes between each two groups (n = 3). B The Venn diagram shows the number of differentially expressed genes (DEGs) in the “V” vs “P” groups and the “P” vs “FP” groups.

Supplementary Figure 3

Total ion chromatogram (TIC) diagram of FZJDD. A The positive ion mode of FZJDD. B The negative ion mode of FZJDD.

Supplementary Figure 4

Inhibitory effect of combined caffeic acid and octyl gallate on GX_P2V infection. Dose-response matrix of serially 2-fold diluted (0-50 μM) caffeic acid and octyl gallate in Vero E6 cells. Vero E6 cells were treated with the indicated concentrations of compounds separately or in combination and were infected with GX_P2V. After 48 h of infection, the virus yield in cell lysates was quantitatively detected by RT-qPCR, and the SynergyFinder website was used for data analysis (https://synergyfinder.fimm.fi/synergy/synfin_docs). A three-dimensional interaction landscape of caffeic acid and octyl gallate was generated by SynergyFinder and a synergistic score was calculated (n = 3). Synergy score: Less than -10: The interaction between two drugs may be antagonistic; From -10 to 10: Interactions between two drugs may be additive; Greater than 10: The interaction between two drugs may be synergistic. Red indicates synergistic action and green indicates antagonistic action between the two drugs.

Supplementary Figure 5

Screening of the compounds in FZJDD anti-SADS-CoV. The inhibition of 134 compounds (10 μM) in FZJDD against SADS-CoV was measured, and the compounds with inhibitory rates greater than 90% were labeled. Compounds with significant inhibition of cytopathic effect induced by SADS-CoV under the microscope were selected for RT-qPCR analysis (n = 3).