Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19

Abstract

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health concern, and effective antiviral reagents are urgently needed. Traditional Chinese medicine theory-driven natural drug research and development (TCMT-NDRD) is a feasible method to address this issue as the traditional Chinese medicine formulae have been shown effective in the treatment of COVID-19. Huashi Baidu decoction (Q-14) is a clinically approved formula for COVID-19 therapy with antiviral and anti-inflammatory effects. Here, an integrative pharmacological strategy was applied to identify the antiviral and anti-inflammatory bioactive compounds from Q-14. Overall, a total of 343 chemical compounds were initially characterized, and 60 prototype compounds in Q-14 were subsequently traced in plasma using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Among the 60 compounds, six compounds (magnolol, glycyrrhisoflavone, licoisoflavone A, emodin, echinatin, and quercetin) were identified showing a dose-dependent inhibition effect on the SARS-CoV-2 infection, including two inhibitors (echinatin and quercetin) of the main protease (M^pro), as well as two inhibitors (glycyrrhisoflavone and licoisoflavone A) of the RNA-dependent RNA polymerase (RdRp). Meanwhile, three anti-inflammatory components, including licochalcone B, echinatin, and glycyrrhisoflavone, were identified in a SARS-CoV-2-infected inflammatory cell model. In addition, glycyrrhisoflavone and licoisoflavone A also displayed strong inhibitory activities against cAMP-specific 3',5'-cyclic phosphodiesterase 4 (PDE4). Crystal structures of PDE4 in complex with glycyrrhisoflavone or licoisoflavone A were determined at resolutions of 1.54 Å and 1.65 Å, respectively, and both compounds bind in the active site of PDE4 with similar interactions. These findings will greatly stimulate the study of TCMT-NDRD against COVID-19.

Keywords: COVID-19; TCMT-NDRD; anti-inflammation; antivirus; traditional Chinese medicine.

Fig. 1.

Chemical identification of Huashi Baidu decoction (Q-14) using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). (A) The chemical base peak ion (BPI) chromatogram of Q-14 in the negative ion mode. (B) The BPI chromatogram of Q-14 in the positive ion mode. (C) Structural classification of compounds contained in Q-14. (D) The number of chemical components for each herb.

Fig. 2.

The compounds with prototype structures in plasma after Q-14 treatment using the UPLC-Q-TOF/MS system. (A) Structural classification of prototype compounds in plasma. (B) The number of prototype compounds in plasma for each herb. (C) Structures of the prototype compounds in plasma.

Fig. 3.

Screening of antiviral compounds from Q-14. (A) Preliminary screening of commercially available prototype compounds with antiviral activity at 100 μM based on a packaging cell line for ectopic expression of the nucleocapsid (Caco-2-N) infected with SARS-CoV-2-like-particles (trVLPs). (B) The 50% effective concentration (EC₅₀) and 50% cytotoxic concentration (CC₅₀) values of the top 10 compounds. The antiviral activity (blue) and cytotoxicity (violet) were measured. (C) SARS-CoV-2 M^pro inhibition activities of quercetin and echinatin by a fluorescence resonance energy transfer (FRET)–based protease assay. (D) The inhibition of RdRp by glycyrrhisoflavone and licoisoflavone A measured using an in vitro polymerase activity assay. (E) Molecular docking of quercetin and echinatin on SARS-CoV-2 M^pro and glycyrrhisoflavone and licoisoflavone A on RdRp. SARS-CoV-2 M^pro and RdRp are shown in gray cartoon representation, essential residues in gray sticks, and four compounds (quercetin, echinatin, glycyrrhisoflavone, and licoisoflavone A) in yellow sticks. The polar interactions between the compounds and SARS-CoV-2 M^pro/RdRp are shown in yellow dashes. Data are expressed as the mean ± SEM. Experiments were repeated in triplicate, independently.

Fig. 4.

Screening of anti-inflammatory compounds from Q-14. (A) Preliminary screening of the anti-inflammatory activity of 30 compounds from Q-14. THP-1 macrophages were incubated with SFTSV (MOI = 5) for 1 h and treated with the 30 compounds in Q-14 at a concentration of 10 μM. Cells and supernatants were harvested 48 h postinfection. P17 levels in supernatants and the expression levels of Pro-IL-1β or NP in cell lysates were determined by western blotting. The inhibition rates were evaluated by analysis of gray values of the P17 bands. (B and C) The anti-inflammatory activity of licochalcone B, glycyrrhisoflavone, and echinatin on the release of P17 induced by SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 (MOI = 0.1) in the presence of licochalcone B, glycyrrhisoflavone, and echinatin at concentrations of 1.1, 3.3, 10, or 30 μM. Cells and supernatants were collected 48 h postinfection. P17 levels in supernatants and expression levels of Pro-IL-1β or NP in cell lysates were determined by western blotting (B). The inhibition rates were evaluated by analysis of gray values of the P17 bands (C). Data are representative of three independent experiments. Error bars represent mean ± SEM. Statistical significance was analyzed by one-way ANOVA. ^###P < 0.001; ^####P < 0.0001.

Fig. 5.

Inhibition of PDE4 by the compounds from Q-14. (A) Preliminary screening of enzymatic inhibition by 30 compounds from Q-14 at 50 μM using the scintillation proximity assay (SPA). (B) The representative IC₅₀ curves of PDE4 inhibition by glycyrrhisoflavone, quercetin, licoisoflavone A, and licochalcone B. The curves were obtained by fitting a four-parameter logistic model to the data points, which represent the mean of single independent experiments. (C) Overview of the structures of glycyrrhisoflavone- and licoisoflavone A–bound PDE4 (PBD codes 7YQF and 7YSX). The protein is shown in cartoon representation. Glycyrrhisoflavone and licoisoflavone A are shown as yellow and magenta sticks, respectively. (I-II) Interactions formed between glycyrrhisoflavone (yellow) or licoisoflavone A (magenta) and surrounding residues (green). Residues and the ligand are shown as sticks, and hydrogen bonds are represented by black dashed lines. 2Fo-Fc electron density maps (yellow and magenta) were contoured at 1.0 σ. (III-IV) Ligplots of all residues making either hydrophobic or hydrogen-bonding interactions with glycyrrhisoflavone (yellow) or licoisoflavone A (magenta). IC₅₀ values were shown as mean ± SEM from three independent experiments.