Salvia miltiorrhiza Bunge as a Potential Natural Compound against COVID-19

Abstract

Salvia miltiorrhiza Bunge, commonly called danshen, is widely used in traditional Chinese medicine for its cardiovascular and neuroprotective effects, which include antioxidative, anti-inflammatory, and antifibrotic properties. The purpose of this study was to evaluate the preclinical potential of S. miltiorrhiza extracts for the treatment of COVID-19. First, the impact of the extract on the binding between SARS-CoV-2 and the cellular ACE2 receptors was assessed using atomic force microscopy (AFM), showing a significant reduction in binding by the extract at concentrations in the µg/mL range. Second, the interference of this extract with the inflammatory response of blood mononuclear cells (PBMCs) was determined, demonstrating potent inhibitory properties in the same concentration range on pro-inflammatory cytokine release and interference with the activation of NFκB signaling. Together, these in vitro data demonstrate the potential of S. miltiorrhiza against COVID-19, consisting first of the blockade of the binding of SARS-CoV-2 to the ACE2 receptor and the mitigation of the inflammatory response from leukocytes by interfering with NFκB signaling. This dataset prompts the launch of a clinical trial to address in vivo the clinical benefits of this promising agent.

Keywords: ACE2; AFM; COVID-19; SARS-CoV-2; atomic force microscopy; binding inhibitor; force spectroscopy; infection; single-molecule; viral entry.

Figure 1

Assessment by AFM of the inhibitory effect of S.miltiorrizha extract on the interaction between the S1-subunit and ACE2. (A) The inhibitory potential of roots S. miltiorrhiza extract is assessed by measuring the binding probability between the S1 subunit immobilized on the AFM tip and the ACE2 receptor grafted on a model surface, before and after incubation with the extracts at increasing concentrations (0, 1, 10, 50, and 100 µM, respectively). (B) Examples of non-adhesive (above) and adhesives (below) recorded between S1-functionalized AFM tips and ACE2-coated model surfaces showing specific adhesion events. (C,D) Histograms showing the binding probability (BP, expressed in %) as a function of the S. miltiorrhiza extract or DMSO concentration. The error bar indicates s.d. of the mean value. One data point belongs to the BP from one map. The figure on panel a was created with Biorender.com.

Figure 2

Assessment of the inhibitory effect of a S. miltiorrizha extract powder on the interaction between the UV-inactivated SARS-CoV-2 virions and ACE2 monitored by AFM. (A) The inhibitory potential of roots S. miltiorrhiza extract is assessed by measuring the binding probability between the UV-inactivated SARS-CoV-2 immobilized on the AFM tip and the ACE2 receptor grafted on a model surface, before and after incubation with the extracts at increasing concentrations (0, 1, 10, 50, and 100 µM, respectively). (B) Examples of non-adhesive (above) and adhesive (below) force distance curves recorded between UV-inactivated SARS-CoV-2 virions-functionalized AFM tips and ACE2-coated model surfaces showing specific adhesion events. (C,D) Histograms showing the binding probability (BP, expressed in %) with the different concentrations of extracts (0, 1, 10, 50, and 100 µM) or DMSO for the control (0, 0.01, 0.1, 0.5, and 1%). The error bar indicates s.d. of the mean value. One data point belongs to the BP from one map. The figure on panel a was created with Biorender.com (accessed on 10 March 2022).

Figure 3

Dose-response release of interleukin (IL)-1β (A), TNF-α (B), IL-6 (C), IL-8 (D), and IFN-α (E–F) following PMBC activation by R848 (0.02, 0.1, 0.5, 1, 2.5, and 5 µg/mL; 0, DMSO control) on PMBC release of interleukin (IL)-1β (A), TNF-α (B), IL-6 (C), IL-8 (D), and IFN-α (E,F). Data are median ± interquartile range (n = 5 separate experiments; * p < 0.05, ** p < 0.01).

Figure 4

Inhibitory effect of S. miltiorrhiza extract (0.5, 1, 5, and 10µg/mL) on PBMC release of IL-1β (A), TNF-α (B), IL-6 (C), IL-8 (D), and IFN-α (E) upon stimulation by R848 (1µg/mL). Data are median ± interquartile range (n = 5; * p < 0.05).

Figure 5

Inhibitory effect of S. miltiorrhiza extract (Ex; 1, 5 or 10 µg/mL) on NF-κB activation following incubation with R848 (1 µg/mL) for 30 min. Representative western blot (A), with quantification data for phosphorylation of p65NF-κB S536 (B) and acetylation of p65NF-κB K310 (C). GAPDH was used as loading control (n = 4; * p < 0.05).