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. 2021 Jun 28;274:114016.
doi: 10.1016/j.jep.2021.114016. Epub 2021 Mar 12.

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants

M S Nair  1 , Y Huang  2 , D A Fidock  3 , S J Polyak  4 , J Wagoner  5 , M J Towler  6 , P J Weathers  7
Affiliations
Free PMC article

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants

M S Nair et al. J Ethnopharmacol. .
Free PMC article

Abstract

Ethnopharmacological relevance: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria.

Aim of the study: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2.

Materials and methods: Using Vero E6 and Calu-3 cells, we measured anti SARS-CoV-2 activity against fully infectious virus of dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as the concentrations that inhibited viral replication by 50%; CC50s were also calculated and defined as the concentrations that kill 50% of cells.

Results: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 μM, 0.01-0.14 μg, and 23.4-57.4 μg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet its hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 μM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 μM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values.

Conclusions: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

Keywords: Amodiaquine; Artemether; Artemisia annua; Artemisinin; Artesunate; Covid-19; Deoxyartemisinin; Dihydroartemisinin; SARS-CoV-2.

Conflict of interest statement

Authors declare they have no competing conflicts of interest in the study.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Compounds used in this study and the plant Artemisia annua L.
Fig. 2
Fig. 2
IC50 inhibition plots of extracts for efficacy against Vero E6 cells infected with SARS-CoV-2 USA/WA1 (MOI 0.1) based on: artemisinin (A); total flavonoids (tFLV) (B); or dry mass of A. annua leaves (C) used in the experiments. SAM -20C = SAM1; SAM 4C = SAM2. Data are plotted from an average of three replicates with ±SE.
Fig. 3
Fig. 3
IC50 inhibition plots and IC50 and IC90 values for four A. annua cultivar extracts for efficacy against Vero E6 cells infected with WT (USA/WA12020) SARS-CoV-2 and variants, B1.1.7 and B1.351 (MOI 0.1) based on their measured artemisinin in the hot water extracts. Data are plotted from an average of three replicates with ±SE.
Fig. 4
Fig. 4
VSV spike pseudovirus in Calu-3 and Vero E6 cells and their viability in response to increasing hot water Artemisia extracts as percent of solvent controls. Artemisia concentration refers to dry leaf mass extracted with hot water. Data plotted using nonlinear regression curve fitting using GraphPad Prism. Data are averages of triplicate samples per condition and error bars are ± SD. Data are a representative experiment that was repeated twice.
Fig. 5
Fig. 5
Cytotoxicity of Vero 6 cells in response to A. annua hot water extracts and to imatinib (inset). Data are plotted from an average of three replicates with ±SE.
Fig. 6
Fig. 6
Comparison of A. annua SAM extracts and other antimalarial and artemisinin compounds against Vero E6 cells infected with SARS-CoV-2 USA/WA1 (MOI 0.1). A full concentration series for all samples, except for the A. annua hot water extract, could not be fully tested due to solvent toxicity, which was also observed for A. annua extracts in dichloromethane (DCM) at higher concentrations. Data are plotted from an average of three replicates with ±SE.
Fig. 7
Fig. 7
Spearman's correlation scatter plots between artemisinin concentration or total flavonoid levels vs. calculated IC50 and IC90 for the hot water extract of each cultivar from data in Table 2.
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