Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants
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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 viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC 50 s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated.
Results
Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC 50 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 the 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 IC 50 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 IC 50 = 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 IC 90 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.
List of compounds studied
Amodiaquine
Artemisinin
Artesunate
Artemether
Deoxyartemisinin
Dihydroartemisinin
Highlights
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Artemisia annua is effective in stopping replication of SARS-CoV-2 including 2 new variants.
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The anti-viral effect does not correlate to artemisinin, nor to the total flavonoid content.
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The anti-viral mechanism does not appear to involve blockade virus entry into cell.
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The plant offers two additional benefits: a decreased inflammatory response and blunting of fibrosis.
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A. annua may provide a safe, low-cost alternative for treating patients infected with SARS-CoV-2.
Article activity feed
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SciScore for 10.1101/2021.01.08.425825: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources The day prior to infection, Vero E6 and Calu-3 cells (ATCC HTB-55) were plated in black, clear-bottomed plates at 10,000 and 30,000 cells/well, respectively, in a final volume of 90 µl. Vero E6suggested: RRID:CVCL_XD71)Calu-3suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)Software and Algorithms Sentences Resources Drug concentrations were log transformed and the concentration of drug(s) that inhibited virus by 50% (i.e., IC50), and the … SciScore for 10.1101/2021.01.08.425825: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources The day prior to infection, Vero E6 and Calu-3 cells (ATCC HTB-55) were plated in black, clear-bottomed plates at 10,000 and 30,000 cells/well, respectively, in a final volume of 90 µl. Vero E6suggested: RRID:CVCL_XD71)Calu-3suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)Software and Algorithms Sentences Resources Drug concentrations were log transformed and the concentration of drug(s) that inhibited virus by 50% (i.e., IC50), and the concentration of drug(s) that killed 50% of cells (i.e., CC50), were determined via nonlinear logistic regressions of log(inhibitor) versus response-variable dose-response functions (four parameters) constrained to a zero-bottom asymptote by statistical analysis using GraphPad Prism 9 (GraphPad Software, Inc.) as described by Hulseberg et al. ( 2019). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Statistical significance of artemisinin and total flavonoid content in hot water extracts was calculated via ANOVA using GraphPad Prism V8.0.2. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2021.01.08.425825: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Indicated dilutions of the drug were incubated for 1 h in wells of well tissue culture plates containing a monolayer of Vero E6 cells seeded the day before at 20,000 cells/well. Vero E6suggested: NoneThe day prior to infection, Vero E6 and Calu-3 cells (ATCC HTB-55) were plated in black, clear-bottomed plates at … SciScore for 10.1101/2021.01.08.425825: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Indicated dilutions of the drug were incubated for 1 h in wells of well tissue culture plates containing a monolayer of Vero E6 cells seeded the day before at 20,000 cells/well. Vero E6suggested: NoneThe day prior to infection, Vero E6 and Calu-3 cells (ATCC HTB-55) were plated in black, clear-bottomed plates at 10,000 and 30,000 cells/well, respectively, in a final volume of 90 µl. Calu-3suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)Software and Algorithms Sentences Resources Drug concentrations were log transformed and the concentration of drug(s) that inhibited virus by 50% ( that killed 50% of cells ( log(inhibitor) versus response-variable dose-response functions (four parameters) constrained to a zero-bottom asymptote by statistical analysis using GraphPad Prism 9 (GraphPad Software, Inc.) as described by Hulseberg et al. ( 2019). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)IC50 and IC90 values were calculated using GraphPad Prism V8.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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