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

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.


Introduction
The global pandemic of SARS-CoV-2 (the etiologic agent of  has infected over 110 million people and killed nearly 2.5 million as of February 19, 2021 (https://coronavirus.jhu.edu/). There is an intense effort to distribute the registered Pfizer/BioNTech, Moderna, and J&J vaccines, but to our knowledge, besides Remdesivir there is no approved, orally deliverable, small-molecule therapeutic and global infections keep rising, and new variants continue to emerge.
The medicinal plant Artemisia annua L. produces the antimalarial therapeutic artemisinin, a sesquiterpene lactone produced and stored in the glandular trichomes located on the shoots and particularly the leaves and flowers of the plant. Both the plant and artemisinin have been used safely for over 2,000 years to treat a variety of fever-related ailments, especially malaria (Hsu, 2006). Artemisinin derivatives (Fig. 1) are front-line therapeutics for treating malaria and are delivered with a second antimalarial drug, such as lumefantrine or amodiaquine, which are formulated as artemisinin-based combination therapies (Blasco et al., 2017). Artemisinins also possess antiviral activity (Efferth 2018). Extracts of A. annua showed anti-SARS-CoV-1 activity, suggesting that they may be active against SARS-CoV-2 (Li et al., 2005).
In a recent small human trial, Li et al. (2020) showed that artemisinin-piperaquine was safe and twice as effective as placebo in completely eliminating the virus 21 days after treatment for seven days. Knowing that artemisinin is much more bioavailable per os when delivered via A. annua (Weathers et al. 2011Desrosiers et al., 2020), we posited that encapsulated powdered dried leaves of A. annua may be a safe, cost-effective therapeutic to combat SARS-CoV-2 infections.
Here we report in vitro results from testing extracts of a diversity of A. annua cultivars against infection of Vero E6 and Calu-3 cells by fully infectious SARS-CoV-2 and two of its recent variants, with correlation analyses of antiviral IC 50 efficacy to artemisinin and total flavonoid contents.

Plant material, extract preparations, and artemisinin and total flavonoid analyses
Batches of dried leaves of various cultivars of Artemisia annua L. with source, age, and voucher identity when known are shown in Table 1. Hot-water extracts (tea infusions) were prepared as follows: dried leaves at 10 g/L were added to boiling water on a stir plate and boiled for 10 min, then poured through a 2 mm stainless steel sieve to retain most solids. Extracts were then cooled and sterile-filtered (0.22 μm) prior to being stored at − 20 • C. Dichloromethane (DCM) extracts of dried leaves were also prepared by extraction of 25 mg in 4 mL DCM for 30 min in a sonicating water bath (Fischer Scientific FS60, 130 W), separating solvent from solids with Pasteur pipets, drying under nitrogen flow, and storing at − 20 • C until analyzing for artemisinin using gas chromatography/mass spectrometry, as detailed in Martini et al. (2020). For artemisinin analysis of tea infusions, two-phase overnight aliquots extracted in DCM in a 1:1 ratio were separated by using Pasteur pipets, dried under nitrogen flow, and stored at − 20 • C until analysis as previously noted (Martini et al., 2020). Total flavonoids were analyzed in DCM extracts via the aluminum chloride method of Arvouet-Grand et al. (1994) and were quantified as quercetin equivalents. Artemisinin and total flavonoid contents of tea infusions are shown in Table 2. The DCM extract of A. annua (cv. SAM) contained a total of 34 mg of artemisinin. After solubilizing in PEG400 containing 5% DMSO the concentration was 8.95 mg/mL.

Viral culture and analyses
Vero E6 cells, obtained from the American Type Culture collection (ATCC CRL-1586), were cultured in Essential Minimal Eagle's Medium (EMEM) containing penicillin-streptomycin (1 × 100 U/mL) and 10% fetal calf serum. SARS-CoV-2 isolate USA/WA12020, UK variant B1.1.7 (CA_CDC_5574/2020), and South African variant B1.351 (hCoV-19/ South Africa/KRISP-EC-K005321/2020) were from BEI Resources (www.beiresources.org). We infected Vero E6 cells with the USA/WA1 isolate according to Liu et al. (2020b). Briefly, infected cells were incubated in flasks until a viral cytopathic effect was observed. The supernatant was then harvested and titered for its tissue culture infective dose (TCID) using an end point dilution method. TCID was calculated using the Reed-Muench proportional distance method (Reed and Muench 1938). Viral aliquots were frozen, then later thawed and used for infection experiments at their desired infectivity multiplicity of infection (MOI).

Assays for determining drug inhibition of SARS-CoV-2
Except for tea infusions that were diluted in water and used directly, amodiaquine, artesunate, artemether, artemisinin, deoxyartemisinin, and dihydroartemisinin compounds were solubilized and diluted in 5% DMSO in PEG400 or 5% DMSO in EMEM enriched with fetal calf serum at a final concentration of 7.5%, prior to testing for efficacy against SARS-CoV-2. Indicated dilutions of the drug were incubated for 1 h in wells of 96 well tissue culture plates containing a monolayer of Vero E6 cells seeded the day before at 20,000 cells/well. Post incubation of the drug with the cells, SARS-CoV-2 USA/WA1 virus was added to each well at a multiplicity of infection of 0.1. Cells were cultured for 3 days at 37 • C in 5% CO 2 and scored for cytopathic effects as detailed in Liu et al. (2020b). Vesicular Stomatitis Virus (VSV)-Spike pseudoviruses were generated as described (Hoffmann et al., 2020;Whitt 2010), using the Spike gene from SARS-CoV-2 containing the D614G mutation (Korber et al., 2020). The construct also contains a deletion of 18 amino acids from the C-terminus, which facilitates loading onto pseudovirus particles. The construct (Δ18 D614G) was kindly provided by Markus Hoffmann and Stefan Pöhlmann (Leibniz-Institut für Primatenforschung, Germany). 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. Cells were then treated with 10 μl of serially diluted Artemisia extract in water and incubated for 1 h prior to infection with 100 μl of VSV-spike Δ18 D614G pseudovirus. At 22 h post-infection, PrestoBlue was added 2 h before the end of assay, so that cell viability in parallel non-infected, drug-treated wells could be measured. Virus-produced Renilla luciferase activity was measured by Renilla-Glo assay at 24 h post-infection. Results were converted into percent of control. Drug concentrations were log transformed and the concentration of drug(s) that inhibited virus by 50% (i. e., IC 50 ), and the concentration of drug(s) that killed 50% of cells (i.e., CC 50 ), 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).

Cell viability assay
To determine the viability of Vero E6 cells post drug treatment, cells were exposed to indicated doses of tea infusions diluted in EMEM containing fetal calf serum at a final concentration of 7.5% and incubated at  Letters in ART and tFLV columns indicate statistical significance at P ≤ 0.05. a SAM1 and SAM2 are replicated hot water extracts from the same batch of A. annua leaves grown and processed from Atelier Temenos; SAM1 was stored at − 20C, thawed and reanalyzed at the same time as SAM2. Data are the average of ≥6 independently extracted leaf samples. ** Quercetin equivalents. 37 • C in 5% CO 2 for 24 h. Cells were then washed and treated with 100 μl XTT reagent premixed with activation agent, followed by incubation for another 2 h at 37 • C in 5% CO 2 . Culture medium was removed, and absorbance measured at 450 nm. The absorbance ratio of treated to untreated cells was plotted as percent viability. Imatinib, an FDAapproved apoptosis inducer and tyrosine kinase inhibitor, was used as a positive control.

Statistical analyses
All in vitro anti-SARS-CoV-2 analyses were done at least in triplicate. Plant extract analyses had n ≥ 6 independent assays. IC 50 and IC 90 values were calculated using GraphPad Prism V8.0 or V9. Correlations between antiviral activity and artemisinin or total flavonoids used Spearman's Rho analysis (Spearman 1904). Statistical significance of artemisinin and total flavonoid content in hot water extracts was calculated via ANOVA using GraphPad Prism V8.0.2.

Artemisia annua hot water extracts have anti-SARS-CoV-2 activity
Hot water extracts of the A. annua cultivars used in the study had significantly different artemisinin contents ranging from 20.1 ± 0.8 to 149.4 ± 4.4 μg/mL (Table 2). Total flavonoid content of leaf material ranged from 7.3 ± 0.2 to 37.2 ± 0.7 μg/mL (Table 2). All cultivars showed anti-SARS-CoV-2 activity ( Fig. 2; Table 2), and IC 50 values calculated on the basis of artemisinin or total flavonoid content ranged from 0.1 to 8.7 μM, or 0.01-0.14 μg/mL, respectively (Table 2). On the basis of leaf dry mass, IC 50 values ranged from 13.5 to 57.4 μg dry weight (DW). On a μg artemisinin/mL tea basis, the IC 50 of the samples ranged from 0.03 to 2.5 μg/mL. Analysis of frozen (SAM -20C) extracts remained potent upon thawing and reanalysis (Table 2, Fig. 2). Leaf samples that were 12 years old were also active with an IC 50 of 32.9 μg DW. Notably two recently isolated variants of SARS-CoV-2 from the UK (B1.1.7) and South Africa (B1.351) that are of concern due to the lowered effect of vaccines and antibodies against them (Wang et al., 2021) were similarly susceptible to A. annua extracts from BUR, MED, A3 and SAM1 (Fig. 3) with IC 50 s and IC 90 s within the range of those values measured for the original isolate from the US ( Table 2).

Hot water extracts are not cytotoxic
When cytotoxicity of the hot water extracts to the Vero E6 and Calu 3 cells was measured, cell viability did not substantially decrease (Figs. 4 and 5) at 24 h post treatment. In comparison, the apoptotic inducer imatinib showed a dose-dependent decrease in viability of the cells by 90% (Fig. 5 inset). At the higher concentrations of hot water extracts, there appeared to be proliferation of Vero E6 cells (Fig. 5).

Activity of antimalarials
In a separate analysis, DCM and hot water extracts of A. annua were compared, yielding IC 50 values of 12.0 and 11.8 μM, respectively ( Fig. 6). However, due to solvent toxicity at higher concentrations of the drug on Vero E6 cells, the IC 50 of the DCM extract had to be estimated at 12 μM. Similar solvent toxicity was encountered with artemisinin that subsequently was estimated to have an IC 50 of 70 μM (Fig. 6). Artemether efficacy also had to be estimated at 1.23 μM and was cytotoxic at concentrations slightly above that level (Fig. 6). Artesunate and dihydroartemisinin were inactive at all tested concentrations. In contrast, amodiaquine showed efficacy at 5.8 μM (Fig. 6).

Anti-SARS-CoV-2 activity of hot water extracts inversely correlated to artemisinin or total flavonoid content
The IC 50 quantifies the antiviral efficacy of a drug or extract. The lower the IC 50 , the more effective a drug or extract. To begin to define the bioactive components in A. annua responsible for suppressing SARS-CoV-2 infection, we correlated IC 50 and IC 90 (the concentrations of drug that inhibits 50% and 90% of virus, respectively) with the artemisinin content of our extracts. A Spearman's Rho analysis showed that both IC 50 and IC 90 values of the hot water extracts increased with artemisinin and total flavonoid content (Fig. 7). If artemisinin was the principle bioactive responsible for suppressing virus infection, then IC 50 and IC 90 concentrations should decrease with increasing concentrations of artemisinin, but they did not. Moreover, results of IC 50 and IC 90 calculations   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.  based on dry leaf mass used to prepare the tea were tightly grouped (Fig. 2). Although cultivar IC 50 ranking from most to least effective on dry weight basis was BUR, MED, A3, #15, PEG01, SAM1, SAM2, and FLV5 (Table 2), the maximum differential was less than 44 μg DW of dried leaves, or ~4.4 μl of tea infusion, an inconsequential difference.
Collectively, the data suggest that artemisinin is not the principal molecule responsible for suppression of SARS-CoV-2 infection.

Human bioavailability
For a preliminary query of the possibility of using dried leaf A. annua (DLA) as a potential therapeutic, we tracked artemisinin as a marker molecule post consumption of per os delivered DLA to one human volunteer. One of us (PJW) consumed 3 g of encapsulated DLA of the SAM cultivar and had blood drawn at 2 and 5 h post consumption, resulting in serum measurements of 7.04 and 0.16 μg artemisinin/mL serum, respectively (See Supplemental Data). Thus, at 2 h post ingestion, 36% of the original DLA-delivered artemisinin was detected in the serum, decreasing to 0.8% at 5 h post ingestion (See Supplemental Material for methodology and data Table S1). This corresponded at 2 h to 2.35 μg artemisinin/mL serum of DLA-delivered artemisinin per gram of DLA consumed.

Discussion
This is the first report of anti-SARS-CoV-2 efficacy of hot water extracts of a wide variety of cultivars of A. annua sourced from four continents. These extracts had an IC 50 corresponding to <12 μM artemisinin, with DCM extracts of A. annua showing similar efficacy. There was a similar response against the two variants B1.1.7 and B1.351 when challenged by four of the extracts including three of the most efficacious. In contrast, artemisinin, when tested as a solo drug, had an estimated IC 50 about sixfold greater (~70 μM), suggesting the plant extracts were more potent against SARS-CoV-2. Furthermore, the anti-SARS-CoV-2 effect was positively correlated to the artemisinin content of the extracts that varied by one to nearly two orders of magnitude for IC 50 and IC 90 values. A lower IC 50 indicates a more active drug. Rather, the Spearman analysis showed that as the IC 50 increased so also did the correlating artemisinin concentration. If artemisinin were the primary active component, one would expect the IC 50 , the concentration of drug that suppresses virus by 50%, to decrease with increasing concentrations of artemisinin, but the reverse occurred. Thus, our results suggest a possible antagonistic role of artemisinin in these extracts.
Total flavonoid content also similarly correlated to antiviral activity; IC 50 and IC 90 values increased with increasing flavonoid content. If total flavonoids were the primary active components, one would expect the IC 50 and IC 90 values to decrease with increasing concentrations of flavonoids, but they did not. One of the cultivar samples was obtained in 2008 and was still active at a level comparable to the most recently harvested cultivar samples, suggesting that the active principles are ubiquitous to different A. annua cultivars and chemically stable during long-term room temperature dry storage. None of the plant extracts were cytotoxic to Vero 6 or Calu-3 cells at concentrations approaching the IC 50 or IC 90 values. Indeed, at higher extract concentrations there was a slight increase in cell growth of Vero E6 cells, a positive response to A. annua extracts that also was reported for other healthy mammalian  Table 2. cells, e.g. fibroblasts (Rassias and Weathers 2019). Finally, the minimal antiviral effects against VSV pseudoviruses containing the SARS-CoV-2 spike protein suggests that A. annua inhibits SARS-CoV-2 infection primarily by targeting a post-entry step.
Although Cao et al. (2020) reported an IC 50 of 10.28 μM for arteannuin B, a metabolite that is formed in a side branch of the artemisinin biosynthetic pathway and that is often present in A. annua extracts, only three of the tested tea extracts had any detectable arteannuin B with SAM having 3.2 μg/mL. Arteannuin B in BUR and MED was barely detectable. Thus, arteannuin B is tentatively eliminated as the principle active component, although if present in an extract, arteannuin B may be providing some antiviral effect as part of the more complex plant extract mixture. Although they can be present in substantial amounts in A. annua Towler and Weathers 2015; see supplemental Table S2), neither artemisinic acid nor deoxyartemisinin, also metabolites in the artemisinin biosynthetic pathway, showed anti-SARS-CoV-2 activity in this study.
There is some discrepancy among IC 50 M values in this and other studies for anti-SARS-CoV-2 efficacy (Table 3). In contrast to Bae, Cao, and Gilmore, we did not observe any anti-SARS-CoV-2 activity for artesunate or dihydroartemisinin. Artemether in our study had an IC 50 of 1.23 μM, while Cao et al. (2020) reported an IC 50 of 73.8 μM but with less toxicity than we observed. In particular, we noted cytotoxicity for artemether. The contrasts are likely the result of differences in how we conducted our viral challenge experiments or solvents used to challenge the virus in Vero E6 cells. For example, our study solubilized our pure artemisinin and other antimalarial compounds in 5% DMSO in PEG400, while the other two studies solubilized compounds in DMSO. Our preliminary experiments indicated that solubilizing in pure DMSO was too toxic to Vero cells to achieve dosing of drug concentrations needed to obtain an IC 50 value. In addition, Cao et al. also had a different viral assay system. We used an endpoint assay to measure the cytopathic effect of the replicating virus at 72 h and estimate the IC 50 values while they collected supernatants to assay the total RNA levels at 24 h post infection using RT-PCR. We recognize that such inherent variations in the biological assays would offset the calculated values. Gilmore et al. (2020) also tested a hot water extract of A. annua and observed IC 50 values ranging from 260 to 390 μg extract/mL. None of these studies reported IC 50 s based on the artemisinin content of their extracts.
Our hot water extracts are not directly comparable to those of Gilmore et al. because we did not dry, concentrate, and then weigh our extracts. Furthermore, we extracted for 10 min in boiling water, while they extracted for 200 min in boiling water. In addition, different viruses were used in our study versus that of Gilmore et al., which could affect the inherent replication kinetics of the assay and in turn affect the specific IC 50 numbers. In Nie et al. (2021), the same team tested hot water extracts prepared for a shorter time, but they still concentrated their extracts before testing. In contrast, we tested our tea extracts directly without any concentration. Interestingly, Nie et al. also reported efficacy of Artemisia afra, a perennial native to South Africa that produces no artemisinin (du Toit and van der Kooy, 2019). Extracts of that species were more effective (IC 50 = 0.65 mg/mL) than pure artemisinin (IC 50 = 4.23 mg/mL) and extracts of four A. annua cultivars (IC 50 = 0.88-3.42 mg/mL) (Nie et al., 2021). Their results further suggest potent nonartemisinin phytochemical(s) present in both Artemisia species.
The high bioavailability of artemisinin after oral consumption of dried-leaf A. annua (DLA) was not surprising considering that a series of earlier studies in rodents showed the drug is > 40 fold more bioavailable when delivered via the plant than in purified form (Weathers et al. 2011. The increased bioavailability is mainly the result of three mechanisms: essential oils in the plant material improving the solubility of artemisinin, improved passage across the intestinal wall, and especially the inhibition of liver cytochrome P450s, 2B6, and 3A4 that are critical in first-pass metabolism Weathers 2016, 2018;Desrosiers et al., 2020). The anti-SARS-CoV-2 IC 90 of the SAM1 and SAM2 cultivar samples ranged from 12.3 to 18.8 μM artemisinin, equal to 1.7-2.6 μg/mL, so 1 g of the SAM cultivar delivered per os yielded 2.6 μg/mL in a patient's serum. Thus, 1 g of DLA could deliver enough artemisinin/DLA to achieve the IC 90 of the hot water extract. While further human studies are required, this hypothetical estimation suggests that reasonable amounts of DLA consumed per os may deliver adequate amounts of the antiviral phytochemicals needed to provide a cost-effective anti-SARS-CoV-2 treatment. Indeed, the broad scale use of both artemisinin and nonartemisinin compound antimalarials including A. annua tea infusions across Africa may help in part explain why despite having anti-SARS-CoV-2 antibodies, Africans have not to date suffered the clinical scourge of SARS-CoV-2 like the rest of the world (Uyoga et al., 2020).

Conclusions
This is a first report of the in vitro activity against SARS-CoV-2 and two of its recent variants, B1.1.7 and B1.351, by hot water extracts of A. annua at concentrations that can be achieved in humans following oral consumption of the plant material. Further work is required to isolate and identify the nonartemisinin bioactive phytochemicals in the plant extracts. If subsequent clinical trials are successful, A. annua could potentially serve as a safe therapeutic that could be provided globally at reasonable cost and offer an alternative to vaccines.

Author contributions
MSNconducted SARS-CoV-2 experiments, helped analyze the data, and contributed to the manuscript.
YHconducted SARS-CoV-2 experiments, helped analyze the data, and contributed to the manuscript.
DAFprovided reagents, helped analyze the data, and edited the manuscript.
SJPhelped plan and analyze pseudovirus data, and contributed to manuscript.

Table 3
Comparative IC 50 s for artemisinin derivatives and partner drug antimalarials. Compound Cao et al.
Gilmore et al.
Bae et al. JWconducted pseudovirus experiments, helped analyze data, and contributed to manuscript.
MJTprepared and analyzed plant extracts and human samples, helped analyze the data, and contributed to the manuscript.
PJWwrote manuscript, conducted the single human PK test, provided reagents, and helped analyze the data.

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