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 Journal of Ethnopharmacology 308 (2023) 116291

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

SARS-CoV-2 omicron variants are susceptible in vitro to Artemisia annua hot

water extracts
M.S. Nair a, Y. Huang a, M. Wang a, P.J. Weathers b, *
a
Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, USA
b
Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, 01609, USA

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr. Thomas Efferth Ethnopharmacological relevance: Artemisia annua L. has >2000 yr of history in treating fever a symptom common
to many infectious diseases including viruses. The plant is widely used as a tea infusion in many areas of the
Keywords: globe to thwart many infectious diseases.
Artemisia annua Aim of the study: The SARS-CoV-2 (COVID-19) virus continues to infect millions while rapidly evolving new
Tea infusions
variants that are more transmissible and evade vaccine-elicited antibodies, e.g., omicron and its subvariants.
Omicron
Having shown potency against all previously tested variants, A. annua L. extracts were further tested against
COVID-19
SARS-CoV-2 highly infectious omicron and its recent subvariants.
WA1 Materials and methods: Using Vero E6 cells, we measured the in vitro efficacy (IC50) of stored (frozen) dried-leaf
BA.1 hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original
BA.2 WA1 (WT), BA.1 (omicron), BA.2, BA.2.12.1, and BA.4. End point virus titers of infectivity in cv. BUR-treated
BA.2.12.1 human lung A459 cells overexpressing hu-ACE2 were determined for both WA1 and BA.4 viruses.
BA.4 Results: When normalized to the artemisinin (ART) or leaf dry weight (DW) equivalent of the extract, the IC50
values ranged from 0.5 to 16.5 μM ART and from 20 to 106 μg DW. IC50 values were within limits of assay
variation of our earlier studies. End-point titers confirmed a dose-response inhibition in ACE2 overexpressing
human lung cells to the BUR cultivar. Cell viability losses were not measurable at leaf dry weights ≤50 μg for any
cultivar extract.
Conclusions: A. annua hot-water extracts (tea infusions) continue to show efficacy against SARS-CoV-2 and its
rapidly evolving variants and deserve greater attention as a possible cost-effective therapeutic.

1. Introduction
Artemisia annua L. has a long ethnobotanical history of use especially
in treating fevers resulting from infectious diseases like malaria (Hsu,
2006; Tu, 2016). Fever is also a typical symptom of viral infections.
Although human viruses are likely very ancient, even predating cellular
origins (Krupovic et al., 2019), they have only recently been recognized
as infectious agents in humans with the first identified in 1901, the YFV
flavivirus that causes yellow fever (Woolhouse et al., 2008). People in
Kenya and Uganda have now also been documented using A. annua tea
as a treatment for viral infection of HIV/AIDS (Hirt et al., 2008; Lubbe
et al., 2012; Willcox et al., 2011). Previously, we and others showed that
extracts of dried leaves of many cultivars of the medicinal plants,
A. annua, which produces the antimalarial sesquiterpene lactone,
artemisinin (ART; Fig. 1), and A. afra Jacq. ex Willd., a related perennial
species lacking artemisinin, prevented SARS-CoV-2 replication in vitro
(Nair et al., 2021, 2022; Nie et al., 2021; Zhou et al., 2021). Although
ART has some anti-SARS-CoV-2 activity, we also showed that antiviral
efficacy was inversely correlated to ART content (Nair et al., 2021).
The novel coronavirus, SARS-CoV-2 (a.k.a., COVID-19), with its
rapidly evolving variants continues to plague the global population with
>650 million cases resulting overall in >6.6 million deaths (https://cor
onavirus.jhu.edu/map.html, accessed June 26, 2022). The past year the
omicron (BA.1) variant of concern (VOC) emerged along with a number
of subvariants, especially BA.4 and BA.5 (Tegally et al., 2022). These are
highly transmissible (Omicron RO ≥ 10, Delta RO = 7 (Burki, 2022))
infecting even vaccinated individuals, albeit with less severe outcomes
(https://www.healthdata.org/covid/COVID-19-vaccine-efficac
y-summary, accessed June 26, 2022). Omicron (BA.1) isolates have

* Corresponding author. Department of Biology and Biotechnology, Worcester Polytechnic Institute, 100 Institute Rd, Worcester, MA, 01609, USA.
E-mail addresses: mn2947@cumc.columbia.edu (M.S. Nair), yh3253@cumc.columbia.edu (Y. Huang), hw2614@cumc.columbia.edu (M. Wang), weathers@wpi.
edu (P.J. Weathers).

https://doi.org/10.1016/j.jep.2023.116291
Received 2 October 2022; Received in revised form 12 February 2023; Accepted 15 February 2023
Available online 18 February 2023
0378-8741/© 2023 Elsevier B.V. All rights reserved.
M.S. Nair et al.
Abbreviations:
ART artemisinin
CC50 concentration of drug(s) that killed 50% of cells
DL drug-likeness
GCMS gas chromatography mass spectrometry
IC50 concentration of drug(s) that inhibited virus by 50%
MOI multiplicity of infection
OB oral bioavailability
PPQ piperaquine
TCID tissue culture infectious dose
VOC variant of concern
Fig. 1. Artemisia annua L. and artemisinin.
shown resistance to neutralization by antibodies in patients who have
had COVID-19 or been vaccinated and even boosted with one of the
widely used vaccines (Cao et al., 2022; Iketani et al., 2022; Liu et al.,
2022). Recently, variants, BA.2.12.1 and BA.4/5 were shown to be 1.8
and 4.2 times, respectively, more resistant to sera from individuals who
were vaccinated and boosted (Wang et al., 2022). Additionally, recent
clinical case studies showed that vaccinated and boosted individuals
who took a course of Paxlovid™ have shown relapse and relapsed in­
dividuals accidentally infect family members (Charness et al., 2022).
This presents an even more pressing need for an expanded diversifica­
tion of therapeutics, which may also serve as prophylactics in a popu­
lation setting.
Although a number of different drugs have been trialed (Sakamuru
et al., 2022) there are few approved small-molecule drugs available to
treat COVID-19. The antiviral drug Paxlovid™ was recently approved as
a per os combination drug of nirmatrelvir (or PF-07321332) with rito­
navir, developed by Pfizer having good anti-SARS-CoV-2 efficacy and
relatively few adverse drug reactions (Hung et al., 2022; Lamb, 2022).
The viral protease inhibitor, nirmatrelvir (Hung et al., 2022), works with
ritonavir. The latter inhibits CYP3A4 to improve the pharmacokinetics
of nirmatrelvir (Owen et al., 2021). Despite this success, access to the
drug may be limited (https://www.nature.com/articles/d41586-022
-00919-5, accessed June 26, 2022) because generic production, while
affordable in developed countries is unaffordable to many worldwide
(https://www.reuters.com/business/healthcare-pharmaceuticals/ge
neric-drugmakers-sell-pfizers-paxlovid-25-or-less-low-income-countries
-2022-05-12/, accessed June 26, 2022). Thus, there remains a need for
more cost-effective antiCOVID-19 therapeutics to treat the global
population.
Here we report in vitro efficacy for four of the seven originally studied
A. annua L. cultivars against omicron (BA.1) and three of its subvariants:
BA.2, BA.2.12.1, and BA.4.
2
Journal of Ethnopharmacology 308 (2023) 116291
2. Materials and methods
2.1. Plant material, extract preparations, and artemisinin analyses
Hot-water extracts (tea infusions) were previously prepared from
vegetative stage dried leaves of Artemisia annua L. (SAM, MASS
00317314; BUR, LG0019527; A3, Anamed; MED, KL/015/6407) In
brief: 10 g dried leaves/L were boiled in water for 10 min, solids
removed via sieving, then 0.22 μm filter-sterilized and stored at − 80 ◦ C
for this study and as detailed in Nair et al. (2021). ART analyses of tea
infusions were by gas chromatography mass spectrometry (GSMS) as
previously described (Martini et al., 2020) using an Agilent 7890 A GC;
MS, Agilent 5975 C; column, Agilent HP-5MS (30 m × 0.25 mm × 0.25
_m) column and He carrier gas at 1 mL/min; injection volume, 1 _μL in
splitless mode; ion source temperature, 230 ◦ C; inlet, 150 ◦ C; transfer
line, 280 ◦ C; oven temperature, 125 ◦ C held for 1 min, then increased to
240 ◦ C at 5 ◦ C/min, and then increased to 300 ◦ C at 30 ◦ C/min. The
natural log transformation of the sum of the three peaks correlating to
the ART standard (two thermal breakdown peaks and one intact mole­
cule peak) was used to generate a standard curve. The natural log of the
total peak area (Y) is plotted against the natural log of the concentration
(X) and a linear equation is obtained (Y = mX + b, where m is slope and
b is y-intercept). The concentration of ART in unknown samples is
calculated from that equation and ART contents were detailed in (Nair
et al., 2021): ART in μg/mL was: 42.5 for A3; 20.1 for BUR; 59.4 for
MED; and 149.4 for SAM. TLC fingerprints and GCMS chromatograms
showing the three ART peaks of the tea infusions of each of the four
tested cultivars are available in Supplemental Materials Figs. S1–S3.
2.2. Vero E6 culture and infection
Cultivation of Vero E6 cells (ATCC CRL-1586) was in Essential
Minimal Eagle’s Medium (EMEM) containing penicillin-streptomycin (1
× 100 U/mL) and 10% fetal calf serum, and viral infections were per­
formed as previously described (Nair et al., 2021). SARS-CoV-2 isolates
(Table 1) were sourced from BEI Resources (www.beiresources.org). To
determine their tissue culture infectious dose (TCID), viruses were
titrated after propagation in Vero E6 cells, aliquoted, and frozen at
− 80 ◦ C until later use. Multiplicity of infection (MOI) was 0.1 as used in
other studies (Liu et al., 2020; Nair et al., 2021, 2022).
2.3. Drug inhibition assays of SARS-CoV-2 and cell viability
Dilutions of extracts were incubated for 1 h in 96-well tissue culture
plates having a monolayer of Vero E6 cells seeded the prior day at
20,000 cells/well. SARS-CoV-2 virus was added to each well 1 h after
extract addition to a final MOI of 0.1. Cells were cultured for 3 days in
5% CO2 at 37 ◦ C and then scored for cytopathic effects as previously
detailed (Liu et al., 2020) and values converted into percent of control.
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; viability), were log-transformed
Table 1
SARS-CoV-2 isolates used in this study.
SARS-CoV-2 BEI Resource Catalogue number
isolate
USA/WA12020 NR-52281 SARS-Related Coronavirus 2, Isolate USA-WA1/2020
Omicron BA.1 NR-56475 SARS-Related Coronavirus 2, Isolate hCoV-19/USA/
HI-CDC-4359259-001/2021
Omicron BA.2 NR-56520 SARS-Related Coronavirus 2, Isolate hCoV-19/USA/
CO-CDPHE-2102544747/2021
Omicron NR-56781 SARS-Related Coronavirus 2, Isolate hCoV-19/USA/
BA.2.12.1 NY-MSHSPSP-PV56475/2022
Omicron BA.4 NR-56806 SARS-Related Coronavirus-2, Isolate hCoV-19/USA/
MD-HP30386/2022
M.S. Nair et al.
and 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. We
already reported viability of Vero E6 cells post extract treatment (Nair
et al., 2021) for the same extracts. To normalize the IC50 values for the
new variants tested or the WT and variants tested previously, dry mass of
leaves and total ART contents measured in the Artemisia extracts as
previously described (Nair et al., 2021).
2.4. Human lung cell line culture and viral infection
Human lung tissue derived cells, line A549 (ATCC CCL-185), engi­
neered to overexpress the human angiotensin converting enzyme
(ACE2) by stable transfection of a hu-ACE2 expressing lentiviral
construct under puromycin selection (Ikhlas et al., 2022) were cultured
in Essential Minimal Eagle’s medium (EMEM) with 10% fetal calf serum
(FCS). These A549 cells stably expressing hu-ACE2 were plated over­
night in 24 well plates to form a monolayer. The monolayer was treated
with two doses of tea extract (cv BUR) on the next day. One dose (50 μg
DW) was higher and the other lower (3.125 μg) than the IC50 of the tea
against the WA1 virus for this cultivar. Virus (either USA/WA1 or Om­
icron BA.4) was added to the treated cells within 15 min post treatment.
Plates were incubated at 37 ◦ C/5% CO2 for 48 h to allow the virus to
replicate. At 48 h, the infected/treated cells were subjected to end-point
virus titration using Vero-ACE2/TMPRSS2 cells. Briefly, the supernatant
was removed, and cells washed three times with phosphate-buffered
saline (PBS) to remove any unbound virus and leftover residual ex­
tracts. Each washed monolayer of cells in the well was treated with 150
μL of 0.25% Trypsin-EDTA (Corning) and incubated for 3 min at
37 ◦ C/5% CO2. Following neutralization of the trypsin with EMEM
containing 10% FCS, the cells were collected from the wells into a tube,
centrifuged at 300×g for 5 min to pellet them. The pellet was recovered
and resuspended in 250 μl of fresh EMEM+10% FCS. A 1:1 dilution in
trypan blue was used to count the cell number per sample using the
automated Biorad TC20 cell counter. Cells were then diluted in EMEM to
set up an endpoint titration in 96 well plates starting at 10,000 cells/­
well. Twelve 3-fold dilutions of the cells were overlaid on Vero cells
expressing ACE2 and TMPRSS2 (Vero/ACE2/TMPRSS2), which are
extremely sensitive to virus replication and show vivid cytopathic ef­
fects. Cells were incubated at 37 ◦ C/5%CO2 for 72 h prior to determining
the viral endpoint titer from each of the treated concentrations of the
extract with eight replicates of titration from each concentration.
A549-ACE2 cells that did not receive any extract and infected with the
virus at same MOI were used as controls to determine the maximal
endpoint titer achievable in the Vero-ACE2-TMPRSS2 cells.
2.5. Chemicals and reagents
Reagents were procured from Sigma-Aldrich (St. Louis, MO). Renilla-
Glo was from Promega (E2720). EMEM (Cat # 30–2003) and XTT re­
agent (Cat # 30-1011 k) were from ATCC.
2.6. Statistical analyses
The anti-SARS-CoV-2 analyses were done at least in triplicate. Plant
hot water extracts had n ≥ 6 independent assays as previously described
(Nair et al., 2021). IC50 values were calculated using GraphPad Prism
V9.4. The endpoint titer of each replicate was scored and plotted using
GraphPad Prism V9.4 and statistics using t-test (Wilcoxon matched-pairs
signed rank test) was performed between the groups to determine the
p-value. The percentage neutralization was calculated and plotted using
GraphPad Prism V9.4 software.
3. Results and discussion
Hot-water extracts of four cultivars of A. annua inhibited the recently
3
Journal of Ethnopharmacology 308 (2023) 116291
evolved omicron and its three tested subvariants of SARS-CoV-2 with
IC50 values calculated and normalized to the ART content of each tested
tea infusion ranging from 0.5 to 16.5 μM ART (Fig. 2; the lower the IC50,
the more potent the drug/extract). When the IC50 values were instead
normalized to the dry mass of the extracted A. annua leaves, values
ranged from 20 to 106 μg (Fig. 3). Although the values for these new
variants are for the most part slightly higher than the IC50 values re­
ported for variants previously reported (Nair et al., 2021, 2022) and all
are summarized in Table 2, they fell within limits of assay variation. As
already reported for extracts used in this study, there was no measurable
loss of cell viability at a dry weight of ≤50 μg for any cultivar extract
(Nair et al., 2021). Others have reported in vitro efficacy of A. annua (Nie
et al., 2021; Zhou et al., 2021) and A. afra (Nie et al., 2021) extracts
against earlier variants of SARS-CoV-2; however, to our knowledge there
are no reports showing efficacy against omicron or its variants.
End point titer was measured for BUR, the most potent of the four
tested cultivars, and results (Fig. 4) confirmed our previous dose-
response studies (Nair et al., 2021, 2022) showing a dose-dependent
response. The highest dose in the study had minimal viral load and
indicated that initial viral loads at 50 μg/mL were almost minimal
(>1000-fold lower) compared to that of the virus controls, even after the
extract was removed. To quantify this further, we measured the fre­
quency of infectivity for each group by calculating the average endpoint
titers of the 8 replicates and averages are shown in Table 3. The fre­
quency of infectivity was inversely proportional to the dry weight
equivalent of the added leaf extract thereby allowing us to conclude that
the cv BUR extracts do have an inhibitory effect on the replication of
SARS-CoV-2 in human A549-ACE2 cells.
The observed dose-response was also proportional to the viral isolate
tested; cv. BUR showed slightly less than two-fold increase in the IC50
value against Omicron BA.4 as compared to the USA/WA1 original
isolate when tested in the Vero E6 cells (Fig. 2). This difference is re­
flected in the current endpoint titer assay using human A549 lung cells,
where the sensitivity of the extracts dropped by four-fold against Omi­
cron BA.4 (endpoint titer 432 ± 101) compared to the WA1 (1759 ±
473) for the highest 50 μg/ml dose and about 1.5-fold against Omicron
BA.4 (endpoint titer 106 ± 41.5) versus WA1 (68.5 ± 16.4) for the
3.125 μg/ml dose.
Using VSV-based spike pseudovirus we previously showed that in­
hibition was not at virus entry into cells, (Nair et al., 2021). Those results
were reconfirmed here wherein none of the VSV-based pseudotyped
viruses (WA1/WT, Omicron BA.1.1 or Omicron BA.4) were inhibited at
any of the doses tested from 100 μg dry weight of the leaves at 5-fold
dilutions (Supplemental Fig. S4). Calculated IC50s of all tea extracts
were greater than the highest dose tested indicating that these extracts
do not have inhibitory activity at the virus entrance into the cell. As an
assay control, serum from a patient who has broad neutralization re­
sponses was tested showing expected data that the more recently
evolved Omicron variants BA.1 or BA.4 are more resilient to serum
antibodies (Fig. S4) than the ancestral WA1 virus. Given that the drop in
potency to the extracts (especially cv BUR) is far lower, future mecha­
nistic studies, should focus on interaction of the Artemisia extracts at
post-entry steps to inhibit viral replication.
Although ART IC50 as well as leaf dry mass IC50 values are shown in
this study, we previously reported that potency was inversely related to
ART concentration (Nair et al., 2021) and others showed that A. afra, a
species lacking ART, was also highly effective in vitro against
SARS-CoV-2 (Nie et al., 2021). Nevertheless, ART has some
anti-SARS-CoV-2 activity as we and others showed (Cao et al., 2020;
Gendrot et al., 2020a, 2020b; Hu et al., 2021; Nair et al., 2021; Nie et al.,
2021; Zhou et al., 2021). Although there have been some clinical studies
using ART, it was used as a combination therapy. For example, in a small
non-randomized controlled trial where patients were treated with
ART-piperaquine (ART-PPQ) or placebo the mean time for recovery
when there was no longer PCR-detectable virus was 10.6 d for ART-PPQ
treated patients vs. 19.3 d for those receiving placebo (Li et al., 2021).
M.S. Nair et al.
Fig. 2. SARS-CoV-2 variant inhibition by four cultivars of A. annua L. hot water extracts normalized to their artemisinin (ART) content and compared to WT. WT,
USA/WA1; omicron and its variants: BA.1, BA.2, BA.2.12.1, and BA.4 at a multiplicity of infection (MOI) of 0.1 in Vero E6 cells. Data are plotted from an average of
three replicates from each of two experiments ± SEM.
All patients treated with ART-PPQ were virus-free after 21 d compared
to 36 d for placebo. In another small trial patients had faster recovery vs.
placebo in those who used ArtemiC, an oral spray containing ART,
curcumin, frankincense, and vitamin C (Hellou et al., 2022). To our
knowledge, however, there are no reports of clinical trials using only
A. annua or its extracts.
Because we and others (Nair et al., 2021; Nie et al., 2021) showed
that ART is not the most likely anti-SARS-CoV-2 therapeutic phyto­
chemical in A. annua extracts, questions remain regarding the identity of
these non-ART phytochemicals. To resolve that question, several groups
have used in silico approaches (Shahhamzehei et al., 2022; Tang et al.,
2022). Tang et al. screened the Traditional Chinese Medicines for sys­
tems Pharmacology Databased and Analysis Platform to identify all
phytochemicals reportedly in A. annua then ranked them according to
oral bioavailability and drug likeness (OB and DL, respectively). That list
was narrowed to 19 compounds within their OB and DL limits of ≥30%
and 0.18, respectively. They concluded that many on the list of 19
compounds had anti-inflammatory, immune regulatory, and therapeutic
properties. Among the top therapeutic candidates were luteolin and
isorhamnetin. Using a ZINC library the Efferth lab also screened an in
silico library of >39,000 natural product compounds including some
from plants with known antiviral activity and narrowed their hits to 33
likely compounds (Shahhamzehei et al., 2022). Of the top 12, three,
isorhamnetin, luteolin, and rosmarinic acid, are present in A. annua and
when tested in vitro had IC50 values of 8.42, 11.81, and 9.43 μM,
respectively, against the main protease in SARS-CoV-2, 3CLpro, a
chymotrypsin-like protease involved in viral replication. Along with
4
Journal of Ethnopharmacology 308 (2023) 116291
reports of anti-SARS-CoV-2 activity of other A. annua phytochemicals, e.
g., quercetin and myricetin against NTPase/helicase (Russo et al., 2020;
Solnier and Fladerer, 2021), many other small molecules, especially
flavonoids, are showing antiviral potential and likely work in combi­
nation (synergistically) in these extracts to achieve the therapeutic
response.
While artemisinin is always of interest when studying the antimi­
crobial efficacy of A. annua, it is not likely the main anti-SARS-CoV-2
molecule. Artemisinin has some antiviral activity, however, it appears
to be antagonistic to the overall anti-SARS-CoV-2 activity of the hot
water extracts (Nair et al., 2021). Of eight different tested cultivars of
A. annua containing different amounts of artemisinin, there was an in­
verse relationship between artemisinin content and extract potency; IC50
values decreased in potency with increasing artemisinin content (Nair
et al., 2021). Further evidence for non-artemisinin anti-SARS-CoV-2
activity in Artemisia sp. was provided by Nie et al. who showed that
A. afra extracts lacking artemisinin were equally potent against the virus
as A. annua (Nie et al., 2021). Clearly, future studies are needed to
isolate, identify and validate the activity of nonartemisinin putative
anti-SARS-CoV-2 therapeutic compounds in A. annua and in combina­
tion with artemisinin.
While authors are not promoting any one type of delivery, in low-
and middle-income countries a traditional tea infusion may be appro­
priate, culturally acceptable, cost-effective, and perhaps the only de­
livery mode available, especially for people in rural areas. For those in
the more developed parts of the world, an encapsulated form of
powdered dried leaves is likely more reasonable. Using ART as a marker
M.S. Nair et al. Journal of Ethnopharmacology 308 (2023) 116291

Fig. 3. SARS-CoV-2 variant inhibition by four cultivars of A. annua L. hot water extracts normalized to their A. annua leaf dry mass (DW) and compared to WT. WT,
USA/WA1; omicron and its variants: BA.1, BA.2, BA.2.12.1, and BA.4 at a multiplicity of infection (MOI) of 0.1 in Vero E6 cells. Data are plotted from an average of
three replicates from each of two experiments ± SEM.

Table 2
Comparative IC50 values of A. annua L. hot-water extracts (10 g/L) against all current and previously tested strains of SARS-CoV-2 based on either artemisinin content
or leaf dry weight (DW).
Cultivar Potency normalized to artemisinin content

IC50 μM artemisinin

WA1a B.1.1.7a B.1.351a P.1b B.1.617.1b B.1.617.2b WA1 BA.1 BA.2 BA.2.12.1 BA.4
(WT) (alpha) (beta) (gamma) (kappa) (delta) (WT) (omicron)

SAM 3.4 4.9 8.4 7.9 7.0 7.0 4.9 8.2 7.6 5.3 16.5
A3 0.8 1.1 2.0 1.9 1.9 2.1 1.2 1.8 1.0 1.5 2.6
BUR 0.4 0.3 0.8 1.2 1.1 1.2 0.7 1.2 0.6 0.5 1.1
MED 2.9 2.0 3.6 2.9 2.5 4.8 4.2 4.7 2.1 3.9 6.4

Cultivar Potency normalized to dry mass of leaves used in tea infusion

IC50 μg leaf DW
WA1a B.1.1.7a B.1.351a P.1b B.1.617.1b B.1.617.2b WA1 BA.1 BA.2 BA.2.12.1 BA.4

SAM 21.5 31.3 53.7 50.7 45.0 45.1 31.4 52.5 48.9 34.1 106.0
A3 15.7 22.1 39.6 38.2 37.0 42.4 23.9 36.7 20.0 30.7 50.9
BUR 15.1 11.0 32.5 50.1 44.7 49.8 27.5 48.9 23.6 22.7 45.2
MED 41.7 28.2 51.5 41.0 37.0 67.7 59.7 67.0 30.0 56.0 90.6

IC50s are values where virus is 50% inhibited. Data are an average of three replicates from each of two experiments.
a
Values taken from Nair et al. (2021).
b
values taken from Nair et al. (2022).

molecule, therapeutically relevant concentrations post-consumption can with a Cmax of 240 μg/L post tea consumption. Delivery as powdered
be delivered from tea infusions (Räth et al., 2004). They showed Artemisia leaves per os, e.g., in a capsule is also possible. Evidence in rats
A. annua tea infusions containing 94.5 mg ART/delivered dose/subject showed that compared to ingestion of pure ART, serum levels of ART 1 h
delivered high levels of ART into the serum of healthy human subjects post oral gavage were 7.4 and 18.7 time greater in males and females,

5
M.S. Nair et al. Journal of Ethnopharmacology 308 (2023) 116291

Fig. 4. Endpoint titer of cv. BUR hot water extract vs. USA/WA1 and omicron BA.4. Titer is shown as relative endpoint infectivity vs. untreated infected controls and
compared at three dosages based on the equivalent dry weight of leaf mass of the applied A. annua extract. N = 8; bars are ± SEM for 3 replicated experiments.

repurposed drugs to treat COVID-19 (Kupferschmidt, 2021). Results are

Table 3
not anticipated until 2023, (last accessed July 11, 2022, https://www.
Comparative averages of endpoint titers and corresponding frequency of infec­
isrctn.com/ISRCTN18066414). Regardless of outcome and based on
tion for USA/WA 1 and Omicron BA.4.
the continuing efficacy of A. annua extracts against all tested variants
Leaf dry USA/WA1 Omicron BA.4 (10 to date), we again urge the WHO to consider including encapsulated
mass (μg)
Endpoint Frequency of Endpoint Frequency of dried leaf A. annua as a separate arm in their trial. While there is no
titer (Mean infection = 1/ titer (Mean infection = 1/ evidence that use of A. annua induces ART resistance (Elfawal et al.,
± SEM) Endpt titer ± SEM) Endpt titer
2015), the plant could be crucial in helping many in the world where
50 1759.26 ± 0.0006 432.10 ± 0.0023 access to vaccines and standard therapeutics is logistically challenging.
473.43 101.70
3.13 68.59 ± 0.0146 106.31 ± 0.0095
16.40 41.46
Funding
Controls 1.35 ± 0.50 0.7387 6.86 ± 2.06 0.1458
Award Number NIH-2R15AT008277-02 to PJW from the National
Center for Complementary and Integrative Health funded phytochem­
respectively, from A. annua than from pure ART, a result consistent ical analyses of the plant material used in this study. The content is
among 6 tested organs including lungs (Desrosiers et al., 2020). Delivery solely the responsibility of the authors and does not necessarily repre­
as powdered Artemisia leaves in a capsule tested in one human had sent the official views of the National Center for Complementary and
serum levels of A. annua-delivered ART at 7.04 μg/mL post consumption Integrative Health or the National Institutes of Health.
of 3 g of powdered and encapsulated A. annua leaves (Nair et al., 2021).
Furthermore, again using ART as a marker molecule, TLC data of MeOH Institutional review board statement
and DCM extracts of leaves, vs. a hot water extract of a tea infusion,
show that tea infusions have an equivalent amount of extracted arte­ Not applicable.
misinin as DCM and MeOH extracts (Supplemental TLC data Fig. S3).
Informed consent statement
4. Conclusions
Not applicable.
Hot-water (tea infusion) extracts of A. annua continue to show ac­
tivity against SARS-CoV-2 and the newest VOCs including omicron and CRediT authorship contribution statement
three of its highly transmissible subvariants. Although the specific
phytochemicals have not yet been identified, there are a number of M.S. Nair: Conceptualization, Methodology, Formal analysis,
possible candidates emerging in the literature. Validation of A. annua Writing – review & editing, Supervision. Y. Huang: Methodology,
extracts against SARS-CoV-2 VOCs in a rodent model are needed as a Formal analysis. M. Wang: Methodology. P.J. Weathers: Conceptuali­
next step towards human trials. Nevertheless, this plant is safe to use, zation, Writing – original draft, preparation, Writing – review & editing.
and we urge testing in clinical trials sooner rather than later. WHO
announced in 2021 that through its COVID-19 Solidarity Therapeutics
Plus Trial that it included intravenous artesunate as one of three

6
M.S. Nair et al.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments:
We thank Prof. David Ho of the Aaron Diamond AIDS Research
Center at Columbia University for supporting the live virus work in his
lab. Dr. Melissa Towler, Worcester Polytechnic Institute, provided crit­
ical review of the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2023.116291.
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