Artemisia annua hot-water extracts show potent activity in vitro against Covid-19 variants including delta

M.S. Nair, Y. Huang, D.A. Fidock, M.J. Towler, P.J. Weathers

doi: https://doi.org/10.1101/2021.09.08.459260

Abstract

Ethnopharmacological relevance For millennia in Southeast Asia, Artemisia annua L. was used to treat “fever”. This medicinal plant is effective against numerous infectious microbial and viral diseases and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria.

Aim of the Study The SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants.

Materials and Methods Using Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five fully infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1).

Results In addition to being effective against the original wild type WA1, A. annua cultivars A3, BUR, MED and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3-8.4 μM and 1.4-25.0 μM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0-67.7 μg DW and 59.5-160.6 μg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of ≤50 μg in the extract of any cultivar.

Conclusions Results suggest that oral consumption of A. annua hot-water extracts (tea infusions), could provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.

1.0 Introduction

The global SARS-CoV-2 (Covid-19) pandemic has infected ~220 million people and killed >4.5 million (https://coronavirus.jhu.edu/map.html). Numerous variants have rapidly evolved (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/). The delta variant is currently the most transmissible to date (RO = 5-7) (Nunes-Vaz and Macintyre, 2021), causing break-through infections in vaccinated individuals (Gupta et al., 2021). Effective and approved small molecule-based therapeutics are still lacking. Recently, we showed that hot-water extracts of dried leaves of seven cultivars of the medicinal plant, Artemisia annua L., used for millennia to treat malaria fever (Hsu, 2006) and sourced from four continents, prevented SARS-CoV-2 replication in vitro (Nair et al., 2021). Recently, anti-SARS-CoV-2 efficacy of A. annua extracts was independently confirmed (Zhou et al., 2021).

Antiviral efficacy inversely correlated to artemisinin (ART) content (Nair et al., 2021). Others also observed that compared to A. annua, A. afra, a related perennial species lacking ART, was equally effective vs. SARS-CoV-2 with IC50 values of 0.9-3.4 and 0.65 mg/mL extract, respectively (Nie et al., 2021). Although these results indicated that both A. annua and A. afra have potent anti-SARS-CoV-2 activity in vitro and that the effect is not ART dependent, it was unclear whether A. annua is effective against emerging variants.

Here we report in vitro efficacy against new variants of four of the seven originally studied A. annua cultivars.

2.0 Methods and Materials

2.1 Plant material, extract preparations, and artemisinin analyses

Hot-water extracts (tea infusions) were prepared from dried leaves of four cultivars of Artemisia annua L. (SAM, MASS 00317314; BUR, LG0019527; A3, Anamed; MED, KL/015/6407) and analyzed as detailed in (Nair et al., 2021). Briefly, hot-water extracts were made from 10 g dried leaves/L that were boiled in water for 10 min, sieved to remove solids, and filter-sterilized (0.22 μm) prior to storage at −20°C. For ART analysis, tea infusions were extracted and analyzed by gas chromatography-mass spectrometry as detailed in (Martini et al., 2020). ART contents in μg/mL were: 42.5 for A3; 20.1 for BUR; 59.4 for MED4; and 149.4 for SAM (Nair et al., 2021).

2.2 Viral culture and infection

Vero E6 (ATCC CRL-1586) cell cultivation and viral infection were detailed in (Nair et al., 2021). SARS-CoV-2 isolates (USA WA1; alpha, B1.1.7; beta, B1.351; gamma, P.1; delta, B.1.617.2; kappa, B.1.617.1) were sourced from BEI Resources (www.beiresources.org). Viruses were titrated upon propagation to determine their tissue culture infectious dose (TCID) in Vero E6 cells, aliquoted and frozen at −80°C until further use. Multiplicity of infection (MOI) was 0.1 as documented by (Liu et al., 2020).

2.3 Assays for determining drug inhibition of SARS-CoV-2 and cell viability

Extract dilutions were incubated for 1 h in wells of 96-well tissue culture plates containing a monolayer of Vero E6 cells seeded the prior day at 20,000 cells/well. Extracts were added at equal ART amounts based on the content of each extract. One hour later SARS-CoV-2 virus was added to each well at a final MOI of 0.1. Cells were cultured for 3 days at 37oC in 5% CO2 and were scored for cytopathic effects as previously detailed (Liu et al., 2020). Results were converted into percent of control. Drug concentrations were log transformed. Concentration of drug(s) that inhibited virus by 50% (i.e., IC50), and 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. Viability of Vero E6 cells post extract treatment was already reported in Nair et al. (2021) for the same extracts.

READ FULL TEXT HERE: https://www.biorxiv.org/content/10.1101/2021.09.08.459260v1.full

5.0 Conclusions

Hot-water (tea infusion) extracts of A. annua are active against SARS-CoV-2 and its variants alpha, beta, gamma, delta, and kappa. In our original report, anti-SARS-CoV-2 activity inversely correlated with ART content. Herein, similar responses are noted for gamma, delta, and kappa wherein the A. annua cultivar with the lowest ART content, BUR, generally had the lowest (most effective) IC50. These results demonstrate the potential of the extracts as treatments in the global fight against this constantly evolving virus. We urge WHO to consider including extracts and encapsulated dried leaves in their announced clinical trials that already include artesunate (Kupferschmidt, 2021). We aim to test preclinical models of SARS-CoV-2 in rodent models (Dinnon et al., 2020; Gu et al., 2020) that could help advance A. annua as an inexpensive therapeutic in parts of the world where logistic issues such as delivery require longer time to achieve vaccination levels that would ultimately quell this pandemic.