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

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 globe to thwart many infectious diseases. AIM OF THE STUDY: The SARS-CoV-2 (COVID-19) virus continues to infect millions while rapidly evolving new variants that are more transmissible and evade vaccine-elicited antibodies, e.g., omicron and its subvariants. Having shown potency against all previously tested variants, A. annua L. extracts were further tested against highly infectious omicron and its recent subvariants. MATERIALS AND METHODS: Using Vero E6 cells, we measured the in vitro efficacy (IC50) of stored (frozen) dried-leaf hot-water A. annua L. extracts of four cultivars (A3, BUR, MED, and SAM) against SARS-CoV-2 variants: original WA1 (WT), BA.1 (omicron), BA.2, BA.2.12.1, and BA.4. End point virus titers of infectivity in cv. BUR-treated human lung A459 cells overexpressing hu-ACE2 were determined for both WA1 and BA.4 viruses. 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.

Multivariate Analysis of Essential Oil Composition of Artemisia annua L. Collected from Different Locations in Korea.

Artemisia annua L. is distributed throughout the world and it is an important medicinal plant in Korea to treat various human diseases. Recently, A. annua has also been considered to be an effective ethnobotanical drug against COVID-19. A. annua contains an appreciable amount of essential oil with different biological properties. However, the composition of essential oils in aromatic plants can be varied depending on several factors, including geographic, genetic, ecological, etc. Hence, the present study aimed to investigate the chemical diversity of essential oils of Korean A. annua collected from different locations in Korea by multivariate analysis. For this purpose, the seeds of A. annua were collected from 112 different locations in Korea and were grown under the same environmental conditions. Except for nine individuals which decayed during the cultivation, essential oils were isolated from the aerial parts of 103 A. annua individuals (AEOs) using the steam distillation extraction method, and their chemical compositions were determined by GC-MS analysis. Furthermore, a multivariate analysis was performed to distinguish the difference between 103 individuals of A. annua based on their essential oil compositions. The yield of A. annua essential oils ranged from 0.04 to 1.09% (v/w). Based on the GC-MS data, A. annua individuals were grouped into six chemotypes such as artemisia ketone, camphor, ß-cubebene, eucalyptol, α-pinene, and ß-selinene. The multivariate analysis results revealed that Korean A. annua could be largely grouped into three clusters such as artemisia ketone, eucalyptol, and ß-selinene. Among 35 components selected for principal component analysis (PCA), PC1, PC2, and PC3 accounted for 82.55%, 8.74%, and 3.62%, respectively. Although all individuals of A. annua were cultivated under the same environmental conditions, there is an intraspecific chemical diversity that exists within Korean native species.

Virtual Screening of Artemisia annua Phytochemicals as Potential Inhibitors of SARS-CoV-2 Main Protease Enzyme.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a human coronaviruses that emerged in China at Wuhan city, Hubei province during December 2019. Subsequently, SARS-CoV-2 has spread worldwide and caused millions of deaths around the globe. Several compounds and vaccines have been proposed to tackle this crisis. Novel recommended in silico approaches have been commonly used to screen for specific SARS-CoV-2 inhibitors of different types. Herein, the phytochemicals of Pakistani medicinal plants (especially Artemisia annua) were virtually screened to identify potential inhibitors of the SARS-CoV-2 main protease enzyme. The X-ray crystal structure of the main protease of SARS-CoV-2 with an N3 inhibitor was obtained from the protein data bank while A. annua phytochemicals were retrieved from different drug databases. The docking technique was carried out to assess the binding efficacy of the retrieved phytochemicals; the docking results revealed that several phytochemicals have potential to inhibit the SARS-CoV-2 main protease enzyme. Among the total docked compounds, the top-10 docked complexes were considered for further study and evaluated for their physiochemical and pharmacokinetic properties. The top-3 docked complexes with the best binding energies were as follows: the top-1 docked complex with a -7 kcal/mol binding energy score, the top-2 docked complex with a -6.9 kcal/mol binding energy score, and the top-3 docked complex with a -6.8 kcal/mol binding energy score. These complexes were subjected to a molecular dynamic simulation analysis for further validation to check the dynamic behavior of the selected top-complexes. During the whole simulation time, no major changes were observed in the docked complexes, which indicated complex stability. Additionally, the free binding energies for the selected docked complexes were also estimated via the MM-GB/PBSA approach, and the results revealed that the total delta energies of MMGBSA were -24.23 kcal/mol, -26.38 kcal/mol, and -25 kcal/mol for top-1, top-2, and top-3, respectively. MMPBSA calculated the delta total energy as -17.23 kcal/mol (top-1 complex), -24.75 kcal/mol (top-2 complex), and -24.86 kcal/mol (top-3 complex). This study explored in silico screened phytochemicals against the main protease of the SARS-CoV-2 virus; however, the findings require an experimentally based study to further validate the obtained results.

Artemisia Extracts and Artemisinin-Based Antimalarials for COVID-19 Management: Could These Be Effective Antivirals for COVID-19 Treatment?

As the world desperately searches for ways to treat the coronavirus disease 2019 (COVID-19) pandemic, a growing number of people are turning to herbal remedies. The Artemisia species, such as A. annua and A. afra, in particular, exhibit positive effects against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and COVID-19 related symptoms. A. annua is a source of artemisinin, which is active against malaria, and also exhibits potential for other diseases. This has increased interest in artemisinin's potential for drug repurposing. Artemisinin-based combination therapies, so-called ACTs, have already been recognized as first-line treatments against malaria. Artemisia extract, as well as ACTs, have demonstrated inhibition of SARS-CoV-2. Artemisinin and its derivatives have also shown anti-inflammatory effects, including inhibition of interleukin-6 (IL-6) that plays a key role in the development of severe COVID-19. There is now sufficient evidence in the literature to suggest the effectiveness of Artemisia, its constituents and/or artemisinin derivatives, to fight against the SARS-CoV-2 infection by inhibiting its invasion, and replication, as well as reducing oxidative stress and inflammation, and mitigating lung damage.

Network pharmacology-based predictions of active components and pharmacological mechanisms of Artemisia annua L. for the treatment of the novel Corona virus disease 2019 (COVID-19).

BACKGROUND: Novel Corona Virus Disease 2019 (COVID-19) is closely associated with cytokines storms. The Chinese medicinal herb Artemisia annua L. (A. annua) has been traditionally used to control many inflammatory diseases, such as malaria and rheumatoid arthritis. We performed network analysis and employed molecular docking and network analysis to elucidate active components or targets and the underlying mechanisms of A. annua for the treatment of COVID-19. METHODS: Active components of A. annua were identified through the TCMSP database according to their oral bioavailability (OB) and drug-likeness (DL). Moreover, target genes associated with COVID-19 were mined from GeneCards, OMIM, and TTD. A compound-target (C-T) network was constructed to predict the relationship of active components with the targets. A Compound-disease-target (C-D-T) network has been built to reveal the direct therapeutic target for COVID-19. Molecular docking, molecular dynamics simulation studies (MD), and MM-GBSA binding free energy calculations were used to the closest molecules and targets between A. annua and COVID-19. RESULTS: In our network, GO, and KEGG analysis indicated that A. annua acted in response to COVID-19 by regulating inflammatory response, proliferation, differentiation, and apoptosis. The molecular docking results manifested excellent results to verify the binding capacity between the hub components and hub targets in COVID-19. MD and MM-GBSA data showed quercetin to be the more effective candidate against the virus by target MAPK1, and kaempferol to be the other more effective candidate against the virus by target TP53. We identified A. annua's potentially active compounds and targets associated with them that act against COVID-19. CONCLUSIONS: These findings suggest that A. annua may prevent and inhibit the inflammatory processes related to COVID-19.

Enhancing artemisinin content in and delivery from Artemisia annua: a review of alternative, classical, and transgenic approaches.

MAIN CONCLUSION: This review analyses the most recent scientific research conducted for the purpose of enhancing artemisinin production. It may help to devise better artemisinin enhancement strategies, so that its production becomes cost effective and becomes available to masses. Malaria is a major threat to world population, particularly in South-East Asia and Africa, due to dearth of effective anti-malarial compounds, emergence of quinine resistant malarial strains, and lack of advanced healthcare facilities. Artemisinin, a sesquiterpene lactone obtained from Artemisia annua L., is the most potent drug against malaria and used in the formulation of artemisinin combination therapies (ACTs). Artemisinin is also effective against various types of cancers, many other microbes including viruses, parasites and bacteria. However, this specialty metabolite and its derivatives generally occur in low amounts in the source plant leading to its production scarcity. Considering the importance of this drug, researchers have been working worldwide to develop novel strategies to augment its production both in vivo and in vitro. Due to complex chemical structure, its chemical synthesis is quite expensive, so researchers need to devise synthetic protocols that are economically viable and also work on increasing the in-planta production of artemisinin by using various strategies like use of phytohormones, stress signals, bioinoculants, breeding and transgenic approaches. The focus of this review is to discuss these artemisinin enhancement strategies, understand mechanisms modulating its biosynthesis, and evaluate if roots play any role in artemisinin production. Furthermore, we also have a critical analysis of various assays used for artemisinin measurement. This may help to develop better artemisinin enhancement strategies which lead to decreased price of ACTs and increased profit to farmers.

Potential Emergence of Plasmodium Resistance to Artemisinin Induced by the Use of Artemisia annua for Malaria and COVID-19 Prevention in Sub-African Region.

Plasmodium resistance to antimalarial drugs is an obstacle to the elimination of malaria in endemic areas. This situation is particularly dramatic for Africa, which accounts for nearly 92% of malaria cases worldwide. Drug pressure has been identified as a key factor in the emergence of antimalarial drug resistance. Indeed, this pressure is favoured by several factors, including the use of counterfeit forms of antimalarials, inadequate prescription controls, poor adherence to treatment regimens, dosing errors, and the increasing use of other forms of unapproved antimalarials. This resistance has led to the replacement of chloroquine (CQ) by artemisinin-based combination therapies (ACTs) which are likely to become ineffective in the coming years due to the uncontrolled use of Artemisia annua in the sub-Saharan African region for malaria prevention and COVID-19. The use of Artemisia annua for the prevention of malaria and COVID-19 could be an important factor in the emergence of resistance to Artemisinin-based combination therapies.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

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

ETHNOPHARMACOLOGICAL RELEVANCE: For millennia, Artemisia annua L. was used in Southeast Asia to treat "fever". This medicinal plant is effective against multiple pathogens and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria caused by Plasmodium parasites. 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 L. extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five 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 (WT) 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 to 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 to 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 potentially provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.

Instant determination of the artemisinin from various Artemisia annua L. extracts by LC-ESI-MS/MS and their in-silico modelling and in vitro antiviral activity studies against SARS-CoV-2.

INTRODUCTION: Numerous efforts in natural product drug development are reported for the treatment of Coronavirus. Based on the literature, among these natural plants Artemisia annua L. shows some promise for the treatment of SARS-CoV-2. OBJECTIVE: The main objective of our study was to determine artemisinin content by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), to investigate the in vitro biological activity of artemisinin from the A. annua plants grown in Turkey with various extracted methods, to elaborate in silico activity against SARS-CoV-2 using molecular modelling. METHODOLOGY: Twenty-one different extractions were applied. Direct and sequential extractions studies were compared with ultrasonic assisted maceration, Soxhlet, and ultra-rapid determined artemisinin active molecules by LC-ESI-MS/MS methods. The inhibition of spike protein and main protease (3CL) enzyme activity of SARS-CoV-2 virus was assessed by time resolved fluorescence energy transfer (TR-FRET) assay. RESULTS: Artemisinin content in the range 0.062-0.066%. Artemisinin showed significant inhibition of 3CL protease activity but not Spike/ACE-2 binding. The 50% effective concentration (EC50 ) of artemisinin against SARS-CoV-2 Spike pseudovirus was found greater than 50 µM (EC45 ) in HEK293T cell line whereas the cell viability was 94% of the control (P < 0.01). The immunosuppressive effects of artemisinin on TNF-α production on both pseudovirus and lipopolysaccharide (LPS)-induced THP-1 cells were found significant in a dose dependent manner. CONCLUSION: Further studies of these extracts for COVID-19 treatment will shed light to seek alternative treatment options. Moreover, these natural extracts can be used as an additional treatment option with medicines, as well as prophylactic use can be very beneficial for patients.

An overview of the anti-SARS-CoV-2 properties of Artemisia annua, its antiviral action, protein-associated mechanisms, and repurposing for COVID-19 treatment.

Artemisia annua and its phytocompounds have a rich history in the research and treatment of malaria, rheumatoid arthritis, systemic lupus erythematosus, and other diseases. Currently, the World Health Organization recommends artemisinin-based combination therapy as the first-line treatment for multi-drug-resistant malaria. Due to the various research articles on the use of antimalarial drugs to treat coronaviruses, a question is raised: would A. annua and its compounds provide anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) properties? PubMed/MEDLINE, Scopus, and Google Scholar were searched for peer-reviewed articles that investigated the antiviral effects and mechanisms of A. annua and its phytochemicals against SARS-CoVs. Particularly, articles that evidenced the herb's role in inhibiting the coronavirus-host proteins were favored. Nineteen studies were retrieved. From these, fourteen in silico molecular docking studies demonstrated potential inhibitory properties of artemisinins against coronavirus-host proteins including 3CLPRO, S protein, N protein, E protein, cathepsin-L, helicase protein, nonstructural protein 3 (nsp3), nsp10, nsp14, nsp15, and glucose-regulated protein 78 receptor. Collectively, A. annua constituents may impede the SARS-CoV-2 attachment, membrane fusion, internalization into the host cells, and hinder the viral replication and transcription process. This is the first comprehensive overview of the application of compounds from A. annua against SARS-CoV-2/coronavirus disease 2019 (COVID-19) describing all target proteins. A. annua's biological properties, the signaling pathways implicated in the COVID-19, and the advantages and disadvantages for repurposing A. annua compounds are discussed. The combination of A. annua's biological properties, action on different signaling pathways and target proteins, and a multi-drug combined-therapy approach may synergistically inhibit SARS-CoV-2 and assist in the COVID-19 treatment. Also, A. annua may modulate the host immune response to better fight the infection.

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

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.