key: cord-0985515-0sw2nkqk
authors: Farmanpour-Kalalagh, Karim; Beyraghdar Kashkooli, Arman; Babaei, Alireza; Rezaei, Ali; van der Krol, Alexander R.
title: Artemisinins in Combating Viral Infections Like SARS-CoV-2, Inflammation and Cancers and Options to Meet Increased Global Demand
date: 2022-02-07
journal: Front Plant Sci
DOI: 10.3389/fpls.2022.780257
sha: ec62b96e3be63a252dcb526edff51b6c3b021a1f
doc_id: 985515
cord_uid: 0sw2nkqk

Artemisinin is a natural bioactive sesquiterpene lactone containing an unusual endoperoxide 1, 2, 4-trioxane ring. It is derived from the herbal medicinal plant Artemisia annua and is best known for its use in treatment of malaria. However, recent studies also indicate the potential for artemisinin and related compounds, commonly referred to as artemisinins, in combating viral infections, inflammation and certain cancers. Moreover, the different potential modes of action of artemisinins make these compounds also potentially relevant to the challenges the world faces in the COVID-19 pandemic. Initial studies indicate positive effects of artemisinin or Artemisia spp. extracts to combat SARS-CoV-2 infection or COVID-19 related symptoms and WHO-supervised clinical studies on the potential of artemisinins to combat COVID-19 are now in progress. However, implementing multiple potential new uses of artemisinins will require effective solutions to boost production, either by enhancing synthesis in A. annua itself or through biotechnological engineering in alternative biosynthesis platforms. Because of this renewed interest in artemisinin and its derivatives, here we review its modes of action, its potential application in different diseases including COVID-19, its biosynthesis and future options to boost production.

Artemisinin is an oxygenated sesquiterpene lactone, mostly produced in glandular trichomes (GTs) of the medicinal plant Artemisia annua L. Wang et al., 2016; Beyraghdar Kashkooli et al., 2018 . Artemisinin and related compounds derived from the biosynthetic pathway (Figure 1 ) have been shown to be effective against malaria caused by the Plasmodium spp. parasite (Klayman, 1985; Tu, 2011) . The action of artemisinin is not only on the Plasmodium itself, but also because of its effect on human physiology. It is the effects of artemisinin on human physiology that relate to its potential uses in other diseases as well. Below the mode of action of artemisinin and related compounds in Plasmodium and humans are discussed, exemplified by its potential use in the fight against COVID-19.

Because of the alternative uses of artemisinin, including the fight against COVID-19, the renewed demand for this compound cannot be met by current production capacity. Therefore, also current production capacity problems and potential solutions are briefly discussed.

Artemisinin contains an endoperoxide bridge that is important for anti-malarial activity. In general, several mechanisms of actions have been proposed to explain the bioactivity of artemisinin against Plasmodium spp. (O'Neill et al., 2010) . The first proposed action is the interference with Plasmodium mitochondrial and plasma functions (Antoine et al., 2014) . Studies have shown that artemisinin/artemisinin semi-synthetic derivatives (totally known as endoperoxides) induce Plasmodium mitochondrial and plasma membrane depolarization Antoine et al., 2014) . These membrane depolarizations are strongly associated with Reactive Oxygen Species (ROS) that are generated by iron bioactivation of the endoperoxides of artemisinin (Antoine et al., 2014; Wang J. et al., 2015; Tilley et al., 2016) . An additional mode of action of artemisinin against the onset of malaria is based on the cleavage of the endoperoxide in the artemisinin molecule, resulting in artemisinin free radicals, which act as alkylation agent for susceptible molecules and proteins in the parasitic cell. For instance, the alkylation of Plasmodium falciparum TCTP, ATP6 (a Ca 2+ transporter) (Shandilya et al., 2013) and PI3K (Mbengue et al., 2015) may interfere with the biological function of these proteins in the infection process. In infected red blood cells, the malaria parasite degrades hemoglobin (as a source of amino acids), resulting in large amounts of free heme molecules. These are potentially toxic to the malaria parasite but are detoxified by the parasite via conversion of heme to hemozoin. The alkylation of heme by activated artemisinin could inhibit this detoxification reaction to hemozoin. In a more general sense, the alkylation of parasitic proteins may also interfere with their correct folding, which in turn may be linked to decreased parasite development. Indeed, treatment with artemisinin results in an upregulation of the Unfolded Protein Response (UPR) (Mok et al., 2015) . In addition to artemisinin, dihydroartemisinin attacks parasites by using a two-pronged process, creating protein damage, and endangering parasite proteasome function. The consequent gradual accumulation of proteasome substrates (i.e., polyubiquitinated and unfolded/damaged proteins) results in the endoplasmic reticulum stress and dihydroartemisinin-mediated death of the parasite. Tests with other specific inhibitors of the proteasome create a similar increase of polyubiquitinated proteins, also causing parasite death (Bridgford et al., 2018) (Figure 2 ).

Besides its role in combating malaria, artemisinin has also been investigated for its potential effect on immune responses under physiological and pathological conditions (Efferth et al., 2002; Aldieri et al., 2003; Xu et al., 2007; Gu et al., 2012; Lai et al., 2015; Yu et al., 2016; Nunes et al., 2017) . Many bacteria and viruses, including the SARS-CoV-2, activate the NF-κB (Nuclear Factor kappa B) signaling pathway in human cells. NF-κB is a transcription factor that regulates multiple aspects of innate and adaptive immune functions and has a central role in inflammatory responses. For instance, NF-κB induces the expression of pro-inflammatory genes like those encoding cytokines and chemokines. NF-κB is a heterodimeric protein complex consisting of p50/p65 which is retained in the cytosol FIGURE 1 | Chemical structure of artemisinin and related compounds: artemisinin (A), dihydroartemisinin, another biosynthetic pathway product and also known as dihydroqinghaosu, or artenimol (B) and artesunate, which is a semi-synthetic chemical derivative of artemisinin biosynthetic pathway product (C). FIGURE 2 | Artemisinin mechanism of action against malaria parasite; (i) production of ROS for depolarization of the parasite's mitochondria, (ii) interference with the heme detoxification pathway of red blood cells (iii) induction of alkylation and inhibition of cellular elements such as PfATPase6 and (iv) via protein damage, and inhibition of parasite proteasome function.

by interaction with IκBα (Verma et al., 1995) . Activation of NF-κB signaling activates the IκB kinase activity which results in the release of p50/p65 from IκBα and subsequent movement of p50/p65 to the nucleus where it leads to the expression of specific genes and the production of pro-inflammatory chemokines and cytokines like Interleukin 6 (IL-6) (Verma et al., 1995; Pahl, 1999; Xiong et al., 2010; Liu et al., 2017;  Figure 2 ). IL-6, is a pleiotropic cytokine that is produced in response to infection, tissue-damaging, cellular immune response, and hematopoiesis to contribute and help the host's defense system (Tanaka et al., 2014; Velazquez-Salinas et al., 2019) . Normally, the production of IL-6 is strictly regulated at transcriptional and post-transcriptional levels. However, certain diseases, like in COVID-19, may cause misregulation of the NF-κB signaling, causing overproduction of IL-6 and other cytokines in a cytokine release syndrome (CRS) (Tanaka et al., 2014; Conti and Younes, 2020; Krishna et al., 2021) . Indeed, the dynamic change of IL-6 level can be used as a potential biomarker for a severe case of COVID-19 Ulhaq and Soraya, 2020; Zhu et al., 2020) . In addition to IL-6, also other factors including interferon γ, tumor necrosis factor (TNF), and Interleukin 1 (IL-1), etc., are over-produced during CRS and contribute to pathophysiological processes and multi-organ dysfunction (MOD) (Krishna et al., 2021) . The cytokine storm (CS) during CRS may be brought under control by artemisinin or artesunate treatment as these block NF-κB signaling by inhibiting IKK activity (Efferth et al., 2002; Aldieri et al., 2003; Xu et al., 2007; Gu et al., 2012; Lai et al., 2015; Nunes et al., 2017; Efferth and Oesch, 2021) . The activation of NF-κB signaling results in the downstream activation of the p50/p65 transcription factors, and artemisinin and artesunate may also act as an inhibitor in the NF-κB signaling pathway by blocking the function of p50/p65 in transcriptional activation of target genes like IL-6.

Artemisinins have been used to combat many different types of cancers and different modes of action have been described ( Table 1) . One of the most important mechanisms is preventing the activation of NF-κB signaling pathway involved in tumor induction, initiation, and progression of many cancerous cell lines. It is noteworthy that artemisinin may affect NF-κB signaling at different levels: it inhibits initiation of nuclear signaling by preventing interaction of p65 and p50 to cytosolic IKK, but in the nucleus it also inhibits interaction of p50 and p65 with target promoters (Tran et al., 2014 ) (see GRAPHICAL ABSTRACT). Also, Helicobacter pylori-induced gastric oncogenesis is inhibited by artemisinins through blocking NF-κB signaling. Remarkably in gastric cancer, artemisinins reverse the IκBα level, prevent NF-κB pathway in a dosedependent manner, and decrease the generation of downstream inflammatory factors such as TNF-α (tumor necrosis factorα) and IL-8 (interleukin-8) (Su et al., 2019) . The artemisinin related compound dihydroartemisinin induces autophagy via suppressing NF-κB pathway in myeloma, colorectal, and cervical cancer cell lines , while the anti-invasive activity of dihydroartemisinin may occur through preventing of PKCa/Raf/ERK and JNK phosphorylation and decreasing NF-κB (Hwang et al., 2010) . Indeed, increasing IkBα protein and blocking p65 subunit in NF-κB pathway is boosted by dihydroartemisinin .

Artesunate attenuates the growth of cancer cells and thus the development of the tumor by targeting NF-κB pathway ( Table 1) . In prostate cancer cells, resistance to androgen receptor antagonists is reduced by using artesunate.

Mechanistically, the combination of artesunate and bicalutamide prevents NF-κB pathway by ubiquitin-mediated proteasomal deterioration (Nunes et al., 2017) . In attempts to treat cervical cancer, the evidence demonstrated that artesunate successfully increases tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-mediated cytotoxicity via pro-survival proteins including X-linked inhibitor of apoptosis protein (XIAP), survivin, and B-cell lymphoma-extra-large (Bcl-xL), and reduces the number of survival proteins in HeLa cells. The downregulation of mentioned proteins can be regulated by repressing activation of serine/threonine-protein kinase and NF-κB signaling. Artesunate further prevents TRAIL-influenced transcriptional activity of NF-κB (Thanaketpaisarn et al., 2011) .

Currently, we are facing the serious challenge of the COVID-19 pandemic, which has disrupted global health and the economy. COVID-19 is a virus-related disease similar to Severe Acute Respiratory Syndrome CoronaVirus (SARS-CoV) and is caused by the SARS-CoV-2 (Lai et al., 2020; Singhal, 2020) . Unlike with SARS-CoV, patients infected with SARS-CoV-2 initially have mild symptoms and continue their daily activities, but in the meantime are infectious to others (Heymann and Shindo, 2020; Zheng et al., 2020) . In some patients, disease symptoms may suddenly increase dramatically due to the development of a CS entitled CRS with hallmarks in the body of inflammation and immunosuppression (Mehta et al., 2020) . CRS in COVID-19 patients may result in respiratory failures that create Acute Respiratory Distress Syndrome (ARDS) and MOD (Krishna et al., 2021) . While worldwide efforts are aimed at vaccines that may prevent infection by COVID-19, additional medicines that can alleviate the severe symptoms of COVID-19 are still a high priority, also because new viral variants may escape vaccine recognition.

Previously, in vitro studies have indicated that the alkylating activity of activated artemisinin (as discussed above in the context of malaria) or the specific structure of artemisinin may have potential in preventing infections by members of the Herpesviridae family (e.g., herpes simplex virus type 1, Epstein-Barr virus, human cytomegalovirus), hepatitis B virus, hepatitis C virus, and bovine viral diarrhea virus (Efferth et al., 2008; Efferth, 2018) . Artemisinin also has received renewed attention to fight emerging new viruses for which no effective antiviral drugs are available (e.g., HIV, dengue virus, chikungunya virus, Ebola virus), against viral strains that have developed drug resistance (e.g., human cytomegalovirus) and most recently against Coronavirus (D'alessandro et al., 2020) . To combat infection by COVID-19, extracts from different medicinal plants (including from Artemisia spp.) have been tested against COVID-19 (Bahrami et al., 2020; Bailly and Vergoten, 2020; Huang et al., 2020;  Kapepula et al., 2020; Koshak and Koshak, 2020; Mani et al., 2020; Verma et al., 2020; Williamson and Kerimi, 2020; Belhassan et al., 2021; Hassanipour et al., 2021; Javed et al., 2021; Lyu et al., 2021; Nazrul Islam et al., 2021; Song et al., 2021) .

In vitro efficacy of artemisinin-based treatments to combating SARS-CoV-2 has indicated that treatment with artesunate, artemether, A. annua extracts, and artemisinin hindered virus infections of human lung cancer A549-hACE2 cells, VeroE6 cells, and human hepatoma Huh7.5 cells. Among these four treatments, artesunate showed the strongest anti-SARS-CoV-2 activity (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). Collectively, time-of-addition experiments in A549-hACE2 cells displayed that artesunate attacked the virus at the post-entry level . In parallel with the previous study, dried-leaf hot-water extracts of A. annua cultivars including SAM, BUR, A3, and MED revealed in vitro anti-SARS-CoV-2 activity against Alpha, Beta, Gamma, Delta, and Kappa variants of the virus. All cultivars in addition to being potent in combating with original wild type WA1 also showed effective potential against mentioned variants. IC90 and IC50 according to measured artemisinin content ranged from 1.4-25.0 µM and 0.3 to 8.4 µM, respectively. Also, the IC90 and IC50 according to dried-leaf weight ranged from 59.5-160.6 µg DW and 11.0 to 67.7 µg DW, respectively (Nair et al., 2022) . Alternatively, Artemisia spp. Extracts and COVID-Organics drink produced in Madagascar hindered in vitro SARS-CoV-2 and Feline coronavirus (FcoV) infections at concentrations that did not influence cell efficacy and viability (Nie et al., 2021) .

In another successful in vitro study, six monomer compounds including artesunate, artemether, arteannuin B, andrographolide, licochalcone B, and echinatin exerted high anti-SARS-CoV-2 and anti-GX_P2V (pangolin coronavirus) activity (Hu et al., 2021) . In addition to the mentioned monomers, it is noteworthy that the quinoline like artemisinins has shown strong anti-SARS-CoV-2 activity (Firestone et al., 2021) . Also, the anti-SARS-CoV-2 activity of nine artemisinin-based compounds experimented in vitro. Results highlighted that arteannuin B, artesunate, and dihydroartemisinin are the most potent agents in inhibiting virus activity. Also, several artemisinins decreased the generating of the virus nucleocapsid (N) proteins in a dose-dependent manner. It can be concluded that targeting N proteins can be considered as one of the possible options to control viral infection. On the other hand, both lumefantrine and arteannuin B suppressed viral infection following SARS-CoV-2 entry toward the host cells (Cao et al., 2020) . In addition to artemisinins monotherapy, in vitro inhibition of SARS-CoV-2 replication by artemisininbased combination therapies (ACTs) in African experimental society indicated that the artesunate-mefloquine exerted high anti-SARS-CoV-2 activity with % inhibition of 72.1 ± 18.3%. Also, other ACTs including artesunate-pyronaridine, artesunateamodiaquine, dihydroartemisinin-piperaquine, and artemetherlumefantrine displayed the same range of inhibition (27.1 to 34.1 %) (Gendrot et al., 2020) . Along with in vitro studies of ACTs, molecular docking studies have also demonstrated the anti-SARS-CoV-2 activity of artemisinin-thymoquinone hybrids against the main protease of the virus (de Oliveira et al., 2021).

While extracts from medicinal plants may show some preliminary efficacy in small scale clinical trials (Dong et al., 2020; reviewed in Orege et al., 2021) , these do not clarify where the activity is coming from. For instance, the antiviral and immunomodulation effects of Artemisia spp. Extracts, as recently been reviewed (Kshirsagar and Rao, 2021) , may not only be due to artemisinins, but also from other potential bioactive compounds like flavonoids, mono-and sesqui-terpenes or tannins in these extracts (Kshirsagar and Rao, 2021) . To address efficiency and potential harmful side effects of plant extracts, a more detailed knowledge on the molecular mode of action of individual bioactive molecules is needed (Cheong et al., 2020; Guastalegname and Vallone, 2020) . Studies indicate that artesunate, dihydroartemisinin, and artemisinin may act at the cell surface by inhibition of the binding of the SARS-CoV-2 spike protein to cell surface receptors, thus potentially preventing both endocytosis of the virus and activation of the NF-κB signaling pathway (Gendrot et al., 2020; Rolta et al., 2020; Sehailia and Chemat, 2020; Uckun et al., 2021) .

However, molecular docking studies indicate that artemisinins may also bind to coronavirus-host proteins such as E protein, helicase protein, N protein, 3CL PRO , S protein, nonstructural protein 3 (nsp3), nsp10, nsp14, nsp15, cathepsin-L, and glucoseregulated protein 78 receptor (Fuzimoto, 2021; Ribaudo et al., 2021) and part of the biological activity of artemisinin against COVID-19 may thus also be partially based on inhibiting the function of these viral proteins.

In addition, artemisinin/and or artesunate may limit CS by inhibiting IKK and thus over-active NF-κB signaling, or it may inhibit the transcriptional activity of p50/p65, released by NF-κB signaling (see GRAPHICAL ABSTRACT). While preliminary studies with artemisinins look promising, researchers have warned that the potential of artemisinins in combating COVID-19 requires further clinical research (Uzun and Toptas, 2020; Krishna et al., 2021) .

A total of 16 trials with Artemisia spp. Extract, artemisinins, and ACTs have been registered in the US National Library of Medicine 1 with clinical trial IDs: NCT04530617, NCT04306497, NCT04701606, NCT04695197, NCT04475107, NCT04532931, NCT04374019, NCT05084911, NCT04502342, NCT04801017, NCT04374084, NCT05004753, NCT04387240, NCT04553705, NCT04802382, NCT04382040, and 3 trials in Chinese Clinical Trial Registry (ChiCTR) database 2 with IDs: ChiCTR2000033049, ChiCTR2000032915, and ChiCTR2000030082 (suspended by the investigator) to combat SARS-CoV-2 infection.

Until December 2021, only one trial registered in https: //clinicaltrials.gov/ has been terminated with ID number: NCT04530617. Preliminary results from this study indicate that the agents such as A. annua and Camostat mesilate may help reduce the number of hospitalized patients and that the use of artemisinin-piperaquine for treatment of COVID-19 is safe . Therefore, the World Health Organization (WHO) has initiated clinical trials on three promising candidate drugs, including artesunate, to evaluate the anti-inflammatory activity against SARS-CoV-2 (Solidarity Trial PLUS is registered at: ISRCTN83971151). As new mutations occur in the SARS-CoV-2, resulting in new variants including Alpha, Beta, Gamma, Delta, Kappa, and Omicron, combating potential downstream effects of COVID-19 infections remains an important aspect of dealing with the ongoing pandemic, especially when this virus has become endemic.

Artemisinin is produced in glandular trichomes in the leaves and ovary of the A. annua . Both the specificity of artemisinin biosynthesis occurring in GTs and the fact that GTs represent about 2% of plant total weight put a FIGURE 3 | A schematic representation of the artemisinin biosynthetic pathway in Artemisia annua L. The precursors for artemisinin biosynthesis (DMAPP and IPP) are produced in the cytosolic Mevalonate (MVA) pathway and the plastidal 2-C-Methylerythritol 4-phosphate pathway (MEP) pathway, respectively (Dewick, 2009; Vranová et al., 2013; Beyraghdar Kashkooli et al., 2019) . IPP+DMAPP are converted to FPP, which is the general precursor for sesquiterpenes (Fuentes et al., 2016) . In sequential enzymatic steps, FPP is converted to amorphadiene, artemisinin alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde, and finally artemisinic acid. The first and key step in the biosynthesis of artemisinin biosynthetic pathway is the conversion of FPP to amorpha 4, 11-diene (known as amorphadiene), which is catalyzed by a well-known terpene cyclase, the amorpha-4,11-diene synthase (ADS) (Mercke et al., 2000; Bertea et al., 2005) . The cytochrome P450 hydroxylase (CYP71AV1) (Teoh et al., 2006) then converts amorphadiene to artemisinic alcohol. CYP71AV1 also oxidizes artemisinic alcohol to artemisinic aldehyde and artemisinic acid, respectively. Artemisinic aldehyde double bond reductase (DBR2) as the branching point and the aldehyde dehydrogenase 1 (ALDH1) convert artemisinic aldehyde to dihydroartemisinic aldehyde and dihydroartemisinic acid, respectively (Bertea et al., 2005; Schramek et al., 2010) . Abbreviation for genes in artemisinin biosynthetic pathway includes; GPPS, geranyl pyrophosphate synthase; FPPS, farnesyl pyrophosphate synthase; ADS, armorpha-4, 11-diene synthase; CYP71AV1, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase; DBR2, artemisinic aldehyde delta-11(13)-double bond reductase; ALDH1, aldehyde dehydrogenase 1.

limit to the bulk production of artemisinin in planta (Judd et al., 2019) . The different steps in artemisinin production are shown in Figure 3 . The end-product of the enzymatic pathway (dihydroartemisinic acid) is presumably toxic to the plant cell and is therefore exported over the plasma membrane and the cell wall to a subcuticular space where it is converted non-enzymatically by light (UV) to artemisinin . In parallel, artemisinic acid, another end-product of the enzymatic pathway may also be exported from the cell, and extracellularly converted to arteannuin B. Typical yield of artemisinin from the Artemisia annua plant is 0.6 to 1.2% but may go up to 2% based on plant dry weight . However, such yield is low and far from the potential world's demand (Judd et al., 2019) . Current supplies of artemisinin are already limiting to treat people for malaria in a cost-effective way, so use in treating the disease of pandemic proportions like COVID-19 will need new approaches to artemisinin production. Several methods have been proposed to increase artemisinin production so far which are treatments impacting A. annua cultivation and physiology and breeding approaches to increase artemisinin yield, engineering artemisinin biosynthetic and transport pathway in the native A. annua plants, and engineering of heterologous (plant and microorganism) systems via ectopic expression of the biosynthetic pathway (Lei et al., 2011; Liu et al., 2011; Parshikov et al., 2012; Fuentes et al., 2016; Kiani et al., 2016; Carqueijeiro et al., 2020) .

Thanks to targeted breeding programs different cultivars of A. annua can be grown in a wide range of climate conditions (temperate, cold temperate, subtropical, and Mediterranean) (Ferreira et al., 2005) . However, the content and composition of secondary metabolites in A. annua plants are determined by numerous interacting factors: geographical conditions, harvesting time, agricultural practices (e.g., fertilization, irrigation, density per unit area) and post-harvest conditions (Mert et al., 2002; Jelodar et al., 2014) . Conventional manipulations of the A. annua plant that may enhance artemisinin production are discussed below and summarized in Figure 4 .

In addition to genetic factors, the artemisinin yield can be influenced by environmental conditions and field management practices (Charles et al., 1991) . Furthermore, the use of fertilizer compounds can also affect the artemisinin content. As studies have shown, manure and chemical fertilizers are effective in the production of secondary metabolites by improving the photosynthetic rate and carbon production (Jha et al., 2011) . Numerous reports have considered the use of nitrogen (N) fertilizer to be somewhat effective in increasing the artemisinin content (Ferreira et al., 2005; Davies et al., 2009; Aftab et al., 2011) . In addition to the important role of macronutrients in increasing the artemisinin production in Artemisia plants, studies showed that the deficiency of micronutrients (iron, copper, zinc, and barium) also plays a significant role in reducing the artemisinin content (Srivastava and Sharma, 1990) .

Similarly, the use of elicitors in plants increases the accumulation of secondary metabolites (Zhao et al., 2005) . For instance, the elicitor of Penicillium chrysogenum extract has increased (up to double) the production of artemisinin in hairy roots of A. annua (Liu et al., 1999) . Additionally, the application of chitosan or of the arbuscular mycorrhizal species Rhizophagus intraradices (as elicitor) was able to increase the content of dihydroartemisinic acid and artemisinin in Artemisia plants (Lei et al., 2011; Mandal et al., 2014) . Since the response to these biotic factors often involves the phytohormone jasmonic acid (JA), the effect of these factors on artemisinin content could be due to activated JA signaling.

The main breeding goals of A. annua are the improvement of artemisinin production by increasing the yield potential of leaves, proliferate the number of shoots, and raising the total number of glandular trichomes per plant (Graham et al., 2010; Jelodar et al., 2014) . Selection through germplasm and genetic modification can be considered as basic strategies for the improvement of artemisinin production in A. annua (Charles et al., 1991; Xie et al., 2016) . A very common breeding technique to increase the secondary metabolites is the manipulation of the ploidy levels in plants (Weathers, 2003) . The production of artificial polyploids as a plant breeding strategy has made it possible to develop new and improved cultivars (Iannicelli et al., 2020) . In this regard, the application of ploidy manipulation techniques has successfully increased the artemisinin production in A. annua. Reports indicate that the amount of artemisinin in tetraploid plants has increased up to 56% compared to diploid plants. Alternatively, induced mutation using chemicals such as sodium azide (NaN 3 ) and ethyl methane sulfonate (EMS) was effective in increasing the artemisinin biosynthesis in native plant (Al-Qurainy and Khan, 2010; Leow et al., 2020) .

In some plant species, the production of some glandular type trichomes is enhanced by JA treatment or UVB light (Yan et al., 2012) . Indeed, the artemisinin content of A. annua is enhanced under the UV treatment at a dosage of 150 gray irradiation (Raymond et al., 2015) and UV-B radiation at 1.44 kJ m −2 d −1 (Pandey and Pandey-Rai, 2014) respectively. This could be due to both an effect on glandular trichome density and enhanced conversion of artemisinic aldehyde to artemisinin.

Various agricultural practices use plant growth regulators (PGRs) to improve artemisinin production. For example, the treatment of A. annua with Salicylic acid increases plant growth, leading to higher biomass (Aftab et al., 2010) , altered plant morphology, artemisinin content and composition (Ma et al., 2009 ). Other studies have shown that PGR GA 3 (Weathers et al., 2005; Zhang et al., 2005; Aftab et al., 2011) and JA can also increase the artemisinin content of A. annua (Zhou and Memelink, 2016) . JA has been shown to boost artemisinin biosynthesis via the releasing of repressors of transcription factor TCP14-ORA at the promoters of double bond reductase 2 (DBR) and aldehyde dehydrogenase 1 (ADH1), two key genes in the artemisinin biosynthetic pathway (see Figure 3 ; Ma et al., 2018) .

Metabolic engineering may be used to improve the production of artemisinin in A. annua itself but is hampered by the difficulties in efficient transformation and regeneration of A. annua plants. Alternatively, the genes that have been isolated from A. annua that are involved in artemisinin production may be expressed in 2 | Introducing artemisinin (ART) pathway genes in Artemisia annua L. to improve the ART production using different strategies.

Artemisia annua L. Overexpression of HMGR and ADS 1.73 mg/g DW Alam and Abdin (2011) Artemisia annua L.

Overexpression of CYP71AV1 and CPR 0.98 ± 0.18 mg/g Shen et al. (2012) Artemisia annua L. Overexpression of FPS,CYP71AV1 and CPR 2.9 mg/g FW Chen et al. (2013) Artemisia annua L.

Overexpression of FPS 1.3% DW Banyai et al. (2010) Artemisia annua L.

Overexpression of HMGR 0.386 ± 0.0332mg/g DW Aquil et al. (2009) Artemisia annua L. Suppressing the expression of SQS 31.4 mg/g DW Zhang et al. (2009) Artemisia annua L.

Overexpression of AaWRKY1 ≥ 14 4 mg/g DW Jiang W. et al. (2016) Artemisia annua L.

Overexpression of DBR2 1.5-2.14 mg/g DW Yuan et al. (2015) TABLE 3 | Introducing artemisinin (ART) pathway genes in planta to improve the ART production using different strategies.

Nicotiana benthamiana Transient expression of ART precursors' genes 0.000220347 mg/g FW Ting et al. (2013) Nicotiana a heterologous host that is easier to transform and grow (Covello, 2008; Ma et al., 2015; Xie et al., 2016; Lv et al., 2017; Ikram et al., 2019) . Options to manipulate artemisinin production in A. annua are briefly discussed below and summarized in Figure 5 . The yield effects of the different transformation efforts of A. annua are summarized in Table 2 .

(1) Artemisia annua has been transformed with Agrobacterium genes that affect endogenous plant hormone levels (rol ABC or ipt), resulting in mild to up to 9 times higher artemisinin levels compared to untransformed (or empty vector transformed) control plants (Sa et al., 2001; Bulgakov, 2008; Dilshad et al., 2015; Kiani et al., 2016) .

(2) Other transformation strategies are aimed at boosting precursors, either by boosting flux through the Mevalonate pathway by ectopic expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or by blocking unwanted side reactions that drain from the precursor pool (e.g., Squalene synthase (SQS), that diverts FPP to squalene) (Liao et al., 2016) . Ectopic expression of HMGR in A. annua can boost artemisinin production (Aquil et al., 2009; Nafis et al., 2011; Ma et al., 2017) , while also suppression of SQS can increase artemisinin levels Yang et al., 2008; Zhang et al., 2009 ; Table 2 and Figure 5) .

(3) Transformation approaches may also be aimed at boosting flux through the artemisinin biosynthetic pathway itself through overexpression of biosynthesis genes. Ectopic overexpression of farnesyl pyrophosphate synthase (FPS) alone or FPS with CYP71AV1 and CPR, increased artemisinin levels in transgenic plants ( Table 2 ; Chen et al., 2000 Chen et al., , 2013 Han et al., 2016; Banyai et al., 2010; Wani et al., 2021) . In another study, upregulated expression of HMGR, FPS, ADS, Aldh1, and ADS in A. annua increased artemisinin level 39-56% fold ; Figure 5 ).

(4) The expression of endogenous biosynthesis genes may also be boosted by ectopic overexpression of relevant transcription factors (TF), provided expression of such TF is limiting for transcription of target genes. Multiple TFs (AP2/ERFs, WRKYs, bHLH, MYCs) have been identified in the regulation of endogenous artemisinin biosynthesis genes (Verpoorte and Memelink, 2002; Yang et al., 2012; Shen et al., 2016; Lv et al., 2017) , but not all of these have been tested for stable transformation of A. annua.

However, ectopic expression of WRKY does result in higher artemisinin production in A. annua (Jiang W. et al., 2016 ; Table 2 and Figure 5 ).

(5) The capacity for extracellular accumulation of dihydroartemisinic acid for extra-cellular conversion to dihydroartemisinin may be of importance for the flux through the biosynthetic pathway to prevent feedback inhibition and possible toxic effects of pathway products. Studies in tobacco have shown that ABC-transporter AaPDR2, in concert with specific LTP AaTLP3 may be required for this function .

In the tobacco assay, AaLTP3 and AaPDR2 prevent dihydroartemisinic acid reflux from the apoplast to the cell, resulting in higher artemisinin levels . However, manipulation of either ABC-transporter or LTP levels in A. annua has not been performed till now. Potentially, overexpression of these proteins could result in enhanced artemisinin production in A. annua (Figure 5 ).

The genes for artemisinin production have also been expressed in other plants, either by transient expression or by stable transformation. To date, in planta artemisinin production has been reported for tobacco (Nicotiana benthamiana) and moss (Physcomitrella patens) ( Table 3) . Transient expression of genes in N. benthamiana leaves is used to characterize gene function and has the advantage that up to 15 genes may be coexpressed at the same time to transiently reconstitute entire biosynthetic pathways (Reed et al., 2017; Carqueijeiro et al., 2020) . Reconstruction of the artemisinin biosynthetic pathway by transient co-expression of pathway genes (Wallaart et al., 2001; Zhang et al., 2011; Ting et al., 2013; Wang et al., 2016) or stable transformation with pathway genes (Farhi et al., 2011) resulted in the first ectopic production of artemisinin in another plant species (Table 3) . Recently, the stable transformation of the moss (P. patens) with artemisinin pathway genes, demonstrated that this compound may also be produced in much more primitive plant species ; Table 3 and Figure 6 ). As a plant-based production platform of artemisinin, moss has been shown to have promiscuous substrate recognition which may be a substitute for some artemisinin biosynthetic pathway genes which are not present in for example N. benthamiana. Substrate promiscuity of sesquiterpenoids pathway from A. annua and Tanacetum parthenium for individual enzymes or pathways is previously reported . Besides, the simple purification step (due to lack of conjugation phenomenon) has been also stated as one of the advantages of this platform compared to the N. benthamiana.

Synthetic biology techniques play an important role in the exploration, overproduction, and structure diversification of phytochemicals (Qi et al., 2015; Muhammad et al., 2020; Alam et al., 2021) . The full set of artemisinin biosynthesis genes have also been introduced into yeast, resulting in substantial production of dihydroartemisinic acid in fermenters (Immethun et al., 2013; Paddon et al., 2013; Tang et al., 2014 ; Table 4 ). This dihydroartemisinic acid can subsequently photo-chemically be converted to (dihydro)artemisinin (Paddon and Keasling, 2014) . Some of the issues that play a role in the potential boosting of artemisinin production in A. annua as discussed above, also play a role in boosting artemisinin production in heterologous hosts. For instance, high activity of HMGR is also important for the ectopic production of artemisinin in yeast (Pitera et al., 2007; Ye and Bhatia, 2012; Tang et al., 2014) (Table 4) .

Whether artemisinin can be used to not only combat malaria, but also other diseases, including those acting on a pandemic scale, will very much depend on further validation of the efficacy of artemisinin in these other diseases and on how this compound can cost-effectively be made available to the community. Multiple and complementary approaches may be necessary to boost synthesis capacity, varying from the transformation of A. annua itself to boost artemisinin yield, to investment into heterologous production platforms that may be easier to scale up. At the same time, we should not ignore the lessons learned from monotherapy in combating disease, as this may result in the emergence of artemisinin-resistance, as currently happening for malaria. Therefore, both for malaria, as for the potential of artemisinin in combating COVID-19 and other viral infections ACTs or triple artemisinin-based combination therapies (TACTs) may be required to prevent the rise of artemisinin resistant disease variants.

ABK conceptualized the review. KF-K, AR, AB, ARK, and ABK wrote the manuscript. ABK, ARK, and AB reviewed the manuscript. KF-K, AR, and ABK did the figures visualization. All authors contributed to the article and approved the submitted version.

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Development of transgenic Artemisia annua (chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNAmediated gene silencing

Artesunate exerts an anti-immunosuppressive effect on cervical cancer by inhibiting PGE2 production and Foxp3 expression

Artesunate inhibits the growth and induces apoptosis of human gastric cancer cells by downregulating COX-2

The production of artemisinin precursors in tobacco

The molecular cloning of artemisinic aldehyde 11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua

Exogenous GA3 and flowering induce the conversion of artemisinic acid to artemisinin in Artemisia annua plants

Artesunate impairs growth in cisplatin-resistant bladder cancer cells by cell cycle arrest, apoptosis and autophagy induction

Elicitor signal transduction leading to production of plant secondary metabolites

Artesunate enhances radiosensitivity of human non-small cell lung cancer A549 cells via increasing NO production to induce cell cycle arrest at G 2/M phase

COVID-19 and the cardiovascular system

Artesunate inhibits angiogenesis and downregulates vascular endothelial growth factor expression in chronic myeloid leukemia K562 cells

Jasmonate-responsive transcription factors regulating plant secondary metabolism

Artesunate induces autophagy dependent apoptosis through upregulating ROS and activating AMPK-mTOR-ULK1 axis in human bladder cancer cells

Artesunate inhibits the growth of gastric cancer cells through the mechanism of promoting oncosis both in vitro and in vivo

Artemisinin derivatives stimulate DR5-specific TRAIL-induced apoptosis by regulating wildtype P53

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

Artesunate suppresses the viability and mobility of prostate cancer cells through UCA1, the sponge of miR-184

Artemisinin reduces cell proliferation and induces apoptosis in neuroblastoma

Clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019

Artesunate induces apoptosis of bladder cancer cells by miR-16 regulation of COX-2 expression

We would like to thank Tarbiat Modares University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.