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Covering: up to 2017-2022Many small molecule drugs are first discovered in nature, commonly the result of long ethnopharmacological use by people, and then characterized and purified from their biological sources. Traditional medicines are often more sustainable, but issues related to source consistency and efficacy present challenges. Modern medicine has focused solely on purified molecules, but evidence is mounting to support some of the more traditional uses of medicinal biologics. When is a more traditional delivery of a therapeutic appropriate and warranted? What studies are required to establish validity of a traditional medicine approach? Artemisia annua and A. afra are two related but unique medicinal plant species with long histories of ethnopharmacological use. A. annua produces the sesquiterpene lactone antimalarial drug, artemisinin, while A. afra produces at most, trace amounts of the compound. Both species also have an increasing repertoire of modern scientific and pharmacological data that make them ideal candidates for a case study. Here accumulated recent data on A. annua and A. afra are reviewed as a basis for establishing a decision tree for querying their therapeutic use, as well as that of other medicinal plant species.

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 (IC 50 ) 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 IC 50 values ranged from 0.5 to 16.5 μM ART and from 20 to 106 μg DW. IC 50 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., Competing Interests: 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., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is a deadly pathogen and causative agent of human tuberculosis, causing ~1.5 million deaths every year. The increasing drug resistance of this pathogen necessitates novel and improved treatment strategies. A crucial aspect of the host-pathogen interaction is bacterial nutrition. In this study, Artemisia annua and Artemisia afra dichloromethane extracts were tested for bactericidal activity against Mtb strain mc 2 6230 under hypoxia and various infection-associated carbon sources (glycerol, glucose, and cholesterol). Both extracts showed significant bactericidal activity against Mtb, regardless of carbon source. Based on killing curves, A. afra showed the most consistent bactericidal activity against Mtb for all tested carbon sources, whereas A. annua showed the highest bactericidal activity in 7H9 minimal media with glycerol. Both extracts retained their bactericidal activity against Mtb under hypoxic conditions. Further investigations are required to determine the mechanism of action of these extracts and identify their active constituent compounds.

Ethnopharmacological Relevance: The Chinese medicinal herb, Artemisia annua L., has been used for >2,000 yr as traditional tea infusions to treat a variety of infectious diseases including malaria, and its use is spreading globally (along with A. afra Jacq. ex Willd.) mainly through grassroots efforts., Aim of the Study: Artemisinin is more bioavailable delivered from the plant, Artemisia annua L. than the pure drug, but little is known about how delivery via a hot water infusion (tea) alters induction of hepatic CYP2B6 and CYP3A4 that metabolize artemisinin., Materials and Methods: HepaRG cells were treated with 10 μM artemisinin or rifampicin (positive control), and teas (10 g/L) of A. annua SAM, and A. afra SEN and MAL with 1.6, 0.05 and 0 mg/g DW artemisinin in the leaves, respectively; qPCR and Western blots were used to measure CYP2B6 and CYP3A4 responses. Enzymatic activity of these P450s was measured using human liver microsomes and P450-Glo assays., Results: All teas inhibited activity of CYP2B6 and CYP3A4. Artemisinin and the high artemisinin-containing tea infusion (SAM) induced CYP2B6 and CYP3A4 transcription, but artemisinin-deficient teas, MAL and SEN, did not. Artemisinin increased CYP2B6 and CYP3A4 protein levels, but none of the three teas did, indicating a post-transcription inhibition by all three teas., Conclusions: This study showed that Artemisia teas inhibit activity and artemisinin autoinduction of CYP2B6 and CYP3A4 post transcription, a response likely the effect of other phytochemicals in these teas. Results are important for understanding Artemisia tea posology., Competing Interests: 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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. IC 50 and IC 90 values based on measured artemisinin content ranged from 0.3 to 8.4 μM and 1.4-25.0 μM, respectively. The IC 50 and IC 90 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 IC 50 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 IC 50 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 IC 50  = 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 IC 90 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Artemisia annua L.is a well-established medicinal herb used for millennia to treat parasites and fever-related ailments caused by various microbes. Although effective against many infectious agents, the plant is not a miracle cure and there are infections where it has proved ineffective or limited. It is important to report those failures., Methods: Here artemisinin, artesunate and dried leaf slurries of A. annua were used daily for 6 days in vivo against Babesia microti in mice 2 days post infection at 100 µg artemisinin/kg body weight. Parasitemia was measure before and 15 days days post treatment. Artemisinin and extracts of A. annua also were tested in vitro against six Candida sp. at artemisinin concentrations up to 180 µM and growth measured after cultures were fed drugs once at different stages of growth and also after repeated dosing., Results: A. annua , artesunate, and artemisinin were ineffective in reducing or eliminating parasitemia in B. microti -infected mice treated at 100 µg artemisinin/kg body weight. Although the growth of exponential cultures of many of the tested Candida sp. was inhibited, the response was not sustained and both artemisinin and Artemisia were essentially ineffective at concentrations of artemisinin at up to 180 µM of artemisinin., Conclusions: Together these results show that artemisinin, its derivatives, and A. annua are ineffective against B. microti and at least six species of Candida ., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/lcm-21-2). PJW serves as an unpaid editorial board member of Longhua Chinese Medicine from Jul 2020 to Jun 2022. PJW reports that she receives Federal and institutional grants, contract fees, and does editorial work for 3 journals. SMR reports a grant from the Center for Clinical and Translational Science (UMassMed/NIH). The authors have no other conflicts of interest to declare.

Ethnopharmacological Relevance: Artemisia annua has a long history of use in Southeast Asia where it was used to treat "fever", and A. afra has a similar history in southern Africa. Since their discovery, A. annua use, in particular, has expanded globally with millions of people using the plant in therapeutic tea infusions, mainly to treat malaria., Aim of the Study: In this study, we used in vitro studies to query if and how A. annua and A. afra tea infusions being used across the globe affect asexual Plasmodium falciparum parasites, and their sexual gametocytes., Materials and Methods: P. falciparumstrain NF54 was grown in vitro, synchronized, and induced to form gametocytes using N-acetylglucosamine. Cultures during asexual, early, and late stage gametocytogenesis were treated with artemisinin, methylene blue, and A. annua and A. afra tea infusions (5 g DW/L) using cultivars that contained 0-283 μM artemisinin. Asexual parasitemia and gametocytemia were analyzed microscopically. Gametocyte morphology also was scored. Markers of early (PfGEXP5) and late stage (Pfs25) gametocyte gene expression also were measured using RT-qPCR., Results: Both A. annua and A. afra tea infusions reduced gametocytemia in vitro, and the effect was mainly artemisinin dependent. Expression levels of both marker genes were reduced and also occurred with the effect mainly attributed to artemisinin content of four tested Artemisia cultivars. Tea infusions of both species also inhibited asexual parasitemia and although mainly artemisinin dependent, there was a weak antiparasitic effect from artemisinin-deficient A. afra., Conclusions: These results showed that A. annua and to a lesser extent, A. afra, inhibited parasitemia and gametocytemia in vitro., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dried-leaf Artemisia annua L. (DLA) antimalarial therapy was shown effective in prior animal and human studies, but little is known about its mechanism of action. Here IC50s and ring-stage assays (RSAs) were used to compare extracts of A. annua (DLAe) to artemisinin (ART) and its derivatives in their ability to inhibit and kill Plasmodium falciparum strains 3D7, MRA1252, MRA1240, Cam3.11 and Cam3.11rev in vitro. Strains were sorbitol and Percoll synchronized to enrich for ring-stage parasites that were treated with hot water, methanol and dichloromethane extracts of DLA, artemisinin, CoArtem™, and dihydroartemisinin. Extracts of A. afra SEN were also tested. There was a correlation between ART concentration and inhibition of parasite growth. Although at 6 hr drug incubation, the RSAs for Cam3.11rev showed DLA and ART were less effective than high dose CoArtem™, 8 and 24 hr incubations yielded equivalent antiparasitic results. For Cam3.11, drug incubation time had no effect. DLAe was more effective on resistant MRA-1240 than on the sensitive MRA-1252 strain. Because results were not as robust as observed in animal and human studies, a host interaction was suspected, so sera collected from adult and pediatric Kenyan malaria patients was used in RSA inhibition experiments and compared to sera from adults naïve to the disease. The sera from both age groups of malaria patients inhibited parasite growth ≥ 70% after treatment with DLAe and compared to malaria naïve subjects suggesting some host interaction with DLA. The discrepancy between these data and in-vivo reports suggested that DLA's effects require an interaction with the host to unlock their potential as an antimalarial therapy. Although we showed there are serum-based host effects that can kill up to 95% of parasites in vitro, it remains unclear how or if they play a role in vivo. These results further our understanding of how DLAe works against the malaria parasite in vitro., Competing Interests: The authors have declared that no competing interests exist.

Ethnopharmacological Relevance: Emergence of drug-resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is a major barrier to tuberculosis (TB) eradication, as it leads to longer treatment regimens and in many cases treatment failure. Thus, there is an urgent need to explore new TB drugs and combinations, in order to shorten TB treatment and improve outcomes. Here, we evaluated the potential of two Asian and African traditional medicinal plants, Artemisia annua, a natural source of artemisinin (AN), and Artemisia afra, as sources of novel antitubercular agents., Aim of the Study: Our goal was to measure the activity of A. annua and A. afra extracts against Mtb as potential natural and inexpensive therapies for TB treatment, or as sources of compounds that could be further developed into effective treatments., Materials and Methods: The minimum inhibitory concentrations (MICs) of A. annua and A. afra dichloromethane extracts were determined, and concentrations above the MICs were used to evaluate their ability to kill Mtb and Mycobacterium abscessus in vitro., Results: Previous studies showed that A. annua and A. afra inhibit Mtb growth. Here, we show for the first time that Artemisia extracts have a strong bactericidal activity against Mtb. The killing effect of A. annua was much stronger than equivalent concentrations of pure AN, suggesting that A. annua extracts kill Mtb through a combination of AN and additional compounds. A. afra, which produces very little AN, displayed bactericidal activity against Mtb that was substantial but weaker than that of A. annua. In addition, we measured the activity of Artemisia extracts against Mycobacterium abscessus. Interestingly, we observed that while A. annua is not bactericidal, it inhibits growth of M. abscessus, highlighting the potential of this plant in combinatory therapies to treat M. abscessus infections., Conclusion: Our results indicate that Artemisia extracts have an enormous potential for treatment of TB and M. abscessus infections, and that these plants contain bactericidal compounds in addition to AN. Combination of extracts with existing antibiotics may not only improve treatment outcomes but also reduce the emergence of resistance to other drugs., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Artemisia annua L. and artemisinin, have been used for millennia to treat malaria. We used human liver microsomes (HLM) and rats to compare hepatic metabolism, tissue distribution, and inflammation attenuation by dried leaves of A. annua (DLA) and pure artemisinin. For HLM assays, extracts, teas, and phytochemicals from DLA were tested and IC 50 values for CYP2B6 and CYP3A4 were measured. For tissue distribution studies, artemisinin or DLA was orally delivered to rats, tissues harvested at 1 h, and blood, urine and feces over 8 h; all were analyzed for artemisinin and deoxyartemisinin by GC-MS. For inflammation, rats received an intraperitoneal injection of water or lipopolysaccharide (LPS) and 70 mg/kg oral artemisinin as pure drug or DLA. Serum was collected over 8 h and analyzed by ELISA for TNF-α, IL-6, and IL-10. DLA-delivered artemisinin distributed to tissues in higher concentrations in vivo, but elimination remained mostly unchanged. This seemed to be due to inhibition of first-pass metabolism by DLA phytochemicals, as demonstrated by HLM assays of DLA extracts, teas and phytochemicals. DLA was more effective than artemisinin in males at attenuating proinflammatory cytokine production; the data were less conclusive in females. These results suggest that the oral consumption of artemisinin as DLA enhances the bioavailability and anti-inflammatory potency of artemisinin., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Artemisia sp., especially A. annua and A. afra , have been used for centuries to treat many ailments. While artemisinin is the main therapeutically active component, emerging evidence demonstrates that the other phytochemicals in this genus are also therapeutically active. Those compounds include flavonoids, other terpenes, coumarins, and phenolic acids. Artemisia sp. phytochemicals also improve bioavailability of artemisinin and synergistically improve artemisinin therapeutic efficacy, especially when delivered as dried leaf Artemisia as a tea infusion or as powdered dry leaves in a capsule or compressed into a tablet. Here results from in vitro, and in vivo animal and human studies are summarized and critically discussed for mainly malaria, but also other diseases susceptible to artemisinin and Artemisia sp. including schistosomiasis, leishmaniasis, and trypanosomiasis.

Background: Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer with poor prognosis. Artemisinin (AN), produced naturally in Artemisia annua L., has anti-cancer activity. Artemisinin delivered as dried leaf Artemisia (DLA) showed efficacy against malaria in rodents and humans., Hypothesis/purpose: DLA is posited as being at least as efficacious as artesunate (AS) in its ability to induce cytotoxicity in NSCLC cells and also inhibit tumor growth in a NSCLC xenograft murine model., Study Design: Three NSCLC cell lines were used, a non-cancerous human fibroblast line, and xenograft murine models to compare efficacy of artemisinin delivered p.o. via DLA, DLA extracts (DLAe), and AS., Methods: DLAe was compared to AS using NSCLC cell lines A549, H1299 and PC9 as well as non-cancerous human dermal fibroblasts (HDF) CCD-1108Sk line. Cell viability, cell migration and cell cycle were compared for AS and DLAe. Westerns measured activated caspases-3, -8 and -9 to determine involvement of intrinsic and/or extrinsic apoptotic pathways. Xenograft murine models of A549 and PC9 cells were used to measure tumor growth inhibition by AS or DLA, with tumor volume the primary endpoint., Results: Both DLAe and AS suppressed A549, H1299 and PC9 cell viability with no inhibition of non-cancerous HDF CCD-1108Sk cells. Caspases-3, -8 and -9 were activated, suggesting cell death was stimulated through both intrinsic and/or extrinsic apoptotic pathways. Both drugs induced G2/M or mitotic arrest in PC9 and H1299 cells, and DLAe induced G1 arrest in A549 cells. AS and DLAe induced DNA damage as double stranded breaks evidenced by phosphorylation of histone H2AX. DLAe inhibited migration of PC9 and A549 cells. In A549 xenografted animals, p.o. AS and DLA inhibited relative tumor growth by 40% and 50%, respectively, compared to controls. AS was ineffective at inhibiting PC9-induced tumor growth, but DLA inhibited relative tumor growth by ∼50% compared to controls., Conclusion: This is the first study demonstrating efficacy of DLA and mechanistic differences of DLAe vs. AS, against NSCLC cells. Compared to AS, DLA possesses qualities of a novel therapeutic for patients with NSCLC., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Ethnopharmacological Relevance: Artemisia annua has been used for > 2000yrs to treat fever and is more recently known for producing the important antimalarial drug, artemisinin., Aim of the Study: Artemisinin combination therapies (ACTs) are effective for treating malaria, but are often unavailable to those in need. Dried leaves of A. annua (DLA) have recently been studied as a cost effective alternative to traditional ACTs. DLA was shown to dramatically increase oral bioavailability compared to pure artemisinin, so more investigation into the mechanisms causing this increased bioavailability is needed., Materials and Methods: In this study, we used a simulated digestion system coupled with Caco-2 cell permeability assays to investigate the intestinal permeability of DLA compared to pure artemisinin. We also determined the effects of different phytochemicals (7 flavonoids, 3 monoterpenes, 2 phenolic acids, scopoletin and inulin) and the cytochrome P450 isoform CYP3A4 on artemisinin intestinal permeability., Results: Artemisinin permeability, when delivered as digested DLA, significantly increased by 37% (P app = 8.03 × 10 -5 cms -1 ) compared to pure artemisinin (P app = 5.03 × 10 -5 cms -1 ). However, none of the phytochemicals tested or CYP3A4 had any significant effect on the intestinal permeability of artemisinin. We also showed that essential oil derived from A. annua negatively affected the intestinal permeability of artemisinin, but only after simulated digestion. Finally, we showed that A. annua essential oil reduced the transepithelial electrical resistance of Caco-2 monolayers, but only in the presence of bile. Although also reduced by essential oils, artemisinin P app subsequently recovered in the presence of plant matrix., Conclusions: These results shed light on the mechanisms by which DLA enhances the oral bioavailability of artemisinin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Dried leaf Artemisia annua (DLA) has shown efficacy against Plasmodium sp. in rodent studies and in small clinical trials. Rodent malaria also showed resiliency against the evolution of artemisinin drug resistance., Purpose: This is a case report of a last resort treatment of patients with severe malaria who were responding neither to artemisinin combination therapy (ACT) nor i.v. artesunate., Study Design: Of many patients treated with ACTs and i.v. artesunate during the 6 mon study period, 18 did not respond and were subsequently treated with DLA Artemisia annua., Methods: Patients were given a dose of 0.5g DLA per os, twice daily for 5d. Total adult delivered dose of artemisinin was 55mg. Dose was reduced for body weight under 30kg. Clinical symptoms, e.g. fever, coma etc., and parasite levels in thick blood smears were tracked. Patients were declared cured and released from hospital when parasites were microscopically undetectable and clinical symptoms fully subsided., Results: All patients were previously treated with Coartem® provided through Santé Rurale (SANRU) and following the regimen prescribed by WHO. Of 18 ACT-resistant severe malaria cases compassionately treated with DLA, all fully recovered. Of the 18, this report details two pediatric cases., Conclusions: Successful treatment of all 18 ACT-resistant cases suggests that DLA should be rapidly incorporated into the antimalarial regimen for Africa and possibly wherever else ACT resistance has emerged., (Copyright © 2017. Published by Elsevier GmbH.)

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Main Conclusion: Roots of plants with high artemisinin-producing leaves increased leaf production of artemisinin in low-producing plants and vice versa indicating roots are involved in controlling artemisinin biosynthesis in shoots. The anti-malarial sesquiterpene, artemisinin, is produced and stored in glandular trichomes (GLTs) of Artemisia annua. Evidence suggested roots, which produce no significant artemisinin nor precursor compounds, regulate production of artemisinin biosynthesis in the leaves. Using grafting, we studied the relationship between rootstock and scion by measuring GLTs and five artemisinic metabolites (artemisinin, deoxyartemisinin, dihydroartemisinic acid, artemisinic acid, arteannuin B) in scions of ungrafted, self-grafted, and cross-grafted plants among three cultivars: S and 15 both having GLTs with artemisinin at 1.49 and 0.57 %, respectively, and G producing neither GLTs nor detectable artemisinin. All artemisinin-producing self-grafts, e.g., S/S (scion/rootstock) and 15/15, produced more artemisinin than ungrafted plants, likely from grafting stress. S/S grafts also produced more GLTs. The 15/S grafts produced more artemisinin than S/15, suggesting rootstocks from high producing S plants stimulated artemisinin production in 15 scions. S/15 grafts yielded less artemisinin than S/S, but more than either 15/15 or ungrafted n15 and nS; S/15 grafts also had a lower density of GLTs than S/S, suggesting rootstock inhibition of the scion. The S rootstock induced trace artemisinin production in G scions, but did not induce GLT formation in G/S grafts. Different grafts exhibited different trichome morphologies and effects on artemisinic pathway flux. This study provides new information regarding the role of roots in GLT development and artemisinin production in this important medicinal plant.

Ethnopharmacological Relevance: Artemisia annua L. produces the antimalarial sesquiterpene lactone, artemisinin (AN), and was traditionally used by the Chinese to treat fever, which was often caused by malaria., Aim of the Study: To measure effects of plant-based and dietary components on release of artemisinin and flavonoids from A. annua dried leaves (DLA) after simulated digestion., Materials and Methods: Simulated digestion was performed on DLA in four types of capsules, or in conjunction with protein, and protein-based foods: dry milk, casein, bovine serum albumin, peanuts, peanut butter, Plumpy'nut(®), and A. annua essential oils. Artemisinin and total flavonoids were measured in the liquid phase of the intestinal stage of digestion., Results: After simulated digestion, peanuts and Plumpy'nut(®) lowered AN and flavonoids, respectively, recovered from the liquid digestate fraction. None of the compositions of the tested capsules altered AN or flavonoid release. Surprisingly, bovine serum albumin (BSA) increased both AN and flavonoids recovered from liquid simulated digestate fractions while casein had no effect. AN delivered as DLA was about 4 times more soluble in digestates than AN delivered as pure drug. Addition of a volume of essential oil equivalent to that found in a high essential oil producing A. annua cultivar also significantly increased AN solubility in simulated digestates., Conclusion: These results indicate encapsulating DLA may provide a way to mask the taste of A. annua without altering bioavailability. Similarly, many peanut-based products can be used to mask the flavor with appropriate dosing. Finally, the essential oil fraction of A. annua contributes to the increased AN solubility in DLA after simulated digestion. Our results suggest that use of DLA in the treatment of malaria and other artemisinin-susceptible diseases should be further tested in animals and humans., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Dried leaves of Artemisia annua show promise as an inexpensive and sustainable antimalarial therapeutic, especially for use in developing countries. Along with the potent terpene, artemisinin, many other small molecules produced by the plant seem to aid in the therapeutic response. However, little is known about the ontogenic and phenological production of artemisinin in the plant, and its plethora of other important secondary metabolites. From a consistently high artemisinin-producing A. annua clone (SAM) we extracted and analyzed by GC/MS 22 different metabolites including terpenes, flavonoids, a coumarin, and two phenolic acids as they varied during leaf development and growth of the plant from the vegetative stage through the reproductive, full flower stage. As leaves developed, the maximum amount of most metabolites was in the shoot apical meristem. Artemisinin, on the other hand, maximized once leaves matured. Leaf and apical tissues (e.g. buds, flowers) varied in their metabolite content with growth stage with maximum artemisinin and other important secondary metabolites determined to be at floral bud emergence. These results indicated that plants at the floral bud stage have the highest level of artemisinin and other therapeutic compounds for the treatment of malaria.

Pharmaceutical monotherapies against human malaria have proven effective, although ephemeral, owing to the inevitable evolution of resistant parasites. Resistance to two or more drugs delivered in combination will evolve more slowly; hence combination therapies have become the preferred norm in the fight against malaria. At the forefront of these efforts has been the promotion of Artemisinin Combination Therapy, but despite these efforts, resistance to artemisinin has begun to emerge. In 2012, we demonstrated the efficacy of the whole plant (WP)--not a tea, not an infusion--as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a rodent malaria model. Here we show that WP overcomes existing resistance to pure artemisinin in the rodent malaria Plasmodium yoelii. Moreover, in a long-term artificial selection for resistance in Plasmodium chabaudi, we tested resilience of WP against drug resistance in comparison with pure artemisinin (AN). Stable resistance to WP was achieved three times more slowly than stable resistance to AN. WP treatment proved even more resilient than the double dose of AN. The resilience of WP may be attributable to the evolutionary refinement of the plant's secondary metabolic products into a redundant, multicomponent defense system. Efficacy and resilience of WP treatment against rodent malaria provides compelling reasons to further explore the role of nonpharmaceutical forms of AN to treat human malaria.

Artemisinin from the plant Artemisia annua (A. annua) L, and used as artemisinin combination therapy (ACT), is the current best therapeutic for treating malaria, a disease that hits children and adults especially in developing countries. Traditionally, A. annua was used by the Chinese as a tea to treat "fever". More recently, investigators have shown that tea infusions and oral consumption of the dried leaves of the plant have prophylactic and therapeutic efficacy. The presence of a complex matrix of chemicals within the leaves seems to enhance both the bioavailability and efficacy of artemisinin. Although about 1000-fold less potent than artemisinin in their antiplasmodial activity, these plant chemicals are mainly small molecules that include other artemisinic compounds, terpenes (mainly mono and sesqui), flavonoids, and polyphenolic acids. In addition, polysaccharide constituents of A. annua may enhance bioavailability of artemisinin. Rodent pharmacokinetics showed longer T 1/2 and T max and greater C max and AUC in Plasmodium chabaudi -infected mice treated with A. annua dried leaves than in healthy mice. Pharmacokinetics of deoxyartemisinin, a liver metabolite of artemisinin, was more inhibited in infected than in healthy mice. In healthy mice, artemisinin serum levels were > 40-fold greater in dried leaf fed mice than those fed with pure artemisinin. Human trial data showed that when delivered as dried leaves, 40-fold less artemisinin was required to obtain a therapeutic response compared to pure artemisinin. ACTs are still unaffordable for many malaria patients, and cost estimates for A. annua dried leaf tablet production are orders of magnitude less than for ACT, despite improvements in the production capacity. Considering that for > 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, the evidence argues for inclusion of affordable A. annua dried leaf tablets into the arsenal of drugs to combat malaria and other artemisinin-susceptible diseases.

Artemisia annua L., long used as a tea infusion in traditional Chinese medicine, produces artemisinin. Although artemisinin is currently used as artemisinin-based combination therapy (ACT) against malaria, oral consumption of dried leaves from the plant showed efficacy and will be less costly than ACT. Many compounds in the plant have some antimalarial activity. Unknown, however, is how these plant components change as leaves are processed into tablets for oral consumption. Here we compared extracts from fresh and dried leaf biomass with compressed leaf tablets of A. annua . Using GC-MS, nineteen endogenous compounds, including artemisinin and several of its pathway metabolites, nine flavonoids, three monoterpenes, a coumarin, and two phenolic acids, were identified and quantified from solvent extracts to determine how levels of these compounds changed during processing. Results showed that compared to dried leaves, artemisinin, arteannuin B, artemisinic acid, chlorogenic acid, scopoletin, chrysoplenetin, and quercetin increased or remained stable with powdering and compression into tablets. Dihydroartemisinic acid, monoterpenes, and chrysoplenol-D decreased with tablet formation. Five target compounds were not detectable in any of the extracts of this cultivar. In contrast to the individually measured aglycone flavonoids, using the AlCl 3 method, total flavonoids increased nearly fivefold during the tablet formation. To our knowledge this is the first study documenting changes that occurred in processing dried leaves of A. annua into tablets. These results will improve our understanding of the potential use of not only this medicinal herb, but also others to afford better quality control of intact plant material for therapeutic use.

Ethnopharmacological Relevance: The Chinese have used Artemisia annua as a tea infusion to treat fever for >2000 years. The active component is artemisinin. Previously we showed that when compared to mice fed an equal amount of pure artemisinin, a single oral dose of dried leaves of Artemisia annua (pACT) delivered to Plasmodium chabaudi-infected mice reduced parasitemia at least fivefold. Dried leaves also delivered >40 times more artemisinin in the blood with no toxicity. The pharmacokinetics (PK) of artemisinin delivered from dried plant material has not been adequately studied., Materials and Methods: Healthy and Plasmodium chabaudi-infected mice were oral gavaged with pACT to deliver a 100 mg kg(-1) body weight dose of artemisinin. Concentrations of serum artemisinin and one of its liver metabolites, deoxyartemisinin, were measured over two hours by GCMS., Results: The first order elimination rate constant for artemisinin in pACT-treated healthy mice was estimated to be 0.80 h(-1) with an elimination half-life (T½) of 51.6 min. The first order absorption rate constant was estimated at 1.39 h(-1). Cmax and Tmax were 4.33 mg L(-1) and 60 min, respectively. The area under the curve (AUC) was 299.5 mg min L(-1). In contrast, the AUC for pACT-treated infected mice was significantly greater at 435.6 mg min L(-1). Metabolism of artemisinin to deoxyartemisinin was suppressed in infected mice over the period of observation. Serum levels of artemisinin in the infected mice continued to rise over the 120 min of the study period, and as a result, the T½ was not determined; the Cmax and Tmax were estimated at ≥6.64 mgL(-1) and ≥120 min, respectively. Groups of healthy mice were also fed either artemisinin or artemisinin mixed in mouse chow. When compared at 60 min, artemisinin was undetectable in the serum of mice fed 100 mg AN kg(-1) body weight. When plant material was present either as mouse chow or Artemisia annua pACT, artemisinin levels in the serum rose to 2.44 and 4.32 mg L(-1), respectively, indicating that the presence of the plant matrix, even that of mouse chow, had a positive impact on the appearance of artemisinin in the blood., Conclusions: These results showed that artemisinin and one of its drug metabolites were processed differently in healthy and infected mice. The results have implications for possible therapeutic use of pACT in treating malaria and other artemisinin-susceptible diseases., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Ethnopharmacological Relevance: Artemisinin (AN) is produced by Artemisia annua, a medicinal herb long used as a tea infusion in traditional Chinese medicine to treat fever; it is also the key ingredient in current artemisinin-based combination therapies (ACTs) effective in treating malaria. Recently we showed that dried leaves from the whole plant Artemisia annua that produces artemisinin and contains artemisinin-synergistic flavonoids seem to be more effective and less costly than ACT oral malaria therapy; however little is known about how digestion affects release of artemisinin and flavonoids from dried leaves., Material and Methods: In the current study we used a simulated digestion system to determine how artemisinin and flavonoids are released prior to absorption into the bloodstream. Various delivery methods and staple foods were combined with dried leaves for digestion in order to investigate their impact on the bioavailability of artemisinin and flavonoids. Digestate was recovered at the end of the oral, gastric, and intestinal stages, separated into solid and liquid fractions, and extracted for measurement of artemisinin and total flavonoids., Results: Compared to unencapsulated digested dried leaves, addition of sucrose, various cooking oils, and rice did not reduce the amount of artemisinin released in the intestinal liquid fraction, but the amount of released flavonoids nearly doubled. When dried leaves were encapsulated into either hydroxymethylcellulose or gelatin capsules, there was >50% decrease in released artemisinin but no change in released flavonoids. In the presence of millet or corn meal, the amount of released artemisinin declined, but there was no change in released flavonoids. Use of a mutant Artemisia annua lacking artemisinin showed that the plant matrix is critical in determining how artemisinin is affected during the digestion process., Conclusions: This study provides evidence showing how both artemisinin and flavonoids are affected by digestion and dietary components for an orally consumed plant delivered therapeutic and that artemisinin delivered via dried leaves would likely be more bioavailable if provided as a tablet instead of a capsule., (© 2013 Published by Elsevier Ireland Ltd.)

KEY MESSAGE : Rooting of Artemisia annua increases trichome size on leaves and helps drive the final steps of the biosynthesis of the sesquiterpene antimalarial drug, artemisinin. Artemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.

Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality.

Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A number of flavonoids including casticin and artemetin from Artemisia annua have shown synergism with artemisinin against Plasmodium falciparum, but it is unclear if the flavonoids are also extracted into a tea infusion of the plant. Using a tea infusion preparation protocol that was reported to be highly effective for artemisinin extraction, we measured casticin and artemetin extraction. There was only a 1.8 % recovery of casticin in the infusion while artemetin was undetectable. After 24 hr storage at room temperature, casticin yield declined by 40 %. These results show that although a tea infusion of the plant may extract artemisinin, the polymethoxylated flavonoids casticin and artemetin are poorly extracted and lost with storage at room temperature and thus, the tea infusion appears to lose synergistic value., (Georg Thieme Verlag KG Stuttgart · New York.)

Sustainable non-food energy biomass and cost-effective ways to produce renewable energy technologies from this biomass are continuously emerging. Algae are capable of producing lipids and hydrocarbons quickly and their photosynthetic abilities make them a promising candidate for an alternative energy source. In addition, their favorable carbon life cycle and a renewed focus on rural economic development are attractive factors. In this review the focus is mainly on the integrated approach of algae culture for bioremediation and oil-based biofuel production with mention of possible other value-added benefits of using algae for those purposes., (Published by Elsevier Ltd.)

Artemisinin is a highly effective sesquiterpene lactone therapeutic produced in the plant, Artemisia annua. Despite its efficacy against malaria and many other infectious diseases and neoplasms, the drug is in short supply mainly because the plant produces low levels of the compound. This review updates the current understanding of artemisinin biosynthesis with a special focus on the emerging knowledge of how biosynthesis of the compound is regulated in planta.

Artemisia annua L. produces the sesquiterpene lactone, artemisinin, a potent antimalarial drug that is also effective in treating other parasitic diseases, some viral infections and various neoplasms. Artemisinin is also an allelopathic herbicide that can inhibit the growth of other plants. Unfortunately, the compound is in short supply and thus, studies on its production in the plant are of interest as are low cost methods for drug delivery. Here we review our recent studies on artemisinin production in A. annua during development of the plant as it moves from the vegetative to reproductive stage (flower budding and full flower formation), in response to sugars, and in concert with the production of the ROS, hydrogen peroxide. We also provide new data from animal experiments that measured the potential of using the dried plant directly as a therapeutic. Together these results provide a synopsis of a more global view of regulation of artemisinin biosynthesis in A. annua than previously available. We further suggest an alternative low cost method of drug delivery to treat malaria and other neglected tropical diseases.

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low-cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2-3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2-3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day(-1) with biomass yield of 12.75 g DW L(-1). This exceeded that in shake flasks at µ = 0.166 day(-1) and 11.10 g DW L(-1). Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L(-1), which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots., (© 2010 Wiley Periodicals, Inc.)

The relationship between the transition to budding and flowering in Artemisia annua and the production of the antimalarial sesquiterpene, artemisinin (AN), the dynamics of artemisinic metabolite changes, AN-related transcriptional changes, and plant and trichome developmental changes were measured. Maximum production of AN occurs during full flower stage within floral tissues, but that changes in the leafy bracts and nonbolt leaves as the plant shifts from budding to full flower. Expression levels of early pathway genes known to be involved in isopentenyl diphosphate and farnesyl diphosphate biosynthesis leading to AN were not immediately positively correlated with either AN or its precursors. However, we found that the later AN pathway genes, amorpha-4,11-diene synthase (ADS) and the cytochrome P450, CYP71AV1 (CYP), were more highly correlated with AN's immediate precursor, dihydroartemisinic acid, within all leaf tissues tested. In addition, leaf trichome formation throughout the developmental phases of the plant also appears to be more complex than originally thought. Trichome changes correlated closely with the levels of AN but not its precursors. Differences were observed in trichome densities that are dependent both on developmental stage (vegetative, budding, and flowering) and on position (upper and lower leaf tissue). AN levels declined significantly as plants matured, as did ADS and CYP transcripts. Spraying leaves with AN or artemisinic acid inhibited CYP transcription; artemisinic acid also inhibited ADS transcription. These data allow us to present a novel model for the differential control of AN biosynthesis as it relates to developmental stage and trichome maturation and collapse.

Isoprenoids are a highly diverse and important group of natural compounds. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) catalyzes a key regulatory step in the non-mevalonate isoprenoid biosynthetic pathway in eubacteria and in plant plastids. For example, in Artemisia annua DXR participates in regulation of the biosynthesis of artemisinin, an important antimalarial drug. We performed phylogenetic analysis using DXR protein sequences from a model prokaryote, Escherichia coli, a picoplanktonic alga, Ostreococcus lucimarinus, and higher plants. The functional domain of DXR was conserved, allowing molecular evolutionary comparisons of both prokaryotic and eukaryotic sequences of DXR. Despite this conservation, for some plant species such as Campthoteca acuminata and Arabidopsis thaliana, phylogenetic relationships of their lineages were consistently violated. Our analysis revealed that plant DXR has an N-terminal transit domain that is likely bipartite, consisting of a chloroplast transit peptide (cTP) and a lumen transit peptide (lTP). Several features observed in the lTP suggest that, while DXR is targeted to the chloroplast, it is localized to the thylakoid lumen. These features include a twin arginine motif, a hydrophobic region, and a proline-rich region. The transit peptide also showed putative motifs for a 14-3-3 binding site with a chaperone phosphorylation site at Thr.

The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.

Despite significant efforts over nearly 30 years, only a few products produced by in vitro plant cultures have been commercialized. Some new advances in culture methods and metabolic biochemistry have improved the useful potential of plant cell cultures. This review will provide references to recent relevant reviews along with a critical analysis of the latest improvements in plant cell culture, co-cultures, and disposable reactors for production of small secondary product molecules, transgenic proteins, and other products. Some case studies for specific products or production systems are used to illustrate principles.

The antimalarial sesquiterpene, artemisinin, is in short supply; demand is not being met, and the role of artemisinin in the plant is not well established. Prior work showed that addition of dimethyl sulfoxide (DMSO) to seedlings increased artemisinin in their shoots and this study further investigated that serendipitous observation. When in vitro-cultured Artemisia annua rooted shoots were fed different amounts of DMSO (0-2.0% v/v), artemisinin levels doubled and showed biphasic optima at 0.25 and 2.0% DMSO. Both artemisinin and its precursor, dihydroartemisinic acid, increased with the former continuing 7 days after DMSO treatment. There was no stimulation of artemisinin production in DMSO-treated unrooted shoots. The first gene in the artemisinin biosynthetic pathway, amorphadiene synthase, showed no increase in transcript level in response to DMSO compared to controls. In contrast, the second gene in the pathway, CYP71AV1, did respond to DMSO but at a level of transcripts inverse to artemisinin levels. When rooted shoots were stained for the reactive oxygen species (ROS), H2O2, ROS increased with increasing DMSO concentration; unrooted shoots produced no ROS in response to DMSO. Both the increases in DMSO-induced ROS response and corresponding artemisinin levels were inhibited by addition of vitamin C. Together these data show that at least in response to DMSO, artemisinin production and ROS increase and that when ROS is reduced, so also is artemisinin suggesting that ROS may play a role in artemisinin production in A. annua.

We compared the growth and productivity of a tobacco line of hairy roots that produces murine interleukin 12 (mIL-12) grown in three different culture systems: shake flasks, an airlift reactor, and a scalable mist reactor. Of the total mIL-12 produced by cultures grown in shake flasks ( approximately 434.8 microg L(-1)), almost 21% was recovered from the medium. In contrast to roots harvested from shake flasks and the mist reactor, roots were not uniformly distributed in the airlift reactor. Roots formed a dense ring around the wall of the reactor and surrounding the central rising column of fine aeration bubbles. Root quality was also better in both the shake flasks and mist reactor than in the airlift reactor. There were more pockets of dark roots in the airlift reactor suggesting some of the roots were nutrient starved. Although the best root growth (7 g DW L(-1)) was in the shake flasks, both reactors produced about the same, but less dry mass, nearly 5 g DW L(-1). Total mIL-12 concentration was highest in the mist reactor at 5.3 microg g(-1) FW, but productivity, 31 microg g(-1) FW day(-1) was highest in shake flasks. Roots grown in the mist reactor produced about 49.5% more mIL-12 than roots grown in the airlift reactor. Protease activity in the media increased steadily during culture of the roots in all three systems. The comparisons of protease activity, protein and mIL-12 levels done in the shake flask system suggest that the increase in proteases associated with progression into stationary phase is most detrimental to mIL-12 concentration. This is the first description of the design and operation of a scalable version of a mist bioreactor that uses a plastic bag. This also the first report of reasonable production levels of functional mIL-12, or any protein, produced by hairy roots grown in a mist reactor. Results will prove useful for further optimization and scale-up studies of plant-produced therapeutic proteins.

Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases.

The potent antimalarial sesquiterpene lactone, artemisinin, is produced in low quantities by the plant Artemisia annua L. The source and regulation of the isopentenyl diphosphate (IPP) used in the biosynthesis of artemisinin has not been completely characterized. Terpenoid biosynthesis occurs in plants via two IPP-generating pathways: the mevalonate pathway in the cytosol, and the non-mevalonate pathway in plastids. Using inhibitors specific to each pathway, it is possible to resolve which supplies the IPP precursor to the end product. Here, we show the effects of inhibition on the two pathways leading to IPP for artemisinin production in plants. We grew young (7-14 days post cotyledon) plants in liquid culture, and added mevinolin to the medium to inhibit the mevalonate pathway, or fosmidomycin to inhibit the non-mevalonate pathway. Artemisinin levels were measured after 7-14 days incubation, and production was significantly reduced by each inhibitor compared to controls, thus, it appears that IPP from both pathways is used in artemisinin production. Also when grown in miconazole, an inhibitor of sterol biosynthesis, there was a significant increase in artemisinin compared to controls suggesting that carbon was shifted from sterols into sesquiterpenes. Collectively these results indicate that artemisinin is probably biosynthesized from IPP pools from both the plastid and the cytosol, and that carbon from competing pathways can be channeled toward sesquiterpenes. This information will help advance our understanding of the regulation of in planta production of artemisinin.

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.

Gas-phase reactors, including the mist reactor, have distinct advantages over liquid-phase reactors including the ability to manipulate the gas composition, to allow effective gas exchange in a densely growing biomass, and to affect secondary metabolite production. Mathematical modeling suggested that roots in a mist reactor are often too sparsely packed to capture mist particles efficiently and cannot, therefore, meet the nutrient demands required to maintain high growth rates. Indeed, growth rates of Artemisia annua hairy roots increased significantly when the initial packing density increased or when a higher sucrose concentration was used in the medium. Growth kinetics for 2, 4, and 6 days, however, showed a decrease or stationary growth rate after only 4 days for both 3 and 5% sucrose feeds. Residual medium analyses indicated that carbon was not exhausted, nor were any of the other major nutrients including phosphate. Increasing the mist duty cycle at constant carbon flux through the reactor reduced the growth rates slightly. In general, the aerosol deposition model correctly predicted how to optimize hairy root growth in the mist reactor., ((c) 2006 Wiley Periodicals, Inc.)

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.

Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale-up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different environments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root-packing density. Surprisingly, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 653-667, 2003.)

The transient growth of Artemisia annua hairy roots was compared for cultures grown in shake flasks and in bubble column and mist reactors. Instantaneous growth rates were obtained by numerically differentiating the transient biomass measurements. Specific sugar consumption rates showed good agreement with literature values. From the growth rate and sugar consumption rate, the specific yield and maintenance coefficient for sugar were determined for all three culture systems. These values were statistically indistinguishable for roots grown in shake flasks and bubble columns. In contrast, the values for roots grown in bubble columns and mist reactors were statistically different, suggesting that sugar utilization by roots grown in these two systems may be different. By measuring respiration rates in the bubble column reactor we also determined the actual biomass yield and maintenance coefficient for O(2) and CO(2). Together with an elemental analysis of the roots, this allowed us to obtain a reasonable carbon balance., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 428-443, 2003.)

We investigated molecular evolution of 1-deoxy-D-xylulose-5-phosphate synthase (DXS), an important regulatory enzyme of the mevalonate-independent pathway involved in terpenoid biosynthesis. Sequence alignment showed that some regions, likely to be functionally important, were highly conserved among all of the plant DXS sequences analysed. Phylogenetic trees were inferred using DXS sequences from 11 species of Angiosperms and showed the division of the sequences into two classes. Clustering of DXS sequences did not correspond to phylogenetic relationships among the plant species studied. There was no consistency in the similarity of the variable regions in the secondary structure of the DXS functional protein except for Capsicum and Lycopersicon, both members of the Solanaceae. Hydrophobicity plots for the functional region of DXS revealed great similarity in their hydrophobic structure, consistent with the phylogenetic trees inferred, and with eight prominent hydrophilic and hydrophobic peaks. We also observed a consistent set of features common to the DXS transit peptides studied. These features were the same hydrophobic slope, a hydrophobic region in residues 35-45, and, in eight of 12 sequences, a Pro-Pro-Thr sequence at the C-terminal end. The transit sequences are likely bipartite and contain features that suggest the DXS protein is not only targeted to the chloroplast, but also to the thylakoid. To our knowledge this is the first suggestion that DXS is located specifically in the chloroplast thylakoid.