Artemisia afra and Artemisia annua Extracts Have Bactericidal Activity against Mycobacterium tuberculosis in Physiologically Relevant Carbon Sources and Hypoxia.

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 mc26230 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.

Artemisinin as a therapeutic vs. its more complex Artemisia source material.

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.

Artemisia extracts differ from artemisinin effects on human hepatic CYP450s 2B6 and 3A4 in vitro.

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.

Artemisia annua and artemisinins are ineffective against human Babesia microti and six Candida sp.

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.

In vitro analyses of Artemisia extracts on Plasmodium falciparum suggest a complex antimalarial effect.

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.

In vitro reduction of Plasmodium falciparum gametocytes: Artemisia spp. tea infusions vs. artemisinin.

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.

Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis.

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.

Dried Leaf Artemisia Annua Improves Bioavailability of Artemisinin via Cytochrome P450 Inhibition and Enhances Artemisinin Efficacy Downstream.

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 IC50 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.

Dried leaf Artemisia annua efficacy against non-small cell lung cancer.

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.

Artemisinin permeability via Caco-2 cells increases after simulated digestion of Artemisia annua leaves.

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% (Papp = 8.03 × 10-5cms-1) compared to pure artemisinin (Papp = 5.03 × 10-5cms-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 Papp 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.

Artemisia annua dried leaf tablets treated malaria resistant to ACT and i.v. artesunate: Case reports.

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.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

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.

Root regulation of artemisinin production in Artemisia annua: trichome and metabolite evidence.

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.

Effect of leaf digestion and artemisinin solubility for use in oral consumption of dried Artemisia annua leaves to treat malaria.

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.

Variations in key artemisinic and other metabolites throughout plant development in Artemisia annua L. for potential therapeutic use.

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.

Dried whole-plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin.

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.

Changes in key constituents of clonally propagated Artemisia annua L. during preparation of compressed leaf tablets for possible therapeutic use.

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 AlCl3 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.

Pharmacokinetics of artemisinin delivered by oral consumption of Artemisia annua dried leaves in healthy vs. Plasmodium chabaudi-infected mice.

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.

Simulated digestion of dried leaves of Artemisia annua consumed as a treatment (pACT) for malaria.

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.