An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use. AIM OF THE STUDY: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium. MATERIALS AND METHODS: We used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound. RESULTS: Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc26230 in both log phase growth and metabolically downshifted hypoxic cultures. CONCLUSIONS: The present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.

Differential Anti-Fibrotic and Remodeling Responses of Human Dermal Fibroblasts to Artemisia sp., Artemisinin, and Its Derivatives.

Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.

A methoxylated flavone from Artemisia afra kills Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current combination therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Artemisia afra is used traditionally in southern Africa to treat malaria and recently has shown anti tuberculosis activity. This genus synthesizes a prodigious number of phytochemicals, many of which have demonstrated human health effects. Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or the well-known antimalarial artemisinin, suggesting other phytochemicals present in A. afra with unique modes of action. A biochemometric study of A. afra resulted in the isolation of a methoxylated flavone (1), which displayed considerable activity against Mtb strain mc26230. Compound 1 had an MIC of 312.5 µg/mL and yielded no viable colonies after 6 days of treatment. In addition, 1 was effective in killing hypoxic Mtb cultures, with no viable cultures after 2 days of treatment. This suggested that A. afra is a source of potentially powerful anti-Mtb phytochemicals with novel mechanisms of action.

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 Artemisia afra tea infusions vs. artesunate-amodiaquine (ASAQ) in treating Plasmodium falciparum malaria in a large scale, double blind, randomized clinical trial.

BACKGROUND AND OBJECTIVE: Prior small-scale clinical trials showed that Artemisia annua and Artemisia afra infusions, decoctions, capsules, or tablets were low cost, easy to use, and efficient in curing malaria infections. In a larger-scale trial in Kalima district, Democratic Republic of Congo, we aimed to show A. annua and/or A. afra infusions were superior or at least equivalent to artesunate-amodiaquine (ASAQ) against malaria. METHODS: A double blind, randomized clinical trial with 957 malaria-infected patients had two treatment arms: 472 patients for ASAQ and 471 for Artemisia (248 A. annua, 223 A. afra) remained at end of the trial. ASAQ-treated patients were treated per manufacturer posology, and Artemisia-treated patients received 1 l/d of dry leaf/twig infusions for 7 d; both arms had 28 d follow-up. Parasitemia and gametocytes were measured microscopically with results statistically compared among arms for age and gender. RESULTS: Artemisinin content of A. afra was negligible, but therapeutic responses of patients were similar to A. annua-treated patients; trophozoites cleared after 24  h, but took up to 14 d to clear in ASAQ-treated patients. D28 cure rates defined as absence of parasitemia were for pediatrics 82, 91, and 50% for A. afra, A. annua and ASAQ; while for adults cure rates were 91, 100, and 30%, respectively. Fever clearance took 48  h for ASAQ, but 24  h for Artemisia. From D14-28 no Artemisia-treated patients had microscopically detectable gametocytes, while 10 ASAQ-treated patients remained gametocyte carriers at D28. More females than males were gametocyte carriers in the ASAQ arm but were unaffected in the Artemisia arms. Hemoglobin remained constant at 11 g/dl for A. afra after D1, while for A. annua and ASAQ it decreased to 9-9.5  g/dl. Only 5.0% of Artemisia-treated patients reported adverse effects, vs. 42.8% for ASAQ. CONCLUSION: A. annua and A. afra infusions are polytherapies with better outcomes than ASAQ against malaria. In contrast to ASAQ, both Artemisias appeared to break the cycle of malaria by eliminating gametocytes. This study merits further investigation for possible inclusion of Artemisia tea infusions as an alternative for fighting and eradicating malaria.

Effect of Artemisia annua and Artemisia afra tea infusions on schistosomiasis in a large clinical trial.

BACKGROUND AND OBJECTIVE: Schistosomiasis (bilharzia), a serious neglected tropical disease affecting millions, has few cost-effective treatments, so two Artemisia wormwood species, A. annua and A. afra, were compared with the current standard praziquantel (PZQ) treatment in an 800 patient clinical trial, August-November of 2015. METHODS: The double blind, randomized, superiority clinical trial had three treatment arms: 400 for PZQ, 200 for A. annua, and 200 for A. afra. PZQ-treated patients followed manufacturer posology. Artemisia-treated patients received 1 l/d of dry leaf/twig tea infusions divided into 3 aliquots daily, for 7 days with 28-day follow-up. RESULTS: Of 800 enrolled patients having an average of >700 Schistosoma mansoni eggs per fecal sample, 780 completed the trial. Within 14 days of treatment, all Artemisia-treated patients had no detectable eggs in fecal smears, a result sustained 28 days post treatment. Eggs in fecal smears of PZQ-treated patients were undetectable after D21. More males than females who entered the trial had melena, but both genders responded equally well to treatment; by D28 melena disappeared in all patients. In all arms, eosinophil levels declined by about 27% from D0 to D28. From D0 to D28 hemoglobin increases were greater in PZQ and A. afra-treated patients than in A. annua-treated patients. Hematocrit increases were greater from D0 to D28 for patients treated with either PZQ or A. annua compared to those treated with A. afra. Gender comparison showed that A. afra-treated males had significantly greater hemoglobin and hematocrit increases by D28 than either PZQ or A. annua-treated males. In contrast, PZQ and A. afra-treated females had greater hemoglobin and hematocrit increases than A. annua-treated females. Both adults and pediatric patients treated with A. annua responded better compared to PZQ treatment. CONCLUSION: Both A. annua and A. afra provided faster effective treatment of schistosomiasis and should be considered for implementation on a global scale.

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.

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.

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.

Dried-leaf Artemisia annua: A practical malaria therapeutic for developing countries?

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 T1/2 and Tmax and greater Cmax 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.

The effect of roots and media constituents on trichomes and artemisinin production in Artemisia annua L.

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.

The flavonoids casticin and artemetin are poorly extracted and are unstable in an Artemisia annua tea infusion.

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.