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Concept: Artemisia annua

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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.

Concepts: Gene, Amino acid, Artemisinin, Leaf, Plant morphology, Antimalarial drug, Artemisia annua, Trichome

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To enhance therapeutic efficacy and reduce adverse effects of traditional Chinese medicine (TCM), practitioners often prescribe a combination of plant species and/or minerals called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang (YCHT), a classical formula and has been shown to be clinically effective for treating hepatic injury (HI) syndrome. The three principal components of YCHT are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G) and rhein ®, respectively. To determine the mechanisms that underlie this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of HI. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect and activates both intrinsic and extrinsic pathways.

Concepts: Protein, Therapeutic effect, Pharmacology, Metabolism, Effect, Effectiveness, Traditional Chinese medicine, Artemisia annua

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Abstract Artemisinins are a family of sesquiterpene trioxane lactone anti-malarial agents originally derived from Artemisia annua L. The anti-malarial action of artemisinins involves the formation of free radicals via cleavage of the endoperoxide bond in its structure, which mediate eradication of the Plasmodium species. With its established safety record in millions of malarial patients, artemisinins are also being investigated in diseases like infections, cancers and inflammation. Artemisinins have been reported to possess robust inhibitory effects against viruses (e.g. Human cytomegalovirus), protozoa (e.g. Toxoplasma gondii), helminths (e.g. Schistosoma species and Fasciola hepatica) and fungi (e.g. Cryptococcus neoformans). Artemisinins have demonstrated cytotoxic effects against a variety of cancer cells by inducing cell cycle arrest, promoting apoptosis, preventing angiogenesis, and abrogating cancer invasion and metastasis. Artemisinins have been evaluated in animal models of autoimmune diseases, allergic disorders and septic inflammation. The anti-inflammatory effects of artemisinins have been attributed to the inhibition of Toll-like receptors, Syk tyrosine kinase, phospholipase Cγ, PI3K/Akt, MAPK, STAT-1/3/5, NF-κB, Sp1 and Nrf2/ARE signaling pathways. This review provides a comprehensive update on non-malarial use of artemisinins, modes of action of artemisinins in different disease conditions, and drug development of artemisinins beyond anti-malarial. With the concerted efforts in the novel synthesis of artemisinin analogues and clinical pharmacology of artemisinins, it is likely that artemisinin drugs will become a major armamentarium combating a variety of human diseases beyond malaria.

Concepts: AIDS, Immune system, Cancer, Infectious disease, Signal transduction, Malaria, Infection, Artemisia annua

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The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. In this study, we cloned an ABA receptor orthologue, AaPYL9, from Artemisia annua L. AaPYL9 is expressed highly in leaf and flower. AaPYL9 protein can be localized in both nucleus and cytoplasm. Yeast two-hybrid assay shows AaPYL9 can specifically interact with AtABI1 but not with AtABI2, AtHAB1 or AtHAB2. ABA can enhance the interaction between AaPYL9 and AtABI1 while AaPYL9-89 Pro→Ser and AaPYL9-116 His→Ala point mutations abolishes the interaction. BiFC assay shows that AaPYL9 interacts with AtABI1 in nucleus in planta. Transgenic Arabidopsis plants over-expressing AaPYL9 are more sensitive to ABA in the seed germination and primary root growth than wild type. Consistent with this, ABA report genes have higher expression in AaPYL9 overexpressing plants compared to wild type after ABA treatment. Moreover, overexpression of AaPYL9 in A. annua increases not only drought tolerance, but also artemisinin content after ABA treatment, with significant enhancement of the expression of key genes in artemisinin biosynthesis. This study provides a way to develop A. annua with high-yielding artemisinin and high drought resistance.

Concepts: Protein, Gene, Gene expression, Seed, Abscisic acid, Plant morphology, Germination, Artemisia annua

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Most terpene synthases (TPSs) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. Here, we report a new sesquiterpene synthase from A. annua, α-bisabolol synthase (AaBOS), which has high sequence identity to amorpha-4,11-diene synthase (AaADS), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (AaBOS-M2). Among the plasticity residues, position 399, located in the substrate binding pocket, is crucial for both enzymes. In AaBOS, substitution of Leu with Thr (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the Thr with Ser (AaADST399S) resulted in a substantial increase of the activity of amorpha-4,11-diene production, likely as a result of accelerated product release. Our work demonstrates that substitution of plasticity residues holds a potential for improving catalytic efficiency of the enzyme.

Concepts: Metabolism, Enzyme, Malaria, Enzyme substrate, Product, Catalysis, Glycosylation, Artemisia annua

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Uveal melanoma is the most common primary intraocular malignancy in adults. So far, there have been no effective targeted therapeutic agents in patients with uveal melanoma. Artesunate is a semi-synthetic derivative of artemisinin extracted from traditional Chinese medicine Artemisia annua L for treatment of severe and multidrug-resistant malaria. Besides its anti-malarial activity, artesunate is identified as an anti-cancer drug due to the inhibition of Wnt/b-catenin pathway in multiple types of cancer. However, the effect of artesunate on uveal melanoma remains unknown.

Concepts: Oncology, Malaria, Chemotherapy, Melanoma, Artemisinin, Uveal melanoma, Traditional Chinese characters, Artemisia annua

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Artemisinin is a substance extracted from the Chinese plant Artemisia annua L. widely used in natural medicine for the treatment of various diseases. Artemether is a substance synthesized from artemisinin, and both drugs are commonly administered in the treatment of malaria. Although considered effective antimalarial drugs, very little is known about the genotoxic, cytotoxic and mutagenic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin (12.5, 25 and 50 µg/mL) and artemether (7.46; 14.92 and 29.84 µg/mL) in cultured human lymphocytes using the comet assay, the micronucleus test and the cytotoxicity assay for detection of necrosis and apoptosis by acridine orange/ethidium bromide staining. Our results showed a significant increase (p < 0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p < 0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. The results demonstrated that these two drugs induce mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

Concepts: Lymphocyte, DNA, Mutation, Malaria, Cytotoxicity, Artemisinin, Antimalarial drug, Artemisia annua

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The anti-malarial drug artemisinin is extracted from the leaves of Artemisia annua L. Due to toxicity to some microorganisms, the release of artemisinin from this medicinal plant in commercial cultivation might produce a potential risk for phosphorus (P) solubilizing bacteria (PSB). Therefore, the growth, P mobilization, and proton and organic acid efflux by two PSB isolates, Bacillus subtilis and Pseudomonas fluorescens, obtained from the soil without growing A. annua L. in history in the region for growing A. annua L., Chongqing, China, were studied through soil and solution incubations with different nominal concentrations of artemisinin (0, 2.5, 5.0, and 10.0 mg/kg or mg/L). Addition of artemisinin into soil and culture solutions decreased significantly the number of PSB except P. fluorescens at a low artemisinin concentration (2.5 mg/L) in culture solution which remained unchanged in comparison with the control (without artemisinin). This suggests high artemisinin inhibited the cell division or led to the death of PSB, and the different species responded differently to artemisinin. Compared with original soil, PSB inoculation significantly increased Olsen P, whilst the addition of artemisinin decreased this P form in soil. There was a positive correlation between the number of PSB and Olsen P content in soils (r2 = 0.824, n = 8), indicating the involvement of PSB in P mobilization of insoluble minerals. Oxalate and acetate were commonly found in the bacterial culture solutions, which accounted for 73.6-84.4% of all organic acids in the culture medium without artemisinin. Malate was detected in the culture solution of B. subtilis, and citrate and succinate in P. fluorescens. The percentage of tricalcium phosphate solubilization (PTPS) positively correlated to the concentrations of protons and all organic acids (r2proton=0.901, n=8, P<0.01; r2organic acids=0.923, n=8, P<0.01). The concentrations of protons, organic acids and soluble inorganic P in culture solutions, and PTPS were decreased simultaneously as nominal artemisinin concentrations increased. For these decreases it implies the metabolic inhibition and the death of PSB caused by artemisinin could be the main reasons for the less efflux of protons and organic acids, presumably resulting in the decreased ability of PSB to mobilize inorganic P. Therefore, artemisinin released from A. annua L. in commercial and continual cultivation could adversely affect the community structure and inorganic P mobilization of PSB in soils.

Concepts: Bacteria, Acid, Chemistry, Soil, Bacillus, Solution, Bacillus subtilis, Artemisia annua

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Currently, the most effective antimalarial is artemisinin, which is extracted from the leaves of medicinal herb Artemisia annua L. (A. annua). Previous studies showed that the complex chemical matrix of A. annua could enhance both the bioavailability and efficacy of artemisinin. The present study aims to evaluate the efficacy and pharmacokinetic properties of a combination therapy based on artemisinin and 3 components from A. annua with high content (arteannuin B, arteannuic acid, and scopoletin). In vivo antimalarial activity was assessed following a 4-day treatment in murine malaria models (Plasmodium yoelii and Plasmodium berghei). Results showed that a much sharper reduction in parasitemia (~93%) was found in combination therapy compared with pure artemisinin (~31%), indicating pharmacodynamic synergism occurring between artemisinin and arteannuin B, arteannuic acid, and scopoletin. Multiple-dose pharmacokinetics further demonstrated that combination therapy results in increased area under the curve (AUC0→∞ ), Cmax , and t1/2 by 3.78-, 3.47-, and 1.13-fold in healthy mice, respectively, and by 2.62-, 1.82-, and 1.22-fold in P. yoelii-infected mice, respectively. The calculated oral clearance of combination therapy in healthy and P. yoelii-infected mice was also reduced. These findings imply that specific components in A. annua might offer a possibility to develop new artemisinin-based natural combination therapy for malaria treatment.

Concepts: Medicine, Malaria, Plasmodium, Apicomplexa, Artemisinin, Pharmacokinetics, Artemisia annua, Pharmacodynamics

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Heat stress (HS) is detrimental to animal origin food production. Artemisia annua L., a natural source of phenolic compounds and flavonoids, exhibits antioxidant property. This study was conducted to evaluate the effects of dietary enzymatically treated Artemisia annua L. (EA) supplementation on meat quality, antioxidant capacity and energy status of breast muscle in heat-stressed broilers.

Concepts: Metabolism, Nutrition, Energy, Antioxidant, Meat, Catechin, Resveratrol, Artemisia annua