In vivo transformations of artemisinic acid in Artemisia annua plants

Artemisinic acid labeled with both C-13 and H-2 at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid-as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation-closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Delta(4,5) double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species. (C) 2007 Elsevier Ltd. All rights reserved.

In vivo transformations of dihydro-epi-deoxyarteannuin B in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydro-epi-deoxyarteannuin B (4a) has been fed to intact Artemisia annua plants via the root and three labeled metabolites (17a-19a) have been identified by 1D- and 2D-NMR spectroscopies. The in vivo transformations of 4a in A. annua are proposed to involve enzymatically-mediated processes in addition to possible spontaneous autoxidation. In the hypothetical spontaneous autoxidation pathway, the tri-substituted double bond in 4a appears to have undergone 'ene-type' reaction with oxygen to form an allylic hydroperoxide, which subsequently rearranges to the allylic hydroxyl group in the metabolite 3 alpha-hydroxy-dihydro-epi-deoxyarteannuin B (17a). In the enzymatically-mediated pathways, compound 17a has then been converted to its acetyl derivative, 3 alpha-acetoxy-dihydro-epi-deoxyarteannuin B (18a), while oxidation of 4a at the 'unactivated' 9-position has yielded 9 beta-hydroxy-dihydro-epi-deoxyarteannuin B (19a). Although all of the natural products artemisinin ( 1), arteannuin K ( 7), arteannuin L ( 8), and arteannuin M ( 9) have been suggested previously as hypothetical metabolites from dihydro-epi-deoxyarteannuin B in A. annua, none were isolated in labeled form in this study. It is argued that the nature of the transformations undergone by compound 4a are more consistent with a degradative metabolism, designed to eliminate this compound from the plant, rather than with a role as a late precursor in the biosynthesis of artemisinin or other natural products from A. annua. (C) 2007 Elsevier Ltd. All rights reserved.

The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao)

The Chinese medicinal plant Artemisia annua L. (Qinghao) is the only known source of the sesquiterpene artemisinin (Qinghaosu), which is used in the treatment of malaria. Artemisinin is a highly oxygenated sesquiterpene, containing a unique 1,2,4-trioxane ring structure, which is responsible for the antimalarial activity of this natural product. The phytochemistry of A. annua is dominated by both sesquiterpenoids and flavonoids, as is the case for many other plants in the Asteraceae family. However, A. annua is distinguished from the other members of the family both by the very large number of natural products which have been characterised to date (almost six hundred in total, including around fifty amorphane and cadinane sesquiterpenes), and by the highly oxygenated nature of many of the terpenoidal secondary metabolites. In addition, this species also contains an unusually large number of terpene allylic hydroperoxides and endoperoxides. This observation forms the basis of a proposal that the biogenesis of many of the highly oxygenated terpene metabolites from A. annua - including artemisinin itself may proceed by spontaneous oxidation reactions of terpene precursors, which involve these highly reactive allyllic hydroperoxides as intermediates. Although several studies of the biosynthesis of artemisinin have been reported in the literature from the 1980s and early 1990s, the collective results from these studies were rather confusing because they implied that an unfeasibly large number of different sesquiterpenes could all function as direct precursors to artemisinin (and some of the experiments also appeared to contradict one another). As a result, the complete biosynthetic pathway to artemisinin could not be stated conclusively at the time. Fortunately, studies which have been published in the last decade are now providing a clearer picture of the biosynthetic pathways in A. annua. By synthesising some of the sesquiterpene natural products which have been proposed as biogenetic precursors to artemisinin in such a way that they incorporate a stable isotopic label, and then feeding these precursors to intact A. annua plants, it has now been possible to demonstrate that dihydroartemisinic acid is a late-stage precursor to artemisinin and that the closely related secondary metabolite, artemisinic acid, is not (this approach differs from all the previous studies, which used radio-isotopically labelled precursors that were fed to a plant homogenate or a cell-free preparation). Quite remarkably, feeding experiments with labeled dihydroartemisinic acid and artemisinic acid have resulted in incorporation of label into roughly half of all the amorphane and cadinane sesquiterpenes which were already known from phytochemical studies of A. annua. These findings strongly support the hypothesis that many of the highly oxygenated sesquiterpenoids from this species arise by oxidation reactions involving allylic hydroperoxides, which seem to be such a defining feature of the chemistry of A. annua. In the particular case of artemisinin, these in vivo results are also supported by in vitro studies, demonstrating explicitly that the biosynthesis of artemisinin proceeds via the tertiary allylic hydroperoxide, which is derived from oxidation of dihydroartemisinic acid. There is some evidence that the autoxidation of dihydroartemisinic acid to this tertiary allylic hydroperoxide is a non-enzymatic process within the plant, requiring only the presence of light; and, furthermore, that the series of spontaneous rearrangement reactions which then convert thi allylic hydroperoxide to the 1,2,4-trioxane ring of artemisinin are also non-enzymatic in nature.

Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

Proteomic analysis of the medicinal plant Artemisia annua: data from leaf and trichome extracts

This article contains raw and processed data related to research published by Bryant et al. [1]. Data was obtained by MS-based proteomics, analysing trichome-enriched, trichome-depleted and whole leaf samples taken from the medicinal plant Artemisia annua and searching the acquired MS/MS data against a recently published contig database [2] and other genomic and proteomic sequence databases for comparison. The processed data shows that an order-of-magnitude more proteins have been identified from trichome-enriched Artemisia annua samples in comparison to previously published data. Proteins known to have a role in the biosynthesis of artemisinin and other highly abundant proteins were found which imply additional enzymatically driven processes occurring within the trichomes that are significant for the biosynthesis of artemisinin.

Effect of Artemisia annua L. leaves essential oil and ethanol extract on behavioral assays

Artemisia annua tem sido utilizada tradicionalmente para o tratamento de malária e febre na China devido à presença do princípio ativo, artemisinina. O presente trabalho avaliou a atividade central de do óleo essencial obtido por hidrodestilação e do extrato etanólico bruto de folhas frescas de A. annua em modelo in vivo como parte de um screening farmacológico dessa espécie. Sono induzido por pentobarbital, nado forçado e o ensaio de campo aberto são modelos de estudo conhecidos para o estudo de fármacos sobre depressão induzida. A administração do óleo essencial ou extrato bruto etanólico de A. annua aumentaram o tempo de imobilidade no teste do nado forçado. Por outro lado, diminuíram outros parâmetros no campo aberto, como ambulação, exploração, o ato de lamber as patas ou se lamber. Ambos produtos aumentaram o tempo de sono induzido por pentobarbital, com o óleo essencial apresentando um efeito superior ao do extrato. Pela análise dos resultados, é possível sugerir que tanto o extrato bem como o óleo essencial podem atuar como depressores do Sistema Nervoso Central (SNC).

In vitro activity of Artemisia annua L (Asteraceae) extracts against Rhipicephalus (Boophilus) microplus

A atividade de extratos vegetais sobre parasitas pode indicar grupos de substâncias de uso potencial no controle de Rhipicephalus (Boophilus) microplus. O objetivo do presente estudo foi investigar a ação in vitro de extratos de Artemisia annua sobre esta espécie. A concentração das lactonas sesquiterpênicas artemisinina e deoxiartemisinina presentes nos extratos vegetais, foi quantificada via cromatografia líquida de alta eficiência. Quatro extratos produzidos a partir do extrato bruto concentrado (EBC) foram avaliados sobre larvas pela metodologia do papel impregnado, com leitura após 24 horas de incubação. As fêmeas ingurgitadas foram imersas por cinco minutos no EBC e nos seus quatro extratos derivados, e incubadas para posterior análise dos parâmetros biológicos. Os extratos não tiveram eficácia sobre as larvas nas concentrações avaliadas (de 3,1 a 50 mg.mL-1). O EBC apresentou melhor eficácia sobre as fêmeas ingurgitadas (CE 50 de 130,6 mg.mL-1 e CE 90 de 302,9 mg.mL-1) que os extratos derivados. Esses resultados tendem a confirmar que a ação da artemisinina sobre as fêmeas ingurgitadas de R. (B.) microplus estaria condicionada à sua ingestão através do sangue. Nesse caso, os métodos in vitro seriam inadequados para a efetiva avaliação da ação de A. annua R.(B.) microplus.

Carbon isotope composition and leaf anatomy as a tool to characterize the photosynthetic mechanism of Artemisia annua L.

Leaves of Artemisia annua L. are a plentiful source of artemisinin, a drug with proven effectiveness against malaria. The aim of this study was to classify the photosynthetic mechanism of A. annua through studies of the carbon isotope composition (δ 13C) and the leaf anatomy. A. annua presented a δ 13C value of - 31.76 ± 0.07, which characterizes the plants as a typical species of the C3 photosynthethic mechanism, considering that the average δ 13C values for C3 and C4 species are -28 and -14, respectively. The leaf anatomy studies were consistent with the δ 13C results, where, in spite of the existence of parenchymatic cells forming a sheath surrounding the vascular tissue, the cells do not contain chloroplasts or starch. This characteristic is clearly different from that of the Kranz anatomy found in C4 species.

Aplicação exógena de artemisinina e indução floral em Artemisia annua L.

The objective of this work was to evaluate the effect of different artemisinin concentrations in the flowering induction of A. annua. Two genotypes of A. annua (CPQBA 239x1V and 3MxPOP) were atomized with four different artemisinin concentrations (0, 500, 5000, and 10000 mg L-1). The application of artemisinin didn't induce the flowering of both genotypes tested, in none of the used concentrations.

Avaliação fitoquímica e microbiológica da espécie Artemisia annua L., submetida a tratamentos de armazenamento e condições de ambiente

Pós-graduação em Agronomia (Energia na Agricultura) - FCA

Avaliação da atividade antiparasitária de Artemisia annua sobre Rhipicephalus (Boophilus) microplus e Haemonchus contortus, in vivo, por meio do fornecimento do material vegetal seco na alimentação animal

Pós-graduação em Medicina Veterinária - FCAV

Caracterização do mecanismo fotossintético e aspectos relacionados à floração de Artemisia annua L

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação fitoquímica e microbiológica da espécia Artemisia annua L. submetida a tratamentos de armazenamento e condições de ambiente

The species Artemisia annua L. (Asteraceae) is native to China and has artemisinin as its main active component, substance that is considered a potent antimalarial drug. With the increased interest in natural active principles, studies related to post-harvest and storage of vegetable material become important for better conservation of its phytotherapic properties. Therefore, the present study had as objectivedefine the best storage conditions to preserve and keep the quality of phytotherapic drugs. Leaves of A. annua dried were stored for six months in polyethylene bags wrapped in Kraft paper and packed in four treatments: at ambient condition, refrigerated at 4 ± 2 oC, using normal packing, and using vacuum packing. Samples were taken for microbiological, moisture content, and level of artemisinin analyses before the experiment begun and at 30, 90, 120 and 180 days. The results of microbiological tests showed no significant contamination, as well as the moisture content of the stored (biological) material, which remain between 5% and 10%, keeping within acceptable parameters. The ambient without vacuum treatment (SVA) was the treatment that better maintained the sample stability during 180 days, however, the refrigerated without vacuum (SVR) showed greater efficiency to conserve the content of artemisinin.

Avaliação da atividade de Aretmisia annua L., Melia azedarach L. e Trichilia claussenii C. sobre nematódeos gastrintestinais de ovinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Metabolomics coupled with proteomics advancing drug discovery toward more agile development of targeted combination therapies

To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations 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, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of 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 hepatic injury. 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.