Artemisia annua L. and photoresponse: from artemisinin accumulation, volatile profile and anatomical modifications to gene expression.

KEY MESSAGE: Blue and yellow light affected metabolism and the morphology. Blue and red promote the DOXP/MEP pathway. ADS gene expression was increased in plants cultivated under blue, promoting artemisinin content. Artemisinin-based combination therapies are the most effective treatment for highly lethal malaria. Artemisinin is produced in small quantities in the glandular trichomes of Artemisia annua L. Our aim was to evaluate the effect of light quality in A. annua cultivated in vitro under different light qualities, considering anatomical and morphological changes, the volatile composition, artemisinin content and the expression of two key enzymes for artemisinin biosynthesis. Yellow light is related to the increase in the number of glandular trichomes and this seemed to positively affect the molecular diversity in A. annua. Yellow light-stimulated glandular trichome frequency without triggered area enhancement, whereas blue light stimulated both parameters. Blue light enhanced the thickness of the leaf epidermis. The B-promoting effect was due to increased cell size and not to increased cell numbers. Green and yellow light positively influenced the volatile diversity in the plantlets. Nevertheless, blue and red light seemed to promote the DOXP/MEP pathway, while red light stimulates MVA pathway. Amorpha-4,11-diene synthase gene expression was significantly increased in plants cultivated under blue light, and not red light, promoting artemisinin content. Our results showed that light quality, more specifically blue and yellow light, positively affected secondary metabolism and the morphology of plantlets. It seemed that steps prior to the last one in the artemisinin biosynthesis pathway could be strongly influenced by blue light. Our work provides an alternative method to increase the amount of artemisinin production in A. annua without the use of transgenic plants, by the employment of blue light.

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

Kinetic parameters for the reaction of 1,2,4-trioxolanes and artemisinin with iron(II): New evidence for the source of antimalarial activity.

The Fenton-like reductive cleavage of antimalarial peroxides like artemisinin by iron(II) species is a chemical reaction whose mechanistic pathway has not been yet fully understood; it is, however, known that there is considerable production of radical species centered at both the oxygen and carbon, which are important to the therapeutical effects of those compounds. This article reports kinetic data for the reaction of artemisinin and two model 1,2,4-trioxolanes with iron(II) species and also a mechanistic interpretation of this reductive cleavage from transition state thermodynamics. The suggestion of the presence of an enhancing specific factor inside the plasmodium is made.

Characterization and comparison of transgenic Artemisia annua GYR and wild-type NON-GYR plants in an environmental release trial.

The anti-malarial drug, artemisinin, is quite expensive as a result of its slow content in Artemisia annua. Recent investigations have suggested that genetic engineering of A. annua is a promising approach to improve the yield of artemisinin. In this study, the transgenic A. annua strain GYR, which has high artemisinin content, was evaluated in an environmental release trial. First, GYR plants were compared with the wild-type variety NON-GYR, with regard to phenotypic characters (plant height, crown width, stem diameter, germination rate, leaf dry weight, 1000-seed weight, leave shape). Second, stress resistance in the two varieties (salt, drought, herbicide, and cold resistance) was evaluated under different experimental conditions. Finally, gene flow was estimated. The results indicated that there were significant differences in several agronomic traits (plant height, stem diameter, and leave dry weight) between the transgenic GYR and NON-GYR plants. Salt stress in transgenic and control plants was similar, except under high NaCl concentrations (1.6%, w/w). Leaf water, proline, and MDA content (increased significantly) were significantly different. Transgenic A. annua GYR plants did not grow better than NON-GYR plants with respect to drought and herbicide resistance. The two varieties maintained vitality through the winter. Third, gene flow was studied in an environmental risk trial for transgenic GYR. The maximum gene flow frequency was 2.5%, while the maximum gene flow distance was 24.4 m; gene flow was not detected at 29.2 m at any direction. Our findings may provide an opportunity for risk assessment in future commercialization of transgenic A. annua varieties.

Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca²âº-ATPase PfATP6.

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae).

Finding an efficient and affordable treatment against malaria is still a challenge for medicine. Artemisinin is an effective anti-malarial drug isolated from Artemisia annua. However, the artemisinin content of A. annua is very low. We used transgenic technology to increase the artemisinin content of A. annua by overexpressing cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes. CYP71AV1 is a key enzyme in the artemisinin biosynthesis pathway, while CPR is a redox partner for CYP71AV1. Eight independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR and Southern blot analyses. The real-time qPCR results showed that the gene cyp71av1 was highly expressed at the transcriptional level in the transgenic A. annua plants. HPLC analysis showed that the artemisinin content was increased in a number of the transgenic plants, in which both cyp71av1 and cpr were overexpressed. In one of the transgenic A. annua plants, the artemisinin content was 38% higher than in the non-transgenic plants. We conclude that overexpressing key enzymes of the biosynthesis pathway is an effective means for increasing artemisinin content in plants.

Mining of miRNAs and potential targets from gene oriented clusters of transcripts sequences of the anti-malarial plant, Artemisia annua.

miRNAs involved in the biosynthesis of artemisinin, an anti-malarial compound form the plant Artemisia annua, have been identified using computational approaches to find conserved pre-miRNAs in available A. annua UniGene collections. Eleven pre-miRNAs were found from nine families. Targets predicted for these miRNAs were mainly transcription factors for conserved miRNAs. No target genes involved in artemisinin biosynthesis were found. However, miR390 was predicted to target a gene involved in the trichome development, which is the site of synthesis of artemisinin and could be a candidate for genetic transformation aiming to increase the content of artemisinin. Phylogenetic analyses were carried out to determinate the relation between A. annua and other plant pre-miRNAs: the pre-miRNA-based phylogenetic trees failed to correspond to known phylogenies, suggesting that pre-miRNA primary sequences may be too variable to accurately predict phylogenetic relations.

Avaliação in vitro de fármacos pró-oxidantes sobre o Schistosoma mansoni / In vitro evaluation of drugs pro-oxidants on Schistosoma mansoni

Segundo a Organização Mundial de Saúde (2002), a esquistossomose é a segunda maior doença tropical, causadora de 200 a 300 mil mortes por ano. Mesmo apresentando alguns quimioterápicos eficazes para o tratamento, como o praziquantel (PZQ) e a oxamniquina (OXQ), ocorrem muitos casos refratários e efeitos colaterais. Diante deste contexto, é necessária a busca racional de novos medicamentos e combinações para o tratamento desta doença. Uma possível solução é o estudo de drogas relacionadas com o estresse oxidativo do patógeno. Dentre estas podem ser analisadas: a artemisinina (ART) , que induz uma maior produção de radicais livres por inibição da formação da hemozoína em Plasmodium falciparum; a butionina sulfoximina (850), que impede a produção da glutationa; além do dietilditiocarbamato de sódio (DDC), que age como inibidor das superóxido dismutases. Portanto, o objetivo deste trabalho foi testar in vitro essas drogas, isoladas e combinadas, analisando a atividade das superóxido dismutases, alterações morfológicas e produção de hemozoína em vermes adultos, além de avaliar a toxicidade em esplenócitos. A ART apresentou efeito esquistossomicida em concentrações elevadas. O DDC mostrou um efeito esquistossomicida satifatório e inibiu as superóxido dismutases. Quanto à associação ART - DDC, esta inibiu a formação da hemozoína, apresentou danos nos tegumentos dos vermes e não apresentou citotoxicidade significativa. Estes resultados indicam que estas drogas são viáveis para estudos in vivo, podendo ser uma nova alternativa quimioterápica para esta patologia.

DFT study of the reductive decomposition of artemisinin.

Artemisinin is a sesquiterpene lactone with an endoperoxide function that is essential for its antimalarial activity. The DFT B3LYP method, together with the 6-31G(d) and 6-31+G(d,p) basis set, is employed to calculate a set of radical anions and neutral species supposed to be formed during the rearrangement of artemisinin from the two radicals (C-centered and O-centered) that are supposed to play a relevant role in the mechanism of action. The B3LYP results show that the primary and the secondary radicals centered on C(4), generated by homolytic break of the C(3)-C(4) bond and by 1,5 hydrogen shift, respectively, are more stable than radicals centered on oxygen. The calculations show that the activation barriers for rearrangements are low, leading to a thermodynamically favorable process. These results reinforce our previous conclusions based on semi-empirical calculations but also give additional information on the reductive decomposition of artemisinin.