yes / Ancient Chinese herbal texts as far back as the 4th Century Zhou hou bei ji fang describe methods for the use of Qing Hao (Artemisia annua) for the treatment of intermittent fevers. Today, the A. annua constituent artemisinin is an important antimalarial drug and the herb itself is being grown and used locally for malaria treatment although this practice is controversial. Here we show that the ancient Chinese methods that involved either soaking, (followed by wringing) or pounding, (followed by squeezing) the fresh herb are more effective in producing artemisinin-rich extracts than the usual current method of preparing herbal teas from the dried herb. The concentrations of artemisinin in the extracts was up to 20-fold higher than that in a herbal tea prepared from the dried herb, but the amount of total artemisinin extracted by the Chinese methods was much less than that removed in the herbal tea. While both extracts exhibited potent in vitro activities against Plasmodium falciparum, only the pounded juice contained sufficient artemisinin to suppress parasitaemia in P. berghei infected mice. The implications of these results are discussed in the context of malaria treatment using A. annua infusions.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-03-31T10:25:55Z No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-31T10:44:27Z (GMT) No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-31T10:44:27Z (GMT). No. of bitstreams: 2 Dissertação - Daniela Cristina da Silva - 2017.pdf: 3223777 bytes, checksum: 67c2832449d10a3a2590e8a834f55b10 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-09 / Entomopathogenic fungi combined with plant extracts may have their development in vitro and their insecticidal activity modified. There are many reports on the activity of entomopathogenic fungi and extracts in insects, but few studies on fungi combined with plant extracts. The objective of this study was to evaluate in vitro the effect of crude ethanol extracts of Artemisia annua, Andrographis paniculata, Curcuma zedoaria, Ginkgo biloba and Rosmarinus officinalis on the Metarhizium anisopliae s.l. IP 46 by the diffusion disc test, to evaluate in vivo susceptibility of Aedes aegypti larvae to extracts by determination the cumulative emergence of adults, development and mortality of larvae for 15 days, then to select the extracts and combine them with IP 46 at 3.3 x 105 conidia.mL-1. There was no significant inhibition germination of IP 46. On the fifteenth day there was a significant effect on cumulative emergence of adults and development of larvae for A. annua at 10 ppm, 33 ppm and 1000 ppm and C. zedoaria at 100 ppm and 333 ppm. There was also a significant effect on accumulated mortality of larvae exposed to G. biloba at 10000 ppm.These extracts with in these concentrations were selected and combined with IP 46 at 3.3 x 105 conidia.mL-1, minus G. biloba which was combined at 5000 ppm after the LC50 and LC90 calculations. These combinations, except for A. annua at 1000 ppm, exhibited a significant increase in larval mortality and/ or showed an effect on development, indicating that combined methods may be more effective in controlling A. aegypti than isolated methods. / Fungos entomopatogênicos combinados a extratos de plantas podem ter o seu desenvolvimento in vitro e sua atividade inseticida in vivo modificada. Existem muitos relatos sobre a atividade de fungos entomopatogênicos e extratos em insetos, porém são poucos os estudos sobre fungos combinados a extratos de plantas. O objetivo do trabalho foi avaliar in vitro o efeito de extratos brutos etanólicos de Artemisia annua, Andrographis paniculata, Curcuma zedoaria, Ginkgo biloba e Rosmarinus officinalis em isolado de Metarhizium anisopliae s.l. IP 46 por meio do teste de disco difusão, avaliar in vivo a suscetibilidade de larvas de Aedes aegypti expostas a esses extratos por meio da avaliação da emergência acumulada de adultos, do desenvolvimento e da mortalidade de larvas, durante 15 dias, para então selecionar os extratos e combiná-los com IP 46 a 3,3 x 105 conídios.mL-1. Observou-se que não houve inibição significativa de IP 46 pelos extratos. No décimo quinto dia houve efeito significativo na emergência acumulada de adultos e no desenvolvimento de larvas expostas a A. annua a 10 ppm, 33 ppm e 1000 ppm e de C. zedoaria a 100ppm e 333 ppm. Também houve efeito significativo na mortalidade acumulada de larvas exposta a G. biloba a 10000 ppm. Esses extratos nessas concentrações foram selecionados e combinados com IP 46 a 3,3 x 105 conídios.mL-1, menos o de G. biloba que foi combinado a 5000 ppm após cálculos da CL50 e CL90. Essas combinações, exceto para A. annua a 1000 ppm, exibiram aumento significativo na mortalidade larval e/ou apresentararam efeito sobre o desenvolvimento, indicando que métodos combinados podem ser mais efetivos no controle de A. aegypti do que métodos isolados.

Artemisia annua L. is a common type of wormwood that grows throughout the world. Artemisinin, a terpene compound in A. annua, has recently been recognized as the most promising antimalaria drug. Artemisinin and other types of terpenoids are synthesized and accumulated in glandualr trichomes that appear on the surface of leaf, stem and flower bud. To identify new genes involved in artemisinin biosynthesis and trichome function in A. annua, a normalized glandular trichome cDNA collection was sequenced by Roche GS FLX pyrosequencing system. Two sequencing runs generated totally 85M nucleotides which were further assembled into 190,377 unigenes (42,678 contigs and 147,699 sigletons). Putative functions were assigned to the unigenes based on Blast search against GeneBank database. Many terpene biosynthesis pathway genes were identified from the pyrosequencing ESTs. Together with other identified A. annua terpene pathway genes, a global view of terpene biosynthesis in glandular trichomes of A. annua were re-established. Meanwhile, a WD repeat protein, AaWD40, which show high amino acid sequence similarity with its Arabidopsis ortholog, AtTTG1 (AT5G24520) was identified. To investigate the functional relevance of AaWD40 to its Arabidopsis counterpart, genetic complementation test using Arabidopsis mutants was conducted. When AaWD40 was transformed into Arabidopsis transparent testa glabrous1 (ttg1-1) mutant, the anthocyanins and proanthocyanidin (PAs) production in seeds were restored, and the trichomeless phenotype of ttg1-1 mutant was rescued. In addition, over-expression of AaWD40 and AtTTG1 modulated the expression of WUS and CLVs genes which are required to maintain the stem-cell niche of Arabidopsis shoot apex. Transcriptomic profiling of transgenic Arabidopsis over-expressing AaWD40, TTG1, or ttg1-1 mutant revealed lists of genes modulated by these two WD40 genes homologue and gene ontology (GO) analysis suggested that the top-ranked categories are defense, stress response and developmental programme. We hypothesize that WD40 repeat protein act as a crucial regulatory factor in a wide variety of cellular functions in A. thaliana. / Wang, Wei. / Advisers: Guo Dianjing; Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 82-105). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Qi, Yan. / Thesis submitted in: December 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 63-79). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.III / ABSTRACT --- p.IV / TABLE OF CONTENTS --- p.VII / LIST OF ABBREVIATIONS --- p.XI / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- the Plant of Artemisia annua L --- p.1 / Chapter 1.2 --- The disease of malaria --- p.3 / Chapter 1.2.1 --- The life cycle of Plasmodium parasites --- p.4 / Chapter 1.2.2 --- The Artemisinin-based combination therapies (ACTs) for the treatment of malaria --- p.5 / Chapter 1.3 --- Artemisinin --- p.8 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.8 / Chapter 1.3.2 --- The mechanism of artemisinin action --- p.9 / Chapter 1.3.2.1 --- The proposed non-specific mechanisms of action --- p.10 / Chapter 1.3.2.2 --- The proposed parasite-specific mechanisms of action --- p.11 / Chapter 1.3.3 --- The biosynthesis of artemisnin in vivo --- p.12 / Chapter 1.3.4 --- The biosynthesis of artemisinin in vitro --- p.16 / Chapter 1.4 --- Trichomes --- p.18 / Chapter 1.4.1 --- Non-glandular trichomes --- p.19 / Chapter 1.4.2 --- Glandular trichome --- p.20 / Chapter 1.4.3 --- Trichomes of Artemisia annua L --- p.21 / Chapter 1.5 --- DNA Sequencing Methods --- p.24 / Chapter 1.5.1 --- The basic principle of pyrosequencing --- p.25 / Chapter 1.5.2 --- 454 pyrosequencing and its application --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Chemicals --- p.32 / Chapter 2.2 --- Plant materials --- p.32 / Chapter 2.3 --- Preparation of the cDNA sample for 454 sequencing --- p.33 / Chapter 2.3.1 --- Scanning electron microscopy --- p.33 / Chapter 2.3.2 --- Isolation of glandular trichomes --- p.34 / Chapter 2.3.3 --- cDNA synthesis and normalization --- p.34 / Chapter 2.4 --- 454-EST SEQUENCING AND PROCESSING --- p.36 / Chapter 2.5 --- Analysis of 454 sequencing data --- p.37 / Chapter 2.6 --- Establishment of regeneration system of A. annua L --- p.37 / Chapter 2.6.1 --- Shoots induction from leaf discs --- p.37 / Chapter 2.6.2 --- The sensitivity of the explants to Kanamycin --- p.38 / Chapter 2.6.3 --- Rooting of the regenerated seedlings --- p.38 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.40 / Chapter 3.1 --- Glandular trichome isolation and cDNA preparation --- p.40 / Chapter 3.1.1 --- The distribution of glandular trichomes on A. annua --- p.40 / Chapter 3.1.2 --- The isolation of glandular trichomes --- p.42 / Chapter 3.1.3 --- The preparation of ds cDNA for 454 sequencing --- p.43 / Chapter 3.2 --- Pre-process of 454 pyrosequencing data --- p.44 / Chapter 3.3 --- Functional annotation of the 454-EST data --- p.47 / Chapter 3.4 --- Comparison of two sequencing runs --- p.49 / Chapter 3.5 --- Analysis of the 454 ESTs involved in secondary metabolisms --- p.50 / Chapter 3.6 --- Selection of the candidate genes --- p.55 / Chapter 3.7 --- Establishment of regeneration system of A. annua L --- p.57 / Chapter 3.7.1 --- Shoots induction from leaf discs --- p.57 / Chapter 3.7.2 --- Roots induction from shoots --- p.57 / Chapter 3.7.3 --- Sensitivity of A. annua to Kan --- p.59 / Chapter CHAPTER 4. --- CONCLUSION --- p.61 / REFERENCES --- p.63

Orientadores: Maria Angela de Almeida Meireles, Paulo de Tarso Vieira e Rosa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T11:45:06Z (GMT). No. of bitstreams: 1 QuispeCondori_Socrates_D.pdf: 2798950 bytes, checksum: 0b1d9044fdb82444a8064327ee12685a (MD5) Previous issue date: 2005 / Resumo: No presente trabalho é apresentado um estudo global do processo de extração de produtos naturais usando dióxido de carbono supercrítico como solvente. As duas etapas do processo foram estudadas usando diversas matrizes vegetais. Na Etapa de Extração (Artemisia annua, Cordia verbenacea e Ocimum selloi) foram medidos dados de rendimento global para os três sistemas. Para os sistemas CO2 + A. annua e CO2 + C. verbenacea foram calculados os parâmetros cinéticos do processo de extração e um modelo matemático foi selecionado para representar as curvas globais de extração. A técnica de extração fracionada foi aplicada para os sistemas CO2 + C. verbenacea e CO2 + Ocimum Selloi. A determinação do rendimento global permitiu visualizar o efeito da temperatura e pressão sobre a solubilidade do extrato, além de proporcionar informações necessárias para modelagem do processo de extração. A condição que maximizou o rendimento em extrato nos sistemas CO2 + A. annua e CO2 + C. verbenacea foi 300 bar / 50 °C. Entretanto, foi demonstrado que o extrato de C. verbenacea obtido nesta condição apresenta menor atividade anticâncer àquele obtido a 200 bar / 40 °C. Para o sistema CO2 + Ocimum Selloi o maior rendimento foi obtido a 200 bar / 40 °C. No estudo da cinética do processo de extração foi verificado que para o sistema CO2 + A. annua a vazão do solvente foi o único fator significante sobre os parâmetros cinéticos. No estudo do efeito da altura do leito para o sistema CO2 + C. verbenacea, verificou-se que os parâmetros cinéticos aumentam com a altura do leito. Na modelagem matemática das curvas globais de extração (OEC) dos sistemas CO2 + A. annua e CO2 + C. verbenacea verificou-se que os modelos de Naik et al. [1989] (empírico), Sovová [1994] e Goto et al. [1993] apresentaram os melhores ajustes aos dados experimentais. Do estudo do efeito da altura do leito na SFE a partir de C. verbenacea, verificou-se que a seleção de um modelo para aumento de escala é difícil de ser realizada, uma vez que o modelo selecionado nem sempre é válido para todos os ensaios. Na Etapa de Separação (Foeniculum vulgare) foi realizado o estudo da influência das condições operacionais (temperatura, pressão e vazão do solvente) na recuperação do extrato. As condições de temperatura e pressão de separação foram determinadas através de modelagem termodinâmica usando-se a equação de estado de Peng-Robinson. A determinação do comportamento da solubilidade dos componentes majoritários do extrato de F. vulgare (anetol e ácido oléico) em CO2 supercrítico permitiu obter uma aproximação das condições ótimas de separação. No estudo da influência das condições operacionais (temperatura e pressão) determinou-se que o aumento da pressão no primeiro separador permite a solubilização do compostos de maior massa molecular, os quais são transferidos para o segundo separador. Entretanto, se o objetivo for o fracionamento do extrato de F. vulgare, observou-se que a melhor condição operacional para separação da fração rica em componentes de óleo volátil foi 80 bar / 40 °C no primeiro separador. Foi verificado também que o aumento da vazão de solvente diminui o rendimento em anetol e funchona e, conseqüentemente, no rendimento global. A otimização das condições de separação da mistura extrato + solvente é uma etapa importante do processo de extração de produtos naturais usando fluidos supercríticos, uma vez que permitirá uma ótima recuperação e/ou fracionamento do extrato / Abstract: In the present work a global study of the extraction of natural products using supercritical carbon dioxide as solvent is presented. The two steps of the SFE process were studied using several raw materials. In the Extraction Step (Artemisia annua, Cordia verbenacea and Ocimum selloi), global yield data for the three plants were determined. Kinetic parameters of the extraction process were calculated for the CO2 + A. annua and CO2 + C. verbenacea systems and a mathematical model to represent the overall extraction curves was selected. The fractional extraction technique was applied for the CO2 + C. verbenacea and CO2 + Ocimum Selloi systems. The determination of the global yield allowed to identify the effect of the temperature and pressure on the solubility of the extract, besides providing necessary information for the modeling of the extraction process. The operational condition that maximized the global yield in the systems CO2 + A. annua and CO2 + C. verbenacea was 300 bar / 50 °C. However, it was demonstrated that the C. verbenacea extract obtained at this condition shows a lower anticancer activity than that obtained at 200 bar / 40 °C. The higher global yield for the CO2 + Ocimum Selloi system was obtained at 200 bar / 40 °C. In the study of the kinetic of the extraction process for the CO2 + A. annua system, it was verified that the flow rate was the only significant factor on the kinetic parameters. In the study of the effect of the extraction bed height for the CO2 + C. verbenacea system, it was verified that the kinetic parameters increase with the bed height. The Naik et al. [1989] (empirical), Sovová [1994] and Goto et al. [1993] models presented the best fittings to the experimental overall extraction curves (OEC) for the CO2 + A. annua and CO2 + C. verbenacea systems. From the study of the effect of the height of the bed for the C. verbenacea, it was verified that the selection of a mathematical model to scale-up is difficult to be accomplished, because the selected model is not always valid for all experiments. In the Separation Step (Foeniculum vulgare) the study of the influence of the operational conditions (temperature, pressure and flow rate) in the recovery of the extract was carried out. The temperature and pressure of the separation step was calculated through thermodynamic modeling using the equation of state of Peng-Robinson. The determination of the solubility behavior of anethole and oleic acid (major compounds of the F. vulgare extract) with supercritical CO2 allowed to obtain an approximation of the optimal condition of the separation step. In the study of the influence of temperature and pressure, it was determined that the increase of pressure in the first separator allows the solubilization of high molecular mass compounds that are transferred to the second separator. However, if the objective is the fractionation of the fennel extract, it was observed that the best operational condition to separate a rich fraction in volatile oil compounds was 80 bar / 40°C. Additionally, it was verified that the increase of flow rate diminishes the anethole and fenchone yields and, consequently, the global yield. The optimization of the operational conditions in the separation of the mixture extract + solvent is an important step in the supercritical extraction of natural products, because it will allow an optimal recovery and/or fractionation of the extract / Doutorado / Engenharia de Alimentos / Doutor em Engenharia de Alimentos

Thesis (M.Sc. Agriculture (Horticulture)) -- University of Limpopo, 2017 / Artemisia annua L. from the family Asteraceae is an annual medicinal plant and has been used to make herbal remedies in Asia for thousands of years. Artemisinin is a sesquiterpene lactone, isolated from aerial parts of Artemisia annua, with the highest concentrations being in flowers and leaves. In addition to potent anti-malarial activity, artemisinin possesses anti-cancer, anti-schistosomiatic, anti-hepatitis B, anti-HIV, anti-leishmanial and herbicidal activities. Low artemisinin production (0.01-2%) from A. annua is a major constraint in commercialisation of the drug for control of malaria. Worldwide, efforts have been underway to improve the concentration of artemisinin using conventional breeding, biochemical, physiological, molecular and hairy-root culture techniques, however all these methods are not economical. Cultural practices like spacing and pruning have limitation in improving artemisinin concentration and these may help in increasing the concentrations of artemisinin. Study was conducted at the experimental farm of the Agricultural Research Council – Vegetable and Ornamental Plants, Roodeplaat Pretoria. The objective of this study was to determine whether spacing, pruning and their interactions would have any effect on the concentrations of artemisinin, growth and yield of A. annua and whether drying methods would have an effect on the concentrations of artemisinin in A. annua. Since there was only one field trial, all sub-objectives were addressed at once (Chapter 3). Fresh seeds of A. annua were obtained from the ARC-VOP gene bank and sown in seedling trays in September 2014. Uniform eight-week-old seedlings were hardened-off, transplanted in November 2014 in 10 cm deep holes and then pruned ten weeks after transplanting. Treatments for Experiment 1, viz., 3 × 4 factorial experiment were laid out in a randomised complete block design, with four replications (n = 48). The two factors of the experiment were (a) spacing [0.5 × 1 m2 (standard: 0.50 m2), 0.5 × 0.7 m2 (small: 0.35 m2) 0.5 × 0.5 m2 (smaller: 0.25 m2) and 0.3 × 0.7 m2 (smallest: 0.21 m2)] and (b) pruning [no pruning (control), removing the apical bud and removing shoots three nodes from the bottom]. The plants were irrigated using overhead sprinklers system for two hours three times per week. Four readings for growth variables (plant height, stem diameter and chlorophyll content) were collected with one week interval. Plants were harvested after 180 days from planting, and leaves, stems and roots were separated weighed and oven dried at 40 ºC for 72 h. In Experiment 2 (drying methods), treatments, namely, 100% sun, 100% shade, 50% shade, freeze and oven drying were arranged in completely randomised design with four replicates (n = 20). The treatments were exposed for a week, to full sunlight, 50% shade-drying under a shade net that allows 50% light penetration, 100% shade under enclosed room at ambient (24-25 ºC) temperature, oven drying for 24 h at 40 ºC, and freeze-drying for three days. Freeze-drying had significant effect on artemisinin concentration of 1.941%. It was followed by oven (1.738%) and 100% shade drying (1.657%) and the lowest artemisinin concentration (1.412%) was obtained from 50% shade drying. The smaller spacing of 0.25 m2 in combination with apical bud removal had a significant effect on artemisinin concentration, producing artemisinin concentration of 0.193%. Spacing had a significant effect on stem diameter, fresh leaf mass and dry leaf mass but had no effect on plant height and chlorophyll content. Pruning had a significant effect on plant height and chlorophyll content and had no effect on stem diameter. The small spacing of 0.35 m2 had the highest fresh and dry leaf mass of 17.99 and 9.62 t/ha. The interaction of spacing and pruning had no significant effect on the growth and yield of A. annua. The results from this study suggested that cultural and processing practices may have direct effects in the concentration of artemisinin, growth and yield of A. annua. The results xiv provided some understanding on how agronomic and processing practices can be used to increase artemisinin content in A. annua and understand the interaction between different agronomic practices and thereby allowing the development of economic methods for A. annua post-harvest handling. Future work should focus on implementing various pruning techniques to trigger stress and indirectly secondary metabolites

A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-12-12T13:51:59Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-12-20T13:18:24Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Made available in DSpace on 2012-12-20T13:18:24Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / A artemisinina é uma substância extraída da planta chinesa Artemisia annua L., sendo bastante utilizada na medicina natural como um terapêutico em várias patologias. Já o artemer é uma substância sintetizada a partir da artemisinina. Estas drogas se enquadram em um grupo especial de moléculas denominadas de lactonas sesquiterpênicas sendo amplamente administradas na terapêutica da malária. Embora sejam considerados eficientes anti-maláricos, muito pouco se sabe sobre os efeitos genotóxicos e citotóxicos destes fármacos. Portanto, no presente trabalho, avaliamos os efeitos citotóxicos, genotóxicos e mutagênicos da artemisinina e do artemeter em cultura de linfócitos humanos por meio do ensaio cometa, do teste do micronúcleo e do ensaio de citotoxicidade para detecção de necrose e apoptose por marcação fluorescente diferencial com laranja de acridina/brometo de etídio (LA/BE), respectivamente. Nossos resultados demonstraram um aumento significativo (p<0,05) no índice de dano do DNA avaliado pelo ensaio do cometa, bem como na frequência de micronúcleos em ambas as substâncias testadas. Foi observado também, que apenas a artemisinina induziu um aumento estatisticamente significativo (p<0.05) no número de células necróticas nos linfócitos em 48 h de tratamento. Desta forma, demonstrou-se em nosso trabalho, que estas duas drogas exercem efeitos citotóxicos, genotóxicos e mutagênicos em culturas de linfócitos humanos, nas condições avaliadas. Nossos dados apontam a necessidade de cautela no uso de tais medicamentos, uma vez que efeitos genotóxicos/mutagênicos podem aumentar o risco de carcinogênese. / Artemisinin is a substance extracted from the Chinese plant Artemisia annua L., and widely used in natural medicine for a treatment of various diseases. Artemether is a substance synthesized from artemisinin. These drugs belong to a special group of molecules called sesquiterpene lactones widely administered in the treatment of malaria. Although considered effective anti-malarial drugs, very little is known about the genotoxic and cytotoxic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin and artemether in cultured human lymphocytes using the comet assay, the micronucleus test and a cytotoxicity assay for detection of necrosis and apoptosis by fluorescent differential acridine orange/ethidium bromide (LA/BE), respectively. 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. Thus, it was shown in our work that these two drugs exert mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes under the conditions evaluated. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported

Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-09-21T19:14:04Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Rejected by Edisangela Bastos (edisangela@ufpa.br), reason: on 2017-10-10T17:00:49Z (GMT) / Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-10-17T18:38:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-11-24T15:00:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) / Made available in DSpace on 2017-11-24T15:00:16Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInVitro.pdf: 1439306 bytes, checksum: 7f71a06de5db3f1824c5314ed19c43b4 (MD5) Previous issue date: 2011-08-12 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O artesunato representa uma das principais drogas utilizadas como antimaláricos em diversos países. É um composto semi-sintético derivado de artemisinina, substancia extraída da planta chinesa Artemisia annua L. Apesar da ampla utilização do artesunato na terapêutica antimalárica, estudos demonstrando seus efeitos genotóxicos e citotóxicos em cultura de linfócitos humanos são ainda hoje quase inexistentes. Portanto, no presente trabalho, avaliamos os efeitos genotóxicos e citotóxicos do artesunato em cultura de linfócitos humanos. Nossos resultados demonstraram aumento significativo (p<0,05) no número de células apoptóticas dos linfócitos, tanto em 24 quanto em 48 h de tratamento. Desta forma, demonstrou-se em nosso trabalho, que o artesunato é uma droga genotóxica e citotóxica em cultura de linfócitos humanos, nas condições avaliadas. / Artesunate is one of the main drugs used as antimalarials in various countries. It is a semi-synthetic compound from artemisinin, a substance extracted from the Chinese plant Artemisia annua L. Despite the widespread use of antimalarial artesunato in malaria treatment, studies demonstrating its cytotoxic and genotoxic effects in human lymphocyte cultures are almost nonexistent. Therefore, in this study, we evaluated possible cytotoxic and genotoxic effects of artesunate on cultured human lymphocytes. A significant increase (p <0.05) in the rate of DNA damage and micronucleus frequency was observed after artesunato treatment. We also observed that artesunato induces a statically significant increase (p <0,05) in the number of apoptotic cells in both 24 and 48 h of treatment. Thus, we conclude in our work that artesunate is a highly cytotoxic and genotoxic drug in cultured human lymphocytes.