Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references. Also available in electronic version.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references. Also available in electronic version.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-08Bitstream added on 2014-06-13T19:08:38Z : No. of bitstreams: 1 aquino_i_me_botib.pdf: 485581 bytes, checksum: 72dc0e003e4ace9410c6185e551add41 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O tratamento para combater a Malária, recomendado pela Organização Mundial da Saúde (OMS), é feito com medicamentos derivados da artemisinina (tais como o artesunato), o princípio ativo extraído da Artemisia annua. Com base nesta informação, foi avaliado neste projeto o potencial genotóxico da artemisinina (ATM) e do artesunato (ART) em células HepG2 (Human hepatocellular liver carcinoma cell line), in vitro, além da investigação do potencial genotóxico e/ou mutagênico do artesunato, em células de camundongos in vivo. Nestas análises foram utilizados o ensaio cometa e o teste de micronúcleo, para a verificação da genotoxicidade e mutagenicidade; nos ensaios in vitro, também foram realizados testes de citotoxicidade e análise da expressão dos genes SOD1 e Casp3. Nos ensaios in vivo, utilizando camundongos tratados com ART, foi verificado através do Ensaio Cometa que, nas células de sangue periférico, apenas a maior dose testada (2000mg/kg) foi genotóxica, quando comparada ao grupo controle negativo. Já nas células de fígado, as doses de 5mg, 50mg e 2000mg/kg mostraram-se genotóxicas, com p<0,05. Quanto à mutagenicidade, os tratamentos com ART nas doses de 50mg, 100mg e 2000mg/kg apresentaram aumento significativo no número de células micronucleadas em relação ao grupo controle. Nos testes in vitro, os resultados do ensaio cometa com a ATM e o ART mostraram um aumento significativo (p<0,001) no número de células com danos no DNA, em relação ao controle negativo, em todos os tratamentos e para ambas as substâncias, o que indica um efeito genotóxico da ATM e do ART. Já na análise da expressão gênica, houve um aumento na expressão de Caspase 3, nas células tratadas com as duas substâncias, porém, a expressão de SOD1 foi maior em células tratadas com ATM, enquanto nas células tratadas com o ART, houve uma diminuição na expressão deste gene.... / The treatment to combat malaria, recommended by the World Health Organization (WHO), is done with drugs artemisinin derivatives (such as artesunate), the active ingredient extracted from Artemisia annua. Based on this information, and the lack of data on genotoxicity of these compounds in mammalian cells, was evaluated in this project the genotoxic potential of artemisinin (ATM) and artesunate (ART) in HepG2 cells (Human Hepatocellular liver carcinoma cell line), in vitro as well as investigating the potential genotoxic and / or mutagenic of artesunate, in mice in vivo. In these tests we used the comet assay and micronucleus test, to check the genotoxicity and mutagenicity, in vitro assays were also performed cytotoxicity tests and analysis of genes expression SOD1 and Casp3. In vivo tests using mice treated with ART, was determined using the Comet assay that in peripheral blood cells, only the highest dose tested (2000mg/kg) was genotoxic when compared to negative control group. Already in the liver cells, doses of 5mg, 50mg and 2000mg/kg were shown to be genotoxic, p <0.05. For mutagenicity, treatment with ART in doses of 50mg, 100mg and 2000mg/kg showed a significant increase in the number of micronucleated cells in the control group. In vitro tests, the results of the comet assay with the ATM and ART showed a significant increase (p <0.001) in the number of cells with DNA damage, compared to negative control in all treatments and for both substances, the indicates that a genotoxic effect of ATM and ART. The analysis of gene expression, there was an increase in the expression of Caspase 3, in cells treated with both substances, however, the expression of SOD1 was higher in cells treated with ATM, whereas in cells treated with ART, there was a decrease in expression of this gene. Although the analysis of gene expression did not reach statistical significance... (Complete abstract click electronic access below)

Wu, Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 56-70). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.Ill / ABSTRACT --- p.V / TABLE OF CONTENTS --- p.IX / LIST OF ABBREVIATIONS --- p.XII / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- THE DISEASE OF MALARIA --- p.1 / Chapter 1.1.1 --- Pathogenesis --- p.2 / Chapter 1.1.2 --- The treatment of malaria --- p.4 / Chapter 1.2 --- THE PLANT OF ARTEMISIA ANNUA L --- p.5 / Chapter 1.2.1. --- Horticulture --- p.5 / Chapter 1.2.2. --- Historical Importance --- p.6 / Chapter 1.3 --- ARTEMISININ --- p.7 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.7 / Chapter 1.3.2 --- The biosynthesis of artemisnin --- p.8 / Chapter 1.4 --- TRICHOMES --- p.13 / Chapter 1.4.1 --- Structure and function of trichomes --- p.13 / Chapter 1.4.2 --- Trichome investigation in A. annua --- p.14 / Chapter 1.5 --- PROTEOMICS --- p.17 / Chapter 1.5.1 --- The basic principle of proteomics --- p.17 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.18 / Chapter 1.5.3 --- Mass Spectrometry --- p.19 / Chapter 1.5.4 --- Gel-free proteomics --- p.20 / Chapter 1.6 --- OBJECTIVES --- p.21 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- CHEMICALS --- p.23 / Chapter 2.2 --- PLANT MATERIALS --- p.23 / Chapter 2.3 --- ISOLATION OF GLANDULAR TRICHOMES --- p.23 / Chapter 2.4 --- PROTEIN EXTRACTION . --- p.25 / Chapter 2.5 --- Two DIMENSIONAL GEL ELECTROPHORESIS --- p.25 / Chapter 2.6 --- IMAGINE ANALYSIS --- p.26 / Chapter 2.7 --- IN GEL DIGESTION AND PROTEIN IDENTIFICAIOTN BY MASS SPECTROMETRY --- p.27 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.29 / Chapter 3.1 --- THE ISOLATION OF GLANDULAR TRICHOMES --- p.29 / Chapter 3.2 --- 2DE PATTERNS OF A. ANNUA LEAVE TRICHOMES AND LEAF TISSUE --- p.32 / Chapter 3.3 --- IDENTIFICATION OF PROTEINS IN GLANDULAR TRICHOMES --- p.34 / Chapter 3.3.1 --- Protein involved in electron transport chain --- p.47 / Chapter 3.3.2 --- Protiens invovled in metabolism --- p.48 / Chapter 3.3.2.1 --- artemisinin biosynthesis --- p.48 / Chapter 3.3.2.2 --- glycolysis --- p.49 / Chapter 3.3.2.3 --- other metabolic enzymes --- p.50 / Chapter 3.3.3 --- Proteins involved in transcription and translation --- p.51 / Chapter 3.3.4 --- Protein involved in proteolysis --- p.51 / Chapter 3.3.5 --- "Detoxificaiton, stress related protein" --- p.52 / Chapter 3.4 --- PERSPECTIVE --- p.53 / Chapter 3.5 --- CONCLUSION --- p.53 / REFERENCES --- p.56

Orientadores: Rolf Dieter Illg, Nicolas Delabays / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-21T13:43:54Z (GMT). No. of bitstreams: 1 Magalhaes_PedroMelillode_D.pdf: 6355659 bytes, checksum: 2d95ce72d59eb7cfb1d57fb1e4c5399c (MD5) Previous issue date: 1996 / Résumé: Le paludisme est aujourd'hui encore l 'une des grandes causes de mortalité et de maladie dans le monde. Au Brésil, i l peuty avoir jusqu'à 500 mille cas de paludisme par ano La maladie, provoquée par le protozoaire Plasmodium, s'est aggravée depuis que l es especes de Plasmodium ont acqui s une rési stance aux remedes traditionnels, chloroquine et mefloquine. L'étude de nouveaux produits a abouti à la découverte des propriétés anti paludéennes d'une lactone sesquiterpenique, appelée Qinghaosu ou Artemisinine, extraite des feuilles de l 'Artemisia annua L., de l a famille dês Asteraceae. Dês composés derives de l 'artémisinine sont encore plus efficaces et sont commercialisés en Chine. Ainsi, la technologie de production et la validation de ces médicaments en Occident sont d'un grand intérêt. L'objectif principal des recherches qui ont composé cette these a été de contribuer à la production des principes actifs de l'espece A. annua L., favorisant ainsi l 'obtention dês médicaments anti-paludéens. Les recherches réalisées à MEDIPLANT CSuisse) et au CPQBA-UNICAMP CBrésil) ont inclus: des aspects de la reproduction, la selection et l¿obtention d¿hybrides pour la culture au Brésil, la fertilisation nitrogén''e et dês tentatives d¿extraction aqueuse de l 'artémisinine. Les résultats ont indiqués que l 'espece, bien que possédant des fleurs completes, présente un taux d'alogamie supérieur à 80%. Ainsi, à travers des croisements controles entre des génotypes sélectionnés dans les populations de base originaires de (hine et du Vietnam, a-t-on obtenu l 'homogénéité des populations et un gain génétique significatif. L'exploitation de la variabilité en biomasse et en teneur d'artémisinine, cette derniére étant connue par controle analytique, a permis de passer de 5Kg d'artémisinine lha pour les populations de base, à 30Kg d'artémisinine lha pour lês popul at i ons amél i orées. La propagat i on végétat i ve, pa r cul ture de tissus ou par boutures, a également été utilisée pour lê programme d'amélioration génétique, essentiellement pour l 'entretien dês progéniteurs et l a fixation des caracteres souhaitables. Deux génotypes, Pl .39 Et P 1 . V , ont été sélectionnés pour la culture en photopériode courte (11 à 12 heures de lumiere), conformément aux conditions du Brésil. Les teneurs en artémisinine et les productions de racines et de feuilles, dans les lignées cultivées en phytotron, ont été plus grandes pour des températures variant entre 22°( et 26°(, par rapport aux rendements obtenus pour des températures variant entre 25°( et 31°(. Le développement des raciness a été directement proportionnel aux teneurs d'artemisine dans les feuilles d'A. annua L.. Les premiers travaux de sélection, menés en des endroits ou les conditions de photopériode n'induisaient pas la floraison de l'espece, a permis la sélection de génotypes plus productifs en biomasse du fait qu'ils expriment tout leur potentiel végétatif. Au Srésil, 1 'évaluation de 1 'hybride Ch. x Viet 55 a attei nt des rendements approximatifs e 30 kg d'artémisinine/ha et par récoltes, deux récoltes par an étant possibles sur une même aire. La culture de cet hybride a été techniquement viable et hautement compétitif grâce aux rendements btenus. L'application de fertilisants azotes àune dose de 90Kg d'azote/ha a augmenté de 28 à 40% es rendements d'artémisinine en fonction de l 'augmentation de la biomasse. La forme nitrate s'est révél ée l a pl us favorabl e. Un possible déplacement de l ' ét a t de plus forte concentration de l 'artémisinine est suggéré. Des extractions aqueuses à partir de feuilles riches en artémi si ni ne présentent une fracti on contenant 50% du pri nci pe actif. On suggere que ce procédé puisse être employé comme pré processus pour des extractions industrielles, ou même servir de base à de nouvelles recherches sur l 'activité anti ¿paludéenne d'infusions. Les études de l'Artemisia annua ont eu pour consequence l ' établ i ssement d'un programme d'amélioration génétique au CPQSA / Resumo: A malária é ainda uma das grandes causas de mortalidade e morbidade no mundo. No Brasil, chegam a ocorrer 500 mil casos de malária por ano. A doença, causada pelo protozoário Plasmodium, tem sido agravada desde que espécies de Plasmodium passaram a adquir resistência às drogas tradicionais, cloroquina emefloquina. O estudo de novos medicamentos resultou no descobrimento das propriedades antimaláricas de uma lactona sesquiterpênica, chamada Qinghaosu ou Artemisinina, extraída das folhas da Artemísia annua L., da família Asteraceae. Compostos derivados da artemisinina são ainda de maior eficácia e vem sendo comercializados na China. Assim, a tecnologia de produção e a validação destes medicamentos no ocidente, são de grande interesse. O objetivo principal das pesquisas que compuseram esta Tese foi de contribuir para a produção dos princípios ativos da espécie Artemisia annua L., favorecendo a obtenção dos medicamentos antimalájicos. As pesquisas realizadas na MEDIPLANT (Suíça) e no CPQBA-UNICAMP (Brasil) envolveram: aspectos da biologia da reprodução; seleção e obtenção de híbridos para cultivo no Brasil; fertilização nitrogenada e ensaios de extração aquosa da artemisinina. Os resultados indicaram que a espécie, embora possua flores completas, apresenta taxa de a 1 oogamia superior a 80%. Assim, através de cruzamentos controlados entre genótipos selecionados nas populações de base, originais da China e do Vietnam, obteve-se a homogeneidade das populações e significativo ganho genético. A exploração da variabilidade em biomassa e em teores de artemisinina, esta última conhecida por monitoramento analítico, possibilitou passar de 5Kg de artemisinina /há nas populações de base, para aproximadamente 30Kg de artemisinina/ha nas populações melhoradas. A propagação vegetativa, por cultura de teci dos ou por estacas, foi também utilizada no programa de melhoramento genético, principalmente para a manutenção dos progenitores e a fixação de caracteres desejáveis. Dois genótipos, Pl.39 e Pl. V, foram selecionados para cultivo em fotoperíodo curto, (11-12 horas de luz) conforme as condições do Brasil. Os teores de artemisinina e as produções de raízes e de folhas, em estirpes cultivadas em fitotrons, foram maiores na faixas de temperatura ambiente de 22 a 26°C, em comparação com os rendimentos obtidos na faixa entre 25 a 31°C. A produção de raízes foi diretamente relacionada com os teores de artemisinina nas folhas de A annua L.. Os primeiros trabalhos de seleção, conduzidos em local onde as condições de fotoperíodo não induzi am o florescimento da espécie, permitiu a seleção de genótipos mais produtivos em biomassa por expressarem todo seu potencial vegetativo. A avaliação, no Brasil, do híbrido Ch. x Viet 55, atingiu rendimentos de aproximadamente 30Kg de artemisinina /há /corte, sendo possível a realização de 2 cultivos por ano, na mesma área o cultivo deste híbrido foi tecnicamente viável e altamente competitivo, devido aos rendimentos alcançados. A aplicação de fertilizantes nitrogenados na dose de 90Kg de nitrogênio / há proporcionou um aumento dos rendimentos de artemisinina que variou entre 28 e 40%, em função de maior biomassa. A forma nitrato foi a mais favorável. Um possível deslocamento do estádio de maior concentração de artemisinina é sugerido. Extrações aquosas, a partir de folhas ricas em artemisinina, apresentaram fração contendo 50% do princípio ativo. Sugere-se que tal procedimento possa servir de base para novas pesquisas farmacológicas e toxicológicas sobre a atividade anti-malárica de infusões. Os estudos com a A. annua L. resultaram no estabelecimento de um programa de melhoramento genético no CPQBA / Abstract: Malaria is still one of the greatest causes of mortality in the world; in Brazil there are over 500.000 reported cases per year. Malaria, caused by the protozoa Plasmodfum, has been aggravated by the increasing resistance of Plasmodfum to the traditional drugs cloroquine and mefloquine. The study of new drugs resulted in the identification of antimalarial activities of an endoperoxide sesquiterpene lactone, called Qinghaosu or Artemisinin, extracted from the leaves of A. annua L., from the Asteraceae family. Compounds derived from artemisinin are highly efficient and are being commercialized in China. The production technology and validation of these drugs in the occidental world are of great interest. The main objective of this thesis was to contribute to the production of the chemical compounds of the species A. annua L., in order to allow the production of anti malarial drugs. The research work developed at MEDIPLANT (Switzerland) and CPQBA-UNICAMP (Brazil) involved: biological aspects of reproduction; selection of hybrids for production in Brazil; nitrogen fertilization and assays of extraction in water of artemisinin. The results indicated that the species, although having complete flowers, presents an alogamy rate superior to 80%. Through controlled hybridization between selected genotypes from China and Vietnam, population uniformity and genetic gain was obtained. Through the analysis of biomass and artemisinin contents (estimated by analytical monitoring), it was possible to increase the artemisinin production of 5Kg/ha for the base population to approximately 30Kg/ha for the genetically bred population. Vegetative propagation, through tissue culture ar stakes, was also utilized in the genetic breeding program, mainly for maintenance of progenitors and fixation of desirable qualities. Two genotypes, Pl.39 and Pl.V were selected for cultivation under short day photoperiod (11-12 hours of light), according to Brazilian conditions. The artemisinin content and the production of roots and leaves in strains cultivated in fitotrons were higher under temperatures ranging from 22 to 26°C, when compared to those cultivated between 25 and 31°C. The root production was directly related to the contents of artemisinin in the leaves of A. annua L.. The first breeding trial, conducted where local photoperiodic conditions did not induce flowering, also allowed the selection of more productive genotype in biomass. In the evaluation for Brazil, the hybrid Ch. X Viet 55 produced approximately 30Kg of artemisinin /ha /cut, with 2cuts possible per year. The cultivation of this hybrid was technically viable end highly competitive, due to the production obtained. The application of Nitrogen fertilizers (90Kg/ha) increased the artemisinin contents in the range of 28 to 40% due to increase in biomass. The nitrate form was the most favorable. It is possible that the period of highest concentration of artemisinin had been delayed. The water extraction of leaves rich in artemisinin produced a fraction containing 50% of active compound. It is suggested that this procedure can be utilized as a base for new pharmacology and toxicology investigations on the antimalarial activity of infusions. The studies with A. annua L. resulted in the establishment of a genetic breeding program at CPQBA / Doutorado / Doutor em Ciências

Malaria is one of the deadliest parasitic diseases worldwide, causing 219 million infections and 435 thousand deaths per year. As such, this mosquito-borne illness is a major target for global eradication efforts. One critical arm of the eradication strategy is chemotherapy. For a therapeutic to advance the eradication agenda, it must cure the patient of infection and eliminate transmission stage parasites (called gametocytes) from the blood, thereby breaking the cycle of transmission. Currently, first-line treatments against malaria infection consist of an artemisinin derivative in combination with another antimalarial drug from a different drug class. Although artemisinin and its derivatives are highly efficacious at curing malaria, these drugs are ineffective at preventing disease transmission. However, recent in vivo studies have suggested that whole plant Artemisia annua (the botanical source of artemisinin) delivered as tea can cure patients of infection and eliminate transmission stage parasites from the bloodstream. To validate these in vivo results in vitro, experiments were performed to measure the killing efficacy of A. annua and A. afra tea infusions against three different stages of the parasite life cycle— one stage of the asexual cycle, immature gametocytes, and mature gametocytes. Killing effects were observed using light microscopy and gametocyte gene-specific RT-qPCR analyses. Results suggested that A. annua tea was nearly as effective as artemisinin at killing all three tested stages of the parasite. A. afra tea, which contains low levels of artemisinin, showed comparable killing efficacy against late stage gametocytes, but not against the other two tested stages. These results supported the notion that A. annua tea is an effective antimalarial and also provides evidence that both A. annua and A. afra teas may be a viable therapeutic option for eliminating gametocytes during human infection.

Many therapies prescribed by traditional healers in Southern Africa include plant combinations to treat infectious diseases. Artemisia afra is one of the most commonly used traditional medicines in African traditional medicine and most often given in combination with other plants. This plant‟s popularity coupled with its wide range of uses in combination serves as the rationale for the bases of this study. In this study, combinations of A. afra (essential oils and plant extracts), which are commonly used for the treatment of respiratory diseases were studied from an antimicrobial perspective in order to determine if a scientific basis exists for their combined use. The plants used often in double or triple combination with A. afra in the treatment of respiratory tract infections are Lippia javanica, Osmitopsis asteriscoides, Agathosma betulina, Eucalyptus globulus, Allium sativum, Leonotis randii, Tetradenia riparia and Zanthoxylum capense. Essential oils from plant samples were analysed using gas chromatography coupled to mass spectroscopy (GC-MS). Compounds found in highest concentrations were camphor (41.0%) in A. afra, linalool (70.7%) in L. javanica, 1,8-cineole (59.0%) in O. asteriscoides, isomenthone (31.4%) in A. betulina, 1,8-cineole (63.0%) in E. globulus and β-caryophyllene (32.4%) in T. riparia. Dichloromethane: methanol extracts and aqueous extracts were prepared for each plant using the dried ground plant material collected. The antimicrobial activities of each sample as well as each combination (including essential oils) were tested using the minimum inhibitory concentrations (MIC) assay against a panel of respiratory tract organisms. The highest sensitivities observed for the essential oils were that of E. globulus against Cryptococcus neoformans with a MIC value of 0.6 mg/ml. The dichloromethane: methanol extracts showed the most activity with E. globulus against Moraxella catarrhalis (MIC value of 0.01 mg/ml). The aqueous extracts showed the best activity with Z. capense against Streptococus agalactiae with a MIC value of 0.4 mg/ml. The 1:1 fractional inhibitory concentration (ΣFIC) values of the combinations of A. afra with L. javanica, A. afra with O. asteriscoides, A. afra with A. betulina, A. afra with E. globulus and A. afra with Z. capense were calculated from the MIC data. Synergy, additivity, indifference and antagonistic interactions within the combinations were then interpreted. The most significant interactions of the double combinations with synergistic ΣFIC values of 0.2 were the combination of the dichloromethane: methanol extracts of A. afra with O. asteriscoides against Streptococcus pyogenes and the combination of the aqueous extracts of A. afra with E. globulus against Streptococcus pneumoniae. Significant antagonism was noted with the combination of the dichloromethane: methanol extracts of A. afra with E. globulus against Enterococcus faecalis. The ΣFIC results of the combinations of A. afra with L. javanica, O. asteriscoides, A. betulina, E. globulus or with Z. capense were used to calculate ratios and plotted on to an isobologram. The isobolograms were interpreted with regard to any synergy, antagonism, or additive interactions present in the combination. Isobolograms revealed the most significant activity with the combination of the aqueous extracts of A. afra with E. globulus against C. neoformans with all the ratios tested being synergistic. The most prominent antagonism (five ratios) noted was in the combination (dichloromethane: methanol extracts) of A. afra with E. globulus against M. catarrhalis. The triple combinations analyzed for their antimicrobial activity were the combinations of A. afra with O. asteriscoides and E. globulus, A. afra with L. randii and E. globulus, A. afra with A. sativum and Z. capense and the combination of A. afra with T. riparia and salt. The most significant synergistic activity was noted for the combination of the essential oils A. afra with T. riparia and salt against Mycobacterium smegmatis with a ΣFIC value of 0.2. The combination of A. afra with O. asteriscoides and E. globulus of (dichloromethane: methanol extracts) displayed the most antagonistic activity against M. catarrhalis. When analysing the combinations that include A. afra, it was noted that adjuncts are an important combination ingredient in the traditional method of preparation. These were also tested for their activity. The combinations that include adjuncts i.e. honey, salt, vinegar, brandy and milk showed mainly indifferent interactions. This indifference noted supports the use of these adjuncts by traditional healers as it serves to verify that these adjuncts are at least not hindering the activity of the plant itself, which is a positive direction for future investigations. Traditional medicine, with regard to A. afra, as prescribed by traditional healers, has commonly employed the use of combinations of more than one plant to treat respiratory conditions. When the antimicrobial activities in combination were examined from a scientific viewpoint, there is evidence of some bases for their traditional use. The results obtained from the testing of the essential oils validate its traditional use as an inhalant. The dichloromethane: methanol extracts showed results varying from synergy to antagonism while the aqueous extracts showed good antimicrobial activity. It is recommended that future studies should be conducted into these interactions to determine the benefits of these combinations for possible use in the commercial and primary health-care sectors.

Malaria är en tropisk sjukdom som orsakas av encelliga organismer, protozoer från Plasmodium släktet. Varje år drabbas ungefär en halv miljard människor av malaria och cirka en miljon av dessa dör. Okomplicerad malaria är en mild form av malaria som enligt WHO rekommendationer ska behandlas med artemisinin baserad kombinationsterapi (ACT). Artemisinin produceras naturligt i låg mängd från växten Artemisia annua. Trots att medicinen har visat sig effektiv mot malaria med färre biverkningar är den höga kostnaden en nackdel. Forskning pågår för att hitta nya syntetiska vägar för framställning av artemisinin i växten genom att studera terpenmetabolism och vilka aktiva enzymer det finns som har en avgörande roll i utbytet av artemisinin i växten. Syftet med denna studie var att med hjälp av genteknik och molekylärbiologiska metoder producera och identifiera två rekombinanta enzymer, seskviterpensyntaser från A. annua. Experimentet inleddes med att transformera klonade T-DNA (AaTS-1 och AaTS-2) som kodar för seskviterpensyntaser från A. annua med hjälp av Agrobacterium tumefaciens vartefter transienta transkriptionen av generna som finns i en binär vektor initierades i blad från växten Nicotiana benthamiana genom infiltration. Totalt RNA extraherades från växten och översattes till cDNA för att sedan studera förhållandet av transient uttryck i bladen med qPCR. Enzymerna extraherades från bladen och inkuberades med farnesyldifosfat övernatt och produkten identifierades följande dag med gaskromatografi-masspektrofotometri (GC-MS). Resultatet blev att inget genuttryck av AaTS-1 och AaTS-2 kunde detekteras i bladen. Resultat från GC-MS visade att ingen proteinprodukt genererades. De negativa resultaten berodde främst på brist av resultat som verifierar att plasmiderna var konstruerade med selektionsgenerna, men även på grund av en icke effektiv transformation, orsakad av bakteriecellklumpar som förhindrade infiltreringsmedium att nå inre delarna av bladen.

Aim of the study: In traditional healing, the burning of selected indigenous medicinal plants and the inhalation of the liberated smoke are widely accepted and a practiced route of administration. This study elucidated the rationale behind this commonly practiced treatment by examining the antimicrobial activity for five indigenous South African medicinal plants commonly administered through inhalation (Artemisia afra, Heteropyxis natalensis, Myrothamnus flabellifolius, Pellaea calomelanos and Tarchonanthus camphoratus). Material and Methods: An apparatus was designed to simulate the burning process that occurs in a traditional setting and the smoke fraction was captured for analysis and bioassay. Methanol and acetone extracts as well as the essential oil (for the aromatic species) were prepared and assayed in parallel with the smoke fraction. Results: Antimicrobial data revealed that in most cases, the ‘smoke-extract’ obtained after burning had lower minimum inhibitory concentration (MIC) values than the corresponding solvent extracts and essential oils. The combustion, acetone and methanol extracts produced different chromatographic profiles as demonstrated for Pellaea calomelanos where several compounds noted in the smoke fraction were not present in the other extracts. Conclusion: These results suggest that the combustion process produces an ‘extract’ with superior antimicrobial activity and provides in vitro evidence for inhalation of medicinal smoke as an efficient mode of administration in traditional healing.