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Student Number : 0000127R - MPharm dissertation - School of Pharmacy and Pharmacology - Faculty of Health Sciences / Traditional medicines form a significant part of the lives of many people around the world and in South Africa almost 60 % of people consult traditional healers in addition to the modern medical services available. Plants form a significant part of traditional healing and hence, selected species of a traditionally used plant genus, Hermannia, were chosen for biological and chemical investigation to determine a scientific basis for the traditional use of these plants. A phytochemical investigation was carried out, firstly using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) and then isolation and identification of compounds from various Hermannia species. TLC analysis indicated significant similarities between the various species with only H. saccifera displaying chemical anomalies. This was further corroborated by the HPLC analysis although very conservative profiles were produced. Isolation of compounds from H. saccifera yielded a novel labdane compound, E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene, as well as two flavones, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin which have previously been isolated. In addition, two commonly found compounds, lupeol and β- sitosterol were isolated from H. cuneifolia and H. salviifolia respectively. This is the first report on the isolation and identification of all five compounds from Hermannia species. Antimicrobial activity was assessed using two methods i.e. minimum inhibitory concentrations as well as the death kinetics assay. Minimum inhibitory concentrations were determined using four Gram-positive and two Gram-negative bacteria as well as two yeasts. All species investigated indicated antimicrobial activity with H. saccifera showing good activity against S. aureus and B. cereus. E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene isolated from H. saccifera indicated activity (MIC = 23.6 μg/ml against S.aureus) although the activity was less than that of the crude extract (MIC = 19.5 μg/ml), thus, demonstrating that there are a number of compound contributing to the promising activity of the crude extract. This was further corroborated by the bioautograms developed of the H. saccifera extract. Time-kill studies on H. saccifera against S. aureus indicated that at concentrations of 0.1, 0.25 and 0.5 % bacteriostatic activity was observed while at 0.75% the extract achieved complete bactericidal activity after 240min. Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Ten of the twelve species indicated good activity with H. cuneifolia demonstrating the most promising activity (IC50 = 10.26 μg/ml for DPPH and 10.32 μg/ml for ABTS). Two of the isolated compound, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin displayed insignificant activity. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of Hermannia species. All species exhibited intermediate activity with the exception of H. cuneifolia (IC50 = 15.32 μg/ml). In addition, four isolated compounds, 5,8- dihydroxy-6,7,4’- trimethoxyflavone, cirsimaritin, lupeol and β-sitosterol showed moderate inhibition of the enzyme indicating that while these compounds do contribute to the activity of the extracts they are not individually responsible for any significant activity. Antimalarial activity was assessed using the titrated hypoxanthine incorporation assay while toxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Only three species indicated any good antimalarial activity i.e. H. saccifera, H. muricata and mostly H. trifurca (IC50 = 25.30, 28.17 and 18.80 μg/ml respectively). However, the activity of H. saccifera and H. trifurca are probably due to a general cytotoxicity as these species exhibited a low safety index. All other species appear safe for use. Several Hermannia species have indicated in vitro biological activity in a number of assays which is related to their use in traditional medicines to treat a number of disease states. Hence, a scientific basis, albeit in vitro, has been established for the use Hermannia species in traditional healing.

Yes / Background: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta ( IC50 = 49.65 nM) and its major alkaloid, cryptolepine ( IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

Malaria is more prevalent today and the death toll is on the increase annually. It is one of the leading causes of morbidity and mortality worldwide and most of these deaths are in the poorest regions of the world. About 500 million cases are reported annually with more than 2 million deaths, and most are children. It is the major killer in the tropics and a major public health problem in developing countries and Papua New Guinea (PNG) is no exception. Resistant strains have been reported. This may be enhanced by inappropriate human behaviour in the use of anti-malarial drugs. Human factors include inappropriate prescribing and patient behaviour in using anti-malarial drugs. Despite the establishment of the standard treatment guidelines for malaria in PNG, three out of every four patients have chloroquine-resistant falciparum malaria and malaria remains a major health problem. The aim of this study was to evaluate the influence of an education program on patients carers' understanding and effective use of anti-malarial drugs for the treatment of uncomplicated malaria in children in general health clinics in PNG. The trial design involved a pre-post intervention study with a control group. The study was undertaken in the National Capital District. Papua New Guinea using one Clinic as the intervention site and another as the control site. The two clinics were similar in characteristics as confirmed in the study by demographic data where there were no significant differences observed. The data collection took placed over the period February to April 2002. It included observation of drug provision at study sites and interviews of patient carers on the first day at the clinic and a follow up seven days later. Three questionnaires were developed to evaluate the process and outcomes of malaria drug treatment in the above health facilities. / Prescribing data were collected from prescriptions and patient carers' interviewed prior to the intervention program. Following the provision of drug information to patient carers. similar drug information and compliance questioning was undertaken. Differences in the pre-post elements of the study and in the control group over the study period were evaluated using Chi-Squared, Kruskal-Wallis, Fisher's Exact or Student's t-tests as appropriate. In excess of 100 patients in the pre- and in the post intervention phases were evaluated for their understanding and effective use of the anti-malarial drugs. In addition, 100 clients were in the control group at another clinic. Patients had attended the clinic up to 8 times in the previous year with a median of 2 visits. Amodiaquine, Fansidar, albendazole and paracetamol made up a total of 60% of the drugs prescribed. The use of medicines was strongly supported with 94.4% indicating no problems with the medication. Only 3% of patients received herbal or local remedies for malaria treatment. 1n patients 10 years or less or their carers, it was found, there was a significant improvement in the carers understanding of the medications. There was a statistically significant improvement in patient outcomes from 57.9% to 92.3% reported as cured following the intervention program. The study has also identified low levels of appropriate administration of antibiotic suspensions in children by patient carers. / For example, incorrect responses recorded for amoxycillin suspension were 80.8% (143). Septrim tablets 92% (23), Septrim suspension 86% (123), erythromycin suspension 100% (26), and chloramphenicol suspension 84.4% (38). In this study the face to face (one-to-one) education program was used to influence patient carers understanding and effective use of drugs. The intervention program involved advising, informing, encouraging, and counselling the patient carers verbally on the appropriate and effective use of medicines. The verbal message was reinforced by a suitable label typed in English and Pidgin-English where instructions were clear, simple and unambiguous. The label was then attached to the envelopes or containers containing the drugs. On feedback, the information on the understanding and effective use of drugs was re-emphasized to the carers to reinforce their understanding for future references. Results showed that the intervention program made an impact in improved patient carers understanding and effective use of drugs and children's health outcomes. In conclusion, it is evident that a patient intervention program designed to improve the dosages and frequency of administration of anti-malarial drugs in PNG had no statistically significant outcome. This may be because the current level of understanding was quite high (>70%) and the study experienced a ceiling effect. However, as shown in the results, the patient carers understanding on the appropriate and effective use of drugs was lower during the pre-intervention and control group. / When compared clinic-pre with clinic-post, there was a significant difference (P < 0.05) in the cured group and the improved cure rate increases from 57.9% to 92.3%. When compared control pre with control post groups, there was no significant difference (P > 0.05) in the cured group. Therefore, the study identified an improvement in patient outcomes with respect to malaria. Hence. the simple intervention program in influencing patient carers understanding of the appropriate and effective use of medications led to a marked improvement in patient outcomes.

Malgré le succès des insecticides pour le contrôle du paludisme, la transmission continue dans la plupart des pays d’Afrique sub-saharienne. La recherche pour de nouveaux moyens de contrôle (plus spécialement la modification génétique des populations vecteurs), ou l'utilisation plus efficace des contrôles actuels, vont nécessiter une recherche sur les structures de populations de moustiques et les processus d’immunisation qui importent pour la transmission du Plasmodium chez les moustiques sauvages. Par ailleurs, l’utilisation des techniques d’association génomique ‘GWAS’ est basée sur un réelle compréhension des structures des populations.Ma thèse inclura une description detaillée du système immunitaire du moustique, basée sur la recherche actuelle et des comparaisons génomiques; ainsi que des descriptions des principales voies immunitaires, et des gênes potentiels mal-caracterisés qui peuvent être trouvés dans une étude GWAS. Ainsi qu’une description des connaissances actuelles des structures des populations, dont la speciation du gambiae / coluzzii, et les effets des grandes variations structurelles.Je présenterai le développement d’un nouveau moyen d'identification des variations structurelles ; utilisant les techniques d’ “apprentissage automatique” permettant d'identifier les karyotypes directement à partir des séquences haut-débit, menant à des résultats d’une précision sans précédent.Je présenterai également la première vraie cartographie génomique du ‘tout-génome’ du moustique. Les colonies sont fondées par des moustiques sauvages; les fondateurs sont controlées par strates, incluant également des sous-espèces et variations structurelles majeures. Avec ces colonies une méthode innovant de cartographie est utilisée: dans un premier temps, une identification des grandes régions au sein des groupes phenotypées par la perte de hétérogénéité; puis dans un second temps, le génotypage individuel ‘Sequenom’ sera utilisé pour une cartographie exacte. Cette méthode est utilisée pour l’identification d’une région avec un effet phenotypique sur la prévalence des infections dans la nature.Enfin, je suggèrerai comment ces techniques peuvent être importantes à l’avenir pour l’application du contrôle génomique dans la nature. / Despite successes in the use of insecticides in the control of malaria, malaria transmission continues in much of sub-saharan Africa. The search for novel methods of control (in particular genetic modification of vector populations), or of superior implementation of the currently available methods will require both greater knowledge of the population structure of the mosquito, and of the immune processes that are important in the wild. It is important to note that the mapping of novel immune genes, via genome wide association studies (GWAS) is predicated on a firm understanding of the population structure.My thesis will include a detailed description of the mosquito innate immune system based on current research and comparative genomics; this will illustrate the major pathways that might be employed in the anti-malarial response, and some potential uncharacterised genes that might be implicated in any GWAS study. It will also include a summary of what is known about the mosquito’s population structure, in particular the gambiae / coluzzii speciation event and the implication of chromosomal inversions in the speciation process.I will present the development of a novel approach to the identification of chromosomal inversions; using machine-learning techniques in order to call inversion karyotypes directly from sequence, leading to calls of unprecedented accuracy.I will also present the first truly genome-wide association study to have been performed in the mosquito. Strata-controlled populations of mosquitoes were derived from the wild, including restriction on the basis of subspecies and chromosomal inversion. A two-stage mapping design was then devised in which loss-of-heterozygosity is used to identify broad regions in phenotype pools, before fine-resolution mapping by Sequenom genotyping in individuals. This was used to identify a novel locus with a phenotypic effect on infection prevalence.Finally I will describe how these techniques and findings could be important in the future application of genetic control in the wild.

Current anti-malarial methods have been effective in reducing the number of malarial cases. However, these methods do not completely block the transmission of the parasite. Research has shown that repeated use of the current anti-malarial drugs, which include artemisinin-based drug combinations, might be toxic to humans. There have also been reports of an emergence of artemisinin-resistant parasites. Finding anti-malarial drugs through the drug discovery process takes a long time and failure results in a great financial loss. The failure of drug discovery projects can be partly attributed to the improper selection of drug targets. There is thus a need for an eff ective way of identifying and validating new potential malaria drug targets for entry into the drug discovery process. The availability of the genome sequences for the Plasmodium parasite, human host and the Anopheles mosquito vector has facilitated post-genomic studies on malaria. Proper utilizationof this data, in combination with computational biology and bioinformatics techniques, could aid in the in silico prioritization of drug targets. This study was aimed at extensively annotating the protein sequences from the Plasmodium parasites, H. sapiens and A. gambiae with data from di fferent online databases in order to create a resource for the prioritization of drug targets in malaria. Essentiality, assay feasibility, resistance, toxicity, structural information and druggability were the main target selection criteria which were used to collect data for protein annotations. The data was used to populate the Discovery resource (http://malport. bi.up.ac.za/) for the in silico prioritization of potential drug targets. A new version of the Discovery system, Discovery 2.0 (http://discovery.bi.up.ac.za/), has been developed using Java. The system contains new and automatically updated data as well as improved functionalities. The new data in Discovery 2.0 includes UniProt accessions, gene ontology annotations from the UniProt-GOA project, pathways from Reactome and Malaria Parasite Metabolic Pathways databases, protein-protein interactions data from. IntAct as well as druggability data from the DrugEBIlity resource hosted by ChEMBL. Users can access the data by searching with a protein identi er, UniProt accession, protein name or through the advanced search which lets users filter protein sequences based on different protein properties. The results are organized in a tabbed environment, with each tab displaying different protein annotation data. A sample investigation using a previously proposed malarial target, S-adenosyl-Lhomocysteine hydrolase, was carried out to demonstrate the diff erent categories of data available in Discovery 2.0 as well as to test if the available data is su fficient for assessment and prioritization of drug targets. The study showed that using the annotation data in Discovery 2.0, a protein can be assessed, in a species comparative manner, on the potential of being a drug target based on the selection criteria mentioned here. However, supporting data from literature is also needed to further validate the findings. / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted

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Acanthospermum australe (Loefl.) O. Kuntze, conhecido popularmente no Brasil como carrapichinho pertence à família Asteraceae e é utilizado na medicina popular como hepatoprotetora, diaforética, antiblenorrágica, antimalárica, entre outras funções. O objetivo do presente trabalho foi estudar a espécie sob o aspecto botânico, químico e farmacológico. Assim, folhas e caules foram analisados macro e microscopicamente, contribuindo no auxílio da diagnose da espécie. Os órgãos em estudo foram submetidos a ensaios preliminares para a pesquisa dos principais grupos de princípios ativos provenientes do metabolismo secundário dos vegetais. Diferentes extratos foram obtidos por percolação e por decocção, sendo alguns deseus componentes isolados por CCD preparativa e identificadas por CG/EM. O óleoessencial do vegetal, coletado em diferentes épocas do ano e variados estágios de desenvolvimento, foi obtido em aparelho de Clevenger modificado, sendo que sua composição também foi analisada por CG/EM. O extrato clorofórmico e o extrato hidroetanólico liofilizado (EHL) foram avaliados quanto à atividade antimalárica e antileishmania. Com o EHL foi realizado também ensaio de atividade antiúlcera em ratos Wistar Hannover fêmeas e atividade antimicrobiana em bactérias e fungos. A espécie em estudo, nas condições deste trabalho, apresentou flavonóides, taninos, saponinas, óleo essencial e mucilagens. O extrato clorofórmico e hidroetanólico liofilizado, nas concentrações de 100 &#181;g/mL, provocaram 100% de morte dos protozoários de Plasmodium chabaudi AJ, porém ambos não apresentaram atividade em promastigotas de Leishmania (L.) chagasi nesta concentração. O EHL na concentração de 10 mg/mL demonstrou significativa atividade antifúngica contra o Aspergillus niger, não apresentando nenhuma atividade para Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli e Candida albicans. O mesmo extrato, na concentração de 400 mg/kg, administrada por via oral, mostrou-se com atividade antiúlcera aguda significativa, reduzindo a Área Total de Lesão (ATL) em 49,13% em relação ao controle. O óleo essencial, nas condições do experimento, apresentou diferenças qualitativas e quantitativas quando comparado a estudos já realizados, sendo constituído em sua maioria por sesquiterpenos dos grupos cadinano (&#945;-cadinol e &#948;-cadineno), cariofilano (&#946;-cariofileno) e, principalmente, germacrano (globulol). As análises permitiram concluir ainda que o isômero do espatulenol é o componente exclusivo e majoritário do óleo essencial de caule e o isômero do globulol, o componente presente em maior quantidade no óleo quando o vegetal encontra-se em fase de floração. / Acanthospermum australe (Loefl.) O. Kuntze, known popularly in Brazil as carrapichinho belongs to the family Asteraceae and it is used in the tradicional medicine as hepatic protector, diaforetic, blenorragic activity, antimalarial activity among others. The objective of the present work was to study this species under the aspect botanical, chemical and pharmacological. Thus, leaves and stems were analyzed macro and microscopic contributing in the aid of the diagnosis of species.The aerial parts in study were submitted to preliminary phytochemical screening for the research of the main groups of secondary metabolites of plants. Difterent extracts were obtained by percolation and for decoction being some of their components, isolated for TLC preparative and identified for GC/MS. The essential oil of the plants, collected at different seasons of the year and different development stadiums, it was obtained in a Clevenger apparatus, and it composition, was also analyzed by GC/MS. The chloroformic extract and liofilized hidroethanolic extract (LHE) were assayed for the antimalarial and antileishmania activities. Using LHE it was also assayed for the anti ulcer activity in mice Wistar Hannover females and antimicrobial activity in bacteria and fungi. The species in study, in conditions of this work, presented flavonoids, tannins, saponnins, essential oil and mucilages. The chloroformic extract and LHE in the concentrations of 100 &#181;g/mL caused 100% of death in the blood forms of Plasmodium chabaudi AJ, however none of them presented activity in blood forms of Leishmania (L.) chagasi in this concentration. LHE in the concentration of 10 mg/mL presented significant antifungal activity against the Aspergillus niger, and no activity against Staphy/ococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The same extract, in the concentration of 400 mg/kg, administered orally, presented significant antiulcerogenic activity, reducing the Total Area of Lesion (TAL) in 49,13% in relation to the control. The essential oil, in the conditions of the experiment, presented qualitative and quantitative difterences when compared to previous studies and constituted in its majority by sesquiterpenes of the cadinano groups (&#945;-cadinol and &#948;-cadinene), caryophyllano (&#946;-caryophyllene) and mainly, germacrano (globulol). The analyses still permit to conclude that the isomer of the spathulenol is the exclusive and mayor component of the essential oil in the stem. Isomer of the globulol is the representative present in mayor amount in the oil when the plant is in flower stadium

Acanthospermum australe (Loefl.) O. Kuntze, conhecido popularmente no Brasil como carrapichinho pertence à família Asteraceae e é utilizado na medicina popular como hepatoprotetora, diaforética, antiblenorrágica, antimalárica, entre outras funções. O objetivo do presente trabalho foi estudar a espécie sob o aspecto botânico, químico e farmacológico. Assim, folhas e caules foram analisados macro e microscopicamente, contribuindo no auxílio da diagnose da espécie. Os órgãos em estudo foram submetidos a ensaios preliminares para a pesquisa dos principais grupos de princípios ativos provenientes do metabolismo secundário dos vegetais. Diferentes extratos foram obtidos por percolação e por decocção, sendo alguns deseus componentes isolados por CCD preparativa e identificadas por CG/EM. O óleoessencial do vegetal, coletado em diferentes épocas do ano e variados estágios de desenvolvimento, foi obtido em aparelho de Clevenger modificado, sendo que sua composição também foi analisada por CG/EM. O extrato clorofórmico e o extrato hidroetanólico liofilizado (EHL) foram avaliados quanto à atividade antimalárica e antileishmania. Com o EHL foi realizado também ensaio de atividade antiúlcera em ratos Wistar Hannover fêmeas e atividade antimicrobiana em bactérias e fungos. A espécie em estudo, nas condições deste trabalho, apresentou flavonóides, taninos, saponinas, óleo essencial e mucilagens. O extrato clorofórmico e hidroetanólico liofilizado, nas concentrações de 100 &#181;g/mL, provocaram 100% de morte dos protozoários de Plasmodium chabaudi AJ, porém ambos não apresentaram atividade em promastigotas de Leishmania (L.) chagasi nesta concentração. O EHL na concentração de 10 mg/mL demonstrou significativa atividade antifúngica contra o Aspergillus niger, não apresentando nenhuma atividade para Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli e Candida albicans. O mesmo extrato, na concentração de 400 mg/kg, administrada por via oral, mostrou-se com atividade antiúlcera aguda significativa, reduzindo a Área Total de Lesão (ATL) em 49,13% em relação ao controle. O óleo essencial, nas condições do experimento, apresentou diferenças qualitativas e quantitativas quando comparado a estudos já realizados, sendo constituído em sua maioria por sesquiterpenos dos grupos cadinano (&#945;-cadinol e &#948;-cadineno), cariofilano (&#946;-cariofileno) e, principalmente, germacrano (globulol). As análises permitiram concluir ainda que o isômero do espatulenol é o componente exclusivo e majoritário do óleo essencial de caule e o isômero do globulol, o componente presente em maior quantidade no óleo quando o vegetal encontra-se em fase de floração. / Acanthospermum australe (Loefl.) O. Kuntze, known popularly in Brazil as carrapichinho belongs to the family Asteraceae and it is used in the tradicional medicine as hepatic protector, diaforetic, blenorragic activity, antimalarial activity among others. The objective of the present work was to study this species under the aspect botanical, chemical and pharmacological. Thus, leaves and stems were analyzed macro and microscopic contributing in the aid of the diagnosis of species.The aerial parts in study were submitted to preliminary phytochemical screening for the research of the main groups of secondary metabolites of plants. Difterent extracts were obtained by percolation and for decoction being some of their components, isolated for TLC preparative and identified for GC/MS. The essential oil of the plants, collected at different seasons of the year and different development stadiums, it was obtained in a Clevenger apparatus, and it composition, was also analyzed by GC/MS. The chloroformic extract and liofilized hidroethanolic extract (LHE) were assayed for the antimalarial and antileishmania activities. Using LHE it was also assayed for the anti ulcer activity in mice Wistar Hannover females and antimicrobial activity in bacteria and fungi. The species in study, in conditions of this work, presented flavonoids, tannins, saponnins, essential oil and mucilages. The chloroformic extract and LHE in the concentrations of 100 &#181;g/mL caused 100% of death in the blood forms of Plasmodium chabaudi AJ, however none of them presented activity in blood forms of Leishmania (L.) chagasi in this concentration. LHE in the concentration of 10 mg/mL presented significant antifungal activity against the Aspergillus niger, and no activity against Staphy/ococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The same extract, in the concentration of 400 mg/kg, administered orally, presented significant antiulcerogenic activity, reducing the Total Area of Lesion (TAL) in 49,13% in relation to the control. The essential oil, in the conditions of the experiment, presented qualitative and quantitative difterences when compared to previous studies and constituted in its majority by sesquiterpenes of the cadinano groups (&#945;-cadinol and &#948;-cadinene), caryophyllano (&#946;-caryophyllene) and mainly, germacrano (globulol). The analyses still permit to conclude that the isomer of the spathulenol is the exclusive and mayor component of the essential oil in the stem. Isomer of the globulol is the representative present in mayor amount in the oil when the plant is in flower stadium

Malaria, caused by the parasite Plasmodium, continues to exact high global morbidity and mortality rate next only to tuberculosis. It causes 300-500 million clinical infections out of which more than a million people succumb to death annually. Worst affected are the children below 5 years of age in sub-Saharan Africa. Plasmodium is a protozoan parasite classified under the phylum Apicomplexa that also includes parasites such as Toxoplasma, Lankestrella, Eimeria and Cryptosporidium. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has worsened recently with the emergence of drug resistance in the parasite. Therefore, deciphering new pathways in the parasite for developing lead antimalarial compounds is the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum has opened up new avenues for the design of new antimalarials as this pathway is different from the one in human hosts. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct intervention of these biochemical pathways has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of the parasite, its metabolic pathways, efforts to use these pathways for antimalarial drug discovery, inhibitors targeting these pathways, introduction to fatty acid biosynthesis pathway, discovery of type II fatty acid biosynthesis pathway in Plasmodium falciparum and prospects of developing lead compounds towards antimalarial drug discovery is given in Chapter 1 of the thesis. In the exploration of newly discovered type II fatty acid biosynthesis pathway of P. falciparum as a drug target for antimalarial drug discovery, one of the enzymes; β-hydroxyacyl- acyl carrier protein dehydratase (PfFabZ) was cloned and being characterized in the lab. The atomic structure of PfFabZ was not known till that point of time. Chapter 2 describes the homology modeled structure of PfFabZ and docking of the discovered inhibitors with this structure to provide a rationale for their inhibitory activity. Despite low sequence identity of ~ 21% with the closest available atomic structure then, E. coli FabA, a good model of PfFabZ could be built. A comparison of the modeled structure with recently determined crystal structure of PfFabZ is provided and design of new potential inhibitors is described. This study provides insights to further improve the inhibition of this enzyme. Enoyl acyl carrier protein reductase (ENR) is the most important enzyme in the type II fatty acid biosynthesis pathway. It has been proved as an important target for antibacterial as well as antimalarial drug discovery. The most effective drug against tuberculosis – Isoniazid targets this enzyme in M. tuberculosis. The well known antibacterial compound – Triclosan, a diphenyl ether, also targets this enzyme in P. falciparum. I designed a number of novel diphenyl ether compounds. Some of these compounds could be synthesized in the laboratory. Chapter 3 describes the design, docking studies and inhibitory activity of these novel diphenyl ether compounds against PfENR and E. coli ENR. Some of these compounds inhibit PfENR in nanomolar concentrations and EcENR in low micromolar concentrations, and many of them inhibit the growth of parasites in culture also. The structure activity relationship of these compounds is discussed that provides important insights into the activity of this class of compounds which is a step towards developing this class of compounds into an antimalarial and antibacterial candidate drugs. Components of the green tea extract and polyphenols are well known for their medicinal properties since ages. Recently they have been shown to inhibit components of the bacterial fatty acid biosynthesis pathway. Some selected tea catechins and polyphenols were tested in the laboratory for their inhibitory activity against PfENR. I conducted docking studies to find their probable binding sites in PfENR. On kinetic analysis of their inhibition, these compounds were found to be competitive with respect to the cofactor NADH. This has an implication that they could potentiate inhibition of PfENR by Triclosan in a fashion similar to that of NADH. As a model case, one of the tea catechins; EGCG ((-) Epigalocatechin gallate) was tested for this property. Indeed, in the presence of EGCG, the inhibition of PfENR improved from nanomolar to picomolar concentration of Triclosan.conducted molecular modeling studies and propose a model for the formation of a ternary complex consisting of EGCG, Triclosan and PfENR. Docking studies of these inhibitors and a model for the ternary complex is described in Chapter 4. Docking simulations show that these compounds indeed occupy NADH binding site. This study provides insights for further improvements in the usage of diphenyl ethers in conjugation or combination with tea catechins as possible antimalarial therapeutics. In search for new lead compounds against deadly diseases, in silico virtual screening and high throughput screening strategies are being adopted worldwide. While virtual screening needs a large amount of computation time and hardware, high throughput screening proves to be quite expensive. I adopted an intermediate approach, a combination of both these strategies and discovered compounds with a 2-thioxothiazolidin-4-one core moiety, commonly known as rhodanines as a novel class of inhibitors of PfENR with antimalarial properties. Chapter 5 describes the discovery of this class of compounds as inhibitors of PfENR. A small but diverse set of 382 compounds from a library of ~2,00,000 compounds was chosen for high throughput screening. The best compound gave an IC50 of 6.0 µM with many more in the higher micromolar range. The compound library was searched again for the compounds similar in structure with this best compound, virtual screening was conducted and 32 new compounds with better binding energies compared to the first lead and reasonable binding modes were tested. As a result, a new compound with an IC50 of 240 nM was discovered. Many more compounds gave IC50 values in 3-15 µM range. The best inhibitor was tested in red blood cell cultures of Plasmodium, it was found to inhibit the growth of the malaria parasite at an IC50 value of 0.75 µM. This study provides a new scaffold and lead compounds for further exploration towards antimalarial drug discovery. The summary of the results and conclusions of studies described in various chapters is given in Chapter 6. This chapter concludes the work described in the thesis. Cloning, over-expression and purification of PanD from M. tuberculosis, FabA and FabZ from E. coli are described in the Appendix.