Three saponin-rich extracts (20, 60, 100% methanol), four 100% methanol subfractions and seven independently acquired fractions (A-G) from guar meal, Cyamopsis tetragonoloba L. (syn. C. psoraloides), were evaluated for antimicrobial and hemolytic activities. These activities were compared against quillaja bark (Quillaja saponaria), yucca (Yucca schidigera), and soybean (Glycine max) saponins in 96-well plates using eight concentrations (0.01 to 1.0 and 0.1 to 12.5 mg extract/mL). Initial guar meal butanol extract was 4.8 ± 0.6% of the weight of original material dry matter (DM). Butanol extract was purified by preparative reverse-phase C-18 chromatography. Two fractions eluted with 20, and one each with 60, and 100% methanol with average yields of 1.72 ± 0.47, 0.88 ± 0.16, 0.91 ± 0.16 and 1.55 ± 0.15% of DM, respectively. Further purification of the 100% methanol fraction using normal-phase silica gel preparatory high pressure liquid chromatography eluted 4 peaks at 16, 39, 44 and 46 min. Only the 100% methanol fraction, its 16 min peak, F and G fractions, and quillaja saponin, exhibited both hemolytic and antibacterial activities against Staphylococcus aureus, Salmonella Typhimurium and E. coli, but 20 and 60% methanol fractions stimulated Lactobacillus spp. growth. Guar meal (0 or 5%) was added to diets fed to chicks from 1 to 21 days of age. Chicks fed both diets were unchallenged or challenged with 5 x 103 Eimeria tenella sporulated oocysts at 10 days. Guar meal diets reduced oocysts shed per gram of feces, body weight, and feed efficiency. Adding 2.5% guar meal, 1% guar gum, or 0.125% saponin-rich guar meal extract to diets fed to chicks to 21 days of age showed that guar meal increased the cfu concentrations of digesta more than controls following a challenge with 107 cfu of Clostridium perfringens at 14 days. Body weights of chicks fed guar meal and saponin-rich extract were significantly lower than control body weights at 21 days of age, whereas the weekly feed to gain ratio of chicks fed saponin-rich extract was higher than controls. Guar meal reduced severity of Eimeria tenella infection and guar saponin-rich extract exhibited antimicrobial activity against several common poultry pathogens.

The purpose of the present study was to evaluate the potential usefulness of tubulin inhibitors when complexed with hydroxypropyl-â-cyclodextrin (ÇPâCD) against a range of protozoan parasites. This approach involved investigations into the complexation of these drugs with ÇPâCD, and subsequent investigations of these drugs and their complexes in regard to cytotoxicity, pharmacokinetics, in vitro efficacy against Giardia, Cryptosporidium and rodent malaria (Plasmodium chabaudi), and their in vivo efficacy against Giardia and malaria. Albendazole (ABZ) is a benzimidazole carbamate with a broad anti-parasite spectrum, while the dinitroanilines trifluralin (TF) and oryzalin (OZ) have recently been found to exhibit activity against certain parasites. All three compounds are microtubule antagonists in either nematodes or weeds and have poor aqueous solubility, with the solubility of ABZ and OZ dependent on pH. Cyclodextrins (CD) have a hydrophobic cavity that allows them to form inclusion complexes with hydrophobic drugs, resulting in increased drug aqueous solubility, and often, improved drug dissolution and bioavailability. Thus the complexation of these drugs with ÇPâCD was investigated. All three compounds exhibited type AL phase solubility diagrams with ÇPâCD complexation, with additional increases in ABZ and OZ solubility achieved through the manipulation of temperature and pH. OZ displayed a stronger interaction with ÇPâCD when ionised over its neutral form. However, insufficient concentrations of the TF/ÇPâCD complex were achieved for drug efficacy studies. The cytotoxicity of the drugs and their complexes was assessed using the assay kit Cytotox 96 with human carcinoma cells. This is a colourimetric assay that measures lactate dehydrogenase release as a consequence of compromised cellular and membrane integrity. Both ABZ and OZ are cytotoxic to rapidly proliferating and differentiating cells but are not cytotoxic to cells in the stationary phase. Complexation did not affect drug cytotoxicity. In pharmacokinetic studies, complexation improved ABZ (and metabolites) bioavailability, but had no significant affect on OZ bioavailability. In vitro drug assessment studies found ABZ to be highly effective against Giardia, and effectiveagainst Cryptosporidium and malaria. OZ on the other hand exhibited no activity against Giardia, but was effective against Cryptosporidium and malaria. Complexation did not improve the antiprotozoal efficacy of either ABZ or OZ. In particular, excess ÇPâCD decreased the antigiardial effects of ABZ, possibly due to competitive complex formation. In addition, complexation did not improve the antiprotozoal effects of ABZ in vivo. However, the cytotoxic effect of the ABZ/ÇPâCD complex was more evident in the treatment of malaria in vivo, resulting in increased anaemia and suppression in weight gain, due to the improved bioavailability of ABZ and metabolites. ÇPâCD alone was found to be cytotoxic at greater than 2.5%, and inhibited Giardia both in vitro and in vivo at greater than 1% and 2% respectively. This was attributed to membrane disruption caused by the dissolution and removal of membrane components. In comparison, malaria grew better in the presence of ÇPâCD in vitro, with no detrimental effect observed at up to 8% ÇPâCD. This was attributed to either the increased solubilization of a necessary media component, or the complexation and removal of an inhibitory compound from the cultivation medium. Therefore ÇPâCD complexation did not improve the antiprotozoal activity of the tubulin antagonists ABZ and OZ. However, the results of the pharmacokinetic studies suggest that anthelmintic activity of ABZ, particularly against systemic infections, may be improved with oral administration of the ABZ/ÇPâCD complex. In addition, the antiparasitic activity of ÇPâCD alone may be promising, especially against intestinal infections. Finally, the improved in vitro cultivation of P. chabaudi in the presence of ÇPâCD presents a promising approach to its potential long term cultivation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Orientador: Alexandre Lima de Andrade / Banca: Alexandre Lima de Andrade / Banca: Flavia de Rezende Eugênio / Banca: Alexandre Pinto Ribeiro / Resumo: A leishmaniose visceral (LV) canina caracteriza-se por uma enorme variabilidade de manifestações clínicas, dentre elas, lesões dermatológicas e oculares. Poucos são os estudos com as alterações do segmento posterior de olhos de cães acometidos pela doença, devido, principalmente, à opacidade do segmento anterior. O objetivo do trabalho foi avaliar e descrever as principais alterações fundoscópicas e eletrorretinográficas observadas em cães naturalmente acometidos por LV, antes e após o tratamento com a miltefosina. Foram selecionados oito cães, que não apresentavam outras doenças infecciosas ou vasculares, e apresentassem meios oculares transparentes. Utilizaram-se registros fotográficos do segmento posterior e a avaliação da funcionalidade da retina através da eletrorretinografia (ERG). Os achados de fundoscopia incluíram atenuação vascular, hiperrefletividade, pigmentação retiniana, embainhamento vascular, hiperpigmentação peripapilar, hemorragia sub-retiniana, turbidez vítrea, papiledema e tortuosidade vascular. Todos os animais se beneficiaram clinicamente da terapêutica medicamentosa, não sendo mais encontradas hemorragia sub-retiniana e turbidez vítrea após tratamento, e somente um animal permanecia com a lesão de embainhamento vascular. A avaliação dos registros de ERG mostrou diminuição da amplitude de onda "b" nas respostas de bastonetes e máxima resposta entre os momentos. Conclui-se que a LV promove alterações no fundo de olhos de cães antes mesmo da ocorrência de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) canine is characterized by a huge variability of clinical manifestations, among them skin and eye damage. Few studies of the changes of the posterior segment of an eye of dogs affected by the disease, mainly due to the opacity of the anterior segment. The objective was to assess and describe the main fundus and ERG changes observed in dogs naturally affected by LV before and after treatment with miltefosine. Eight dogs were selected which showed no vascular or other infectious diseases, and to produce clear ocular media. They used photographic records of the posterior segment and evaluation of retinal function by electroretinography. The findings included fundus vascular attenuation, increased reflective, retinal pigmentation, vascular sheathing, peripapillary hyperpigmentation, subretinal hemorrhage, atrophy of the optic disk, vitreous turbidity, papilledema, vascular tortuosity. All animals were clinically benefit of drug treatment, no longer found bleeding subretinal and vitreous turbidity after treatment, only one animal was to vascular sheathing. The evaluation of the ERG records showed decreased amplitude wave "b" in the responses of rods and maximum response between times. It concludes that the LV promotes changes in the dog's eyes background even before the occurrence of visual loss and miltefosine is effective in treating the disease, but provided few changes of fundus findings and ERG. / Mestre

Protozoan infections such as Malaria, Leishmaniasis, Toxoplasmosis, Chaga’s disease and African trypanosomiasis caused by the Plasmodium, Leishmania, Toxoplasma and Trypanosoma genuses respectively; inflict a huge economic, health and social impact in endemic regions particularly tropical and sub-tropical regions. The combined infections are estimated at over a billion annually and approximately 1.1 million deaths annually. The global burden of the protozoan infections is worsened by the increased drug resistance, toxicity and the relatively high cost of treatment and prophylaxis. Therefore there has been a high demand for new drugs and drug targets that play a role in parasite virulence. Cysteine proteases have been validated as viable drug targets due to their role in the infectivity stage of the parasites within the human host. There is a variety of cysteine proteases hence they are subdivided into families and in this study we focus on the clan CA, papain family C1 proteases. The current inhibitors for the protozoan cysteine proteases lack selectivity and specificity which contributes to drug toxicity. Therefore there is a need to identify the differences and similarities between the host, vector and protozoan proteases. This study uses a variety of bioinformatics tools to assess these differences and similarities. The Plasmodium cysteine protease FP-2 is the most characterized protease hence it was used as a reference to all the other proteases and its homologs were retrieved, aligned and the evolutionary relationships established. The homologs were also analysed for common motifs and the physicochemical properties determined which were validated using the Kruskal-Wallis test. These analyses revealed that the host and vector cathepsins share similar properties while the parasite cathepsins differ. At sub-site level sub-site 2 showed greater variations suggesting diverse ligand specificity within the proteases, a revelation that is vital in the design of antiprotozoan inhibitors.

Urginea sanguinea, Aloe marlothii, Elephantorrhiza elephantina and Rhoicissus tridentate are all plants utilized for the management of tick borne diseases in the Madikwe area of North-west province. These plants, in certain concoctions, are believed to be effective against “seme”, “gala” and “Bolwetsi jwa mothlapo o moshibidu” which we have assumed to represent heartwater, gallsickness and redwater from circumstantial epidemiological data available. To obtain a representative extract, which would be indicative of the general activity of the plant, only acetone or methanol extracts were tested for the presence of antimicrobial, antiparasitic or anti-oxidant activity within that specific plant. Activity in all cases made use of either an in vitro biological assay or more specific chemical tests, which were validated in all cases. Ehrlichia ruminantium, Babesia caballi and Theileria equi, all grown in specific cell cultures, were used as a model for evaluating the efficacy against the common protozoan and rickettsial diseases caused by these organisms in livestock. Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli, four human nosocomial infectious agents, were used as an indicator for the presence of antibacterial activity against these common animal bacterial pathogens. Diphenyl-picrylhydrazyl and the trolox equivalent anti-oxidant chemical assays were used to determine anti-oxidant activity, which although not curative, may aid in the recovery from an infection by stimulating the immune system. The activities demonstrated among the various plants and organisms were not consistent. E. elephantine extracts were the most effective, with activity demonstrable in all biological and chemical screening assays. Although R. tridentate demonstrated poor activity (> 100 ìg/ml) against the tick-borne parasites, the plant extract did demonstrate significant anti-oxidant activity. U. sanguinea extracts showed good activity in both the antibacterial and anti-rickettsial assays (EC50 = 44.49 ng/ml), which may be due to the presence of the toxic bufadienolides present within the plant. A. marlothii possessed significant anti-rickettsial activity (EC50= 111.4 µg/ml) and to a lesser degree antibacterial activity. The results of the study support the use of these plants against heartwater, gallsickness and redwater, which gives credence for the traditional use against “Seme, Gala, and Bolwetsi jwa mothlapo o moshibidu”. Further studies are required to isolate and determine the structure of the active compounds of these plants as well as to confirm the safety and efficacy of the extracts against disease conditions in livestock. Copyright / Urginea sanguinea, Aloe marlothii, Elephantorrhiza elephantina and Rhoicissus tridentata word tradisioneel gebruik vir die bekamping van siektes deur bosluise oorgedra in die Madikwe gebied van die Noordwes provinsie. Ekstrakte van hierdie species word gebruik teen “seme”, “gala” en “Bolwetsi jwa mothlapo o moshibidu” wat waarskynlik op hartwater, galsiekte and rooiwater dui volgens die beskikbare epidemiologiese data. Asetoon en metanol ekstrakte is gebruik vir die bepaling van antimikrobiese, antiparasitiese en antioksidant aktiwiteite in verskillende species deur gevalideerde in vitro metodes. Selkulture van Ehrlichia ruminantium, Babesia caballi en Theileria equi, , is in ‘n model gebruik om die doeltreffendheid van ekstrakte teen algemene siektes deur protozoa en ricketsias te bepaal. Vier algemene menslike nosokomiale patogene Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli, is gebruik om antibakteriese aktiwiteit van ekstrakte te bepaal. Difeniel-pikrielhidrasiel en die trolox ekwivalente anti-oksidant essajeermetode is gebruik om anti-oksidantaktiwiteit te bepaal. Antioksidante mag herstel na infeksies bespoedig deur stimulering van die immuunstelsel. Daar was ‘n groot verskil in die aktiwiteite tussen die verskillende ekstrakte en organismes. E. elephantina ekstrakte was die mees doeltreffende met die biologiese and chemiese bepalings. R. tridentata het sterk anti-oksidantaktiwiteit gehad, maar het lae aktiwiteit (> 100 µg/ml) teen bosluis-oorgedraagde parasiete gehad. U. sanguinea ekstrakte was aktief in beide die antibakteriese en anti-riketsiale bepalings (EC50 = 44.49 ng/ml), wat moontlik toegeskryf kan word aan die giftige bufadienoliede teenwoordig in hierdie species. A. marlothii ekstrakte het betekenisbolle anti-riketsiale aktiwiteit (EC50 = 111.4 µg/ml) maar slegs geringe antibakteriese aktiwiteit gehad. Hierdie resultate bevestig die moontlike waarde van hierdie species teen hartwater, galsiekte and rooiwater, en ondersteun die tradisionele etnoveterinêre gebruik teen “Seme, Gala, and Bolwetsi jwa mothlapo o moshibidu”. Verdere studies word benodig om die 5 aktiewe verbindings te isoleer en te karakteriseer en om die veiligheid en doeltreffendheid van ekstrakte teen hierdie siektes in vee te bevestig. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2004. / Paraclinical Sciences / unrestricted

<p>Within this doctoral thesis, eighteen new compounds, polycyclic polyprenylated<br />acylphloroglucinols, were isolated from petrol ether extract of <em>Hypericum barbatum</em><br />plant. The compounds are named hyperibarbins A&ndash;R. For hyperibarbins A&ndash;D,<br />cytotoxicity towards carcinogenic cells and antibacterial activity were evaluated. All<br />four compounds exhibited intermediate activity towards tumor cells in vitro. Although<br />inactive towards Gram negative bacteria, examined compounds have expressed<br />extraordinary bacteriostatic activity towards Gram positive bacterial strains.<br />Hyperibarbins C, E, N and K were, since having endoperoxide bridge in their structures,<br />tested for antiprotozoal activity, but shown intermediate activity towards the examined<br />parasite strains. All goals set for this doctoral thesis at the beginning have been fully<br />met, with isolation of new compounds, described andcompletely chemically<br />characterized for the first time.</p> / <p>U okviru ove doktorske disertacije izolovano je osamnaest novih jedinjenja, policikličnih&nbsp; poliprenilovanih acilfloroglucinola, iz petroletarskog ekstrakta biljke <em>Hypericum&nbsp; barbatum</em>. Imena jedinjenja su hiperibarbini A&ndash;R. Za hiperibarbine A&ndash;D, ispitana je&nbsp; citotoksičnost prema kancerogenim ćelijama i antibakterijska aktivnost. Sva četiri&nbsp; jedinjenja su ispoljila srednju aktivnost prema tumorskim ćelijama <em>in vitro</em>.&nbsp; Iako neaktivni&nbsp; prema Gram negativnim bakterijama, ispitana jedinjenja su ispoljila izuzetnu bakteriostatsku aktivnost prema sojevima Gram pozitivnih bakterija. Hiperibarbini C, E, N i K su, zbog posedovanja endoperoksidnog prstena u svojoj strukturi, testirana na antiprotozoalnu aktivnost, ali su pokazali srednju aktivnost prema ispitanim sojevima parazita. Svi ciljevi postavljeni na početku izrade ove doktorske disertacije su do kraja ispunjeni, sa izolacijom novih, prvi put opisanih i u potpunosti hemijski okarakterisanih jedinjenja.</p>

Il y a plus de cinquante ans, la pactamycine a été isolée en tant qu’agent antitumoral potentiel. Il a été réalisé plus tard qu’il s’agissait en fait d’un agent antibactérien capable d’inhiber la synthèse de protéines lors du procédé de traduction. Récemment, il a même été démontré que certains de ses analogues possèdent des propriétés antiprotozoaires prometteuses. La présente thèse détaille la première synthèse totale de la pactamycine, entreprise au sein du groupe Hanessian, ainsi que la préparation d’une sélection d’analogues testés pour leurs propriétés biologiques. En outre, la daphniglaucine C appartient à une vaste famille de composés naturels isolés des feuilles du daphniphyllum au cours des dix dernières années. Bien que relativement peu d’information soit connue par rapport à l’activité biologique de la daphniglaucine C, la synthèse de celle-ci représente certainement un défi intéressant pour un chimiste organicien. Au passage, nos efforts vers la synthèse totale du composé cible auront permis d’explorer l’emploi de plusieurs méthodes en vue de la formation de centres quaternaires. De plus, un réarrangement réductif atypique, catalysé au palladium à partir d’alcools allyliques non-activés, a été étudié et employé afin de générer une sélection de pyrrolidines polysubstituées. / Although pactamycin was first isolated as a potential antitumoral drug, further studies highlighted its capacities in inhibiting protein synthesis, and thus its potency as an antibacterial agent. Furthermore, it was recently discovered that some of its analogs display promising antiprotozoal activity. The present thesis reports and details the first total synthesis of pactamycin, pursued in the Hanessian lab over the last few years, as well as the preparation of a selection of analogs thereby tested for their biological properties. Daphniglaucin C belongs to a large family of natural compounds isolated from the leaves of daphniphyllum over the last decade. Although relatively little is known as to the biological activity of daphniglaucin C, its synthesis poses an obvious and interesting challenge for organic chemists. En route towards its total synthesis, the use of several methods for the formation of quaternary centers was explored. Moreover, an atypical reductive allylic transposition, catalyzed by palladium from unactivated allylic alcohols, was studied and used to generate a variety of polysubstituted pyrrolidines.

Na busca por espécies vegetais com atividade antileishmania, selecionaram-se, para o estudo, duas espécies bem conhecidas da família Asteraceae: Artemisia annua L. e Bidens pilosa L. Ambas são reconhecidamente utilizadas na medicina popular, como antiprotozoárias. Apesar de terem sido amplamente estudadas em diversos aspectos, alguns permaneceram inexplorados, até o momento, e foram abordados, neste trabalho. As duas espécies foram analisadas quanto aos aspectos químico e biológico de extratos (hidroetanólico e infuso) e frações orgânicas selecionados, em função da atividade antileishmania in vitro, frente às formas promastigotas de Leishmania amazonensis. Os extratos foram obtidos a partir de órgãos vegetais, em estados de conservação diferentes (in natura, droga) e coletados em fenofases distintas. Extratos e frações orgânicas das espécies estudadas mostraram promissora atividade antileishmania in vitro e baixo nível de citotoxicidade in vitro em células epiteliais humanas (HEP-2). No estudo químico dos extratos e frações bioativos, realizaram-se análises qualitativas e/ou quantitativas de terpenos, flavonóides e de marcadores específicos (artemisinina, quercetina e rutina), avaliando-se a variação da composição dos mesmos, nas diferentes fenofases consideradas. Discutiram-se as possíveis relações existentes entre a composição química e a atividade biológica verificada. Aspectos inéditos do estudo morfoanatômico de partes aéreas de A. annua foram descritos. / Plants are potential sources of new antileishmanial drugs. Two well-known antiprotozoal species were selected, from the Asteraceae family, for this study: Artemisia annua L and Bidens pilosa L. Despite the traditional and scientific accumulated knowledge, some aspects were not investigated before and were the subject of this work. Several extracts (infusions and ethanol 96 &#176;GL) and selected fractions from both species were evaluated according to the different parameters, such as: plant organs and/or parts, growth stages and drying state of starting materials (fresh, drug). Fractions were selected among those more active against promastigotes of Leishmania amazonensis. Ethanol extracts and their fractions showed a high level of in vitro antileishmanial activity and a low cytotoxicity on epithelial human cells (HEP-2). Qualitative and/or quantitative analysis of extracts and fractions were performed for terpenes, flavonoids and selected markers (artemisinin, quercetin and rutin) in order to characterize them and evaluate variations during the different growth stages. Correlations of the chemical composition and the biological activity were discussed. The main anatomical characters of the aerial parts of A. annua were described for the first time and illustrated by photomicrographs.

Il y a plus de cinquante ans, la pactamycine a été isolée en tant qu’agent antitumoral potentiel. Il a été réalisé plus tard qu’il s’agissait en fait d’un agent antibactérien capable d’inhiber la synthèse de protéines lors du procédé de traduction. Récemment, il a même été démontré que certains de ses analogues possèdent des propriétés antiprotozoaires prometteuses. La présente thèse détaille la première synthèse totale de la pactamycine, entreprise au sein du groupe Hanessian, ainsi que la préparation d’une sélection d’analogues testés pour leurs propriétés biologiques. En outre, la daphniglaucine C appartient à une vaste famille de composés naturels isolés des feuilles du daphniphyllum au cours des dix dernières années. Bien que relativement peu d’information soit connue par rapport à l’activité biologique de la daphniglaucine C, la synthèse de celle-ci représente certainement un défi intéressant pour un chimiste organicien. Au passage, nos efforts vers la synthèse totale du composé cible auront permis d’explorer l’emploi de plusieurs méthodes en vue de la formation de centres quaternaires. De plus, un réarrangement réductif atypique, catalysé au palladium à partir d’alcools allyliques non-activés, a été étudié et employé afin de générer une sélection de pyrrolidines polysubstituées. / Although pactamycin was first isolated as a potential antitumoral drug, further studies highlighted its capacities in inhibiting protein synthesis, and thus its potency as an antibacterial agent. Furthermore, it was recently discovered that some of its analogs display promising antiprotozoal activity. The present thesis reports and details the first total synthesis of pactamycin, pursued in the Hanessian lab over the last few years, as well as the preparation of a selection of analogs thereby tested for their biological properties. Daphniglaucin C belongs to a large family of natural compounds isolated from the leaves of daphniphyllum over the last decade. Although relatively little is known as to the biological activity of daphniglaucin C, its synthesis poses an obvious and interesting challenge for organic chemists. En route towards its total synthesis, the use of several methods for the formation of quaternary centers was explored. Moreover, an atypical reductive allylic transposition, catalyzed by palladium from unactivated allylic alcohols, was studied and used to generate a variety of polysubstituted pyrrolidines.