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.

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>