Chemical and biological aspects of thiarubrine, a highly antifungal dithiacyclohexadiene polyine, were investigated. A tissue culture system for the production of thiarubrines was developed by culturing hairy roots of Chaenactis douglasii induced by Agrobacterium rhizogenes strain TR7. One culture line accumulated two times the levels of thiarubrines of nontransformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced less thiarubrine B relative to thiarubrine A compared to controls. Thiarubrine synthesis appears to be closely correlated with degree of tissue differentiation; it is suggested that it may be more practical to improve the growth rate of thiarubrine-producing root cultures by transformation rather than seek to induce synthesis in fast-growing suspension cultures. The biosynthetic relation between thiarubrines and the always co-occurring thiophenes was investigated by performing ³⁵S tracer experiments with C. douglasii hairy root cultures. It is possible that the thiophenes are not actively synthesized by the roots but rather are products of thiarubrine decomposition resulting from the extraction procedures and other manipulations of the cultures. The in vitro conversion of thiarubrine to thiophene can be induced by light, heat and other agents. No turnover of thiarubrines could be detected in the cultures in late logarithmic or stationary phases of the growth cycle. I Thiarubrines show strong light-independent antibacterial and antifungal activity. The mechanism of action of thiarubrine against E. coli and S. cerevisiae was investigated using comparative disk bioassays. A very similiar polyine from Rudbeckia hirta was as active as thiarubrine in the dark, indicating the central role of the disulfide ring in toxicity of the compounds. Visible light enhanced this activity suggesting that decomposition of the disulfide ring is important for its antibiotic effects. The photodegradation product, a thiophene, is phototoxic, probably via both type I and type II photosensitization mechanisms. The root culture extracts of Rudbeckia hirta yielded a new isomer of a known dithiacyclohexadiene polyine. MS and NMR analyses confirmed the cis configuration of this isomer. / Science, Faculty of / Botany, Department of / Graduate

M.Sc. (Chemistry) / Please refer to full text to view abstract

Virginiamycin (V) and Ionophores, such as Poulcox (active ingredient monensin sodium), are antimicrobial feed additives approved for use in cattle to improve performance. The effect of virginiamycin on Gram positive bacteria is similar to that of monensin (M) although the modes of actions differ. Very little information is available on the potential synergistic effects of V and M, especially in dairy cattle diets. The objectives of this study were to investigate the effect of combinations of V and M on the performance of dairy cows. Forty high producing Holstein cows were blocked according to previous milk production and randomly allocated to one of the following lucerne based total mixed diets: 1) Control, no medication (C); 2) Control plus 20 ppm virginiamycin (V); 3) Control plus 15 ppm monensin (M); 4) Control plus 20 ppm virginiamycin and 15 ppm monensin (V+M). The experimental period was from 21 days prepartum until 60 days postpartum. Data were analysed according to a randomized block design, using the model GLM procedure (SAS, 2001). Dry matter intake varied from 23.6 kg/d to 25.4 kg/d and did not differ between treatments (P>0.10). Milk production was higher (P<0.10) for cows receiving V+M (41.2 kg/d) when compared to cows receiving only V (36.6 kg/d), but did not differ from other treatments (P>0.10). Milk fat % was lower for cows receiving M (3.42 %) and the control (3.62 %) when compared to treatment V+M (3.86 %) (P<0.10). Milk protein and MUN did not differ. Body weight loss for the period from calving until day 60 postpartum, tended (P<0.15) to be less for cows receiving V+M (-8.1 kg) when compared to the control (-34.2 kg) and M (-31.9 kg) treatments. Both treatments M and V respectively, decreased blood BHBA and treatment M increased blood glucose (P<0.10) when compared to the control diet. Results suggest a complementary effect between the two additives monensin and virginiamycin when supplemented to early lactation cows. / Dissertation (MSc(Agric))--University of Pretoria, 2008. / Animal and Wildlife Sciences / unrestricted

Silybin is the main component of silymarin, a standardized extract obtained from the seeds of milk thistle (Silybum marianum). Flavonolignan silybin has antioxidant, hepatoprotective, chemoprotective and antitumor activities. Natural silybin occurs as an approximately equimolar mixture of two diastereoisomers - silybin A and silybin B. Analytical separation of these diastereoisomers is possible but preparative separation is complicated. The biological activity of the silybin A is different from the silybin B. Silybin diastereoisomers are mainly conjugated to glucuronides and sulfates in organism. A mixture of both silybin diasteroisomers is used in the majority of reported biological, chemical and pharmacokinetic studies. For the first time, optically pure silybin A and silybin B were used for pharmacokinetic study in this thesis. The object of this work was determination of the concentration of free and total silybin in rats plasma in relation to time. Theoretical introduction describes the current state of the problem of chemistry, pharmacology and pharmacokinetics of silybin diastereoisomers. Second part is focused on the selection of appropriate analytical column, chromatographic method and suitable procedure for preparation of biological samples for the determination of the silybin...

Nicotinic acid (NA) is a hypolipidemic agent with pleiotropic effects on plasma lipoproteins. Its medicamentous form with delayed secretion leads to pyrimidine metabolites, which make increased risk of hepatotoxicity and should be monitored. The aim of the presented study was to introduce HPLC-MS method for the determination of NA, nicotinamide (NAM), nicotinuric acid (NUA), 1-methyl-nicotiamide (MNA), nicotinamid-N-oxide (NNO), 1-methyl-2-pyridon-5-carboxamide (2-Pyr) and 1-methyl- 4-pyridon-5-carboxamide (4-Pyr) in blood plasma useful for the monitoring of hypolipidemic therapy. We have compared calibration dependences of individual metabolites using two columns - Hypercarb (grapfite carbon) and Hypersil Silica (silicagel). Both columns revealed linear calibration dependences for all mentioned analytes except MNA in the concentration range 10-2000 ng/ml for chemical standards; calibration dependence in human blood plasma measured with the column Hypersil Silica was linear in the range 20-4000 ng/ml for all tested analytes. Correlation coefficient varied between the value 0,9400 and 0,9997. Analysis time was 15 min with the column Hypercarb and 27 min with Hypersil Silica. Biological samples were extracted using solid phase with sulphonyl group. Reproducibility of the results of biological samples...

Extract from milk thistle (Silybum marianum (L.) Gaertn., synonym Carduus marianus L., Asteraceae) silymarin contains among others primarily bioactive flavonolignans. They have hepatoprotective and antioxidative effects and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activity. This thesis focuses on the preparation of metabolites of the second phase of biotransformation unexplored flavonolignans 2,3-dehydrosilybin (DHSB), silychristin (SCH), 2,3-dehydrosilychristin (DHSCH). Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate (p- NPS) as a donor. Flavonolignans yield exclusively monosulfates at the position C- 20 (C-19 in the case of silychristin and 2,3-dehydrosilychristin), except for 2,3- dehydrosilybin that gives also the 7,20-disulfated derivatives. For all samples were made antioxidant tests - DPPH assay (the highest activity had 2,3-dehydrosilychristin sulfate: IC50= 7,87 µM), Folin-Ciocalteau reduction assay (the highest activity had 2,3-dehydrosilychristin: 1,58 ekvivalents of gallic acid), ABTS+ scavenging (the highest activity had silychristin: 1,50 ekvivalents of vitamin C), inhibition of microsomal lipid peroxidation (the highest activity had 2,3-dehydrosilybin: IC50 = 10,6 µM),...

Hemoglobin and serum albumin, two prevalent proteins in human blood, contain unbound cysteine thiol moieties, creating a nucleophilic site with the potential for covalent modification by reactive chemical species. These covalent modifications, called “adducts”, are stable entities that accumulate during acute and chronic exposure and remain covalently bound for the life-span of the protein. Despite their current use as exposure markers for a variety of compounds, the use of adducts in assessing exposure to drugs of abuse has not yet been explored. The goal of this work was to examine the in vitro adduct forming capability of selected drugs of abuse with hemoglobin to provide additional proof of principle for the development of a real-world detection and monitoring analysis method. This goal was accomplished by first analyzing the binding capabilities of the drugs of interest with glutathione, a smaller tripeptide. Use of protein adducts as biomarkers of drug exposure may allow for an increased window of detection, from several days to several months, as compared to current blood analysis methods. In total, there were 16 drugs analyzed in the research, and they covered a wide range of abused drugs, including cocaine, methamphetamine, and Δ 9 -THC. Results from the glutathione trials showed that 10 of the 16 the drugs of interest were able to form covalent adducts with the free thiol moiety, with four drugs forming more than one novel adduct. The MS results for hemoglobin showed 11 adducts formed for five of the drugs under investigation. Additional MS/MS confirmatory data was obtained for two of those 11 adducts. I successfully identifyied adducts formed between drugs of abuse and glutathione and hemoglobin, which have the potential to be used as long-term biomarkers of exposure.

Methods were developed for the detection and quantification of the sulfone drug, 4,4'-diaminodiphenyl sulfone, or dapsone (DDS) and its metabolites, monoacetyldapsone (MAD) and diacetyldapsone (DAD). The work involved the synthesis of those two metabolites; confirmation of them by NMR and mass spectrometry; separation and identification of mixtures of DDS, MAD and DAD using HPLC with a uv detector and/or a mass spectrometric detector. The HPLC was operated at 1 m1/min at 285 nm, using a C-18 reversed-phase column (AXXI CHROM, 5 micron, 25cm x 2.1mm). The isochratic mobile phase consisted of 67% aqueous pH 6 phosphate buffer, 23% acetonitrile and 10% methanol, or 23% acetonitrile and 77% water. The limit of determination using a 5 pl sample was 0.2 pg/ml (0.004 nmole) for DDS, 0.3 pg/ml (0.005 nmole) for MAD and 0.4pg/ml (0.006 nmole) for DAD. The LC/MS work was done using an electrospray interface between the HPLC and the quadrupole mass spectrometer. The LC/MS method gave crucial information to confirm the identity of the three compounds measured. Overall, a combination of the two techniques is a superior approach for the determination of DDS and its metabolites with the MS confirming the identity of the metabolites and the HPLC separating and quantitating the analyses. Since only a small amount of sample is required in the experiment, these methods could be applied in studies involving analysis of metabolites from urine and plasma, facilitating future pharmacokinetics analysis of DDS and its metabolites.

No description available.

As antimicrobial resistance persistently disrupts the treatment of microbial infection, identifying novel drugs with novel modes of action is critical to getting ahead of resistance. The primary goal of this project is to extract and identify novel chemical products produced by Arthrobacter sp. TAJX1902, particularly antimicrobial metabolites. Although underexplored, Arthrobacter sps. have been shown to produce bioactive compounds of great versatility; one such is a depsipeptide with quorum-sensing inhibitory activity.1 In this research, Arthrobacter sp. TAJX1902 isolated from a soil sample showed inhibitory activity against a filamentous indicator-type bacterium and a violacein-producing Janthinobacterium sp. A. sp. TAJX1902 was cultured using rich medium broth and agar and extracted with solvents of varying polarity. Characterization of purified bioactive compounds from A. sp. TAJX1902 was done via spectroscopic techniques, including 1D and 2D-NMR spectroscopy, FTIR, and GCMS analysis. The A. sp. TAJX1902 was found to produce pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl), and five other bioactive cyclic dipeptides (CDP).