There is an urgent need to discover new antimicrobial and antiviral compounds owing to increasing problems of resistance to drugs encountered in many pathogenic organisms. There are also problems with currently used drugs in terms of side effects and expense. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive results. In particular, plants with antimicrobial properties are the subject of much investigation. This study focuses on isolating the compounds responsible for biological activity in one such medicinal plant, Combretum paniculatum, extracts of which have been shown to possess antimicrobial activity. Members of the genus Combretum are widely used for medicinal purposes by many groups in Africa, to treat various conditions. Other researchers have discovered antifungal, antibacterial, anti-inflammatory and molluscicidal effects of these plants. One species of this genus, C. paniculatum, has been reported in the literature to have antiviral activity against HIV-2 with a promising selectivity index. It is important to exclude highly toxic effects of potential antimicrobial preparations. C. paniculatum extracts also displayed good antibacterial activity and some anti-inflammatory activity in other studies. Although many active compounds, especially antibacterial and antifungal, have been isolated from other Combretum species, little is known about the identity of compounds responsible for activity in C. paniculatum. In the initial stages of this project, the crude extracts of leaves of C. paniculatum were investigated for antiviral and cytotoxic activity. It was found that the acetone and water extracts of C. paniculatum leaves reduced the cytopathic effect of feline herpesvirus type 1 by 3.0 log10, a very promising result. Investigations were carried out to determine the best solvent to use for extracting the active components. It was found that acetone was the best extractant in terms of the number of compounds extracted from the plant after analysis using thin layer chromatography (TLC) and the number of bioactive compounds using bioautography against bacteria. Water extracted a large quantity of material. Different plant parts, namely stem bark, root bark and leaves, were screened for antiviral and antibacterial activity and the leaves and stem bark showed good activity. The test organisms were feline herpesvirus type 1 (FHV-1) for antiviral testing, and a range of Gram-positive and Gram-negative bacteria for antibacterial activity. Cytotoxicity against African green monkey kidney (Vero) cells was observed only at a relatively high concentration of 0.28 mg/ml. Based on availability and sustainability, the leaves were chosen for further work especially since leaves were used in the published data. Isolation of active compounds from a 70% acetone extract of a large quantity of C. paniculatum leaf material was carried out using bioassay-guided fractionation. The bioassay used to select the active fractions for further fractionation was an antibacterial assay since it is easier and more rapid to detect antibacterial activity than antiviral activity. Various techniques including column chromatography and high performance liquid chromatography (HPLC) were used to fractionate the extract to result in pure compounds. The isolated compounds were structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. Nine compounds were identified as cholest-5-en-3-ol, 2-phyten-1-ol, isoquercitrin, p-coumaric acid, 2, 3, 8-tri-O-methylellagic acid, beta-sitosterol, gallocatechin, apigenin and apigenin-7-glucoside. The compounds were subjected to various bioassays to evaluate their biological activity. The isolated compounds had a broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens, as well as some antifungal and antimycobacterial activity. Cholest-5-en-3-ol, 2-phyten-1-ol, gallocatechin and apigenin were active against Escherichia coli (Gram-negative) and Mycobacterium vaccae, and against the fungi Sporobolomyces salmonicolor and Penicillium notatum. Cholest-5-en-3-ol and 2-phyten-1-ol were also active against Bacillus subtilis (Gram-positive). None of the compounds showed substantial antiviral activity against coxsackievirus strain B3 Nancy, influenzavirus type A strain Hong Kong and herpes simplex virus type 1 strain K1. The compounds were generally moderately cytotoxic to the HeLa cell line but were less toxic to the Madin-Darby Canine Kidney (MDCK) and Vero cell lines. The results obtained confirm the ethnobotanical use of C. paniculatum. Nine compounds with various biological activities were isolated from the leaf extract. The constituents responsible for antiviral activity still remain to be isolated and further work should be carried out making use of antiviral assay-guided isolation. These compounds may be present in low concentrations in C. paniculatum. Synergistic effects of isolated compounds on biological activity, particularly antiviral activity, could be investigated. The results reported here confirm that the presence of antibacterial activity in plant extracts is not an indicator of antiviral activity. Although the crude extracts of C. paniculatum had both antibacterial and antiviral activity, different compounds are responsible for antibacterial and antiviral activity respectively. / Thesis (PhD (Paraclinical Science))--University of Pretoria, 2007. / Paraclinical Sciences / PhD / unrestricted

This thesis describes a study in petroleum geochemistry and specifically of the application of Land-plant derived hydrocarbons to elucidating source matter type, depositional environment and thermal maturity of crude oils. Large volumes of petroleum have been generated from land-plant organic matter in many parts of the world, but especially in the Tertiary deltaic provinces of the USA, Canada, Africa, Southeast Asia, Australia and New Zealand. Compared to oils from other regions, these oils have a relatively complex pattern of biomarkers or molecular fossils, a feature reflecting the diversity of deltaic depositional environments and the advanced state of plant evolution during late Mesozoic and Tertiary time. The molecular and carbon isotopic composition of these oils is useful in identifying the character of their source beds and also in understanding the conditions that give rise to oil-prone sediments generally.Despite their widespread occurrence in oils and sediments, land-plant biomarkers have been used less frequently than compounds of algal origin as markers for source, depositional environment and thermal maturity. The present work sets out to extend the range of land-plant biomarker and isotope tools available to the petroleum geochemist and to provide a firmer foundation for some of the existing tools. A systematic study of the biomarker and n-alkane carbon-isotope composition of a set of late Mesozoic/Tertiary-age oils was carried out as a preamble to more focussed investigations. A comparison of the biomarker and n-alkane isotope data showed that parameters such as the oleanane/hopane ratio overestimate the higher plant contribution to marine deltaic oils. The abundance of bicadinane isomers was found to be much more variable than that of the oleananes, probably because of their close association with the highly resinous, dipterocarp hardwoods of ++ / Southeast Asia. However, small amounts of bicadinanes were found in an oil from outside the paleogeographic range of the dipterocarp family, indicating a more general source. Retention indices based on the regular hopane series were calculated for the main bicadinane and methyl bicadinane isomers and for a group of oleanoid triterpanes.Source rock depositional setting proved to be the primary control on the shape of the n-alkane isotope profile, with negatively sloping curves being characteristic of fluvio-deltaic and marginal lacustrine oils and flat or positively sloping curves typical of marine oils. The difference is probably related to the bacterial reworking of higher plant matter in the fluvio-deltaic environment. A slight isotopic anomaly at n-C(subscript)17 correlates with the abundance of algal-derived steranes and may indicate a minor marine contribution to the source of an fluvio-deltaic oil. A study of sediments from the South Sumatra Basin and New Zealand showed that the n-alkane isotope profile is determined mainly by source matter type and is little influenced by thermal maturity or depositional environment.Based on the results of the work described above, three aspects were chosen for further examination. These were: a) the factors controlling the carbon isotopic composition of n-alkanes and of resin-derived compounds in terrigenous oils; b) the influence of source, maturity and biodegradation on the abundance of bicadinanes, especially the effect of maturity on the isomer distribution, and c) the factors controlling the abundance of oleananes and rearranged oleananes in oils and Ancient sediments, especially the role of depositional environment.A preliminary study using sediments, from the Visayan Basin (Philippines) showed the bicadinane isomeric distribution to be sensitive to maturity. This suggested new maturity parameters which would be ++ / resistant to even severe biodegradation and largely immune to interference from diagenetic effects. A more detailed study of a marine-fluvio-deltaic depositional sequence from the South Sumatra Basin confirmed that the maturity indices based on bicadinanes and aromatic analogs were less subject to non-maturity influences than those based on the steranes and aromatic hydrocarbons. The values obtained for the bicadinane indices were compared with the maturity required for oil generation as estimated by kinetic modelling and with conventional maturity indicators such as sterane epimerisation and the methyl phenanthrene index. Values for several of the indices were also measured for 17 Tertiary-age oils from Southeast Asia, Papua New Guinea, New Zealand and Australia. The main bicadinane maturity indicator (BMI-1) continues to change into the oil window and hence is useful in ranking the relative maturity of oils as well as sediments. Values of BMI-1 for two oils having unusually low sterane maturities were found to be normal and, in the light of this observation, the conventional explanation for low sterane epimerisation of many Tertiary-age oils was critically evaluated.The oleananes, as markers for the angiosperms, provide valuable source and age information when present in an oil. Nevertheless, their abundance is not quantitatively related to the land plant input and indeed their presence results from a small "leak" in diagenetic processes leading primarily to aromatic oleanoids. Evidence is presented that contact of plant matter with seawater during early diagenesis enhances the expression of oleananes in a mature sediment or oil. Oleananes are absent or present at very low concentrations in samples from the base of an Eocene coal seam affected by post-depositional seawater intrusion. However, their abundance increases toward the top of the seam in correlation with ++ / % organic sulphur, dibenzothiophene/phenanthrene and the homohopane index. Similarly, in deltaic sediments from the South Sumatra Basin, oleanane/hopane is strongly correlated with indicators of marine influence such as C[subscript 27]/C[subscript 29] steranes and of oxic/anoxic conditions such as the homohopane index. In each case, increasing oleanane abundance is accompanied by a reduction in the extent of aromatisation and, for the South Sumatra Basin, the proportion of A-ring contracted oleananes. An angiosperm-derived Miocene coal from the Philippines, deposited under freshwater conditions, shows abundant aromatic oleanoids but no oleananes. These results show that oleananes need to be used with caution as age and source markers in fluvio- deltaic and lacustrine petroleum systems. On the other hand, their sensitivity to early diagenetic conditions may make them useful in locating effective source rocks in such systems.Compounds derived from plant resins are major components of some terrigenous oils. Although recent studies have elucidated the molecular structure of resinites, very little information was available on the carbon isotope composition of resinites prior to the present study. No carbon isotope studies of resin-derived compounds in oils had been performed. Hence, carbon stable isotope analyses were carried out on a set of modern and fossil resins of diverse origins and compound specific isotope analysis was used to characterise individual hydrocarbons in resin pyrolysates and oils derived from resinitic source matter. The results showed that "Class V' resinites derived from gymnosperms are enriched in the heavy carbon isotope compared with the angiosperm-derived "Class W' resinites. Furthermore, both the fossil resinites themselves and individual hydrocarbons derived from them are isotopically heavy compared with modern plant resins. The reasons for ++ / these differences and their implications for petroleum geochemical studies are discussed.

<p dir="ltr">Plants emit an amazing diversity of volatile organic compounds (VOCs) that in addition to being utilized by humans for a multitude of applications, allow plants to communicate with their environment, and play numerous roles in plant growth and development. Plants must be able to perceive and distinguish between VOC cues mediating plant-plant, plant-insect, and plant-microbe interactions to appropriately respond to stimuli. Due to the plethora of biological processes dependent on VOCs, significant progress has been made towards understanding the biosynthesis of plant VOCs and their regulation, and, in recent years, the molecular mechanisms involved in VOC emission. However, to date, little is known about how VOCs are perceived by plants and trigger cellular response(s). In animals, VOCs are recognized by odorant receptors known as G-protein-coupled receptor (GPCR) proteins. However, the few GPCR genes identified in plants appear to have different functions and the lack of a reliable marker for VOC perception has hampered research in this field.</p><p dir="ltr">The discovery of natural fumigation of terpenoids in petunias provides a means of studying VOC perception and the downstream signaling pathways by providing a visual indicator of perception. Transcriptomic analysis of wild-type and transgenic petunias deficient in terpenoid synthesis revealed a link between terpene perception in pistils with the karrikin-like signaling pathway. By utilizing biochemical, computational, and in planta experiments, we demonstrate that of the four petunia karrikin-insensitive receptors (PhKAI2), one of the Lamiid-specific KAI2 intermediate clade receptors, PhKAI2ia, can stereo-specifically perceive the (−)-germacrene D signal emitted from the floral tubes, triggering a KAI2-mediated signaling cascade and affecting plant fitness. Downregulation of PhKAI2ia results in significantly smaller stigmas compared to wild-type, and the phenotype cannot be complemented by the treatment of pistils with (−)-germacrene D, indicating that PhKAI2ia transgenic plants are acting as deaf receptors. We also show that the binding of (−)-germacrene D to PhKAI2ia is sufficient to induce complex formation with more axillary growth 2 (PhMAX2) and the subsequent degradation of suppressor of MAX2 (PhSMAX1a).</p><p dir="ltr">Altogether, our research uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).</p>