Browsing by Author "Osborne, Roy."
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Item A chemical investigation of a KwaZulu-Natal medicinal plant, Momordica foetida Schum. & Sond.(1994) Sewram, Vikash.; Osborne, Roy.; Mulholland, Dulcie Aca.Momordica foetida Schum. & Sond. (Cucurbitaceae), locally known as iNtshungu, is widely used by the Zulu people of Natal-KwaZulu for the treatment of a variety of ailments. The dried leaves leaves and stems of this plant was subjected to soxhlet extraction by refluxing with hexane, chloroform and methanol successively. Thin layer chromatography of the chloroform extract of the leaves revealed a multiplicity of compounds. The chloroform extract was further partitioned with sodium hydroxide resulting in an organic and aqueous phase. The organic phase, containing extract A, afforded two compounds, viz. compounds 1 and 2. The basic aqueous fraction was neutralised and re-extracted with chloroform to give extract B, affording five compounds, viz. compounds 3,4,5,6 and 7. Structural elucidation was accomplished by techniques such as IH and 13C NMR spectroscopy, HETCOR, COSY, FTIR and High Resolution Mass Spectrometry. Compounds 1 and 2 were identified as cucurbitane triterpenoids known as momordicines which had been previously discovered in the related species of this plant, Momordica charantia L. The remaining five compounds were identified as novel compounds, although natural derivatives of compounds 6 and 7 had been isolated previously from Momordica charantia L. Compounds 3-7 were each isolated as an epimeric mixture but it was possible to select the resonances corresponding to the major epimer. These five epimers were respectively identified as 5, 19-epoxy-19(R)-hydroxy-25-methoxy-5β- cucurbita-6,23-diene-3β-ol [102], 5,19-epoxy-19(R),25-dihydroxy-5β-cucurbita- 6,23-diene-3β-ol [103], 5,19-epoxy-19(R)-methoxy-25-hydroxy-5β-cucrbita- 6,23-diene-3β-ol [104], 5,19-epoxy-25-methoxy-5β-cucurbita-6,23diene- 3β-ol [105] and 5,19-epoxy-19(R),25-dimethoxy-5β-cucurbita-6,23diene- 3β-ol [106]. Appropriate reactions were performed, where possible, on the compounds isolated in order to confirm their identity.Item An investigation into in vitro culture and phytochemical aspects of some members of the order Cycadales.(1988) Osborne, Roy.; Van Staden, Johannes.The present-day cycads represent the diverse, modified remnants of a much larger group of gymnosperms which flourished in the Mesozoic Era. The approximately 148 surviving species of the Cycadales are sparsely distributed through tropical and sub-tropical floras in a variety of habitats. About one-half of the extant taxa are considered endangered, vulnerable or rare and, because of their scarcity and decorative appeal, have attracted much public interest. Their slow growth rate, the paucity of viable seeds and limited potential for vegetative reproduction severely limit both the natural regeneration and the controlled propagation of cycads. Over the past 40 years, various attempts have been made to establish in v~o systems for cycad culture but none has been successful in establishing a functional protocol for the artificial propagation of these plants. The author has made renewed attempts to establish in vitro cultures from a range of haploid and diploid tissues from South African Encephalartos and Stangeria species. Callus proliferation was readily obtained from most explant sources of most species using a variety of media. Addition of the growth factors 2,4-dichlorophenoxyacetic acid and kinetin in the 10[-7] to 10[-6]M range was beneficial but not essential. Culture vessels which allowed relatively free gaseous interchange were advantageous and dark conditions were marginally better than constant light. Explants from cycad taxa which are mesic in habit gave a more rapid response than similar explants from xeric plants. Attempts to induce any form of differentiation other than, or after, callus formation were unsuccessful in all Encephalartos cultures, but two forms of morphogenesis were obtained from Stangeria-derived material. Megagametophytic tissue occasionally developed spherical outgrowths analogous to coralloid root primordia. More significantly, primary root cultures after callus formation, subculture and transfer to a light environment, regularly gave rise to meristematic zones and subsequent leaf emergence. This is the first recorded case of in vitro morphogenesis of a South African cycad. The order Cycadales shows several distinctive phytochemical features, principally the presence of the unique methylazoxymethanol glycoside toxins and a-amino-s-methylaminopropionic acid together with some unusual phenolic compounds, flavanoids, carotenoids and cyclitols. Stangeria differs from other cycads in at least two phytochemical aspects; the absence of biflavonoids in the leaves and the absence of rhamnose and methyl rhamnose in the hydrolysed mucilages. These diffe.rences may indicate broader physiological differences which would in turn explain the observed differences in morphogenetic competence of tissues from Stangeria and Encephalartos. Analyses of various tissues from these and other cycad taxa were performed with respect to moisture, protein, enzyme, toxin and hydrocarbon content. Significant differences, both between organs and between taxa, were noted. The results of peroxidase analyses were particularly important in that high levels of this enzyme correlate with the rapidity of callus formation in vitro. Furthermore, a sharp increase in peroxidase activity signals the onset of callogenesis in Stangeria megagametophyte cultures. An important incidental aspect of the phytochemical analyses is that of potential value of these data to the taxonomist. In particular, the leaf wax hydrocarbon profiles appear to be species-specific and are ideally suited to processing by numerical taxonomy computer programmes. It is anticipated that extension of this work will make a significant contribution to the resolution of existing problems in cycad taxonomy and, additionally, provide a means to construct phylogenetic sequences in the order.Item Toxic compounds in cycads.(1990) Nair, Jerald James.; Osborne, Roy.The present-day cycads comprise the diverse, modified, remnants of a much larger group of gymnosperms which flourished in the Mezozoic era. The systematic position that the cycads occupy in the botanical hierarchy is significant in that they represent relatively unchanged survivors from prehistory. The present global complement of 182 species occur in tropical and mild temperate regions in both hemispheres. Despite the density of proliferation of species, about one-half of the extant taxa are considered endangered, vulnerable or rare. Apart from characteristic features such as differences in growth forms, variation in reproductive structures and anatomical details, cycads are distinguished from all other plant groups by the unique phytotoxins, azoxyglycosides, which they possess. The toxicity of cycads is well-documented in cases which refer to both man and animals. Cycasin, which together with macrozamin represent the major azoxyglycosides occurring in cycads, has been reported to elicit responses similar to those that have been observed during carcinogenicity, mutagenicity and neurotoxicity assays. It has become apparent that the mechanism by which azoxyglycosides manifest their toxicity involves deglucosylation, by enzyme systems, which releases the aglycone, methylazoxymethanol (MAM), and sugar moieties. Metabolic activation of MAM succeeds deglucosylation to generate methylene carbene units (: CH2) which are capable of methylating macromolecules including DNA, RNA and protein. During this investigation, macrozamin was extracted from seed kernels of Encephalartos transvenosus and cycasin was tentatively identified in seed kernels of Cycas thouarsii . The hexa-acetate derivative of macrozamin was prepared whereas the tetra-acetate derivative of cycasin was not secured in a pure form. The spectroscopic techniques employed for identification include DV-absorption, infra-red and nuclear magnetic resonance spectroscopy, all of which are useful for detecting signals which arise as a result of the azoxy function. A kinetic study was carried out to determine the rate of hydrolysis of macrozamin with 4M sulphuric acid, and to allow calculation of the activation energy for the process. A comparison of the kinetic parameters determined for the above process with those derived for the hydrolysis of cycasin and methylazoxymethanol under similar conditions followed. The rates of hydrolysis increase in the order macrozamin, cycasin, methylazoxymethanol since the molecules contain two, one and zero glycosidic linkages respectively. Additional glycosidic bonds are observed to decrease the rate of reaction. Consequently, activation energies for hydrolysis of the above molecules decrease in the above-stated order. Macrozamin and cycasin were quantified in cycad material by high performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) and by the chromotropic acid assay. The results of the quantitative analysis has highlighted certain limitations of the methods of detection, and has been found to be applicable to a taxonomic evaluation and a proposal for the biosynthesis of the azoxyglycosides.