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Doctoral Degrees (Research Centre for Plant Growth and Development)

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    Alkaloids from three South African Crinum species.
    (2000) Elgorashi, Esameldin Elzein.; Van Staden, Johannes.; Drewes, Siegfried Ernst.
    The alkaloid content of three Crinum species namely C. bulbispermum, C. macowanii and C. moorei was investigated. The ethanolic extracts of C. bulbispermum yielded seven compounds. The new alkaloids 8α-ethoxyprecriwelline, N-desmethyl-8α-ethoxypretazettine and N-desmethyl-8β-ethoxypretazettine were isolated for the first time from a natural source. In addition, the known alkaloids bulbispermine, crinamine, 6-hydroxycrinamine and 3-O-acetylhamayne were isolated in this study. The ethanolic extracts of C. moorei were found to contain Iycorine, 1-O-acetyllycorine, crinine, 3-O-acetyllycrinine, epibuphanisine, powelline, crinamidine, undulatine, epivittatine, 1-epideacetylbowdensine, cherylline and the new alkaloids mooreine and 3-[4'-(2'-aminoethyl)phenoxy]bulbispermine. The alkaloids crinine, lycorine, bulbispermine, cherylline and hamayne were obtained from the ethanolic extracts of C. macowanii. In addition, the amine tyramine was identified during the isolation process. Dilute HCl solution extraction followed by GC analysis was used to investigate organ-to-organ and seasonal variation of alkaloids in each Crinum species, as well as the interspecific variation in these alkaloids over two consecutive years. Twelve alkaloids were identified, including crinine, epibuphanisine, powelline, crinamine, crinamidine, 6-hydroxycrinamine, 1-epideacetylbowdensine, 3-O-acetylhamayne, undulatine, Iycorine, 1-O-acetyllycorine and cherylline. Alkaloids were detected in all organs of C. moorei and C. macowanii. However, alkaloids were not detected in the leaves of C. bulbispermum. Organ-to-organ and seasonal variations in the total yield and total ring types of these alkaloids were noticed. Organ-to-organ and seasonal statistical variations were also detected for some of the individual alkaloids detected in each of these species. The results also showed that C. moorei had the highest levels of all individual alkaloids except crinamine when compared to C. bulbispermum and C. macowanii. Quantitatively, the detected alkaloids chemotaxonomically separated C. moorei from C. bulbispermum and C. macowanii. The results also indicated that C. macowanii is more closely related to C. bulbispermum. Qualitatively, Iycorine, 1-O-acetyllycorine, cherylline, crinamidine, 1-epideacetylbowdensine, crinine, crinamine and 3-O-acetylhamayne were detected in both C. moorei and C. macowanii, indicating the close relationship of these species.
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    Evaluation of anthelmintic, antiamoebic and antibacterial activity in traditional South African medicinal plants.
    (2001) McGaw, Lyndy Joy.; Van Staden, Johannes.; Jäger, Anna Katharina.
    Traditional medicine in southern Africa draws upon a vast selection of plants to treat gastrointestinal disorders such as diarrhoea and intestinal parasites. The evaluation of these plants for biological activity is necessary, both to substantiate the use of these plants by healers, and also a possible lead for new drugs or herbal preparations. After a survey of the existing ethnobotanical literature, plants used to treat stomach ailments such as diarrhoea, dysentery or intestinal worm infestations were selected and submitted to bioassays according to their traditional uses. Extracts of the chosen plants were made using the solvents hexane, ethanol and water, to ensure the extraction of compounds with a wide range of polarity. In total, 138 extracts were tested for antibacterial activity, 72 for anthelmintic activity, and 42 for antiamoebic activity. Antibacterial activity was evaluated using the disc-diffusion assay, and Minimal Inhibitory Concentration (MIC) values were determined using a microdilution assay. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Ethanolic extracts showed the greatest activity and Gram-positive bacteria were the most susceptible microorganisms. The free-living nematode Caenorhabditis elegans, which is morphologically similar to parasitic nematodes, was used in two different assays to evaluate anthelmintic activity. A microdilution technique was employed to investigate antiamoebic activity against the enteropathogenic Entamoeba histolytica, the causal organism of amoebic dysentery. These assays were suitable for the screening of a large number of extracts at one time. Several plants exhibited significant activity against these test organisms. Many species of plants belonging to the family Combretaceae are used in southern African traditional medicine against a variety of ailments, including abdominal complaints, bilharzia and diarrhoea. Extracts of powdered leaf material of 24 species belonging to the Combretaceae were prepared using the solvents ethyl acetate, acetone, methanol and water. These extracts were screened for anthelmintic activity. Significant activity was exhibited by C. apiculatum, C. hereroense and C. mossambicense. The most anthelmintic activity was shown by acetone extracts, followed by ethyl acetate, water and then methanol extracts. The aromatic rhizomes of Acarus calamus L. are used extensively in traditional medicine worldwide. They reportedly relieve stomach cramps and dysentery, and are used as anthelmintics. Rhizome extracts of A. calamus growing in KwaZulu-Natal, South Africa, exhibited anthelmintic and antibacterial activity in the initial general screening. Using bioassay-guided fractionation, the phenylpropanoid β-asarone was isolated from the rhizome. This compound possessed both anthelmintic and antibacterial activity. It has previously been isolated from A. calamus, and a related species, A. gramineus. Different varieties of A. calamus exhibit different levels of β-asarone, with the diploid variety containing none of the compound. Mammalian toxicity and carcinogenicity of asarones has been demonstrated by other researchers, supporting the discouragement of the medicinal use of Acarus calamus by traditional healers in South Africa. Schotia brachypetala was another plant to show good antibacterial activity in the initial screening. The roots and bark of S. brachypetala are used in South African traditional medicine as a remedy for dysentery and diarrhoea. The lack of pharmacological and chemical data on this plant prompted a further investigation into its antibacterial activity. The differences in activity of ethanol and water extracts with respect to plant part, season and geographical position were analysed. No extreme fluctuations in activity were noted. Two other Schotia species, S. afra and S. capitata, were included in the study, and both displayed good antibacterial activity. The storage of the plant, either as dried, ground plant material at room temperature, or as an extract residue at -15°C, had little effect on the antibacterial activity. Preparing the extracts from fresh or dry material also did not notably affect the activity. In general, the ethanolic extracts were more active than the aqueous extracts. The chemical profiles on TLC chromatograms were compared and found to be very similar in the case of ethanol extracts prepared in different months of the year, and from different trees. The extracts of the three species, and of the leaves stored under various conditions, as well as extracts prepared from fresh or dry material, also showed similar TLC fingerprints. However, various plant parts of S. brachypetala showed distinctly different chemical compositions. The leaves of S. brachypetala showed slightly higher antibacterial activity than the roots. Fractionation of the ethanol extract of the dried leaves using liquid-liquid partitioning and chromatographic techniques yielded 9,12,15-octadecatrienoic (linolenic) acid and methyl-5, 11,14,17-eicosatetraenoate. These fatty acids displayed antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and activity to a lesser extent against the Gram-negative Escherichia coli and Klebsiella pneumoniae. Linolenic acid is known to have antibacterial activity. The screening of plants for biological activity yielded valuable preliminary information about the plants used by traditional healers to treat gastrointestinal illnesses. The isolation of biologically active compounds from two highly active plants was achieved.
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    The role of smoke as a germination cue.
    (2006) Light, Marnie Elizabeth.; Van Staden, Johannes.
    No abstract available.
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    Root-stimulating activity from various gelling agents used in tissue culture.
    (2003) Arthur, Georgina Dede.; Van Staden, Johannes.; Stirk, Wendy Ann.
    Extracts of gelling agents have been shown to stimulate rooting and this study was initiated to investigate the presence of root stimulating substances in gelling agents. After screening a number of gelling agents, four were selected, namely; Agar Bacteriological, Agar Commercial Gel, Difco Bacto Agar and Gelrite were selected and examined for the presence of root-stimulating substances using mungbean bioassay. Water extracts of Agar Bacteriological, Agar Commercial Gel and Difco Bactol Agar stimulated rooting of mungbean cuttings. Addition of Charcoal neither reduced nor increased rooting produced by the water extract of the first two agars but when added in conjunction with Difco Bacto Agar rooting was reduced. Autoclaving, however reduced rooting in extracts of the gelling agents. The possibility that root-stimulating substances may not be the same in all the gelling agents can not be excluded. Extraction of Gelrite with water was problematical and was therefore excluded. IBA solution and water extracts of the gelling agents separately promoted good rooting in mungbeans cuttings. Rooting in extracts of autoclaved frozen-thawed gelling agents was poor, however, IBA + gelling agents gave high rooting at the 100% concentration and this could possibly be due to an additive effect of the IBA. Addition of charcoal reduced rooting significantly in extracts of IBA + gelling agents. Using 80% acidic methanol, reasonable levels of rooting substances were obtained from the residue extract of this complex (IBA + gelling agent+charcoal) of all the gelling agents except Gelrite indicating that root-promoting substances were adsorbed by charcoal. The low rooting in the presence of the Gelrite extract was attributed to the matrix of the polymer of the Gelrite. Ethyl acetate fractionated extracts (EA-pH 8.0; EA-pH 3.0; and Aqueous) obtained from the four gelling agents stimulated rooting indicating the presence of numerous root promoting substances. Gelrite gave good rooting with both the 50 and 100% concentrations of all the fractions. Purified water and ethanol extracts of the gelling agents exhibited auxin-like activity when separated by paper chromatography and compared with IBA and IAA standards. Using HPLC, IAA was quantified in all the gelling agents with Difco Bacto Agar and Agar Commercial Gel having the highest IAA concentration and Gelrite the lowest IAA concentration. IAA concentration in Agar Bacteriological was a third of the level detected in Difco Bacto Agar. The information from this work may enable researchers to consider gelling agents as sources of auxin-like compounds and other plant hormones as well as support media for use in tissue culture procedures and also increase the enthuse for further research into the nutrient types and levels in gelling agents.
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    Micropropagation and pharmacological evaluation of Boophone disticha.
    (2013) Cheesman, Lee.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.; Light, Marnie Elizabeth.
    Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa.