Polyacrylamide gel electrophoresis was employed in an investigation of peroxidase isozymes extracted from the mature fruits of seventeen individuals belonging to the genusThe purpose of this study was to develop procedures which could supplement existing information contributing to the biosystematics of this genus.Samples were detected on 5%-20% linear gradient gels in a sodium borate - boric acid buffer (pH, 9.0). Following electrophoresis, incubation was carried out in a solution containing benzidine dihydrochioride and hydrogen peroxide in an acetate buffer (pH, 4.5).Resulting zymograms were analyzed and eight different isozyme bands identified, based on width, staining characteristics and relationship to a horseradish peroxidase standard zymogram. Composite zymograms were drawn for each Species, based on frequency of occurrence of individual bands. Within each species, some isozymes were observed in 100% of the individuals, while other bands appeared in 20% to 92% of the gels examined. Three of the seven species investigated exhibited unique zymograms. Similarities were noted in isozyme patterns among species. Three groups of species were established based on the presence or absence of certain variable bands. Two of these groups had some correspondence to series groupings based on observable morphological characteristics. A third group consisted of species not related in traditional classification.

The Menispermaceae comprises some 75 genera and 520 species and is widespread in tropical and subtropical countries. Approximately 25 genera with 101 species are found in Africa. The southern African Menispermaceae comprises 7 genera and 13 species, of which one genus (Antizoma) and one species (Cissampelos capensis) are endemic. Field and herbarium surveys succeeded in expanding the knowledge on the geographical distribution of the South African species and a taxonomic synopsis is presented. A key to the 13 species was developed in which they can clearly be distinguished according to leaf morphology. The generic status of Antizoma was always somewhat problematic. Based on morphological features and growth habit, the two species of Antizoma seem to be adaptations to xerophytic conditions and are hardly different to Cissampelos capensis. Cladistic analysis of morphological and alkaloid data indicated strongly that Antizoma is nested within the genus Cissampelos. It is therefore proposed that the genus Antizoma be subsumed within Cissampelos. The family Menispermaceae is highly specialized in its rich diversification of bisbenzylisoquinoline alkaloids. Because of this richness the family is used worldwide in traditional medicines to treat a wide variety of ailments. From the 13 species Cissampelos capensis [(dawidjies (wortel)] is the best known and most used medicinal plant especially by the Khoisan and other rural people in the western region of South Africa. An ethnobotanical survey had confirmed the known medicinal uses and added several additional anecdotes for this species. Very few medicinal uses were hitherto recorded for the other 12 species and this study now adds several new records. The ethnobotanical survey also indicated that Cissampelos is the most widely and frequently used genus in this family in South Africa, Africa and the rest of the World. Seventeen alkaloids from seven species were isolated, using column chromatography, and identified with nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). No new alkaloids were identified during this study. Chemotaxonomic comparisons between species, provenances (different localities) and different plant parts of the same species, were done by using analytical crude alkaloid extracts. Alkaloids in these samples were identified withanalytical thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) using pure reference standards obtained from own isolations. Significant differences in the presence and concentration of alkaloids in different plant parts and between populations indicated that alkaloids can be a useful chemotaxonomic tool, but an appropriate sampling strategy is a pre-requisite. Clear alkaloid patterns emerged for the seven species studied. The occurrence of alkaloids in plants was affected by locality as was confirmed by the difference in alkaloid patterns between coastal and inland forms of Cissampelos capensis. Antimalaria and anticancer screening were done on all 13 species and the significant positive results obtained support the ethnobotanical knowledge that some Menispermaceae species are used as antimalarial and anticancer medicines. The biological activities of some of the identified alkaloids also support the rationale behind the medicinal uses of the 13 South African species. Most of them are used as bitter tonics (“amara”) or stimulants. This study therefore succeeded in contributing to the knowledge about the ethnobotany, chemotaxonomy and biological activities of the South African members of this important and interesting family. / Prof. B.E. van Wyk

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Student Number : 0009899J - PhD thesis - School of Therapeutic Sciences - Faculty of Health Sciences / The genus Eriocephalus commonly known as ‘wild rosemary’, ‘Cape snow bush’, or ‘kapokbos’ is a member of the family Asteraceae (tribe Anthemideae). The genus is endemic to southern Africa, with the highest concentration of species in the Western and Northern Cape. The genus comprises 32 species and a total of 42 taxa, which are distributed in South Africa, Namibia, Botswana, and Lesotho. The characters used in species delimitation are purely based on morphological variation in floral and foliar parts and are highly homoplastic due to phenotypic plasticity. In many cases these features are not sufficiently distinctive, as some taxa tend to exhibit dimorphism in some character states such as the presence of opposite and alternate leaves. In some species there is extensive intergrading of the major diagnostic characters leading to uncertainty in species delimitation. Both chemical and molecular characters were used in this study in an attempt to evaluate current species delimitations in the genus, along with species-level relationships and affinities. The genus is also economically important with some of its members used as medicinals, fodder, perfumes, and cosmetics. This warrants investigation into the phytochemistry and biological activity of these species in order to determine a scientific rationale for their traditional uses. For this reason, the antimicrobial, antiinflammatory, antioxidant activities, and inhibition of acetylcholinesterase by the volatile oils and leaf extracts of the genus, which are relatively unknown for most members of the genus, were also investigated. Representatives of 22 species of the genus, eight of which were from Namibia and 14 from South Africa were collected from wild populations. In most cases multiple collections per population per species were considered. Aerial plant parts were hydrodistilled to obtain the essential oils, and phenolics were extracted from leaves using acetone. Essential oils were analysed by thin layer chromatography (TLC), gas chromatography (GC), gas chromatography coupled to mass spectroscopy (GC/MS), and phenolics were analysed using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC/UV). Biological assays were carried out using the 5-lipoxygenase enzyme to evaluate antiinflammatory activity; disc diffusion and microtitre plate dilution assays were used to assess antimicrobial activities of selected fungi and bacteria; the TLC-DPPH and DPPHmicrotitre methods were used to investigate antioxidant activities and a TLC-bioautographic assay was used for testing the inhibition of the acetylcholinesterase enzyme. Total genomic DNA was extracted from silica dried leaf material. The non-coding plastid DNA regions, the psbA-trnH intergenic spacers and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were amplified, and sequenced and analysed using the parsimony algorithm. The essential oils are largely comprised of acyclic, monocyclic, and bicyclic regular and irregular mono- and sesquiterpenes of various structural groups. Two hundred compounds were noted in the essential oils with some of the common constituents being; α- and β-pinene, yomogi alcohol, ρ-cymene, 1,8-cineole, camphor, 4-terpineol, spathulenol, caryophyllene oxide, α-copaene and β-caryophyllene. Most of the species have a relatively high content of 1,8-cineole and camphor. Twenty-two chemotypes were noted and the potential for commercial development in the flavour, fragrance and pharmaceutical industries has been recorded. Among the favourable chemotypes noted includes the camphor, 1,8-cineole, bisabolol oxide B and nerolidol rich oils. However, due to the extensive variability in the essential oil profiles, standardization of oils in commercial development is crucial. The leaf extracts comprised of flavonoids with the flavones and flavanones as the major structural types present in most species. The terpene and flavonoid chemistry of the genus is highly divergent even among multiple individuals of the same species and hence not a good taxonomic marker for specific delimitation as no coherent groups was evident although some phytochemical congruence has been noted between some of the taxa. The DNA sequence data revealed lack of variability in the non-coding regions psbA-trnH and trnL-F among species of the genus. The nuclear DNA region (ITS) was variable but the number of characters separating taxa was too few for resolution of relationships between taxa. Presence of highly divergent paralogous repeats of ITS were also noted in some taxa. The combination of molecular and chemical data did not resolve the species delimitation problems due to the highly variable distribution of characters within a single species. The patterns of variation observed in the genus may be attributed to chemical convergence, divergence, hybridisation, differential gene expression, polymorphism and allelochemical diversification among other factors. The lack of coherence in the phylogenetic and phenetic groupings of the various taxa implies that the current species boundaries may not be a true reflection of natural taxonomic entities. The use of multiple taxa in taxonomic studies is strongly recommended due to the extensive variability noted in the chemical profiles of the taxa that is also depicted in the phylogenetic histories. It also implies that caution should be taken in bioprospecting for new natural products for commercial development, as plant chemical profiles especially from the same species can be very variable. This implies carrying out exhaustive population and genetic studies for evaluation of diversity in the study group. In the antimicrobial assay, the oils were more active against the Gram-positive bacteria (2-16 mg/ml) and yeasts (1-16 mg/ml). Bacillus cereus and Cryptococcus neofomans were the most susceptible pathogens to the oils. The extracts exhibited low activity against the test pathogens except E. aromaticus and E. pinnatus with activity of 0.2 mg/ml against Staphylococcus aureus and Bacillus cereus respectively. The susceptibility of the fungal pathogens Cryptococcus neoformans and Candida albicans and the Gram-positive bacteria Bacillus cereus to the oils and extracts is an indication of the potential for use of the members of the genus as natural antibiotics. The essential oils exhibited antiinflammatory activities with IC50 values ranging between 19.0-98.6 μg/ml. The oils did not show antioxidant activity at the starting concentration of 100 μg/ml but the acetone leaf extracts exhibited antioxidant activities with IC50 values ranging between 21.5-79.6 μg/ml. The essential oils showed inhibitory activity against acetylcholinesterase enzyme. The biological activity of the oils indicates that most of the traditional uses are influenced by the presence of the oils. The in vitro biological activity of the essential oils and extracts against the test pathogens provides a scientific basis for the use of some of the members in traditional herbal remedies and validates the use of some of the members of the genus for treatment of respiratory tract infections, gastro-intestinal disorders, mental conditions, dermal infections, and inflammation. The study records the biological activities for some of the species for the first time and their potential for use in flavourings, perfumery, cosmetics, as sources of antimicrobial drugs, permeability enhancers in pharmaceutical formulations and for use as industrial oils.

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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Taxonomy, according to Lawrence (1951) is a science that includes identification, nomenclature ahd classification of objects, and is usually restricted to objects of biological origin. He, further says that the plant taxonomy is fundamentally based on morphology vdth the support of all interrelated sciences. [...]

This investigation attempts to verify the identification of the anthocyanin in A. amplectens by spectral means. An attempt was also made to identify the sugars, and possibly their locations on the heterocyclic ring system, of the anthocyanins.

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