Thesis (M. Sc. (Botany)) -- University of Limpopo, 2018 / The aim of the study was to investigate medicinal plants used for the treatment of various ailments by the traditional healers and local people and to determine antifungal activities against animal fungal pathogens. Ethnobotanical survey was conducted to identify medicinal plants used by local people and traditional healers to treat various ailments in Makhado Local Municipality, Vhembe District, Limpopo, South Africa. A questionnaire was designed to gather information on the local name of plants, plant parts used and the methods of preparation and administration by the traditional healers. In our findings, sixty-three medicinal plants belonging to thirty-three families were identified to be used for treatment of various diseases such as chest complaint, sexual transmitted infections, headache, swollen legs, hypertension, blood purification, asthma, and infertility. Specific parts of the plant used for medicinal purposes vary from species to species and from one traditional healer to another. The dominant families were Fabaceae, Celastraceae and Euphorbiaceae. Of the sixty-three plants species identified, trees were the most predominant plant form (53%), followed by shrubs (23%), herbs (14%), and climbers (10%). Root, fruit, bark, leaves, seeds and in some instances the whole plant are used for the preparation of medicine while decoction and infusion were the general methods of preparation. The mode of administration of medicine was mainly oral. The most frequently used plant species were Warbugia salutaris (Bertol.f.) Chiov, Sclerocarya birrea (A.Rich) Hochst and Eleondron transvaalense (Burtt Davy) R.H. Archer. Eight plant species (Asparagus buchananii Bak., Albuca seineri (Engl. & K.Krause) J.C Manning & Goldblatt, Elephantorrhiza elephantina (Burch.) Skeels, Indigofera circinnata Benth, Maerua juncea Pax, Pentarrhinum insipidum E. Mey., Senna italica Mill. and Schinus molle L.) were selected based on the information given by the local people and the traditional healers for further phytochemical analysis and microbiological assays. Antifungal activities of the selected plant species were determined against three fungal pathogens namely, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Of the tested plant species, hexane leaf extracts of M. juncea, ethyl acetate leaf extracts of S. italica, A. buchananii and E. elephantina were the most active against Candida albicans, Cryptococcus v neoformans and Aspergillus fumigatus with Minimum inhibitory concentration (MIC) values ranging between 0.02 mg/ml and 0.08 mg/ml. Bioautography assay was used to determine the number of active compounds in the plant extracts. No active compounds were observed in some plant extracts against the tested animal fungal pathogens indicating possible synergism. The most promising plant species were: A. buchananii, A. seineri and M. juncea, all had shown good activity with 4 compounds against A. fumigatus. Acetone and methanol extracts had the same active compounds visible on bioautograms. Most of the active compounds were observed in TLC chromatograms developed Benzene: ethanol: ammonia hydroxide (BEA) eluent solvent system. Based on excellent antifungal activity against the tested microorganisms, leaf extracts of A. buchananii, A. seineri M. juncea, P. insipidum and root extracts of I. circinnata were also tested for cytotoxicity against the Vero kidney cells. All plant extracts investigated were relatively not toxic against the cells with LC50 ranging between 0.131 mg/ml and  1 mg/ml. Water extracts of A. buchananii, A. seineri and M. juncea had LC50  1 mg/ml. The leaf aqueous extracts of P. insipidum were less toxic than root aqueous extracts of I. circinnata with LC50 of 0.65 mg/ml and 0.49 mg/ml against the Vero kidney cells respectively. The results indicate that the local people and traditional healers in Makhado Local Municipality use medicinal plants and their indigenous knowledge on the treatment of fungal infections and related ailments. / University of Limpopo and National Research Foundation (NRF)

Made available in DSpace on 2016-12-23T13:56:03Z (GMT). No. of bitstreams: 1 dissertacao-simonebrmaifrede.pdf: 1273260 bytes, checksum: d5439bcf28dc75da348359e5efa529ef (MD5) Previous issue date: 2009-03-06 / INTRODUÇÃO: Onicomicose é a infecção da unha causada por amplo espectro de espécies fúngicas, incluindo leveduras e fungos filamentosos dermatófitos e nãodermatófitos. Devido à variável suscetibilidade dos diversos agentes etiológicos às drogas antifúngicas, o diagnóstico laboratorial vem sendo considerado uma ferramenta importante para se estabelecer a etiologia e auxiliar na escolha do tratamento da onicomicose. Com base na elevada porcentagem de falha terapêutica no tratamento da onicomicose, tem-se evidenciado o interesse na padronização de testes de suscetibilidade in vitro de fungos filamentosos. OBJETIVOS: Estabelecer a freqüência das onicomicoses em relação a outras dermatomicoses; definir a etiologia das onicomicoses através do isolamento e identificação dos fungos; comparar o padrão de suscetibilidade entre fungos dermatófitos e não-dermatófitos às drogas fluconazol, cetoconazol, itraconazol, miconazol, ciclopirox, terbinafina e griseofulvina. MÉTODOS: As amostras clínicas foram colhidas através de raspagem e/ou fragmentação da unha e o exame microscópico direto foi realizado através do tratamento destas amostras com hidróxido de potássio (KOH) a 20% e tinta Parker. As culturas foram realizadas nos meios de ágar Sabouraud dextrose adicionado de 0,05 mg.mL-1 de cloranfenicol e ágar Mycose, incubados à temperatura ambiente e por um período de até 15 dias. A identificação dos fungos filamentosos foi baseada na observação de suas características macroscópicas e microscópicas e os testes de suscetibilidade in vitro a drogas antifúngicas foram baseados no Documento M38-A do CLSI. RESULTADOS: O diagnóstico laboratorial das dermatomicoses foi estabelecido em 69% dos 1.008 pacientes com lesões sugestivas de dermatomicoses encaminhados ao Laboratório de Diagnóstico Micológico do Depto. de Patologia / UFES, no período de 12/03/2004 a 14/08/2008. Onicomicose foi diagnosticada em 333 pacientes e os grupos de fungos mais isolados foram: leveduras 55,6%, fungos filamentosos não-dermatófitos 27,2% e dermatófitos 17,3%. Fungos dermatófitos foram mais inibidos in vitro que fungos não-dermatófitos. As drogas fluconazol e griseofulvina inibiram apenas fungos dermatófitos, enquanto a terbinafina foi a droga que mais inibiu os dois grupos de fungos e em baixas concentrações. A quantificação do inóculo por contagem em hemocitômetro e em placas de ágar Sabouraud revelou que o acerto do inóculo em espectrofotômetro pode ter uma boa correspondência se estabelecido em faixas de transmitância diferenciadas para os diversos tipos de fungos. CONCLUSÃO: É necessário estabelecer o diagnóstico laboratorial das onicomicoses, já que estas podem ser causadas por diversos agentes etiológicos e com diferentes suscetibilidades in vitro a várias drogas antifúngicas. / INTRODUCTION: Onychomycosis is the nail infection caused by a wide spectrum of fungi species, including yeasts, dermatophyte and nondermatophyte mould. Due to the variable susceptibility of the several etiologic agents to the antifungal drugs, the laboratorial diagnosis is being considered an important tool to establish the etiology and to help in the choice of the treatment of onychomycosis. Based on the high percentage of therapeutic flaw in the treatment of onychomycosis, there has been some evident interest in the standardization of the susceptibility tests in vitro of filamentous fungi. OBJECTIVES: To establish the frequency of the onychomycosis in relation to other dermatomycosis; to define the etiology of the onychomycosis by the isolation and identification of the fungi; to compare the pattern of susceptibility among dermatophytes and nondermatophytes mould to drugs such as fluconazole, cetoconazole, itraconazole, miconazole, ciclopirox, terbinafine and griseofulvine. METHODS: The clinical samples were collected by the scratching and/or fragmentation of the nail and the direct microscopic examination was made by the treatment of these samples with potassium hydroxide (KOH) at 20% and Parker ink. The cultures were made in dextrose agar Sabouraud with 0,05 mg.mL-1 of cloranphenicol and agar Mycosel, incubated to room temperature and for a period of up to 15 days. The identification of the filamentous fungi was based on the observation of its macroscopic and microscopic characteristics and the tests of susceptibility in vitro to the antifungal drugs were based on CLSI M38-A reference method. RESULTS: The laboratorial diagnosis of the dermatomycosis was established in 69% of the 1.008 patients with lesions that suggested dermatomycosis sent to the Laboratory of Mycologic Diagnosis from the Dept. of Pathology / UFES, in the period of 03/12/2004 to 08/14/2008. Onychomycosis was diagnosed in 333 patients and the groups of more isolated fungi were: yeasts 55,6%, nondermatophyte mould 27,2% and dermatophytes 17,3%. Dermatophytes were more inhibited in vitro than nondermatophytes ones. Drugs such as fluconazole and griseofulvine inhibited just dermatophytes fungi while terbinafine was the drug which most inhibited both groups of fungi and in low concentrations. The quantification of the inoculum for counting in haemocytometer and in plates of agar Sabouraud revealed that the correctness of the inoculum in espectrofotometer may have established a good correspondence in transmission bands differentiated for the various types of fungi. CONCLUSION: It is necessary to establish the laboratorial diagnosis of the onychomycosis, as these may be caused by several etiologic agents and with different susceptibilities in vitro to several antifungal drugs.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The global rise in microbial resistance, ranging from the agricultural industry to the medical sector, has created the urgent need for novel or supplementary antibiotics. Antimicrobial peptides or “nature’s antibiotics” may be the answer to this major problem. In this study a group of antimicrobial peptides, cyclic decapeptides named tyrocidines, produced by the soil bacterium Bacillus aneurinolyticus, was investigated for their antifungal activity, possible mode of antifungal action and potential applications. The study illustrated that the tyrocidines have significant antifungal activity against a range of phytopathogens, including Fusarium solani and Botrytis cinerea, as well as the human pathogen Candida albicans. The activity of the tyrocidines is influenced by the identity of both the target organism and the media environment. Further evidence was obtained in support of the hypothesis that the tyrocidines are extremely sensitive to their environmental conditions and that they tend to self-assemble to form oligomers. The assessment of a small tyrocidine library and analogues, comprised of eight peptides, revealed no overt structure-activity relationships against fungal pathogens, except for the importance of a tyrosine residue. This indicated an important role for the conserved sequence of the tyrocidines, NQYVOLfP, together with the tendency of the tyrocidines to oligomerise into higher-order active structures in their antifungal activity. The tyrocidines were found to be membrane active toward the fungal pathogens. However, supporting evidence was also obtained for additional mode(s) of antifungal action for the tyrocidines which inter alia induces morphological abnormalities in filamentous fungal target cells. Furthermore, the results also indicated that the membrane activity of the tyrocidines may be influenced by additional factors to that of the composition of the target cell membrane, for instance components of the fungal cell wall. This investigation also indicated the significant potential of the tyrocidines to be developed for the commercial sector. The potent activity of the tyrocidines against agronomically important phytopathogens (significantly higher than the commercial fungicide bifonazole) together with their relative salt stability bodes well for their development as bio-fungicides for the agricultural sector. The tyrocidines also exhibited an overt sinergistic effect on the in vitro candidacidal activity of two key antifungal drugs, caspofungin and amphotericin B. Furthermore, tyrocidine A and caspofungin exhibited synergistic activity in vivo which had a significant positive effect on the survival of C. albicans infected Caenorhabditis elegans. Latter results highlighted their potential to serve as candidates for combinatorial treatment in the medical industry. / AFRIKAANSE OPSOMMING: Die globale verskynsel van mikrobiese weerstand, wat strek vanaf die landbou sektor tot in die mediese bedryf, het ’n dringende behoefte vir die ontwikkeling van nuwe antmikrobiese middels geskep. Antimikrobiese peptiede of “die natuur se antibiotika”, kan moontlik die antwoord op hierdie ernstige problem wees. Tydens hierdie studie is ‘n groep sikliese antimikrobiese peptiede, naamlik die tirosidiene wat deur die grondbakterium Bacillus aneurinolyticus geproduseer word, vir hulle antifungiese aktiwiteit, hulle moontlike meganisme(s) van antifungiese werking en hulle potensiёle aanwendings bestudeer. Hierdie studie het getoon dat die tirosidiene uitsonderlike antifungiese aktiwiteit teen ‘n reeks fitopatogene, insluitend Fusarium solani en Botrytis cinerea, asook teen die mens patogeen Candida albicans het. Die aktiwiteit van die tirosidiene is deur beide die identiteit van die teikenorganisme sowel as die mediumomgewing beїnvloed. Daar is ook verdere bewyse verkry wat die hipotese dat tirosidiene uiters sensitief is tot hulle omgewing en dat hulle neig om te oligomeriseer, ondersteun. Die studie van die klein tirosidien-biblioteek, saamgestel uit agt tirosidiene en analoё, het geen ooglopende struktuur-aktiwiteit verwantskappe opgelewer nie, behalwe vir die oёnskynlike invloed van die tirosien-residu. Laasgenoemde het die belangrikheid van die gekonserveerde aminosuurvolgorde van die tirosidiene, NQYVOLfP, asook die neiging van tirosidiene om hoё-orde aktiewe strukture te vorm deur self-verpakking, beklemtoon. Tydens die studie is daar gevind dat die tirosidiene membraan-aktiewiteit toon teenoor fungiese patogene. Daar is egter ook goeie bewyse vir alternatiewe meganisme(s) van antifungiese werking, wat ondermeer tot morfologiese abnormaliteite in filamentagtige fungi-teikenselle lei, vir die tirosidiene verkry. Die resultate het verder ook daarop gewys dat die membraan-aktiwiteit van die tirosidiene ook deur ander faktore, soos deur komponente van die fungiese selwand, en nie net deur die samestelling van die fungiese membraan beїnvloed word nie. Hierdie ondersoek het ook die aansienlike potensiaal van die tirosidiene vir kommersiёle ontwikkeling en gebruik uitgelig. Die merkwaardige aktiwiteit van die tirosidiene teen fitopatogene van agronomiese belang (wat selfs beter as diè van die kommersiёle swamdoder bifonazole was) tesame met die relatiewe sout stabiliteit van die tirosidiene, is belowende tekens om die tirosidiene as bio-swamdoders vir die landbou sektor te ontwikkel. Die tirosidiene het ook ‘n uitgesproke sinergistiese effek op die in vitro candidasidiese aktiwiteit van twee sleutel antifungiese middels, caspofungin en amphotericin B, getoon. Verder is daar in vivo sinergistiese aktiwiteit gewys deur die kombinasie van tirosidien A en caspofungin wat ’n beduidende positiewe effek op die oorlewing van C. albicans geïnfekteerde Caenorhabditis elegans gehad het. Laasgenoemde dui op die potensiaal van die tirosidiene om in die mediese bedryf as kandidate vir kombinasie-behandeling te dien.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Salvia africana lutea is one of 26 Sage species indigenous to Southern Africa from a total of 900 worldwide. The genus Salvia belongs to the Lamiaceae family. Labeled a ‘broad spectrum remedy’ S. africana lutea amongst other sage species is medicinally important. Reports are many highlighting its benefits, which include from alleviating coughs and colds to gynaecological complaints. Studies have revealed in vitro antimicrobial, anti-cancer and antioxidant activity. Plant secondary metabolites fundamentally have a strong bearing on the phytochemical activities a plant may possess. Consequently the environment indirectly affects the phytochemical properties as it influences the variation in the plant metabolome via plant-environment interactions. Five S. africana lutea plant populations, within the Western Cape province of South Africa were sampled and chemotypes and bioactivity tested. Four populations were wild growing in protected areas namely; Brackenfell, Koeberg, Silwerstroomstrand and Yzerfontein, while the fifth was a garden growing population from Stellenbosch. Using gas chromatography hyphenated with mass spectrometry (GC-MS), compounds such as monosaccharides, carboxylic acids and fatty acids were detected. Variation of compounds identified with 80% certainty was compared across all populations. Stellenbosch population showed some compounds that were not present in the other four sites. These compounds were namely; propanoic acid, rythronic acid, 2-keto-1-gluconic acid and 1,3-dibromobicyclon, while this population also did not have xylitol that was detected in all the other four populations. To consolidate the GC-MS findings, analysis on the metabolite profiles (utilizing liquid chromatography linked with mass spectrometry (LC-MS) and nuclear magnetic resonance (1H NMR)) was done. Principal component analysis (PCA) was applied to the NMR data. The partial least squaresdiscriminant analysis (PLS-DA) was used to integrate LC-MS and NMR data sets. All statistics were performed with the SIMCA-P+ 12.0 software. By integrating LC-MS and 1H NMR analyses, large chemotype differences leading to samples grouping by site, suggested strong plant-environment interactions as factors influencing metabolite composition. Signals distinguishing the Stellenbosch profile were in the aromatic part of the 1H NMR spectra. Antimicrobial activity was tested against two Fusarium species. Fusarium is a plant pathogenic species that causes large agricultural losses particularly in the maize crop, one of the staple foods in the African continent. Some species also produce mycotoxins in infected crop and lead to a significant increase in the risk factor of cancers when contaminated foods are consumed. Four high-mycotoxin producing strains from two species F. verticillioides (MRC 826 and MRC 8267); F. proliferatum (MRC 7140 and MRC 6809) were utilized in all in vitro antifungal assays in this study. A preliminary assay using dichloromethane: methanol (1:1 v/v) crude plant extracts of the five populations; Stellenbosch, Brackenfell, Koeberg, Silwerstoomstrand and Yzerfontein, from 2009 and 2011. All test samples exhibited good activity as the minimum inhibitory concentrations (MIC) values ranged from 0.031 mg ml-1 to 0.5 mg ml-1, values below the latter are regarded as strong inhibitors. The Stellenbosch extracts were the most active for both 2009 and 2011 collections, with the best activity against F. verticillioides MRC 8267 and MRC 826 at 0.031 mg ml-1. While the least activity, albeit still a strong inhibitor, was observed from the Yzerfontein extracts with an MIC value of 0.5 mg ml-1. Generally comparison between the two years revealed that samples collected in 2011 were more potent than those in 2009, possibly due to prolonged storage that may have resulted in chemical decomposition. As the Stellenbosch population had shown the best activity as well as a relatively different chemical profile, leaves from these plants were then introduced into tissue culture conditions. Leaf explants were placed on solid plant growth regulator (PGR)-free Murashige and Skoog media and that supplemented with hormones in various combinations. (two concentrations of benzyl adenine (BA) utilized individually and in combination with naphthalene acetic acid (NAA) 4.4 and 8.8, while for NAA 0.27, 2.7 and 5.4.) Namely: 4.4 BA, 8.8 BA, 0.27 NAA: 4.4 BA, 2.7 NAA: 4.4 BA, 5.4 NAA: 4.4 BA, 2.7 NAA: 8.8 BA and 5.4 NAA: 8.8 BA. The PGR combinations did not induce shooting nor rooting, only callus on PGR-free MS media. Antifungal activity of the callus extract was in the same range as the whole plant extracts from which the leaf explants were harvested from, showing no ‘loss’ of activity after introduction to tissue culture conditions. Metabolite profiles using LC-MS, however, did reveal qualitative and quantitative differences, though they appear to not have any bearing on the activity. A bioassay-guided fractionation was then conducted on samples collected from Stellenbosch. This led to the identification of carnosol and carnosic acid being involved in the anti-Fusarium activity of S africana-lutea. A combinational study revealed no synergistic activity of the two compounds against four Fusarium test strains, with fractional inhibitory concentration (FIC) values of 1.5 and 3.0. Antifungal activity of carnosol and carnosic acid was observed to be in the same range (strong inhibitor) as was the callus and whole plant extracts. The study showed variation in population chemotypes and identified two compounds that are involved in S. africana-lutea activity against Fusarium species. It also provided a tissue culture system onto which mass production of the two bioactives may be achieved from, in the development of new fungicides. / AFRIKAANSE OPSOMMING: Salvia Africana lutea is een van die 26 Salie spesies wat inheems aan Suid-Afrika is uit ‘n totaal van 900 spesies wêreldwyd. Die genus Salvia hoort tot die Lamiaceae familie. S. africana lutea word geklassifiseer as ‘n “breë spektrum geneesmiddel”, en medisinaal as belangrik geag tussen die ander salie spesies, want volgens verslag word dit gebruik vir die verligting van hoes en verkoues tot selfs ginekologiese ongesteldhede. Definitiewe biologiese aktiwiteit eksperimente het anti-mikrobiese, antikanker en anti-oksidant aktiwiteite aan die lig gebring. Plant sekondêre metaboliete het fundamenteel ‘n baie sterk verband met die fitochemiese aktiwiteite van ‘n plant. Gevolglik affekteer die omgewing indirek die fitochemiese eienskappe, want dit beïnvloed die variasie in die plant metaboloom deur die interaksies van die plant met die omgewing. In vyf streke binne die Wes-Kaap van Suid-Afrika waar S. africana lutea bevolkings voorkom, is steekproewe gedoen en chemotipes en bioaktiwiteit getoets. Vier bevolkings was wild-groeiende bevolkings in beskermde areas, naamlik; Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, terwyl die vyfde uit ‘n tuingroeiende bevolking in Stellenbosch geneem is. Deur gas chromatografie gekoppel met massa spektrometrie te gebruik, is primêre samestellings soos monosakkariede, karboksielsure en vetsure gevind. Variasies van samestellings wat met 80% sekerheid geïdentifiseer is, is oorkruis met al die bevolkings vergelyk. Die Stellenbosch bevolking het ‘n paar samestellings geopenbaar wat nie aanwesig was in die ander vier terreine nie. Hierdie samestellings was: propanoësuur, erythroniese suur, 2-keto-1-glukoniese suur en 1,3-dibromobicyclon. Verder het hierdie bevolking geen xylitol gehad nie en dit is in al vier die ander bevolkings gevind. Verdere studies was gedoen met die gebruik van vloeibare chromatografie gekoppel met massa spektrometrie (LC-MS) sowel as kern magnetiese resonansie (1H NMR). Chemiese profiele het hoë variasies getoon, en dus deur te fokus op die aromatiese samestelling streke, het die Stellenbosch terrein duidelik merkbare verskille en punte op die PLS-DA aangetoon. Met die koppeling van NMR data met LC-MS data, is daar gevind dat onderskeidende punte van die NMR PLS-DA wat gegroepeer is met retensie tye die skeiding van die Stellenbosch terrein van ander terreine gedryf het. Dit het onweerlegbaar bewys dat daar variasie binne die vyf bevolkings voorkom en dat Stellenbosch die mees noemenswaardige chemotipe variasie het. Dit blyk uit die anti-mikrobiese eksperimente dat aktiwiteit teen Fusarium heel nuwe belangstelling wek. Fusarium is ‘n plant-patogeniese spesie wat groot landbou verliese veroorsaak veral in die mielie gewasse, een van die stapelvoedsels van die Afrika kontinent. Dit produseer ook mikotoksiene in aangetaste gewasse en hierdie kan lei tot die ontstaan van kankers wanneer besmette voedsel op groot skaal verbruik word. Vier hoë-mikotoksien produserende swamlyne van twee spesies, naamlik F. verticillioides (MRC 826 en MRC 8267) en F. proliferatum (MRC 7140 en MRC 6809) is gebruik in alle in vitro anti-swam ondersoeke in hierdie studie. Die eerste analise het dichloromethan: methanol (1:1 v/v) ongesuiwerde plant ekstrakte bevat van die vyf bevolkings: Stellenbosch, Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein, geneem gedurende 2009 en 2011. Al hierdie toets monsters het goeie aktiwiteit getoon waar die minimum beperkende konsentrasie (MIC) waardes van 0.031 mg ml-1 tot 0.5 mg ml-1 gevarieer het. Waardes laer as laasgenoemde word beskou as sterk inhibeerders. Die Stellenbosch ekstrakte was die mees aktief vir albei jare hierbo genoem, met die beste aktiwiteit teen F. verticillioides MRC 8267 en MCR 826 by 0.031 mg ml-1. Die minste aktiwiteit (hoewel nog ‘n sterk inhibeerder) waargeneem was van die Yzerfontein ekstrakte, met ‘n MIC waarde van 0.5 mg ml-1. Oor die algemeen het ‘n vergelyking tussen die twee jare aangetoon dat die monsters wat in 2011 versamel is veel sterker was dan dié van 2009, moontlik te wyte aan ‘n verlengde bewaringstyd wat moontlik ‘n chemiese dekomposisie ten gevolge gehad het. Omdat die Stellenbosch bevolking die beste aktiwiteit getoon het sowel as ‘n relatief afwykende chemiese profiel, is blare van hierdie plante toe bekendgestel aan weefselkultuur kondisies. Blaar eksplante is op soliede hormoonvrye Murashige en Skoog media geplaas en dit is aangevul met sintetiese auksien Naftaleen asynsuur (NAA) en sitokien Bensiel adenien (BA) individueel en in verskillende kombinasies. Geen wortels of uitloopsels is waargeneem in al die hormoon kombinasies nie maar in die hormoonvrye media het daar egter Kallus in twintig persent van die eksplante voorgekom. Kallus is toe as subkultuur van hormoonvrye MS media gekweek en saamgevoeg en dichloromethan: methanol (1:1v/v) ekstrakte is getoets teen die volgende Fusarium swamlyne MRC 826; MRC 8267; MRC 7140 en MRC 6809. MIC waardes het sterk inhiberende eienskappe getoon met die laagste waarde as 0.025 mg ml-1 teen drie swamlyne: MRC 1740, MRC 8267 en MRC 826, en die hoogste was 0.25 mg ml-1 na 48 uur. Die minimum inhiberende konsentrasie waardes het gestyg na 0.5mg ml-1 na 60 uur, wat ‘n fungistatiese aksie getoon het. Maar van 60 tot 92 uu het waardes egter ‘n swamdodende aksie aangetoon met geen verandering van 0.5mg ml-1 nie. In die identifisering van die bioaktiewe komponente, is die ekstraksie van Stellenbosch se bevolking in dichloromethan: methanol (1:1 v/v) uitgevoer, en met gebruik van vyftig gram van die ekstrak is bioanalise-geleide fraksionering gedoen deur gebruik van ‘n VersaFlash®. Die mees aktiewe fraksie is verder gefraksioneer deur die gebruik van ‘n konvensionele silikajel kolom. Aktiewe fraksies is getoets deur LC-MS te gebruik, en twee verbindings, carnosol en carnosic suur, is geïdentifiseer. Voorbereidende TLC is gebruik om identiteit te bevestig, want fraksies was naas die kommersiele standaarde van die twee verbindings getoets. Sinergistiese aktiwiteit van die twee samestellings is ondersoek deur ‘n antiswam ontleding teen die vier swamlyne uit te voer. Hierdie studie het dus die veronderstelde bestaan van verskillende chemotipes tussen die bevolkings waarvan voorbeelde geneem is, bekend gestel. Veral die Stellenbosch se bevolking het die meeste verskil, heel moontlik omrede die verlengde en hoër versteurings deur die nabyheid van mense. Plant– omgewing interaksies speel ‘n belangrike rol in die metaboloom van plante, wat dan indirek hul eienskappe verander, en in hierdie geval die antiswam aktiwiteit. Die tuingroeiende bevolking was die mees aktief, heel moontlik omrede hierdie aspek. Nietemin was geen bioaktiwiteit verloor waar die mees kragtige bevolking met weefsel kultuur kondisies in aanraking gebring is nie. Dus is dit ideaal vir kommersialisering. Een nuwe belangrike bevinding was die carnosol en carnosic suur wat twee welbekende samestellings is wat meesal geassosieer is met Rosmarinus officinalis en gedokumenteer is vir antioksidant aktiwiteit. Hier dui laasgenoemde samestellings ‘n antiswam aktiwiteit aan teen die getoetste Fusarium swamlyne. Met ‘n gevestigde weefsel kultuur sisteem alreeds in plek, voorsien dit ‘n beginpunt vir die bestudering van hoe hierdie bioaktiewe komponente in massa geproduseer kan word in die ontwikkeling van nuwe swamdodende produkte.

Candida species were discovered more than a century ago as a causative organism of oral thrush. In HIV patients, the presence of oral candidiasis has been shown to be the earliest opportunistic infection. Candidiasis lesions associated with HIV infections are primarily a reflection of the specific change of the host's immune response caused by the virus. Studies of AIDS all over the world show that 58-81% of all patients contract a fungal infection at some time during the primordial stage or after developing AIDS and 10-20% have died as a direct consequence of fungal infections. Twenty four South African medicinal plants were screened using a modification of the NCCSL broth microdilution antifungal test against Candida albicans standard strain ATCC 10231 and two clinical isolates from a 5-month- old baby and an adult. This assay was performed in order to find a traditional remedy to treat oral candidiasis. Of all the screened plants Allium sativum L., Glycyrrhiza glabra L., Polygala myrtifolia L. and Tulbaghia violacea L. aqueous extracts were found to have the best activity. Allium sativum and Tulbaghia violacea aqueous bulb extracts had MIC values of 0.56 mgml-1 and 3.25 mgml-1 respectively, whilst Polygala myrtifolia leaf extracts and Glycyrrhiza glabra rhizome extracts had MIC values of 1.56 mgml-1 and 3.25 mgml-1 respectively when tested against the isolate from a 5-month-old baby, which was the most susceptible of the isolates used. All the extracts had higher MIC values against the standard strain (ATTC 10231), which was the least susceptible to the extracts used. Stability testing was performed on fresh aqueous extracts of A. sativum, G. glabra, T. violacea and P. myrtifolia stored at 4°C, 23°C and 33°C over a period of one week, to determine the stability of the extracts in solution. All A. sativum extracts maintained stability for three days in solution, whilst T. violacea extracts remained stable for only two days in solution. TLC fingerprinting of A. sativum and T. violacea extracts indicated the presence of the known antibacterial and antifungal compound allicin. The activity of allicin and other active compounds was observed by using the bioautographic assay, which was performed on these extracts. P. myrtifolia and G. glabra extracts lost stability 24 hours after preparation at all tested temperatures. However, it was clear with the four plant extracts tested that storage of solutions at higher temperatures reduced their activity and stability. The unpleasant taste and smell of A. sativum and G. glabra could however not be masked, since the intake of these two extracts would result in HIV patients being recognised. These two plants where therefore not considered for further investigation. G. glabra and P. myrtifolia are both saponin containing plants. These could be the active constituents responsible for the anticandidal action. G. glabra is known for its biological activity as an antibacterial agent, whilst other Polygala species have been reported to possess antifungal saponins. Although P. myrtifolia and G. glabra are not stable for more than 24 hours, they do not have an unpleasant smell or taste. These plants are therefore further investigated for use as oral mouthwash in clinics and homes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined. Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate. The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens. The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.

Volatile organic compounds (VOCs) are organic chemicals typically characterized as having low molecular weight, low solubility in water, and high vapor pressure. Consequently, they readily evaporate from liquid to the gaseous phase at standard temperature and pressure. VOCs are produced by many microorganisms as a result of both uninduced and induced metabolic pathways. Volatile-based microbial inhibition in environments such as soil is well founded, with numerous antimicrobial VOCs and formulations having been identified. Inhibitory VOCs are of particular interest as microbial control agents, as low concentrations of gaseous VOCs have been observed to elicit significant antimicrobial effects. It is believed that this contact-independent antagonism may present unique advantages over traditional microbial control methods, particularly where contact-dependent treatment methods are either impractical or inconvenient. This method may be of particular benefit for managing infections where disease may become pervasive in the population, such as with white-nose syndrome (WNS) among bats. A list of potential antifungal compounds and formulations was compiled by referencing the scientific literature. Screening of compounds and formulations was conducted through toxicity analyses and antimicrobial susceptibility testing for the in vitro ability of VOCs and formulations to inhibit growth of select pathogenic fungi. A dispersal system was developed that entailed electrical circuit and software engineering as well as quantitative analysis to validate consistent and accurate dispersal of potential treatment compounds and formulations. Successful completion of these goals culminated in exposure trials involving live bats to determine any significant toxicological effects. Ex and in situ treatment trials were conducted to determine efficacy of promoting the reduction of disease severity and increasing survivorship of infected bat populations. The identification of volatile-based inhibitory compounds, in conjunction with a novel method for accurate and automated delivery, could prove a promising treatment and prophylactic in combatting microbial pathogenesis and contamination.

Une grande variété de curcuminoïdes Cs et de tétrahydrocurcuminoïdes THCs a été synthétisée et leurs propriétés antioxydantes ont été étudiées en détail par la méthode du radical DPPH°. Pour la première fois, il a été montré que les propriétés antioxydantes supérieures des THCs par rapport à leurs homologues curcuminoïdes sont dues à la présence d’atomes d’hydrogène benzyliques dans les THCs. L’association de THCs avec un biopolymère, le chitosane, a permis l’élaboration de films à propriétés antioxydantes et antibactériennes. Des interactions entre les THCs et le chitosane ont été mises en évidence par spectroscopie d’absorption UV-visible. Les THCs et plus particulièrement la tétrahydrocurcumine, se sont révélés capables à la fois d’inhiber le développement de Fusarium proliferatum, champignon qui s’attaque aux céréales, et de limiter la production de mycotoxines issues de cette souche. / A large variety of curcuminoids Cs and tetrahydrocurcuminoids THCs was synthesized and their antioxidant properties were studied in detail by the radical DPPH° method. For the first time, it was shown that the higher antioxidant properties of THCs compared to their counterparts’ curcuminoids are due to the presence of benzylic hydrogen atoms in THCs. The association of THCs with a biopolymer chitosan, allowed the preparation of films presenting antioxidant and antibacterial properties. Interactions between THCs and chitosan were evidenced by UV-visible absorption spectroscopy. THCs and more particularly tetrahydrocurcumin inhibited development of Fusarium proliferatum fungi in cereals, and limited the production of associated mycotoxins.

Invasive fungal infections are a major threat to immune-compromised patients. There is a critical need to develop new antifungal agents because of increasing resistance to the common antifungal drugs. In the first part of this dissertation, methods for preparation of novel barbiturate saponin as antifungals and their biological activities would be described. Barbiturates and steroidal saponins have shown remarkable antifungal activity in the biological assays. Therefore, attempts were directed to combine the barbiturate with the steroid to give novel antifungal agents. The need for extensive SAR studies and to better understand these compounds efforts were directed to synthesize novel saponin barbiturates. Glycosylation of barbiturates was achieved under basic conditions to synthesize mono and disaccharide barbiturates. Saccharide molecules were directly introduced into the barbiturate without requiring protection and deprotection of saccharides. Efficient methods were developed for synthesis of 3β derivatized steroid derivatives containing ether, carbonate, ester and carbamate linker. Synthesized mono and disaccharide barbiturates were incorporated into the steroidal skeleton to give the novel antifungal agents. Several reaction conditions were explored to give the best yield under the most efficient reaction conditions. However, a better understanding and extensive SAR study needs to be done in order to develop more promising and potent antifungal compounds. The second part of this dissertation describes the progress towards monocysteine metal complex synthesis and their biological activities. In this attempt, several protection deprotection strategies were explored and some novel protective groups were designed for peptide synthesis.