Thesis (PhD (Phytomedicine, Paraclinical Sciences))--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (MSc (Phytomedicine, Paraclinical Sciences))--University of Pretoria, 2007. / Includes bibliographical references.

Submitted by Romulus Lima (romulus.lima@ufpe.br) on 2015-03-12T17:46:46Z No. of bitstreams: 2 DISSERTAÇÃO José Robson Neves Cavalcanti Filho.pdf: 853299 bytes, checksum: f5df497c46cc3c25f784a5b6906e7e46 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T17:46:46Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO José Robson Neves Cavalcanti Filho.pdf: 853299 bytes, checksum: f5df497c46cc3c25f784a5b6906e7e46 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / A frequência de infecções fúngicas invasivas devido a fungos patógenos oportunistas aumentou claramente nos últimos anos, sendo Candida albicans responsável por grande parte dos casos. Esta levedura pode causar infecções superficiais de pele e mucosas, é a espécie mais encontrada nas infecções vaginais. Assim, é importante a busca de novas fontes de moléculas bioativas anti-C. albicans. Buchenavia tetraphylla (Combretaceae) é uma espécie neotropical, distribuída desde Cuba ao Rio de Janeiro, sendo uma planta etnomedicinal utilizada por comunidades tradicionais da região Nordeste do Brasil. Em pesquisa recente utilizando folhas da B. tetraphylla foi comprovada sua atividade antimicrobiana. O objetivo deste estudo foi avaliar a atividade antimicrobiana de extratos orgânicos de folhas de Buchenavia tetraphylla frente a Candida albicans, além de analisar o potencial citotóxico e antioxidante do extrato mais ativo. A triagem fitoquímica foi realizada por cromatografia em camada delgada, onde foram observada a presença de hidrocarbonetos, açúcares, glicosídeos, terpenos, taninos e flavonóides. A atividade antimicrobiana, foi determinada pelo método de microdiluição em caldo utilizando 10 linhagens de C. albicans. Os valores de CMI dos extratos variaram a partir de 0,15625 mg/mL a 2,5 mg/mL. Como o extrato metanólico apresentou melhor resultado anti-C. albicans, sua ação combinada com fluconazol foi testada sendo obsevado efeitos aditivos e sinérgicos. Nas células tratadas com o extrato metanólico no valor do CMI foram observadas modificações externas, apresentando maiores rachaduras e encolhimento celular que podem ser atribuído à perda do volume citosólico. Em relação a atividade hemolítica do mesmo extrato testado acima, foi observado um IC50 igual a 3,935 ±0,3088 mg/mL e para atividade antioxidante um IC50 de 64,66 (±1,81 μg/mL). Assim, estes resultados estimulam novas pesquisas sobre aspectos farmacológicos e citotóxicos dos extratos de B. tetraphylla a fim de apoiar a sua aplicação como agente antimicrobiano.

Several investigations into the antimicrobial activity of members of the Combretaceae have been undertaken in recent years. Although the antibacterial properties of various species of Combretum, Terminalia and Pteleopsis have been investigated in depth, this is not the case for their antifungal properties. Due to the increasing importance of fungal infections the aim is to address this by focusing on antifungal activities of Combretaceae species. This was done by focusing on the following objectives: <ol> <li>Developing minimum inhibitory concentration (MIC) and bioautography procedures for fungi to be used in the laboratory in order to screen Combretum andTerminalia species for antifungal activity.</li> <li>Selecting three or four species for further investigation based on antifungal activity and availability.</li> <li>Isolating the antifungal compounds from one or more of the selected species.</li> <li>Determining the chemical structure and in vitro biological activity of the antifungal compound.</li> <li>Developing and applying a protocol and determining in vivo antifungal activity of Combretum and Terminalia extracts and isolated compounds in rats infected with different fungal pathogens.</li> </ol> Leaves of 24 Combretum and 6 Terminaliaspecies were collected in the Lowveld National Botanical Gardens (LNBG) in Nelspruit. After the dried plants were milled to a fine powder, they were extracted with hexane, dichloromethane, acetone and methanol. Chemical constituents of the 120 extracts were analyzed by thin layer chromatography (TLC). The TLC plates were developed with one of the three eluent systems developed in our laboratory that separate components of Combretaceae extracts well i.e.: Ethyl acetate/methanol/water (40:5.4:5) [EMW] (polar/neutral), Chloroform/ethyl acetate/formic acid (5:4:1) [CEF] (intermediate polarity/acidic) and Benzene/ethanol/ammonia hydroxide (90:10:1) [BEA] (non-polar/basic). To detect the separated compounds, vanillin-sulphuric acid-methanol was sprayed on the chromatograms and heated at 110 °C to optimal colour development. Methanol was the best extractant, extracting a greater quantity of plant material than any of the other solvents. There was similarity in the chemical composition of the non-polar compounds of extracts using extractants of varying polarity Qualitative analysis of antioxidant activity, the 2, 2,diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates was used as a screen test for the radical scavenging ability of the compounds present in the different 120 extracts. TLC-DPPH screening method indicated the presence of antioxidant compounds in some of the extracts tested, with C. woodii and C. hereroense showing the most prominent antioxidant activity. Methanol and acetone extracted the most antioxidant compounds based on DPPH TLC. In vitro</li> studies coupled with the phytochemical analysis confirm that the extracts had antioxidant activity. The solvent tolerance of the microorganisms was tested using the following solvents; DMSO, acetone, methanol and ethanol. In order to determine the maximum concentration at which different solvents would allow the test microorganisms to reach normal growth, different concentrations from 10 to 100% were used. Uninhibited growth was evaluated as no toxic effects of the solvent. Methanol and ethanol were found to be toxic. The growths of the fungi were not affected by DMSO and acetone concentrations up to 60%. A serial microdilution assay was used to determine the minimum inhibitory concentration (MIC) values for plant extracts using tetrazolium violet reduction as an indicator of growth. This method had previously been used only for antibacterial activities. To apply it to measuring antifungal activities, a slight modification was made to suit fungal growth conditions. The following fungal pathogens were used: yeasts (Candida albicans and Cryptococcus neoformans), thermally dimorphic fungi (Sporothrix schenckii) and moulds (Aspergillus fumigatus and Microsporum canis). To determine MIC values, growth was checked after 24 and 48 hours to determine the end point. The MIC values of most of the extracts were in the order of 0.08 mg/ml and some had values as low as 0.02 – 0.04 mg/ml after 24 hours incubation. TLC plates were loaded with 100 ㎍ (5 ㎕ of 20 mg/ml) of each of the extracts. The prepared plates were developed in the three different mobile systems used: CEF, BEA and EMW. The chromatograms were dried for a week at room temperature under a stream of air to remove the remaining solvent. The TLC plates developed were inoculated with a fine spray of the concentrated suspension containing approximately 109 organisms per ml of actively growing fungi e.g. conidia for A. fumigatus and yeast cells (blastocysts) for the other fungi in a Biosafety Class II cabinet (Labotec, SA) cupboard. The plates were sprayed until they were just wet, and after drying were sprayed with a 2 mg/ml solution of INT. White areas indicate where reduction of INT to the coloured formazan did not take place due to the presence of compounds that inhibited the growth of tested fungi. During this study we experienced a number of difficulties. Firstly I found that preparing cultures some days before spraying them makes it difficult to get good results, possibly due to quick mycelial overgrowth and blockage of the spray gun with mycelia. The new method was developed. This procedure led to reduced overgrowth of the mycelia. In the study of biologically active compounds from extracts, it was indicated that the extracts had antifungal compounds. Fractionation and bioassay-guided isolation of the antifungal compounds was undertaken on the crude extracts of C. nelsonii, based on very low MIC’s of the crude extracts on all tested pathogens, it had several compound which are active against all pathogens, lastly it is one of the Combretum species which have never being worked on. Antifungal compound was successfully isolated from the leaves of C. nelsonii. The structure was elucidated. After structure elucidation bioassays of isolated active compounds was done to confirm that the compound isolated is the one expected, and how active the compound is, on its own. The compound was very active against all tested pathogens. Cytotoxicity of the acetone extracts of C. imberbe, C. nelsonii, C. albopunctactum and T. sericea were evaluated using Brine shrimp (Artemia salina assay and tetrazolium-based colorimetric assay (MTT assay) on Vero monkey kidney cells. These four extracts were chosen because of the good in vitro antifungal activity of crude extracts and there was intention of using them in in vivo studies in animal models. The results on brine shrimps indicated that the four leaf extracts have LC50 values above 20 ㎍/ml, the recommended cut-off point for detecting cytotoxic activity. Using MTT assay it was found that the four extracts did not suppress mitochondrial respiration in monkey kidney cells. Only C. imberbe was closer to the cut-off value (200 µg/ml), which was used by other authors. In searching for cytotoxic activity to the criteria of the American National Cancer Institute, the LC50 limit to consider a crude extract promising for further purification is lower than 30 µg/ml. In vivo antifungal activity was investigated on the wound irritancy and efficacy of the four most promising, Combretum nelsonii, Combretum imberbe, Combretum albopunctactum and Terminalia sericea extracts applied topically to skin wounds in fungal infected skin wound of rat model. Wound irritancy and wound healing were evaluated by macroscopical, physical and histological methods. Aspects evaluated include wound healing, erythema, exudate formation and possible toxic effects of the extracts. Twenty rats were used in two pilot studies (Exploratory studies and Infection with different pathogens). During the pilot studies rats were not irritated by treatment of infection. The wound healed within three weeks. Only one rat was terminated due to weight loss and it was found that nasal discharge was due to external factors, which were not related to the experiment. The clinical treatment of skin infected with pathogens continues to be a major problem especially in immuno-compromised patients. Therapeutic agents selected for the treatment of infected wounds had ideally shown antifungal activity on in vitro studies. I investigated whether these agents would improve phases of wound healing without producing deleterious side effects. All the parameters showed that the crude extracts and amphotericin B were effective in decreasing formation of the exudate, increasing crust formation and that they have antifungal activities used in in vivo studies. Acetone extract of leaves of C. nelsonii, C. albopunctactum, C. imberbe and T. sericea possessed remarkable growth inhibitory activities against fungal pathogens. Acetone extracts of leaves and isolated compound demonstrated wound healing properties comparable with that of antibiotic powder (amphotericin B). The results of this study in general indicate that the Terminalia and Combretum species possess substantial antifungal properties. This explains the use of these plants in folk medicine for the treatment of various diseases related to fungal infections. / Thesis (PhD (Phytomedicine))--University of Pretoria, 2006. / Paraclinical Sciences / unrestricted

Made available in DSpace on 2015-05-14T13:00:00Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3920642 bytes, checksum: 700cc99c707640a6e78c710c821073b2 (MD5) Previous issue date: 2013-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The family Combretaceae comprises of genera with over 600 species, in which Combretum and Terminalia are the two largest genera, with about 250 species each. Aiming to contribute to the chemotaxonomic study of the family Combretaceae and the genus Combretum, the leaves of Combretum duarteanum were subjected to phytochemical and pharmacological study. The essential oil (2.0 mL) was obtained from fresh leaves by steam distillation of the species, and subsequently analyzed by Gas Chromatography coupled to Massass Spectrometry. The extracts and phases obtained were subjected to usual chromatographic methods for the isolation and purification of the chemical constituents. For the identification of the isolated substances, spectroscopic methods such as infrared, uni-dimensional and bidimensional spectroscopic 1H NMR and 13C NMR for mass spectrometry were used in addition to the comparison with published data. Chemical studies of the C. duarteanum leaves resulted in the identification of twelve substances: cycloartane- 24-ene-1α, 3β-diol (Cd-1), isolated for the first time in the investigated family; 2α, 3β- urs-12-en-28-oic acid (Cd-2); 4α-carboxy-3β-hydroxy-24-cycloartenol (Cd-3), isolated for the first time in the genus Combretum; 2α, 3β, 28-trihidroxiurs-12-ene (Cd-4); 2α, 3β acid, 23α-trihidroxiurs-12-ene-28-oic acid and 2α, 3β, 23α-trihydroxyolean-12-ene- 28-oic acid (Cd-5); stigmasterol (Cd-6); stigmasterol and spinasterol (Cd-7); methyl gallate (Cd-8); 3',5,7-trihydroxy-3, 4'- dimethoxyflavone (Cd-9); 5,7,3',4'- tetrahydroxyflavone-3-O-galactopyranoside (Cd-10), catechin (Cd-11) and kaempferol-3-O-β-D-glucopyranoside (Cd-12). The chemical composition of the essential oil showed twenty-eight components, among which spathulenol (15.1%) and β-caryophyllene (14.1%) were the major compounds. Hexane, chloroform, ethyl acetate, methanol extract phases, as well as the Cd-2, Cd-5, and CD-10 substances were tested against bacterial strains of Gram positive and Gram negative, strains of the Candida genera. All phases, extracts and substances tested did not show any significant antibacterial and antifungal activity. Substances Cd-2, Cd-5, Cd-9 and Cd- 10 were tested in vitro during the cytotoxicity assay on Ehrlich ascites carcinoma cells, in order to evaluate antitumor activity. The substances Cd-2 (IC50 = 17.82 μg/mL) and Cd-5 Cd-2 (IC50 = 31.23 μg/mL) showed potent cytotoxic activity. / A família Combretaceae compreende 18 gêneros com mais de 600 espécies, sendo os dois maiores gêneros Combretum e Terminalia, com aproximadamente de 250 espécies cada. Visando contribuir para o estudo quimiotaxonômico da família Combretaceae e do gênero Combretum, as folhas de Combretum duarteanum foram submetidas ao estudo fitoquímico e farmacológico. O óleo essencial (2,0 mL) foi obtido das folhas frescas da espécie através da hidrodestilação, sendo posteriormente analisada por Cromatografia Gasosa acoplada à Espectrometria de Massass. Os extratos e fases obtidos foram submetidos a métodos cromatográficos usuais, para a purificação e isolamentos dos constituintes químicos. Para a identificação das substâncias isoladas, utilizaram-se métodos espectroscópicos, tais como Infravermelho, Ressonância Magnética Nuclear de 1H e 13C uni e bidimensionais e espectométricos, como espectometria de massas, além de comparação com dados da literatura. O estudo químico das folhas de C. duarteanum resultou na identificação de doze substâncias: cicloartan-24-ene-1α,3β-diol (Cd-1), isolada pela primeira vez na família em estudo; ácido 2α,3β-urs-12-en-28-óico (Cd- 2); 4α-carboxi-3β-hidroxi-24-cicloarteno (Cd-3), isolada pela primeira vez no gênero Combretum; 2α, 3β, 28-trihidroxiurs-12-eno (Cd-4); ácido 2α,3β,23α-trihidroxiurs-12- ene-28-óico e ácido 2α,3β,23α-trihidroxiolean-12-ene- 28-óico (Cd-5); estigmasterol (Cd-6); estigmasterol e espinasterol (Cd-7); galato de metila (Cd-8); 3',5,7-triidroxi- 3,4 -dimetoxiflavona (Cd-9), 5,7,3 ,4 -tetrahidroxiflavona-3-O-galactopiranosídeo (Cd- 10), catequina (Cd-11) e Canferol-3-O-β-D-glicopiranosídeo (Cd-12). A composição química do óleo essencial apresentou vinte e oito componentes, dentre os quais, espatulenol (15,1%) e β-cariofileno (14,1%), foram os compostos majoritários. As fases hexânica, clorofórmica, acetato de etila, o extrato metanólico, bem como as substâncias Cd-2, Cd-5, e Cd-10 foram testadas frente cepas de bactérias Gram positivas e Gram negativas, cepas de fungos do gênero Candida. Todas as fases, extrato e substâncias testadas não mostraram qualquer atividade antibacteriana e antifúngica considerável. As substâncias Cd-2, Cd-5, Cd-9 e Cd-10 foram testadas in vitro no ensaio de citotoxicidade em células de carcinoma ascítico de Ehrlich para avaliar a atividade antitumoral. As substâncias Cd-2 (CI50 = 17,82 μg/mL) e Cd-5 Cd- 2 (CI50 = 31,23 μg/mL) mostraram uma potente atividade citotóxica.

L'étude phytochimique de plantes médicinales suscite depuis toujours beaucoup d'intérêt à cause de la variété de métabolites secondaires à activités biologiques intéressantes fabriqués par les plantes. Dans ce travail de thèse, notre contribution à l'amélioration de la connaissance de quelques plantes médicinales ivoiriennes par l'isolement et la caractérisation de leurs molécules a été rapportée. Les espèces Gouania longipetala, Ventilago africana (Rhamnaceae), Combretum racemosum (Combretaceae) et Glyphaea brevis (Malvaceae) ont été investiguées. Soixante métabolites secondaires ont été identifiés par les techniques spectroscopiques (RMN et SM). Une première partie présente l'investigation phytochimique des parties aériennes de G. longipetala qui a conduit à l'isolement de 23 composés dont neuf saponosides à squelette de type dammarane et quatre hétérosides flavoniques de structures nouvelles. Dans un second temps, nos travaux ont mené à l'élucidation de 12 flavonoïdes dont deux nouveaux et un acide sesquiterpénique à partir des feuilles de V. africana. Ces flavonoïdes ont été identifiés à la rhamnocitrine substituée en position 3 par une chaîne diosidique ou triosidique. Les racines de Combretum racemosum ont fourni 16 triterpènes dont un à squelette de type 18-oléanène de structure nouvelle. Finalement, un dérivé du cinnamide et dix nouveaux C-glycosides de phényl nonane substitués en position 9 par un iminosucre ont été caractérisés à partir des racines de G. brevis. Cette étude correspond à la première identification structurale de métabolites secondaires des espèces Gouania longipetala, Ventilago africana (Rhamnaceae) et Combretum racemosum (Combretaceae). / The phytochemical study of medicinal plants has always raised a lot of interest because of the variety of secondary metabolites with interesting biological activities produced by plants. In this thesis, our contribution to improving the knowledge of some ivorian medicinal plants by the isolation and characterization of their molecules has been reported.Species Gouania longipetala, Ventilago africana (Rhamnaceae), Combretum racemosum (Combretaceae) and Glyphaea brevis (Malvaceae) were investigated. Sixty secondary metabolites were identified by spectroscopic techniques (NMR and MS).The first part presents the phytochemical investigation of the aerial parts of G. longipetala which led to the isolation of 23 compounds including nine new saponins with skeleton type dammarane and four new flavonoids glycosides.In a second step, our work has led to the elucidation of 12 flavonoids including two original and a sesquiterpene acid from the leaves of V. africana. These flavonoids have been identified as rhamnocitrin substituted at position 3 by a disaccharide or trisaccharide chain. The roots of Combretum racemosum provided 16 triterpenes and one of them is a new compound with a skeleton type 18-oleanene. Finally, a derivative of cinnamide and ten new alkyl phenyl C-glycosides substituted at position 9 by iminosugar have been characterized from the roots of G. brevis.This study represents the first structural identification of secondary metabolites of the species Gouania longipetala, Ventilago africana (Rhamnaceae) and Combretum racemosum (Combretaceae).

This thesis describes a phytochemical investigation of four medicinal plants, namely, Agave attenuata (Agavaceae), Balanites maughamii (Balanitaceae), Astrotrichilia parvifolia (Meliaceae) and Combretum fragrans (Combretaceae). Investigations of A. attenuata and B. maughamii were undertaken as biological studies have shown that these plants possess anti-bilharzial properties. Schistosomiasis is an important public health issue in South Africa and attempts to deal with infected rural communities have forced scientists to focus their attention on snail control using plant molluscicides. A. attenuata yielded two glycosides, timosaponin A III (compound I) and 5?-pregn-16-en-20one- 3?-O-tetrasaccharide (compound II), while a coumarin, scopoletin (compound III) and a sterol, stigmasterol (compound IV) were isolated from B. maughamii A dammarane, shoreic acid (compound V) was isolated from A. parvifolia, this formed part of an ongoing investigation into the Meliaceae of Madagascar. Plants of the family Combretaceae are widespread in Africa and are used by traditional healers for a wide range of illnesses. The leaves and bark are used abundantly, however, the winged fruits are never eaten as they are highly toxic to animals and humans. The leaf surface is covered with epidermal scale-like trichomes through which acidic triterpenoid mixtures are secreted. Six lupane-type triterpenoids were isolated from C. jragrans, namely, lupeol (compound VI), lupenone (compound VII), lupeol 3?- docosanoate (compound VIII), lupeol 3?-eicosanoate (compound IX) hennadiol (compound X) and 30hydroxylupenone (compound XI). All the above compounds were isolated by column chromatography and the structures were elucidated by means of NMR spectroscopy, mass spectrometry and infra red spectroscopy. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

Thesis (MSc.(Pharmacology)--Faculty of Health Sciences)-University of Pretoria, 2002.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Pharmacology))--University of Pretoria, 2007. / Pharmacology / unrestricted

MSc (Botany) / Department of Botany / See the attached abstract below