Thesis (Ph.D. (Botany)) -- University of Limpopo, 2023 / The use of medicinal plants as a source of treatment for various ailments including fungal infections is still practised in South Africa and across the globe. Fungal infections especially of Cryptococcus, Candida and Aspergillus species are the main cause of morbidity and mortality worldwide, particularly in developing countries. Traditional medicine is used as a source of remedies worldwide and has contributed extensively towards the development of modern medicine. Twelve selected medicinal plants (Kleinia longiflora DC. Berchemia discolor (Klotzsch) Hemsl., Persea americana Mill., Sansevieria hyacinthoides (L.) Druce, Dichrostachys cinerea (L.) Wright &Arn, Withania somnifera Dunal (Ashgandh), Momordica balsamina L., Lonchocarpus capassa, Pappea capensis, Rhus lancea L. fil, Peltophorum africanum, Maytenus heterophylla (Eckl. & Zeyh.) Robson) were analysed qualitatively for antifungal activities against Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. The plant materials were extracted with solvents of various polarities such as acetone, dichloromethane, methanol, hexane, and water. Methanol extracted the highest amount of crude extracts from all the plant species as compared to other organic solvents. Chemical components of the extracts were analysed using aluminum backed Thin Layer Chromatography (TLC) plates and developed using three different eluent systems: Ethyl acetate: methanol: water [EMW], Chloroform: ethyl acetate: formic acid [CEF] and Benzene: ethanol: ammonia hydroxide [BEA]. CEF was the best eluent solvent system since it separated more compounds from plant extracts. This indicates that the active compounds were relatively non-polar. More chemical compounds were observed in TLC chromatograms separated with CEF, followed by BEA and EMW. All plant extracts had shown different chemical components when separated from the three solvent systems. The bioautography and serial dilution assays were used to determine the biological activity of plant extracts against the tested microorganisms, respectively. All the tested plant extracts revealed some varying degrees of fungal inhibition, with minimum inhibitory concentrations (MIC) values ranging between 0.02 mg/ml and 2.5 mg/ml. The aqueous extracts had shown some activity against the tested microorganisms. Noteworthy, antifungal activity was observed in acetone, DCM, hexane, and methanol root extracts of D. cinerea against the three tested microorganisms with MIC values ranging between 0.02 mg/ml and 0.04 mg/ml. Furthermore, acetone extracts of D. cinerea and P. africanum had excellent activity against three fungal pathogens with MIC values of 0.02 mg/ml and 0.08 mg/ml. Active compounds were observed in dichloromethane extracts of W. somnifera with Rf values of 0.40 and 0.64. In TLC chromatograms separated with BEA, active compounds were observed in acetone, hexane, and methanol leaf extract of P. americana, this indicates that the fungal compounds were relatively non-polar. No active compounds were observed in plant extracts of K. longiflora. Active compounds were visible in all extracts of P. capensis in TLC chromatograms developed in CEF and EMW. The antioxidant present in plants prevents the free radicals from causing various diseases in humans by inhibiting the oxidation of free radicals at the cellular level. The qualitative and quantitative 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods were used to determine the antioxidant activities of plant extracts. The presence of antioxidant compounds was indicated by yellow bands against the purple background on the TLC plates. More antioxidant compounds were observed in acetone and dichloromethane extracts of S. hyacinthoides developed in BEA compared to other plant species tested. Methanol, hexane, and water extracts of L. capassa revealed good antioxidant activity against DPPH by having a high percentage of inhibition compared to other solvents. Noticeably, extracts of P. africanum possess strong antioxidant activity as compared to other plant species. Solvent-solvent fractionation using column chromatography of the acetone extract led to the isolation of six compounds. The biological activity of the isolated compounds of L. capassa was investigated against the tested pathogenic fungi. The isolated compounds revealed some varying degrees of inhibition to the fungal pathogens. The largest quantity was isolated from compound 1 (80 mg), compound 4 (39 mg), compound 3 (27 mg), compounds 2 and 5 (14 mg) and the least was compound 6 (4.8 mg). However only three compounds were successfully identified as Lupeol (compound 1), Friedelin (compound 3) and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”- dimethylpyrano-[2”,3”:7,8]-flavanone (compound 4). Compounds 2, 5 and were not identified due to some impurities. More importantly, the isolated compounds exhibited good antioxidant activity in qualitative and quantitative scavenging assays, which indicates that isolated compounds of L. capassa can scavenge the free radicals causing fungal infections in humans. The results support the traditional use of the selected plants to combat fungal infections and related ailments by the local people and traditional health practitioners in Vhembe District, Limpopo Province. The (3-(4,5-dimethylthiazol) -2,5-diphenyltetrazoliumbromide) (MTT) assay was used to determine the toxic effects of the plant crude extract and isolated compounds. Lupeol and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”-dimethylpyrano-[2”,3”:7,8]- flavanone revealed the same degree of cytotoxicity against the Vero monkey kidney cells. All the compounds were not toxic with an LC50 value of ˃ 0.2 mg/ml. / University of Limpopo and National Research Foundation

Studies of biodegradation in lignins by basidiomycetes yeasts show the conversion of lignin in various degradation products among which vanillin, a valuable substance, suggested to be a strong inhibitor of both fermentation and growth of yeasts, stands. Sporidiobolales yeasts used in these experiments were aimed to be identified by their highly conserved ITS region as well as studied in vanillinsupplemented medium through, vanillin-supplemented plates, TLC and Neubauer’s chamber to find out which, among the several isolates tested, were the most resistant ones, understand how they take up vanillin and how their growth is affected by the presence of the phenolic compound. Two strains were identified as Rhodotorula babjevae. One of them, L4, together with LS22, Rhodosporidium kratochvilovae, could withstand and biodegrade high concentrations of vanillin, producing biodegradation products with Rf values similar to the ones know for vanillic acid and vanillyl alcohol. Better growth in medium supplemented with small doses of vanillin was found, as well as disparity among the same species and their metabolic features, therefore, herbicides resistance was suggested as a reason for strains divergence. Further morphological-species comparison could also describe if there exist a relation between them. / Estudios sobre la biodegradación de ligninas por levaduras basidiomicetes muestran la conversión de lignina en distintos productos de degradación, entre los cuales se encuentra la vainillina, un fuerte inhibidor de la fermentación y el crecimiento de levaduras. Las levaduras Sporidiobolales utilizadas en estos experimentos han intentado ser identificadas a través de la región ETI, muy conservada, además de estudiadas en medios suplementados con vainillina mediante placas suplementadas con vainillina, CCF y cámara de Neubauer para averiguar cuáles son las cepas más resistentes, entender cómo metabolizan la vainillina y cómo su crecimiento se ve afectado por la presencia de dicho compuesto. Dos cepas fueron identificadas como Rhodotorula babjevae. Una de ellas, L4, junto con con la cepa LS22, Rhodosporidium kratochvilovae, pudieron soportar y biodegradar elevadas concentraciones de vainillina, originando productos de biodegradación con valores de Rf similares a los del ácido vanílico y alcohol vanílico previamente conocidos. Se encontró un crecimiento mejor en medios suplementados con pequeñas dosis de vainillina además de una disparidad entre mismas especies y sus características metabólicas, así, herbicidas han sido sugeridos como una posible causa en dicha divergencia. Una futura comparación morfología-especie podrá describir si existe relación entre ambos.

Ce projet vise à étudier, par une approche pluridisciplinaire, l’influence des la cristallisation des protéines membranaires (PM) en prenant pour protéine modèle le complexe photosynthétique RC-LH1-pufX de Rhodobacter blasticus. Des cristaux de ce complexe avaient été obtenus en présence de dodécyl-!-maltoside (DDM) et avaient diffractés à 8 Å de résolution. L’objectif final est de pouvoir améliorer, de façon rationnelle, la qualité des cristaux du complexe RC-LH1-pufX grâce à une meilleure compréhension des mécanismes mis en jeu. Dans un premier temps, trois tensioactifs dérivés du DDM ont été conçus et synthétisés. L’intérêt est d’augmenter la rigidité et le caractère lipophobe des parties hydrophobes des tensioactifs par rapport au DDM, pour les rendre moins déstabilisants envers la protéine: soit par l’incorporation d’un groupement bicyclohexyle (PCC-maltoside), soit par l’ajout d’un segment fluoré de longueur modulable (F4H5- et F2H9-maltoside). Nous avons inclus également le F8TAC, tensioactif fluoré utilisé depuis une vingtaine d’années pour le maintien en solution des PM, et les "tripodes", amphiphiles faciaux dont la géométrie particulière n’avaient jamais été testée. Nous avons ensuite réalisé la caractérisation physico-chimique, en solution, de ces tensioactifs et du DDM en terme de CMC (concentration micellaire critique), nombre d’agrégation, taille (par diffusion de la lumière dynamique, DLS), facteur de forme (par diffusion des rayons X aux petits angles, SAXS) et facteur de structure (par mesure du second coefficient du viriel, indicateur du potentiel des tensioactifs à initier la cristallisation)afin de déterminer les caractéristiques importantes au maintien en solution et à la cristallisation des PM. Le PCC-malt présentant le même comportement que le DDM,nous l’avons sélectionné pour réaliser une étude en présence de la protéine.Après avoir mis au point une méthode de dosage des tensioactifs par HPTLC (HighPerformance Thin Layer Chromatography) et identifier les lipides présents dans les de Rhodobacter blasticus, nous avons pu quantifier les quantités de lipides et de tensioactifs associés à la protéine en présence de DDM et de PCC-malt.Enfin, dans une dernière partie, nous avons réalisé des essais de cristallisation du complexe RC-LH1-pufX en présence des tensioactifs sélectionnés pour faire le lien entre les conditions de cristallisation et l’étude physico-chimique des micelles en solution. / Membrane proteins (MPs) are involved in the regulation of various fundamental cellular functions, such as cell recognition, receptor-mediated signal transduction and selective transportation of metabolites. However despite their huge importance, researches in MPs are relatively limited. For example MPs represent approximately 30% of the human proteome and less than 1% of current Protein Data Bank entries. Indeed, the presence of hydrophobic domains in MPs makes them not soluble in water. Therefore surfactants are used to extract MPs from their native environment and substitute for lipids around the transmembrane domain of the protein, forming water-soluble complexes. However MPs are often unstable in surfactant solution because of the intrusion of the alkyl chain of the surfactant into the transmembrane domain and/or the dissociation of stabilizing lipids, cofactors or subunits. Our project aims to study, through a multidisciplinary approach, the influence of surfactants for MP crystallization. Since dodecylmaltoside (DDM) is the most common gentle detergent used for MPs crystallization, we synthesized three new structurally DDM-derivative surfactants whose designs were expected to limit MPs inactivation. The objective was to increase the rigidity and the lipophobic behavior of the hydrophobic moiety by adding a bicyclohexyl group (PCC-maltoside) or using different lengths of fluorinated segments (F4H5- and F2H9-maltoside). Comparison of these surfactants with DDM occurs on:Physico-chemical properties: Surfactants are characterized by their CMC, molar mass (SEC-MALS, SAXS), hydrodynamic size (DLS), form factor (SAXS) and structure factor (A2, indicator of surfactant potential to lead to crystallization) in order to determine their best characteristics for MPs crystallization. Biochemical properties: We chose the RC-LH1-Puf X complex from Rhodobacter blasticus as model protein because of its biological interest. Besides this membrane protein has already been crystallized in DDM giving a low diffraction resolution (8Å). A better understanding of mechanisms involved in crystallization is a prerequisite for the development of rational approaches to increase crystals quality. Therefor protein complexes are characterized by quantifying lipids and surfactants bound to the transmembrane domain. Surfactant and lipid assays are performed by High Performance Thin Layer Chromatography (HPTLC). Crystallization trials: we show the link between crystallization and surfactants physico-chemical properties

Die Rückstandsanalytik tierischer Lebensmittel ist eine anspruchsvolle Aufgabe aufgrund des hohen Lipidanteils der Proben sowie des sich stetig vergrößernden Wirkstoffspektrums. Heutzutage werden für die Probenaufarbeitung die DFG S 19 Methode, mit der vorrangig unpolare Analyten nachgewiesen werden und zunehmend die QuEChERS Methode eingesetzt, die insbesondere auf die Erfassung polarer Pestizide abzielt. In dieser Arbeit wurde eine moderne Multirückstandsmethode für Hühnereier entwickelt, um sowohl gas- als auch flüssigkeitschromatographisch (GC, LC) erfassbare Wirkstoffe zu analysieren. Dazu gehören unpolare PCBs, Pyrethroide und Organochlorpestizide, aber auch polarere Organophosphate, Triazole und Carbamate. Das Verfahren basiert auf der Extraktion mittels Matrix Solid Phase Dispersion, der Reinigung auf Grundlage einer modifizierten Gelpermeationschromatographie (GPC) und zwei verschiedenen Festphasenextraktionen (SPEs) für GC- und LC-erfassbare Pestizide sowie der Quantifizierung mittels GC- und LC-MS/MS. Dünnschichtchromatographisch wurde die effektive Entfernung hochmolekularer Lipide durch die modifizierte GPC und niedrigmolekularer Fette durch die SPEs belegt. Laut der für Ei durchgeführten Validierung erfüllten 164 der 172 untersuchten Pestizide und alle sechs PCBs die Leistungskriterien für die amtliche Rückstandskontrolle - zumeist am niedrigsten validierten Level (5 µg/kg bzw. 0,5 µg/kg). Ausnahmen bildeten sehr polare LC-Pestizide (z.B. Aminopyralid, Clopyralid, MCPA, Quinmerac) und pH-Wert-abhängige GC-Analyten (Nicotin, Tolylfluanid, Dichlofluanid), die auch mit den etablierten Verfahren schwierig zu analysieren sind. Weiterhin verdeutlichte die erfolgreiche Untersuchung von verschiedenen Ringversuchsmaterialien, dass die ursprünglich für Eier entwickelte Methode auch für mageres Geflügelfleisch und Sahne genutzt werden kann. Gegenüber den etablierten Verfahren wies die neue Methode deutliche Vorzüge auf. So belegte die Dünnschichtchromatographie, dass mit der neuen Methode Cholesterin, aber auch freie Fettsäuren besser abgetrennt werden als mit den etablierten Verfahren. Die neue Methode verbrauchte im Vergleich zur DFG S 19 Methode 46 % weniger Lösungsmittel und ermöglichte eine Verdopplung des Probendurchsatzes innerhalb von 8 h. Zudem eignete sich das entwickelte Verfahren laut den Validierungsdaten für GC-Analyten deutlich besser als die QuEChERS Methode und etwas besser als die DFG S 19 Methode (v.a. für Pyrethroide). Hinsichtlich der LC-Analyten unterschieden sich die neue und die QuEChERS Methode nur bei wenigen Analyten. Mit dem neuen Verfahren konnten folglich im Gegensatz zu den etablierten Methoden sowohl unpolare GC- als auch polare LC-Analyten sicher erfasst werden.

University of Macau / Institute of Chinese Medical Sciences

Ce projet vise à étudier, par une approche pluridisciplinaire, l'influence des la cristallisation des protéines membranaires (PM) en prenant pour protéine modèle le complexe photosynthétique RC-LH1-pufX de Rhodobacter blasticus. Des cristaux de ce complexe avaient été obtenus en présence de dodécyl-!-maltoside (DDM) et avaient diffractés à 8 Å de résolution. L'objectif final est de pouvoir améliorer, de façon rationnelle, la qualité des cristaux du complexe RC-LH1-pufX grâce à une meilleure compréhension des mécanismes mis en jeu. Dans un premier temps, trois tensioactifs dérivés du DDM ont été conçus et synthétisés. L'intérêt est d'augmenter la rigidité et le caractère lipophobe des parties hydrophobes des tensioactifs par rapport au DDM, pour les rendre moins déstabilisants envers la protéine: soit par l'incorporation d'un groupement bicyclohexyle (PCC-maltoside), soit par l'ajout d'un segment fluoré de longueur modulable (F4H5- et F2H9-maltoside). Nous avons inclus également le F8TAC, tensioactif fluoré utilisé depuis une vingtaine d'années pour le maintien en solution des PM, et les "tripodes", amphiphiles faciaux dont la géométrie particulière n'avaient jamais été testée. Nous avons ensuite réalisé la caractérisation physico-chimique, en solution, de ces tensioactifs et du DDM en terme de CMC (concentration micellaire critique), nombre d'agrégation, taille (par diffusion de la lumière dynamique, DLS), facteur de forme (par diffusion des rayons X aux petits angles, SAXS) et facteur de structure (par mesure du second coefficient du viriel, indicateur du potentiel des tensioactifs à initier la cristallisation)afin de déterminer les caractéristiques importantes au maintien en solution et à la cristallisation des PM. Le PCC-malt présentant le même comportement que le DDM,nous l'avons sélectionné pour réaliser une étude en présence de la protéine.Après avoir mis au point une méthode de dosage des tensioactifs par HPTLC (HighPerformance Thin Layer Chromatography) et identifier les lipides présents dans les de Rhodobacter blasticus, nous avons pu quantifier les quantités de lipides et de tensioactifs associés à la protéine en présence de DDM et de PCC-malt.Enfin, dans une dernière partie, nous avons réalisé des essais de cristallisation du complexe RC-LH1-pufX en présence des tensioactifs sélectionnés pour faire le lien entre les conditions de cristallisation et l'étude physico-chimique des micelles en solution.

The paper aims to mediate thin film chromatography analysis by means of drugs commonly found in households (analgesic-antipyretics) to secondary school and grammar students, or to college students. For this purpose, laboratory tasks have been designed to cover all levels of the Bloom pyramid so that the educational potential is as high as possible. This is closely related to the topic of drug expiration and treatment. Pupils and students should take on both the competences of knowledge, skills, and attitudes. Keywords: thin layer chromatography; TLC; chromatography in education; educational experiment; expiratory medication; drug handling; analgesic- antipyretics, acetylsalicylic acid, paracetamol

Made available in DSpace on 2014-07-29T15:16:27Z (GMT). No. of bitstreams: 1 Dissert Leslivan Ubiratan de Moraes.pdf: 723767 bytes, checksum: 88adcd404b3c30cc9f22d95b44bd13b3 (MD5) Previous issue date: 2008-04-16 / The jurubeba was identified that such species was Solanum scuticum M. Nee, from which pharmacognostic and morphanatomic data were not available to Goiás. Because of that, we tried some complementary data. For the morphoanatomic evaluation of the leaves some cuts were carried out as described by Kraus and Arduin (1997). For the phytochemical trial, it was used some methodologies as described by Costa (2001). With the phytochemical screening, were identified alkaloids, flavonoids, coumarins, anthraquinones and saponins in prospecting phytochemical. Both in phytochemical trial, as in the phytochemical prospection, it was found alkaloids. Due to the biological activities of these secondary metabolites, we obtained the alkaloid fraction, using the dust of the leaves, and we got the ethanol extract. The fractions were divided by polarity and they were evaluated by thin-layer chromatography. Microbiological evaluation was carried out to verify possible contaminants. Such evaluation did not reveal the presence of microorganisms, and it was raised the possibility of antimicrobiotic activity of the raw extract in twenty three strains of bacteria and in two yeasts. According to the antimicrobial tests, the extract presented some features, as the difficult of solubility in aqueous medium, and the best dilution was achieved in dimethyl sulfoxide (DMSO) 50%. These tests also demonstrated the difficult of the solubilization of the fractions, which it will be used in the experiments of Vero cells cultures. It was verified the low antimicrobial activity of the raw hidroethanolic extract. Of all the fractions, the alkaloid fraction presented the best solubility in DMSO 0.3%. So, it was proceeded the dilution and the evaluation of the alkaloid fraction in Vero cells, and it was evaluated morphology, viability and cellular proliferation. We finally verified the citotoxicity of the alkaloid fraction in the statistical analyses, and in the vitality test carried out by Trypan blue, and also in the morphological alterations compatible to citotoxity alterations. / A espécie de jurubeba estudada no presente trabalho é Solanum scuticum M. Nee, da qual não eram disponíveis dados farmacognósticos e nem morfoanatômicos para Goiás. Para a avaliação morfoanatômica das folhas S. scuticum realizaram-se cortes à mão livre e cortes permanentes como descrito por Kraus e Arduin (1997). Para realização de triagem fitoquímica utilizou-se as metodologias descritas por Costa (2001). Com na triagem fitoquímica, foram identificados alcalóides, flavonóides cumarinas, heterosídios antraquinônicos e heterosídios saponínicos na prospecção fitoquímica. O extrato etanólico das folhas foi obtido e apartir do mesmo procedeu-se o fracionamento por diferença de polaridade, e realizaram-se avaliações dos seus componentes através de cromatografia de camada delgada. Testou-se a esterelidade da droga em pó e a possível atividade antimicrobiana do extrato bruto hidoalcoólico de S. scuticum. Tal avaliação não revelou a presença de microrganismos, sendo levantada a possível atividade antimicrobiana do extrato bruto frente a vinte e três cepas de bactérias e duas de leveduras. Os testes demonstraram também a dificuldade de solubilização das frações, as quais seriam utilizadas nos experimentos de cultura de células da linhagem Vero. Sendo averiguada baixa atividade anti-microbiana do extrato hidroalcóolico bruto. Tendo em vista as atividades biológicas do grupo dos alcalóides procedeu-se à obtenção da fração alcaloídica a partir do pó das folhas. Pelos testes antimicrobianos o extrato apresentou características de difícil solubilidade em meio aquoso, a melhor diluição para emprego do extrato foi obtida em dimetilsulfóxido (DMSO) 50%. Das frações a que possuiu melhor solubilidade em DMSO foi à alcaloídica, na concentração de 0,3%. Assim procedeu-se a diluição e a avaliação da atividade da fração alcaloídica em células da linhagem Vero, avaliando-se a morfologia, viabilidade e proliferação celular frente aos tratamentos realizados. Verificando-se atividade citotóxica da fração alcaloídica tanto nas avaliações estatísticas do teste de vitalidade por azul de Trypan, quanto nas alterações morfológicas compatíveis com alterações citotóxicas.

This diploma thesis is focused on the study of structure and physicochemical properties of organic fractions of humic substances, which were obtained by the method of thin-layer chromatography. Humic substances, which were used in this study, were isolated from a sample of Slovak alginite based on the procedure of the International Humic Substances Society (IHSS). The following analytical techniques were selected for the characterization of isolated humic substances: thermogravimetric and elemental analysis, molecular absorption spectroscopy (UV/Vis), Fourier transform infrared spectroscopy (FTIR) and steady-state fluorescence spectroscopy. Each organic fraction of humic substances were characterized by molecular absorption spectroscopy (UV/Vis) and steady-state fluorescence spectroscopy. Using steady-state fluorescence spectroscopy, humic substances were found to be composed of fluorophores of humic and non-humic (protein) character. Organic fractions corresponding to the humic fluorophores were characterized by a higher content of oxygen substituents on the aromatic nukleus, a higher degree of aromaticity and also a higher molecular weight. The aim of this diploma thesis was to design and optimize the process of organic matter fractionation for the purpose of detailed understanding of the structure and properties of humic substances, which were isolated from the sedimentary rock alginite. Based on this, the practical applicability of the thin layer chromatography method to significantly reduce the molecular heterogenity of the studied humic substances was evaluated.

(EN) In the first part of this work, analytical methods for determination of impurities of active pharmaceutical ingredients (API) in combined pharmaceutical dosage forms were developed and validated. Development of the methods covered both the optimization of sample preparation procedure and chromatographic conditions. The methods were validated according to International Conference on Harmonization guideline and both of them were confirmed to be able to analyze stability samples. Impurities in paracetamol, codeine phosphate hemihydrate and pitophenone hydrochloride in the presence of fourth API fenpiverinium bromide were separated by using ion-pair reversed phase chromatography with gradient elution. Symmetry C18, 250 x 4,6 mm, 5 µm heated to 35 řC was used as a separation column. A diode array detector was used. The detection wavelengths were set as follows: 220 nm for paracetamol impurity K, 245 nm for paracetamol and its other impurities and 285 nm for codeine, pitophenone and their impurities. Impurities in valsartan, amlodipine besylate and hydrochlorothiazide were separated by reversed phase UHPLC method with gradient elution. Chromatographic column Zorbax Eclipse C8 RRHD, 100 x 3,0 mm, 1,8 µm heated to 30 řC and spectrophotometric detection were used. The detection wavelengths were set as...