Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-09-27T12:20:20Z No. of bitstreams: 1 TeseRNF.pdf: 5131887 bytes, checksum: 5f32fa62636719671f0675a379d2cd24 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:55:39Z (GMT) No. of bitstreams: 1 TeseRNF.pdf: 5131887 bytes, checksum: 5f32fa62636719671f0675a379d2cd24 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:55:44Z (GMT) No. of bitstreams: 1 TeseRNF.pdf: 5131887 bytes, checksum: 5f32fa62636719671f0675a379d2cd24 (MD5) / Made available in DSpace on 2016-09-27T19:55:52Z (GMT). No. of bitstreams: 1 TeseRNF.pdf: 5131887 bytes, checksum: 5f32fa62636719671f0675a379d2cd24 (MD5) Previous issue date: 2016-01-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The search for new sustainable alternative energy sources has followed the increasing concerns with common welfare and fossil fuel shortage. In this context, Bioethanol is a good option and lignocellulosic biomass is an interesting way of obtaining it. The enzymatic conversion of lignocellulosic biomass in fermentable sugars still is a costly process, which makes characterization mechanisms indispensable to make it economically viable. Being of great importance in the lignocellulosic biomass convertion, β-glucosidases catalyzed reaction is the last step in the saccharification processes. Beta glucosidase hydrolyze non-reduced β-D-glycoside terminals, releasing β-D-glucose. GH 1 and GH 3 are the families of those most studied enzymes. However, structural and functional data from this GH 3 family of enzymes are still scarce. This work aimed at the biochemical and structural characterization of β-glucosidase from Bifidobacterium adolescentis (BaBgl). This enzyme has a catalytic domain (CCD) and a fibronectin III-like domain (FnIII) whose function is still unknown. Biochemical data showed optimal conditions for enzyme activity at pH from 6.0 to 6.5, temperature at 45 ° C and synthetic substrate specificity of 4-nitrophenyl- -Dglucopyranoside (pNPG). The values of kinetic parameters, KM and Vmax, were 0.32±0.03 mM e 0.37±0.01 nmol/min, respectively. The enzyme doesn’t have transglycosylation mechanisms, indicating only hydrolytic activity. Some monosaccharides such as xylose and galactose increased the enzyme activity significantly, while glucose and arabinose inhibited it. The crystal structural model of the BaBgl revealed an N-terminal domain with fold like a TIM barrel, an intermediate sandwich α / β domain and a third C-terminal like FnIII domain. In this work we also studied the transglycosylation mechanisms of two β-glucosidases from Trichoderma harzianum (ThBgl1 and ThBgl2). Both enzymes exhibit transglycosylation reaction but the ThBgl1 showed a hydrolysis/transglycosylation ratio lower than the one for ThBgl2. Crystallographic structures shows a typical folding for GH family 1 β-glucosidases, folding in the form of a TIM barrel (α / β)8. However, ThBgl2 has a more polar active site and therefore, favorites the interaction with water molecules, promoting better the hydrolysis reaction when compared to ThBgl1. / A preocupação ambiental e com a qualidade de vida da população aliados com o esgotamento dos combustíveis fósseis, tem aumentado a busca por energias alternativas e sustentáveis. Neste contexto, a hidrólise da biomassa lignocelulósica é uma opção interessante para obtenção de bioetanol. A utilização de enzimas para conversão da biomassa lignocelulósica a açúcares fermentescíveis ainda é um processo de custo elevado, o que torna imprescindível os estudos de caracterização dos mecanismos dessas enzimas afim de torná-las economicamente mais viáveis. A reação catalisada por β-glicosidases é a última etapa da sacarificação da celulose, sendo de grande relevância na conversão da biomassa ignocelulósica. β- glicosidases hidrolisam terminais não reduzidos β-D-glicosil liberando β-D-glicose e GH 1 e GH 3 são as famílias dessas enzimas mais estudadas. Entretanto dados estruturais e funcionais das enzimas da família GH 3, ainda são escassos. O presente trabalho apresenta a caracterização bioquímica e estrutural de uma β-glicosidase de Bifidobacterium adolescentis (BaBgl). Essa enzima possui um domínio catalítico (CCD) e um domínio do tipo fibronectina III (FnIII) cuja função ainda é desconhecida. Os dados bioquímicos revelaram condições ótimas para atividade da enzima em pH entre 6,0 e 6,5, temperatura de 45 °C e especificidade pelo substrato sintético 4- nitrofenil-β-D-glicopiranosídeo (pNPG). Os parâmetros cinéticos KM e Vmáx apresentaram valores de 0,32±0,03 mM e 0,37±0,01 nmol/min respectivamente. A enzima não apresentou mecanismos de transglicosilação, indicando apenas atividade hidrolítica. Ensaios com monossacarídeos como xilose e galactose aumentaram significativamente a atividade enzimática enquanto que glicose e arabinose inibiram sua atividade. O modelo da estrutura cristalográfica da BaBgl revelou um domínio Nterminal enovelado como um barril TIM, um domínio intermediário na forma de sanduíche α/β e um terceiro domínio C-terminal do tipo FnIII. Neste trabalho também foram estudados os mecanismos de tranglicosilação de duas β-glicosidases de Trichoderma harzianum (ThBgl1 e ThBgl2), sendo que ambas realizam reação de transglicosilação, porém a ThBgl1 possui relação hidrólise/tranglicosilação menor que a ThBgl2. As estruturas cristalográficas demonstram um enovelamento típico para as β-glicosidases da família GH 1, com o enovelamento na forma de um barril TIM (α/β)8. Contudo, a ThBgl2 apresenta sítio ativo mais polar e portanto propício à interação com moléculas de água, favorecendo a reação de hidrólise quando comparada à ThBgl1.

A Doença de Gaucher (DG) é a Doença Lisossômica de Depósito mais frequente com prevalência de 1:50.000. Ela é causada pela deficiência da betaglicosidase ácida (GBA), gerando acúmulo de glicosilceramidas (glicocerebrosídio) nos lisossomos. Outra enzima relacionada é a quitotriosidase (QT), onde a atividade está aumentada nos pacientes em até 1000 vezes. Atualmente o diagnóstico é baseado na atividade específica de enzimas em leucócitos ou fibroblastos. O uso de sangue impregnado em papel filtro (SPF) vem sendo ampliado, porém somente como forma de triagem, pois não há forma validada de estabelecer a atividade específica da enzima. A utilização de sangue em papel filtro tem diversas vantagens, tais como: fácil transporte, fácil armazenagem das amostras, menor volume de reação e segurança de manipulação das amostras. Esse estudo tem como objetivo validar técnicas fluorimétricas para o estabelecimento da atividade específica da GBA e QT em sangue impregnado em papel filtro eluido com tampão universal (20 mmol/L de fosfato de sódio, pH 7,0) através da correção do volume amostral pela quantificação de proteínas totais. Além disso, foram miniaturizadas as técnicas padrão para a triagem em SPF e as técnicas confirmatórias padrão ouro em leucócitos da DG. Foram estabelecidos novos valores de referência para todas as técnicas estabelecidas. Foi possível diferenciar os controles saudáveis dos pacientes com DG utilizando as técnicas miniaturizadas em SPF e leucócitos. A atividade específica em SPF se mostrou valida e os coeficientes de variação foram considerados aceitáveis. A estabilidade enzimática foi analisada por 21 dias de armazenamento a 4°C e foi observado que a há um decaimento da atividade da GBA, mas não da QT. Deste modo a atividade específica em SPF pode ser utilizada de forma confiável como método de triagem e confirmação do diagnóstico de DG. / Gaucher disease (GD) is the most frequent Lysosomal Storage Disorder with a prevalence of 1:50,000. It is caused by the deficiency of acid betaglucosidase (GBA), generating glucosylceramide (glucocerebroside) accumulation in the lysosomes. Another enzyme related to GD is chitotriosidase (CT), which activity is increased in patients up to 1000 times. Currently the diagnosis is based on the specific activity of enzymes in leukocytes or fibroblasts. The use of dried blood spots (DBS) has been extended, but only as screening, because there is no validated specific activity of the enzymes. The use of DBS has several advantages, such as easy transport and easy storage of samples, smaller reaction volume and safety of handling samples. This study aims to validate fluorometric techniques for establishing specific activity of the GBA and CT in DBS eluted with universal buffer (20 mmol/L sodium phosphate, pH7.0) by correcting for sample volume quantification of total protein. Moreover, standard screening techniques in DBS and standard diagnosis techniques in leukocytes for GD have been miniaturized. New reference values and cut-off points were established for all techniques. It was possible to differentiate healthy controls from patients with GD using miniaturized techniques in DBS and leukocytes. The specific activity of DBS proved valid and coefficients of variation were acceptable. The enzymatic stability was analyzed by 21 days of storage at 4° C and it was observed that there is a decrease of the activity of GBA, but not of CT. Thus the specific activity on DBS can be used as a reliable screening method and as diagnosis of GD.

A busca por novas tecnologias que visam à produção de biocombustíveis renováveis, especialmente bioetanol e outros biomateriais, tem se intensificado nos últimos anos. Há um interesse mundial crescente na limitação dos impactos ambientais e mudanças climáticas através da substituição de produtos petroquímicos por análogos ambientalmente corretos, a fim de alcançar uma economia mais sustentável. Além disso, as plataformas biorrefinarias lignocelulósicas necessárias para a produção de bioetanol representam uma oportunidade de estimular novos mercados para o setor agrícola e aumentar os empregos locais, contribuindo para o desenvolvimento das economias emergentes. No entanto, a maioria dos processos de conversão são baseados no conhecimento empírico, exigindo estudos mais aprofundados sobre os fatores envolvidos na hidrólise enzimática da celulose, tais como características biomassas, a otimização da etapa de pré-tratamento, bem como das atividades das enzimas e seus mecanismos de ação. Assim, com o objetivo de contribuir para a viabilização e implantação das tecnologias de produção do etanol lignocelulósico, na primeira parte deste trabalho de doutorado, foi realizada a purificação da β-glicosidase do fungo Aspergillus Níger (NaBgl1), principal enzima do coquetel comercial Novozymes 188, e sua caracterização bioquímica e biofísica. As análises de espalhamento de raios-x a baixo ângulo revelaram uma organização multidomínios desta enzima, com uma estrutura molecular de girino semelhante ao encontrado para as celulases. A sua estrutura é composta por um domínio catalítico N-terminal e um domínio fibronectina de tipo III (FnIII) na região C-terminal, conectados entre si por um longo linker com uma inserção de 100 resíduos de aminoácidos numa conformação estendida. Apesar desta estrutura molecular incomum, os ensaios de eletroforese capilar revelaram um perfil processividade característico de β-glucosidases, e os ensaios enzimáticos confirmaram, também, a ausência de atividade em substratos poliméricos. Nos ensaios adosrção com diferentes compostos poliméricos, a enzima β-glicosidase mostrou uma capacidade de adsorção elevada em lignina. Os mecanismos de ligação FnIII-lignina foram elucidados por simulações de dinâmica molecular, que confirmaram apresença de vários sítios de ligação à lignina no domínio FnIII da enzima. Como segunda parte da presente tese, diferentes biomassas, como bagaço de cana, resíduos de casca de eucalipto e gramíneas (Panicum maximum, Pennisetum purpureum e Brachiaria brizantha) foram submetidas a vários métodos de pré-tratamento (ácido diluído, alcalino, sulfito e água quente) em diferentes condições de tratamento e avaliadas quanto ao seu potencial para a produção de bioetanol. As biomassas in natura e pré-tratadas foram caracterizadas quanto à sua composição química por métodos cromatográficos, ressonância magnética nuclear e espectroscopia de infravermelho por transformada de Fourier; o índice de cristalinidade das amostras foi determinado por método químico e difração de raios-x; as análises morfológicas foram realizadas por microscopia eletrônica de varredura; e os resultados da caracterização foram correlacionados com os perfis de sacarificação enzimática encontrados para cada uma delas. / The search for new technologies aimed at the production of renewable biofuels, specially bioethanol, and other biomaterials has intensified in recent years. There is an increasing world-wide interest in the limitation of environmental impact and climate change by replacing petrochemical products with environment-friendly analogues in order to move towards a sustainable economy. In turn, the lignocellulosic biorefining platforms required for ethanol production present an opportunity to stimulate new markets for the agriculture sector and increase domestic employment, contributing to the development of emerging economies. However, most of conversion processes are based on empirical knowledge, demanding thorough studies about the factors involved on enzymatic hydrolysis of cellulose, such as biomasses characteristics, optimization of pretreatment steps and enzymes activities and molecular action mechanisms. Aiming to contribute for the viability and establishment of lignocellulosic ethanol technologies, on the first part of the present thesis, we performed the purification of main Aspergillus niger β-glucosidase (AnBgl1) from the commercial cocktail Novozymes 188 and its biochemical and biophysical characterization. The small angle x-ray scattering analysis revealed a multidomain organization, with a tadpole-like molecular shape similar to that found for cellulases. Its structure is composed by a N-terminal catalytic domain and a fibronectin type III-like (FnIII) C-terminal domain, connected by a long linker with a 100 aminoacids residues insertion in a extended conformation. In spite of this uncommon molecular structure, capilar zone electrophoresis assays revealed a processivity profile characteristic of β-glucosidases and the enzymatic assays confirmed no-activity on polymeric substrates. On the pull-dowm assays with different polymeric compounds, the β-glucosidase showed a high adsorption ability to lignin. The FnIII-lignin binding mechanisms were elucidated by molecular dynamics simulations, confirming the multiple binding sites to lignin in the enzyme FnIII domain. As a second part of the present thesis, different biomasses such as sugarcane bagasse, eucalyptus bark residues and grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha) were submitted to several pretreatment methods (diluted acid, alkaline, sulfite and hot water) at various conditions and evaluated about their potential to bioethanol production. The raw and pretreated biomasses were characterized about their chemical composition by chromatographic methods, nuclear magnetic ressonance and Fourier transformed infrared spectroscopy; the crystallinity index was determined by chemical method and x-ray diffraction; morphological features were analysed by scanning electron microscopy; and the characterization results were correlated to their enzymatic saccharification profiles.

A Doença de Gaucher (DG) é a Doença Lisossômica de Depósito mais frequente com prevalência de 1:50.000. Ela é causada pela deficiência da betaglicosidase ácida (GBA), gerando acúmulo de glicosilceramidas (glicocerebrosídio) nos lisossomos. Outra enzima relacionada é a quitotriosidase (QT), onde a atividade está aumentada nos pacientes em até 1000 vezes. Atualmente o diagnóstico é baseado na atividade específica de enzimas em leucócitos ou fibroblastos. O uso de sangue impregnado em papel filtro (SPF) vem sendo ampliado, porém somente como forma de triagem, pois não há forma validada de estabelecer a atividade específica da enzima. A utilização de sangue em papel filtro tem diversas vantagens, tais como: fácil transporte, fácil armazenagem das amostras, menor volume de reação e segurança de manipulação das amostras. Esse estudo tem como objetivo validar técnicas fluorimétricas para o estabelecimento da atividade específica da GBA e QT em sangue impregnado em papel filtro eluido com tampão universal (20 mmol/L de fosfato de sódio, pH 7,0) através da correção do volume amostral pela quantificação de proteínas totais. Além disso, foram miniaturizadas as técnicas padrão para a triagem em SPF e as técnicas confirmatórias padrão ouro em leucócitos da DG. Foram estabelecidos novos valores de referência para todas as técnicas estabelecidas. Foi possível diferenciar os controles saudáveis dos pacientes com DG utilizando as técnicas miniaturizadas em SPF e leucócitos. A atividade específica em SPF se mostrou valida e os coeficientes de variação foram considerados aceitáveis. A estabilidade enzimática foi analisada por 21 dias de armazenamento a 4°C e foi observado que a há um decaimento da atividade da GBA, mas não da QT. Deste modo a atividade específica em SPF pode ser utilizada de forma confiável como método de triagem e confirmação do diagnóstico de DG. / Gaucher disease (GD) is the most frequent Lysosomal Storage Disorder with a prevalence of 1:50,000. It is caused by the deficiency of acid betaglucosidase (GBA), generating glucosylceramide (glucocerebroside) accumulation in the lysosomes. Another enzyme related to GD is chitotriosidase (CT), which activity is increased in patients up to 1000 times. Currently the diagnosis is based on the specific activity of enzymes in leukocytes or fibroblasts. The use of dried blood spots (DBS) has been extended, but only as screening, because there is no validated specific activity of the enzymes. The use of DBS has several advantages, such as easy transport and easy storage of samples, smaller reaction volume and safety of handling samples. This study aims to validate fluorometric techniques for establishing specific activity of the GBA and CT in DBS eluted with universal buffer (20 mmol/L sodium phosphate, pH7.0) by correcting for sample volume quantification of total protein. Moreover, standard screening techniques in DBS and standard diagnosis techniques in leukocytes for GD have been miniaturized. New reference values and cut-off points were established for all techniques. It was possible to differentiate healthy controls from patients with GD using miniaturized techniques in DBS and leukocytes. The specific activity of DBS proved valid and coefficients of variation were acceptable. The enzymatic stability was analyzed by 21 days of storage at 4° C and it was observed that there is a decrease of the activity of GBA, but not of CT. Thus the specific activity on DBS can be used as a reliable screening method and as diagnosis of GD.

A soja (Glycine max) contém uma variedade de compostos com comprovada atividade biológica, tais como as isoflavonas, que estão presentes em diferentes formas, glicosiladas e agliconas. Além disso, a soja contém uma grande quantidade de proteínas, que são consideradas fontes de peptídeos bioativos. As isoflavonas agliconas, daidzeína e genisteína, possuem maior atividade antioxidante que as glicosiladas, daidzina e genistina. No entanto, os grãos de soja são ricos nas formas glicosiladas das isoflavonas. Estudos mostram que a biotransformação da soja, por micro-organismos e enzimas, leva ao aumento dos teores das isoflavonas agliconas, as quais são liberadas pela ação de enzimas -glicosidases, que clivam as ligações -glicosídicas das isoflavonas glicosiladas, e também pode possibilitar a hidrólise das proteínas da soja. Além disso, pesquisadores têm demonstrado aumento na atividade antioxidante e na prevenção e/ou supressão de certos cânceres após biotransformação da soja. Neste contexto, foi realizada a biotransformação da soja pelo fungo A. awamori, e por uma mistura enzimática, proveniente do processo fermentativo deste fungo na soja. Os extratos da soja biotransformada, não biotransformada, e o extrato comercial isoflavin beta®, rico em isoflavonas, foram avaliados quanto aos perfis cromatográficos, teores de daidzeína, genisteína, proteínas, aminoácidos e/ou peptídeos, potencial antioxidante e atividade citotóxica frente a células de fibroblasto e melanoma. O modo de morte celular das células de melanoma, necrose ou apoptose, também foi avaliado. A biotransformação da soja, pelos dois processos, resultou em extratos enriquecidos com isoflavonas agliconas e aminoácidos e/ou peptídeos, e com maior atividade antioxidante que o extrato da soja não biotransformada. Os dois processos de biotransformação da soja resultaram em extratos com características químicas e biológicas diferentes. O conteúdo de daidzeína, proteínas, aminoácidos e/ou peptídeos encontrados no extrato da soja biotransformada pelo fungo foram 6%, 56% e 357%, respectivamente, superiores ao extrato da soja biotransformada pela mistura enzimática. Ao contrário do observado para o teor de genisteína que foi 48% maior no extrato da soja biotransformada pela mistura enzimática. O extrato da soja biotransformada pelo fungo apresentou maior atividade antioxidante que o extrato da soja biotransformada pela mistura enzimática, além disso, foi o único dos extratos aqui estudados que apresentou citotoxicidade seletiva para as células de melanoma, induzindo morte celular por apoptose destas células cancerosas. Sendo assim, os resultados obtidos pelo extrato da soja biotransformada pelo fungo A. awamori fornecem boas perspectivas para futura utilização deste extrato como antitumoral. / Soybean (Glycine max) contains a variety of compounds with proven biological activity, such as isoflavones, which are present in different forms, glycosides and aglycones. In addition, soybean contains a lot of proteins, which are considered sources of bioactive peptides. The aglycone isoflavones, daidzein and genistein, have higher antioxidant activity than the glucoside ones, daidzin and genistin. However, soybean grains are rich in the glycosylated forms of isoflavones. Studies have shown that the soybean biotransformation, by microorganisms and enzymes, lead to increased levels of aglycone isoflavones, which are released by the action of -glycosidase enzymes, which cleave the -glycosidic bonds of isoflavone glucosides, and can also allow the hydrolysis of soybean proteins. Additionally, researchers have shown an increase in the antioxidant activity and in the prevention and/or suppression of certain cancers after soybean biotransformation. In this context, it was performed the biotransformation of soybeans with the fungus A. awamori, and with an enzyme mixture, from the fermentation process of the fungus in soybean. The biotransformed, the non biotransformed soybean extracts and the marketed isoflavin beta® extract rich in isoflavones, were evaluated regarding their chromatographic profiles, levels of daidzein, genistein, proteins, amino acids and/or peptides, the antioxidant potential and the cytotoxic activity against melanoma cells and fibroblasts. The mode of cell death of melanoma cells, necrosis or apoptosis, was also evaluated. The biotransformation of soybean by the two processes resulted in extracts enriched with aglycone isoflavones and aminoacids and/or peptides, and with antioxidant activity higher than the non biotransformed soybean extract. The two processes of soybean biotransformation resulted in extracts with different chemical and biological characteristics. The contents of daidzein, proteins, aminoacids and/or peptides found in soybean extract biotransformed by the fungus were 6%, 56% and 357%, respectively, higher than the soybeam extract biotransformed by the enzyme mixture. Contrary to what was observed with the genistein content that was 48% higher in the soybean extract biotransformed by the enzyme mixture. The soybean extract biotransformed by the fungus had a higher antioxidant activity than the soybean extract biotransformed by the enzyme mixture, moreover, it was the unique extract among the ones studied in this work that showed selective cytotoxicity to melanoma cells, inducing cell death by apoptosis of these cancer cells. Thus, the obtained results of the soybean extract biotransformed by the fungus A. awamori provide good prospects for future use of this extract as antitumoral.

No description available.

Type 2 diabetes mellitus (T2DM) is an increasing global health concern, currently affecting an estimated 382 million individuals. There is no cure for T2DM and the search for new and improved treatments is ongoing. Presently, various pharmacological regimens are available to treat T2DM, but with varied success. Thousands of traditional herbs are also used to treat T2DM, but mainly without scientific validation. The aim of this study was to assess the polyphenolic content, antioxidant capacity, as well as in vitro toxicity and hypoglycaemic activity of a commercial ‘antidiabetic’ tea mixture (Diabetea) and its individual constituents: Achillea millefolium L. (Yarrow), Agathosma betulina Bartl. & Wendl. (Buchu), Salvia officinalis L. (Sage), Taraxacum officinalis L. (Dandelion), Thymus vulgaris L. (Thyme), Trigonella foenum-graecum L. (Fenugreek) and Urtica urens L. (Nettle). All herbs were tested as crude extracts, prepared using hot water (HW) and dichloromethane (DCM). The total polyphenolic content of each extract was determined using the Folin-Ciocalteau and aluminium trichloride methods. The non-cellular antioxidant activity was assessed using 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods. The cell-based antioxidant activity was measured against p-chloranil-induced generation of reactive oxygen species (ROS) in Ea.hy926 cells, using the fluorescent dye, 2',7'-dichlorfluorescein-diacetate (DCFH-DA). The effect of each extract on the viability of C2C12 myotubes, Ea.hy926 endothelial cells and human lymphocytes (HL) was determined using sulforhodamine B (SRB). The in vitro hypoglycaemic activity was assessed against α-amylase and α-glucosidase activity using 3,5-dinitrosalicylic acid (DNSA) and p-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. The type of inhibition exerted on these enzymes was determined using the Michaelis-Menten enzyme kinetics model, expressed as mixed, competitive, non-competitive and uncompetitive. Glucose uptake activity was measured using the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) fluorescent analogue. T. vulgaris and S. officinalis had the highest amount of polyphenols of all extracts tested. The HW extracts of T. vulgaris and S. officinalis showed significant (p < 0.05) cell-free antioxidant activity and cell-based radical scavenging activity. In addition, U. urens (HW) also limited cell-based ROS generation (p < 0.05). The Diabetea extracts presented with poor antioxidant activities, of which some had a pro-oxidant effect on Ea.hy926 cells. The positive linear relationship between antioxidant activity and polyphenolic content was shown to be dependent on the solvent type used. All of the DCM extracts had low antioxidant activity and polyphenolic content. None of the extracts produced < 50% cell density at the concentrations tested (1.3 - 20 μg/mℓ). In general, the DCM extracts showed a greater decrease in cell density than the HW extracts. The Ea.hy926 cells were the least affected by the extracts in terms of decreased cell density. The DCM extract of U. urens inhibited α-amylase activity in a mixed manner, which was comparable to the percentage inhibition exerted by the commercial drug, acarbose. Both the HW and DCM extracts of U. urens caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. The HW extract of T. vulgaris had a significant (p < 0.05) inhibitory activity against α-glucosidase (mixed). It also caused the uptake of glucose into C2C12 myotubes, which was significantly (p <0.05) more active than insulin. S. officinalis (DCM extract) also inhibited α-glucosidase activity (p < 0.05) in a mixed manner. Its HW extract displayed potent hypoglycaemic potential by causing glucose uptake into C2C12 myotubes, which was more significant (p < 0.05) than the activity of the positive control, insulin. The DCM extract of A. betulina was active against α-glucosidase (non-competitive), which was comparable to the activity of acarbose. Its HW extract also showed a significant (p < 0.05) glucose uptake activity. Furthermore, the DCM extracts of T. officinalis, A. millefolium, Diabetea and HW extracts of T. foenum-graecum and T. officinalis also caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. This study provides evidence for the antidiabetic potential of T. vulgaris and S. officianlis, in terms of antioxidant capacity and potential to prevent of post-prandial hyperglycaemia and alleviate hyperglycaemia by mimicking the action of insulin. In addition, the organic preparation of U. urens is also a potent α-amylase inhibitor. All herbs tested in this study exerted some form of in vitro antidiabetic activity. The Diabetea mixture, as a traditional preparation, did not have a significant antidiabetic capacity. In vitro observations from this study do not support the use of Diabetea as an antidiabetic preparation and reveal that some of the individual extracts prove more efficacious than the herb mixture. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Pharmacology / MSc / Unrestricted

Diabetes mellitus (DM) is a group of non-infectious diseases that cause hyperglycemia. DM symptoms were first clinically described by ancient Greek physicians whose prescriptions included plant-based remedies. Today, DM affects >400 million people globally and prevalence rates are rapidly increasing in developing countries where basic healthcare relies on local knowledge of botanical remedies. Many developing countries are home to diverse peoples and plants—providing fodder for varied plant-selection strategies and unique botanical pharmacopoeias. I addressed the plant-selection strategies used in a multi-ethnic, developing country, Trinidad and Tobago (T&T), to ascertain their role in shaping the local antidiabetic pharmacopoeia and to assess their benefits and risks in identifying safe and useful remedies. Using literature reviews, field surveys, and laboratory bioassays, I completed three categories of analysis. Ethnobotanical analyses showed that T&T’s antidiabetic pharmacopoeia is primarily of recent origin as >50% of the 48 historical DM remedies were Neotropical natives, including congenerics of well-known medicinal Paleotropical genera. Nevertheless, conservative knowledge transmission was also evident as several Paleotropical species of T&T’s pharmacopoeia, including Momordica charantia and Catharanthus roseus were also used in Africa, India and across the Caribbean. Paleotropical natives with a long history of use are likely to be safer remedies. Ethno-medicinal analyses of the pre- and post-2000 DM remedies of T&T, totaling 99 species, suggest that the centuries-old hot/cold folk disease-model was the model predominantly used in plant-selection. Parallels found between T&T folk concepts and biomedical mechanisms of DM provide probable bases for efficacy but the chronic use of purgatives and bitter-tasting plants is likely to be risky. Phytochemical analyses revealed that 69% of the tested plant extracts contained phenolic compounds, with more than half producing >80% alpha-glucosidase inhibition. Phenolic content and alpha-glucosidase inhibition were strongly correlated among food plants used as medicines, suggesting higher probability of selection as a result of non-target effects. The medicinal use of food plants may provide the best margins of safety and efficacy in identifying antidiabetic remedies. Together, these analyses showed how culture-specific plant-selection strategies can identify safe, useful remedies for developing countries to address their increasing DM prevalence in a cost-effective and sustainable manner.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR

Silver nanoparticles (AgNPs) are expected to enter aquatic systems, but there are limited data on how they might affect microbial communities in pathogen impaired streams. We examined microbial community responses to citrate-AgNP (10.9 ± 0.7 nm) and polyvinylpyrrolidone (PVP)-AgNP (11.0 ± 0.7 nm) based on microbial concentration and enzyme activity in sediment from a pathogen impaired stream. Addition of each nanoparticle to sediment caused at least a 69% decrease in microbial concentration (1,264 ± 93.6 to 127 ± 29.5 CFU/g) and a 62% decrease in β-glucosidase activity (11.7 ± 2.1 to 1.3 ± 0.3 μg/g/h). Each AgNP reduced alkaline phosphatase activity but their effects were not statistically significant. Sediment exposed to 0.108 mg Ag/kg of AgNO3 resulted in a 92% decrease in microbial concentration and a reduced enzyme activity which was not statistically significant. Measured total silver in sediments treated with AgNPs which exhibited significant inhibition effects on the microbial community ranged from 0.19 ± 0.02 to 0.39 ± 0.13 mg Ag/kg. These concentrations tested in this study are much lower than the expected concentrations (2-14 mg Ag/kg) in freshwater sediments. The results of this study demonstrate that AgNPs can alter microbial community activity and population size, which may lead to false negative fecal indicator bacteria detection and enumeration using methods that rely on β-glucosidase activity. We conclude that the presence of AgNPs in impaired streams and recreational waters can influence pathogen detection methods, potentially affecting public health risk estimates.