This thesis describes bioanalytical methods for drug determination in biological matrixes, with drugs in focus used against diseases largely affecting low-income countries. Solid-phase extraction is used for sample cleanup, and processed samples are analyzed by liquid chromatography. Developed bioanalytical methods are validated according to international guidelines. Eflornithine (DFMO) is a chiral drug, used for treating human African trypanosomiasis. A bioanalytical method for determination of DFMO enantiomers in plasma is presented. The enantiomers are detected by evaporative light-scattering detection. The method has been applied to determination of D-DFMO and L-DFMO in rats, after intravenous and oral administration of racemic DFMO. It is concluded that DFMO exhibits enantioselective absorption, with the more potent enantiomer L-DFMO being less favored. Sulfadoxine (SD) and sulfamethoxazole (SM) are sulfa-drugs used for malaria and pneumonia respectively. Two methods are described for simultaneous determination of SD and SM in capillary blood sampled on filter paper. The former method allows direct injection of extracts from dried blood spots (DBS), while for the latter method solid-phase extraction is added. Pre-analytical factors contributing to measurement uncertainty is also discussed, and it is concluded that it is of high importance that homogeneity in type of sampling paper and sampling volume is assured. Piperaquine (PQ) is an antimalarial, increasingly used in artemisinin combination therapy. A method for determination of piperaquine in DBS is presented. By using a monolithic LC column, a very short LC analysis of two minutes per sample is achieved. A method for simultaneous determination of three antiretroviral drugs i.e. lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP), in DBS samples is described. The method is applied to drug determination in two subjects after receiving standard antiretroviral treatment. Conclusion is that the method is suitable for determination of 3TC and NVP, and to some extent for AZT.

A series of experiments were conducted to determine nutritional strategies to improve the efficiency of N utilization in dairy cows when feeding co-products including wheat-based (W-DDGS) and corn-wheat blend distillers grains with solubles (B-DDGS), and dried whey permeate (DWP). In Experiment 1, the objective was to determine the effects of replacing canola meal (CM) as the major protein source with W-DDGS on ruminal fermentation, microbial protein production, omasal nutrient flow, and animal performance. Cows were fed either a standard barley silage-based total mixed ration containing CM as the major protein supplement (0% W-DDGS, control) or diets formulated to contain 10, 15 and 20% W-DDGS (dry matter [DM] basis), with W-DDGS replacing primarily CM. Diets were isonitrogenous (18.9% crude protein [CP]). Inclusion of W-DDGS to the diet did not negatively affect ruminal fermentation, microbial protein production, and omasal nutrient flow. However, there was a 0.7- to 2.4-kg increase in DM intake, and a 1.2- to 1.8-kg increase in milk yield after the addition of W-DDGS in place of CM. In Experiment 2, the objective was to delineate the effects of including either W-DDGS or B-DDGS dried distillers grains with solubles as the major protein source in low or high CP diets fed to dairy cows on ruminal function, microbial protein synthesis, omasal nutrient flows, urea-N recycling, and milk production. The treatment factors were type of distillers co-product (W-DDGS vs. B-DDGS) and dietary CP content (15.2 vs. 17.3%; DM basis). The B-DDGS was produced from a mixture of 15% wheat and 85% corn grain. All diets were formulated to contain 10% W-DDGS or B-DDGS on a DM basis. Feeding up to 10% of dietary DM as B-DDGS or W-DDGS as the major source of protein did not have negative effects on metabolizable protein (MP) supply and milk production in dairy cows. However, reducing dietary CP content from 17.3 to 15.2% decreased milk production. This response was attributed to an insufficient supply of ruminally degradable protein (RDP) that suppressed microbial nonammonia N (NAN) synthesis in the rumen, thus decreasing intestinal MP supply. In Experiment 3, the objective was to determine the effects of replacing barley or corn starch with lactose (as DWP) in diets containing 10% W-DDGS on ruminal function, omasal nutrient flow, and lactation performance. The treatment factors were source of starch (barley vs. corn) and dietary inclusion level of DWP (0 vs. 6%; DM basis) as a partial replacement for starch. Diets were isonitrogenous (18% CP) and contained 3 or 8% total sugar. The starch content of the low sugar diet was 24% compared to 20% for the high sugar diet. Dry matter intake, and milk and milk component yields did not differ with diet. However, partially replacing dietary corn or barley starch with sugar up-regulated ruminal acetate and propionate absorption, and reduced ruminal NH3-N concentration, but had no effect on ruminal pH, microbial protein synthesis, omasal nutrient flow and production in dairy cows. In summary, data presented in this thesis indicate that W-DDGS and B-DDGS can be included as the major source of protein in dairy cow diets without compromising ruminal function, nutrient supply and milk production in dairy cows. Feeding medium to low CP diets, and partial replacement of starch with sugar in diets containing W-DDGS and B-DDGS can improve N utilization efficiency in dairy cows. Additionally, an upregulation of facilitated transport of acetate and propionate across epithelial cells possibly prevents the occurrence of ruminal acidosis when lactose partially replaces starch in cow diets.

This study was conducted to determine if nutrient utilization and enteric methane (CH4) emissions could be improved in overwintering beef cows consuming low-quality forage supplemented with protein in the form of dried distillers grain with solubles (DDGS) in thermal-neutral and cold-stressed environments. Thirty mature, dry and non-pregnant beef cows were divided into three treatment groups and fed diets consisting of low-quality (6.0% crude protein; CP) forage with no DDGS (control, CON), 10% DDGS (borderline sufficient CP, 8.7% CP), or 20% DDGS (excess CP, 11.6% CP). Cold acclimatization did not appear to affect nutrient intake and digestibility by beef cows, but increased N and P excretion by 1.2x and 2.5x, respectively. Cold acclimatized cows reduced energy excretion by 26.8% (7.1 vs. 5.2 ± 0.30% GEI in fall and winter, respectively; P < 0.0001) in accordance with a 33.8% increase in rumen fluid rate of passage (ROP). Supplementation with DDGS improved digestibility of N and P (40.6 vs. 61.2 ± 2.45% N and -23.9 vs. 5.7 ± 5.95% P for CON and 20%DDGS, respectively; P < 0.0001) by increasing digestible substrate in the diet. Protein supplementation increased rumen NH3-N concentrations (1.5, 2.1 and 3.1 ± 0.15 mg 100 mL-1; P < 0.0001) enough to increase rumen fermentation efficiency, resulting in 18.5% lower enteric CH4 emissions when CP was fed in excess of animal requirements. Total excretion of N and P were increased two- and 45-fold, respectively, when excess CP was fed. Reduced enteric CH4 emissions as a result of cold acclimatization suggest an advantage for the Canadian beef herd in terms of environmental sustainability. Supplementing CP in excess of cow requirements may improve nutrient utilization and rumen fermentation efficiency, and mitigate enteric CH4 emissions in beef cows fed low-quality forage diets, but may also contribute to greater N and P loading of soil and ground water.

This study was conducted to determine if nutrient utilization and enteric methane (CH4) emissions could be improved in overwintering beef cows consuming low-quality forage supplemented with protein in the form of dried distillers grain with solubles (DDGS) in thermal-neutral and cold-stressed environments. Thirty mature, dry and non-pregnant beef cows were divided into three treatment groups and fed diets consisting of low-quality (6.0% crude protein; CP) forage with no DDGS (control, CON), 10% DDGS (borderline sufficient CP, 8.7% CP), or 20% DDGS (excess CP, 11.6% CP). Cold acclimatization did not appear to affect nutrient intake and digestibility by beef cows, but increased N and P excretion by 1.2x and 2.5x, respectively. Cold acclimatized cows reduced energy excretion by 26.8% (7.1 vs. 5.2 ± 0.30% GEI in fall and winter, respectively; P < 0.0001) in accordance with a 33.8% increase in rumen fluid rate of passage (ROP). Supplementation with DDGS improved digestibility of N and P (40.6 vs. 61.2 ± 2.45% N and -23.9 vs. 5.7 ± 5.95% P for CON and 20%DDGS, respectively; P < 0.0001) by increasing digestible substrate in the diet. Protein supplementation increased rumen NH3-N concentrations (1.5, 2.1 and 3.1 ± 0.15 mg 100 mL-1; P < 0.0001) enough to increase rumen fermentation efficiency, resulting in 18.5% lower enteric CH4 emissions when CP was fed in excess of animal requirements. Total excretion of N and P were increased two- and 45-fold, respectively, when excess CP was fed. Reduced enteric CH4 emissions as a result of cold acclimatization suggest an advantage for the Canadian beef herd in terms of environmental sustainability. Supplementing CP in excess of cow requirements may improve nutrient utilization and rumen fermentation efficiency, and mitigate enteric CH4 emissions in beef cows fed low-quality forage diets, but may also contribute to greater N and P loading of soil and ground water.

Thesis (MSc Food Sc )--Stellenbosch University, 2000. / ENGLISH ABSTRACT: This study shows Fourier transform near infrared (FT-NIR) spectroscopy's application on wine, fruit and dried fruit for quantitative determinations or as a discriminative method for classification purposes. During wine production optimum yeast growth, resulting in healthy alcohol fermentation rates, is monitored by the amount of free amino nitrogen (FAN) present in the must. The status of malolactic fermentation (MLF) in Chardonnay wines is monitored by determining the degree of conversion of malic to lactic acid. Ethyl carbamate (EC), a suspected carcinogen, is mainly formed during ageing of wine and is restricted by legislation in some countries. It is therefore necessary to determine the EC content in wine. Fourier transform near infrared spectroscopy can be used on wine as a rapid method to measure the °Brix (residual sugars) content of must and to discriminate between different must samples in terms of their FAN values. It can also be used as a rapid method to discriminate between Chardonnay wine samples in terms of the MLF status and between table wine samples in terms of the EC content. Calibrations were derived and it was found that a very strong correlation existed in the sample set for the FT-NIR spectroscopic predictions for °Brix (r = 0.99, SEP = 0.31%), but poorer correlations for the FAN (r = 0.405, SEP = 275%), malic acid (r = 0.64, SEP = 1.02%), lactic acid (r = 0.61, SEP = 1.35%) and EC predictions (r = 0.47, SEP = 3.6%). When soft independent modelling by class analogy (SIMCA) was applied as a discriminative method, the must and wine samples were classified in terms of their FAN and EC values and MLF status, respectively, obtaining results with recognition rates exceeding 80%. The canning of peaches has become a significant industry in South Africa, producing approximately 4.1 million cartons per year. Fourier transform near infrared spectroscopy was applied as an alternative non-destructive method for the quantitative determination of the total soluble solid (TSS) content of whole fresh peaches. The TSS content of fresh clingstone peaches is an indication of the internal quality, maturity and perceived sweetness of the fruit for the peach canning industry. By determining the TSS, fresh peaches can be graded and the farmers compensated accordingly. Results obtained by building QUANT+™calibrations for the TSS content (r = 0.96, SEP = 0.55%) showed acceptable accuracy and can replace the present destructive methods. Peak periods during the harvesting season necessitate storage of peaches for up to three weeks before canning. Approximately 5 - 10% of the peaches stored, disintegrate during canning due to loose skin, large stone cavities, soft flesh and rot. The storage potential of fresh clingstone peaches can be successfully predicted with FT-NIR and SIMCA models, using subjective internal quality evaluations. Results with recognition rates exceeding 80% were obtained in most cases and this method proved useful as a non-destructive method of quality assessment. By applying this method, losses caused when storing peaches with poor storage quality will be reduced. The golden sultana industry plays an important role in the dried fruit exporting market of South Africa. Due to the large numbers of consignments that must be checked upon arrival, and the need for rapid decision making during processing, it is essential to replace the present time-consuming analytical methods. Fourier transform near infrared spectroscopy was used as a rapid, analytical technique to determine whether the S0₂ and moisture contents of sultanas are within specifications upon arrival at the factory and during processing. High positive correlation was found between the measured values and those predicted by FT-NIR spectroscopy for S0₂ (r = 0.99, SEP = 24.09%) and moisture (r = 0.99, SEP = 0.051 %) contents. / AFRIKAANSE OPSOMMING: Hierdie studie dui op Fourier transformasie naby-infrarooi (FT-NIR) spektroskopie se toepassing op wyn, vrugte en droëvrugte vir die uitvoer van kwantitatiewe bepalings of vir klassifikasie doeleindes om as 'n diskriminasie metode te dien. Gedurende wynproduksie word die optimum groei van giste wat lei tot 'n gesonde alkohol fermentasie gemonitor deur die hoeveelheid vry-aminostikstof (VAS) wat in die mos teenwoordig is te bepaal. Die status van appelmelksuurgisting (AMG) in Chardonnay wyne word gemonitor deur die mate van omskakeling van appelsuur na melksuur te bepaal. Etielkarbamaat (EK), 'n vermoede karsinogeen wat hoofsaaklik in verouderde wyne voorkom, word in sekere lande deur wetgewing beperk en dus die bepaling van die EK inhoud van wyne noodsaak. Fourier transformasie naby-infrarooi spektroskopie kan op mos toegepas word as 'n vinnige metode vir die bepaling van °Brix (residuele suiker) en om tussen die monsters te onderskei in terme van hulle VAS inhoud. FT-NIR kan ook gebuik word as 'n vinnige metode om tussen Chardonnay monsters te onderskei op grond van die status van AMG en tussen tafelwyn monsters op grond van die EK inhoud. Kalibrasies is ontwikkel en daar is gevind dat baie sterk korrelasies bestaan in die monsterstel vir die FT-NIR spektroskopiese voorspelling van °Brix (r = 0.99, SEP = 0.31%), maar swakker korrelasies vir die VAS (r = 0.405, SEP = 275%), appelsuur (r = 0.64, SEP = 1.02%), melksuur (r = 0.61, SEP = 1.35%) en EK voorspellings (r = 0.47, SEP = 3.6%). Met die toepassing van soft independent modelling by class analogy (SIMCA) as diskriminasie metode, is die mos- en wynmonsters geklassifiseer op grond van hul VAS en EK waardes en die status van AMG, en herkenningswaardes van bo 80% is onderskeidelik behaal. Die inmaak van perskes het 'n beduidende industrie in Suid-Afrika geword en produseer jaarliks ongeveer 4.1 miljoen kartonne. Fourier transformasie nabyinfrarooi spektroskopie is toegepas as alternatiewe, nie-beskadigende metode om kwantitatiewe bepalings van die totale oplosbare vastestowwe (TOV) inhoud van heel vars perskes, te doen. Vir die perske inmaak-industrie is die TOV inhoud van vars taaipitperskes 'n aanduiding van interne kwaliteit, rypheid en die soetheid van die vrugte. Vars perskes kan gradeer word deur die TOS te bepaal en sodoende kan boere oreenkomstige vergoeding ontvang. Resultate' wat verkry is deur QUANT+™ kalibrasies vir TOS inhoud te ontwikkel (r = 0.96, SEP = 0.55%), dui op aanvaarbare akkuraatheid en kan die huidige metodes vervang. Tydens oestyd kom piektye voor wanneer dit soms nodig is om perskes vir tot drie weke op te berg voordat dit ingemaak kan word. Ongeveer 5 tot 15% van hierdie opgebergte perskes disintegreer tydens inmaak omdat opberging lei tot defekte in die perskes soos skille wat loskom, groot pitholtes, sagte vleis en vrot. Die opbergingspotentiaal van vars taaipitperskes kan suksesvol voorspel word deur FTNIR en SIMCA modelle te bou en subjektiewe interne kwaliteitsevaluerings daarop toe te pas. Herkenningsresultate wat 80% in die meeste gevalle oorskry, is behaal wat hierdie metode as 'n suksesvolle nie-beskadigende kwaliteitbepalingsmetode bewys. Hierdie metode sal verliese kan beperk wat voorkom as gevolg van opberging van perskes met swak opbergingskwaliteit. Die goue sultana industrie speel 'n belangrike rol in die droë vrugte uitvoermark van Suid-Afrika. As gevolg van die hoeveelheid monsters wat gelyktydig getoets moet word en besluite wat vinnig geneem moet word tydens prosessering, is dit belangrik om die huidige tydrowende analitiese metodes te vervang. Fourier transformasie naby-infrarooi spektroskopie is gebruik as 'n vinnige, analitiese tegniek om tydens ontvangs by die fabriek en gedurende prosessering te bepaal of die S0₂- en voginhoud van goue sultanas binne die spesifikasies val. Goeie positiewe korrelasie is gevind tussen die bepaalde en voorspelde FT-NIR spektroskopiese waardes vir SO₂- (r = 0.99, SEP = 24.09%) en voginhoud (r = 0.99, SEP = 0.051 %).

As folhas de Maytenus ilicifolia são amplamente utilizadas na medicina tradicional brasileira no tratamento de distúrbios gástricos. Após a comprovação de sua eficácia e segurança, a referida droga vegetal tem sido objeto de diversos estudos com a finalidade de estabelecer parâmetros para o controle de qualidade. Neste sentido, foi avaliada a viabilidade do desenvolvimento de um método analítico fundamentado na precipitação de taninos empregando povidona como agente seqüestrante. Os dados revelaram que a remoção de taninos em solução é satisfatória quando empregada a povidona insolúvel. Foi observado que o polímero também é capaz de precipitar outros polifenóis além dos taninos presentes na solução extrativa de M. ilicifolia. Entretanto, as interações do polímero ocorrem preferencialmente com os derivados catéquicos. Adicionalmente foi desenvolvido e validado um método por CLAE para separação e quantificação de catequina e epicatequina em produtos derivados de M. ilicifolia. Não foram detectadas variações nas áreas dos picos ou nos tempos de retenção durante os ensaios de avaliação do método, sugerindo que as condições cromatográficas empregadas são satisfatórias. Para viabilizar a preparação de produto seco por aspersäo (PSA) em torre de secagem piloto munida de aspersor rotatório, foi necessária a concentração da solução extrativa. A operação foi realizada sob pressão reduzida até alcangar um teor de sólidos mínimo de 15 % (m/m). A operação de secagem da solução concentrada apresentou rendimento de 90 % e o PSA obtido apresentou partículas esféricas, com superfície rugosa e tamanho médio de 22,5 um. A caracterização tecnológica do material revelou que as suas propriedades tecnológicas, tais como, densidades, compressibilidade e fluxo, foram incrementadas quando comparados com o produto preparado em equipamento de bancada. Entretanto, estas modificações não foram suficientes para viabilizar sua compressão direta. Assim sendo, o material foi compactado em máquina de comprimir e em rolos para produção de grânulos. A análise do perfil compressional do PSA e dos granulados, empregando o modelo de Heckel, revelou que o aumento da força de compactação durante a operação de granulação foi o principal responsável pela diminuição no potencial de deformação plástica dos grânulos. Como conseqüência, a recompressão dos grânulos em máquina de comprimir originou compactos com dureza inferior àqueles preparados diretamente do complexo farmacêutico. Enquanto o PSA apresentou reduzida tendência ao rearranjo particular nos estágios iniciais da compressão, os grânulos sofreram intensa reacomodação decorrente da fragmentação e formação de novas pontes, assumindo comportamento plástico em pressões de compressão mais elevadas. Por fim, comprimidos contendo teor elevado de granulado de PSA foram preparados utilizando como adjuvantes a celulose microcristalina, o dióxido de silício coloidal e a croscarmelose sódica. A influência da concentração de dióxido de silício coloidal e croscarmelose sódica na formulação, sobre as respostas de dureza, friabilidade e tempo de desintegração dos comprimidos foram avaliadas através de um Desenho Composto Central. Os dados experimentais foram analisados estatisticamente, utilizando os modelos matemáticos para gerar superfícies de resposta. Os resultados indicaram que a concentração de dióxido de silício coloidal foi inversamente relacionada às respostas de dureza e friabilidade, enquanto que a concentração de croscarmelose sódica foi a principal responsável pela redução no tempo de desintegração. As condições ótimas foram selecionadas através da sobreposição dos gráficos, buscando uma formulação com menor tempo de desintegração, menor friabilidade e o máximo de dureza. A formulação eleita deve conter 1,2 % (m/m) de dióxido de silício coloidal e 5,0 % (m/m) de croscarmelose sódica. Nestas condições os comprimidos apresentaram dureza de 107,9 N, friabilidade de 0,56 % (m/m) e desintegração máxima em 6,8 min. / The leaves of Maytenus ilicifolia are widely used within the traditional Brazilian medicine due to its properties against gastric disorders. Once proved its efficacy and safety, this vegetable drug has been the object of several studies to establish the basis for quality control. Taking this into consideration, it was necessary to evaluate the feasibility of developing an analytical method based on the precipitation of tannins, by using polyvinylpyrrolidone for precipitation. The data have shown that the removal of the tannins in solution was successful when insoluble polyvinylpyrrolidone was used. Hereby it was observed that the polymer has also the ability to precipitate other polyphenols besides the tannins from the aqueous solution of M. ilicifolia. However, the interactions of the polymer happened mostly with the condensed tannins. Further it was developed and also validated an HPLC method to separate and quantify catechin and epicatechin present in aqueous extracts of M. ilicifolia. No significant variations of peak areas or retention times were observed when the evaluation of the method was executed, showing satisfactory chromatographic conditions. In order to make a spray dried extract (SDE) using a spray-drier fitted out with a rotary atomizer, it was necessary to concentrate the extractive solution. This operation had been performed under reduced pressure until a content of 15 % solids was achieved. The drying of the concentrated solution lead to a yield of 90 %, and the obtained SDE presented spherical particles, which have a rough surface and a mean particle size of 22,51.1.m. The technological characterization of the SDE showed that attributes such as densities (tap and bulk), compressibility and flow, have been improved when compared to the product prepared using a mini spray-drier supplied with a pneumatic nozzle. However, these modifications were not enough to enable its direct compression. Therefore, the material was compacted to produce granules after slugging or roller compaction, The compressional analysis of the SDE and each granule batch using the Heckel equation proved that the increase in the compaction force once processing the granulation was the main factor to reduce the material's ability to undergo plastic deformation. Consequently, the recompression of the granules in a tablet press led to compacts with a lower crushing strength compared to that prepared by direct compression of the pharmaceutical complex. Whereas the SDE has shown a reduced tendency to particle rearrangement at early stage of compression, the granules showed an intense fragmentation and rebound behavior, presenting a plastic behavior when higher compaction pressures were used. Finally, tablets containing a high dose of granulated spray dried extract of M. ilicifolia were prepared by using the following excipients: microcrystalline cellulose (filler/binder), colloidal silicon dioxide (glidant and moisture adsorber) and cross-linked carboxymethylcellulose (disintegrant). The influences of colloidal silicon dioxide (CSD) and cross-linked carboxymethylcellulose (CMC-Na) on the tablet hardness, disintegration time and friability were evaluated by a central composite design. The data were analyzed statistically and mathematical models were used to create response surfaces. The results have indicated that the concentration of CSD shows an inverse relation to the responses of hardness and friability, while the concentration of CMC-Na was the most important factor that caused a reduction of the time necessary for total disintegration. The optimal conditions for processing were chosen by the overlapping of graphics, taking into consideration that the formulation should present a minimum of disintegration time, lower friability and a maximum of hardness. Thus, it was found that the best formulation should have a content of 1.2% (w/w) of CSD and 5.0% (w/w) of CMC-Na. Regarding these conditions, the tablets have shown a hardness of 107.9 N, friability of 0.56% (w/w) and a disintegration time of 6.8 min.

As folhas de Maytenus ilicifolia são amplamente utilizadas na medicina tradicional brasileira no tratamento de distúrbios gástricos. Após a comprovação de sua eficácia e segurança, a referida droga vegetal tem sido objeto de diversos estudos com a finalidade de estabelecer parâmetros para o controle de qualidade. Neste sentido, foi avaliada a viabilidade do desenvolvimento de um método analítico fundamentado na precipitação de taninos empregando povidona como agente seqüestrante. Os dados revelaram que a remoção de taninos em solução é satisfatória quando empregada a povidona insolúvel. Foi observado que o polímero também é capaz de precipitar outros polifenóis além dos taninos presentes na solução extrativa de M. ilicifolia. Entretanto, as interações do polímero ocorrem preferencialmente com os derivados catéquicos. Adicionalmente foi desenvolvido e validado um método por CLAE para separação e quantificação de catequina e epicatequina em produtos derivados de M. ilicifolia. Não foram detectadas variações nas áreas dos picos ou nos tempos de retenção durante os ensaios de avaliação do método, sugerindo que as condições cromatográficas empregadas são satisfatórias. Para viabilizar a preparação de produto seco por aspersäo (PSA) em torre de secagem piloto munida de aspersor rotatório, foi necessária a concentração da solução extrativa. A operação foi realizada sob pressão reduzida até alcangar um teor de sólidos mínimo de 15 % (m/m). A operação de secagem da solução concentrada apresentou rendimento de 90 % e o PSA obtido apresentou partículas esféricas, com superfície rugosa e tamanho médio de 22,5 um. A caracterização tecnológica do material revelou que as suas propriedades tecnológicas, tais como, densidades, compressibilidade e fluxo, foram incrementadas quando comparados com o produto preparado em equipamento de bancada. Entretanto, estas modificações não foram suficientes para viabilizar sua compressão direta. Assim sendo, o material foi compactado em máquina de comprimir e em rolos para produção de grânulos. A análise do perfil compressional do PSA e dos granulados, empregando o modelo de Heckel, revelou que o aumento da força de compactação durante a operação de granulação foi o principal responsável pela diminuição no potencial de deformação plástica dos grânulos. Como conseqüência, a recompressão dos grânulos em máquina de comprimir originou compactos com dureza inferior àqueles preparados diretamente do complexo farmacêutico. Enquanto o PSA apresentou reduzida tendência ao rearranjo particular nos estágios iniciais da compressão, os grânulos sofreram intensa reacomodação decorrente da fragmentação e formação de novas pontes, assumindo comportamento plástico em pressões de compressão mais elevadas. Por fim, comprimidos contendo teor elevado de granulado de PSA foram preparados utilizando como adjuvantes a celulose microcristalina, o dióxido de silício coloidal e a croscarmelose sódica. A influência da concentração de dióxido de silício coloidal e croscarmelose sódica na formulação, sobre as respostas de dureza, friabilidade e tempo de desintegração dos comprimidos foram avaliadas através de um Desenho Composto Central. Os dados experimentais foram analisados estatisticamente, utilizando os modelos matemáticos para gerar superfícies de resposta. Os resultados indicaram que a concentração de dióxido de silício coloidal foi inversamente relacionada às respostas de dureza e friabilidade, enquanto que a concentração de croscarmelose sódica foi a principal responsável pela redução no tempo de desintegração. As condições ótimas foram selecionadas através da sobreposição dos gráficos, buscando uma formulação com menor tempo de desintegração, menor friabilidade e o máximo de dureza. A formulação eleita deve conter 1,2 % (m/m) de dióxido de silício coloidal e 5,0 % (m/m) de croscarmelose sódica. Nestas condições os comprimidos apresentaram dureza de 107,9 N, friabilidade de 0,56 % (m/m) e desintegração máxima em 6,8 min. / The leaves of Maytenus ilicifolia are widely used within the traditional Brazilian medicine due to its properties against gastric disorders. Once proved its efficacy and safety, this vegetable drug has been the object of several studies to establish the basis for quality control. Taking this into consideration, it was necessary to evaluate the feasibility of developing an analytical method based on the precipitation of tannins, by using polyvinylpyrrolidone for precipitation. The data have shown that the removal of the tannins in solution was successful when insoluble polyvinylpyrrolidone was used. Hereby it was observed that the polymer has also the ability to precipitate other polyphenols besides the tannins from the aqueous solution of M. ilicifolia. However, the interactions of the polymer happened mostly with the condensed tannins. Further it was developed and also validated an HPLC method to separate and quantify catechin and epicatechin present in aqueous extracts of M. ilicifolia. No significant variations of peak areas or retention times were observed when the evaluation of the method was executed, showing satisfactory chromatographic conditions. In order to make a spray dried extract (SDE) using a spray-drier fitted out with a rotary atomizer, it was necessary to concentrate the extractive solution. This operation had been performed under reduced pressure until a content of 15 % solids was achieved. The drying of the concentrated solution lead to a yield of 90 %, and the obtained SDE presented spherical particles, which have a rough surface and a mean particle size of 22,51.1.m. The technological characterization of the SDE showed that attributes such as densities (tap and bulk), compressibility and flow, have been improved when compared to the product prepared using a mini spray-drier supplied with a pneumatic nozzle. However, these modifications were not enough to enable its direct compression. Therefore, the material was compacted to produce granules after slugging or roller compaction, The compressional analysis of the SDE and each granule batch using the Heckel equation proved that the increase in the compaction force once processing the granulation was the main factor to reduce the material's ability to undergo plastic deformation. Consequently, the recompression of the granules in a tablet press led to compacts with a lower crushing strength compared to that prepared by direct compression of the pharmaceutical complex. Whereas the SDE has shown a reduced tendency to particle rearrangement at early stage of compression, the granules showed an intense fragmentation and rebound behavior, presenting a plastic behavior when higher compaction pressures were used. Finally, tablets containing a high dose of granulated spray dried extract of M. ilicifolia were prepared by using the following excipients: microcrystalline cellulose (filler/binder), colloidal silicon dioxide (glidant and moisture adsorber) and cross-linked carboxymethylcellulose (disintegrant). The influences of colloidal silicon dioxide (CSD) and cross-linked carboxymethylcellulose (CMC-Na) on the tablet hardness, disintegration time and friability were evaluated by a central composite design. The data were analyzed statistically and mathematical models were used to create response surfaces. The results have indicated that the concentration of CSD shows an inverse relation to the responses of hardness and friability, while the concentration of CMC-Na was the most important factor that caused a reduction of the time necessary for total disintegration. The optimal conditions for processing were chosen by the overlapping of graphics, taking into consideration that the formulation should present a minimum of disintegration time, lower friability and a maximum of hardness. Thus, it was found that the best formulation should have a content of 1.2% (w/w) of CSD and 5.0% (w/w) of CMC-Na. Regarding these conditions, the tablets have shown a hardness of 107.9 N, friability of 0.56% (w/w) and a disintegration time of 6.8 min.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

Luiz Gonzaga tornou-se um dos maiores intérpretes e compositores populares de sua época. Sua voz, sua vestimenta, seu modo particular de falar, sua entonação fizeram com que se transformasse em um grande ícone nacional, o representante de uma identidade regional. Considerado um dos maiores divulgadores da cultura, costumes e crenças do Nordeste, suas canções tentam representar o contexto sociocultural e sociopolítico da região Nordeste. Alguns pesquisadores consideram suas canções, até hoje, como uma marca legítima e mantedora das raízes culturais e folclóricas da região. Após leitura parcial da literatura sobre a sua obra percebemos uma carência de estudos críticos lingüísticos vistos numa perspectiva da Semiótica-Social. Este estudo investiga de que maneira as canções de Luiz Gonzaga contribuem na constituição de uma identidade nordestina. A relevância dessa abordagem, consiste em colaborar para a construção do conhecimento acerca dos processos de constituição da identidade nordestina, através de uma leitura crítica das canções, rompendo com as práticas discursivas que favoreçam a criação de estereótipos nordestinos e conseqüentemente a unificação de uma identidade cultural regional. Foram escolhidas, de forma aleatória, vinte canções da obra musical de Luiz Gonzaga, as quais foram analisadas numa perspectiva da Análise Crítica do Discurso (ACD), segundo o modelo tridimensional de Norman Fairclough (2001). As categorias utilizadas na análise foram: Transitividade na perspectiva da Lingüística Sistêmica Funcional (LSF), Ethos/Identidade culturais, Intertextualidade e a Interdiscursividade. Nas canções observam-se articulações dos traços culturais da região com a natureza e seus componentes particularmente regionais, instituindo sentidos à construção de uma realidade social. A seca, nessas canções, transforma-se no único grande problema do Nordeste. Dessa maneira, Luiz Gonzaga reproduz a prática discursiva estabelecida pela sociedade da época, aderindo à ordem do discurso do seu entorno. Assim, contribui para reforçar uma visão estereotipada, reduzindo a identidade nordestina ao flagelado da seca. As canções de Luiz Gonzaga além de reforçar esse estereótipo nordestino, reproduzem a ordem social da época em que viveu Luiz Gonzaga. Suas canções, de certa forma, faziam com que o Governo mandasse cada vez mais subsídios para socorrer a região, o que gerou a indústria da seca. O nordestino é representado como um povo escravizado e dependente economicamente do Governo, incapaz de perceber as lutas hegemônicas da sua região e reagir a elas. / Luiz Gonzaga has become one of the most important composers and interpreters of Brazilian popular music of his time. His voice, his clothing, his particular way of talking and his intonation made him become a great national symbol, a representative of a regional identity. Luiz Gonzaga is considered one of the most active men who spread out culture, costumes and beliefs of the Brazilian Northeast, and his songs try to portrait the socio-cultural and sociopolitical context of the Northeast. This very day, some researchers consider his songs as a genuine mar k that also keeps alive cultural and folkloric roots of the region. After reading part of the literature on his work, we realized that there were few linguistic critical studies based upon Social Semiotics. This study investigates the way Luiz Gonzaga.s songs contribute to establish a Brazilian northeastern people identity. This approach becomes relevant while it helps to improve the knowledge on the processes of construction of that identity, through a critical reading of the songs opposite to those discursive practices which tend to favor the creation of stereotypes related to Brazilian northeastern people, and so to the unification of a cultural identity in the region. We chose at random twenty songs by Luiz Gonzaga, which were analyzed according to Critical Discourse Analysis (CDA), and based upon the threedimensional model proposed by Norman Fairclough (2001). The categories for the analysis were: transitivity - according to Systemic Functional Linguistics (SFL), Ethos/Cultural identities, intertextuality and interdiscourse. In the songs, it.s possible to see a connection between cultural aspects of the region, nature and its regional elements, what confers meanings to the construction of a social reality. The drought, in those songs, appears as the only big problem in the Brazilian Northeast. Thus, Luiz Gonzaga represents the discursive practice of the society of that time and adheres to the discursive order that surrounds him. Then, he reinforces a stereotyped point-of-view which reduces Brazilian northeastern people identity to those of who suffered with the drought. In addition, Luiz Gonzaga.s songs represent the social order of the time he lived. His songs in a certain way incited the Government to send more and more supplies to help people in the region, an attitude that generated the Brazilian drought industry. Brazilian Northeastern people are represented as slaves and economically dependent on the Government, unable to perceive hegemonic struggles of the region and to combat them.

O interesse mundial por produtos derivados de plantas medicinais cresceu vertiginosamente nos últimos anos. Produtos derivados de plantas medicinais são misturas multicomponentes e sua eficácia pode estar associada a mais de um composto tornando o seu controle da qualidade tarefa árdua. Apesar de grandes investimentos em pesquisa terem sido feitos para a comprovação da eficácia e segurança dos medicamentos derivados de plantas medicinais, poucos são os trabalhos científicos que dizem respeito à manutenção da qualidade desses produtos. Extratos secos de plantas medicinais possuem efeito farmacológico devido à presença de substâncias ativas que estão sujeitas a reações de degradação. A velocidade dessas reações pode ser prevista através do estudo de estabilidade que envolve o armazenamento dos extratos em diversas condições (diferentes temperatura e umidade relativa do ar). Por esse motivo, o objetivo dessa tese foi o estudo da influência do processo de obtenção, das condições de armazenamento e das propriedades físico-químicas sobre a estabilidade de extratos secos padronizados de plantas medicinais. Visando o entendimento dos fatores que afetam a estabilidade de produtos naturais, os extratos foram caracterizados em relação à composição (presença de açúcares redutores, proteínas, lipídeos, carboidratos de alta e baixa massa molecular, acidez titulável), análise térmica, determinação de isotermas de adsorção (ou absorção), espectroscopia de Infravermelho e difração de raios-X. Após caracterização, os extratos secos foram armazenados em câmara climática visando avaliar a variação das propriedades físicas e químicas em função do período e condições de armazenamento. Para um entendimento mais amplo dos fatores que proporcionam maiores influências na estabilidade de produtos derivados de plantas medicinais, extratos secos foram obtidos a partir de três plantas medicinais amplamente utilizadas na medicina popular brasileira: Bauhinia forficata (hipoglicemiante), Maytenus ilicifolia (anti-úlcera gástrica) e Passiflora alata (sedativo e ansiolítico). Os extratos secos foram obtidos a partir de dois distintos processos de secagem o spray-drying (secagem por nebulização) e o processo leito de jorro. Os resultados obtidos indicam que todos os extratos secos analisados apresentaram algum tipo de alteração dos padrões de qualidade inicialmente observados. O que não foi possível mostrar é o quanto essas perdas poderiam influenciar na atividade biológica desses produtos. Por se tratar de uma mistura multicomponente é pouco provável que se estabeleçam técnicas que possam assegurar que todos esses componentes sejam mantidos durante todo o período de armazenamento. Porém, fenômenos importantes para a atividade farmacológica como a cristalização das substâncias, (demonstrada através dos espectros de raios-X), ou a degradação de marcadores (CLAE e espectrofotometria) puderam ser monitorados e previstos. / Nowadays, the global interest for herbal drugs has increased significantly. Herbal medicinal products are a complex mixture of substances and their efficacy might be associated with one or more compounds. For this reason, the quality control of herbal products is a challenging task. In spite of a great increase in researches concerning the pharmacological and toxicological aspects of herbal drugs, there is a lack of scientific works related to the quality maintenance of these products. Among all the herbal medicinal products the standardized dried extracts are gaining more interest as a way of controlling dosage and increasing safety of the phytopharmaceuticals. Herbal dried extracts are effective due to the presence of active substances, and these substances may degrade. The rate of degradation can be measured through stability testing what is done through the storage of the product in different storage conditions (temperature and relative humidity). For this reason, the aim of this thesis was the study of the influence of the obtainment process, storage conditions and physical and chemical properties in the standardized dried extracts stability. For the understanding of the factors which affects the stability of the natural products, the extracts were characterized in relation to their composition (reducing sugars, proteins, lipids, low and high molecular weight carbohydrates, and titrable acidity), the calorimetric profile, determination of sorption isotherms, near infrared spectroscopy and X-ray diffraction analysis. Furthermore, the dried extracts were stored in a climatic chamber for the evaluation of the physical and chemical properties variations in relation to the time and storage conditions. Herbal dried extracts obtained from three plant species commonly used in Brazil were used in this study: Bauhinia forficata (hypoglycemiant), Maytenus ilicifolia (antiulcerogenic) and Passiflora alata (sedative and anxiolytic). The dried extracts were produced trough two different drying processes the spray drying and the spouted bed drying. In conclusion, the results obtained showed that all the studied extracts presented some deviation of the initial standards owing to its great sensibility to the environmental conditions. However, it was not possible to show how this lost would affect the effectiveness of these products. Moreover, for being such a complex mixture of components it is almost impossible the establishment of techniques that will assure that all this components will remain unchanged during the storage period. Nevertheless, some important factors for the pharmacological activity of substances as the crystallization (X-ray diffraction) or the degradation of the chemical markers (high performance liquid chromatography and spectrophotometry) can be evaluate and previewed