Tesis sobre el tema "Herbs Thin layer chromatography"

This research was initiated after a 100-year flood caused an oil spill on the San Jacinto River (Houston, Texas) in October of 1994. After the floodwaters subsided the released petroleum floating on the water was deposited on the surrounding lands. The petroleum spill was used as an opportunity to research intrinsic petroleum biodegradation in a 9-acre petroleum impacted estuarine wetland. The first phase of this research (Phase I) began in December 1994, approximately 1.5 months after the spill of opportunity and involved the study and quantification of in-situ petroleum biodegradation. The second phase of the research (Phase II) began in March 1996 with a controlled oil release to study and evaluate the success of two bioremediation treatments versus natural biodegradation. The study of in-situ petroleum hydrocarbon degradation and the evaluation of bioremediation amendments were successfully quantified using GC-MS analytical techniques. However, the GC-MS technique is limited to the analyses of hydrocarbon compounds, a disadvantage that precludes the overall characterization of petroleum degradation. The research presented here details an analytical technique that was used to provide a full characterization of temporal petroleum biodegradation. This technique uses thin layer chromatography coupled with flame ionization detection (TLC-FID) to characterize the saturate and aromatic (hydrocarbon) fractions and the resin and asphaltene (non-hydrocarbon, polar) fractions. Other analysis techniques, such as HPLC-SARA analysis, are available for the full characterization of the four petroleum fractions. However, these techniques do not lend themselves well to the application of large sample set analysis. A significant advantage of the TLC-FID analysis to other petroleum analysis techniques is the ability to analyze several samples concurrently and quickly with relative ease and few resources. For the purposes of the Phase I and Phase II research the TLC-FID analysis method was evaluated, refined and applied to quantify the temporal biodegradation and bioremediation of petroleum. While the TLC-FID analysis produces a full characterization, it cannot supplant the GC-MS analysis for petroleum bioremediation research. However, it can be used in conjunction with the GC-MS to expand the knowledge of petroleum bioremediation and remediation strategies.

Recently, high performance thin layer chromatography, or HPTLC has seen considerable growth as an analytical technique. Most often perceived as a semi-quantitative or preliminary technique, in reality HPTLC is an analytical technique in its own right. Modern instrumentation, improved stationary phases, and automated techniques have found their way into the realm of HPTLC, vastly improving the scope of the technique. Today, HPTLC is a very popular analytical technique, possessing many advantages such as ease of use, high throughput, high sensitivity, and low cost. HPTLC is also applicable to a wide variety of compounds in a wide variety of matricies. Despite these advantages, HPTLC has still not received the recognition it deserves as a true quantitative analytical technique. The following chapters will describe improvements in various stages of the technique. Chapter 1 will discuss the general theory behind HPTLC, highlighting its advantages and disadvantages, as well as areas needing improvement. Chapter 2 will discuss a new application technique for HPTLC that greatly improves upon current methodologies for sample application. Chapters 3 and 4 will discuss a novel detection technique used to image the entire plate simultaneously and give excellent quantitative information of analytes on the HPTLC plate. Chapter 5 will discuss the use of an infrared focal plane array as a detection technique that gives both qualitative and quantitative information. Finally Chapter 6 will discuss the use of a CCD home video camera as an inexpensive alternative to scientifically operated CCD's.

This dissertation describes advances in the microfabrication of thin layer chromatography (TLC) plates. These plates are prepared by the patterning of carbon nanotube (CNT) forests on substrates, followed by their infiltration with an inorganic material. This document is divided into ten sections or chapters. Chapter 1 reviews the basics of conventional TLC technology. This technology has not changed substantially in decades. This chapter also mentions some of the downsides of the conventional approach, which include unwanted interactions of the binder in the plates with the analytes, relatively slow development times, and only moderately high efficiencies. Chapter 2 focuses primarily on the tuning of the iron catalyst used to grow the CNTs, which directly influences the diameters of the CNTs grown that are produced. Chapter 3 focuses on the atomic layer deposition (ALD) of SiO2 from a silicon precursor and ozone onto carbon-nanotubes to obtain an aluminum free stationary phase. This approach allowed us to overcome the tailing issues associated with the earlier plates prepared in our laboratory. Chapter 4 is a study of the hydroxylation state of the silica in our TLC plates. A linear correlation was obtained between the SiOH+/Si+ time-of-flight secondary ion mass spectrometry (ToF-SIMS) peak ratio and the isolated silanol peak position at ca. 3740 cm-1 in the diffuse reflectance infrared spectroscopy (DRIFT) spectra. We also compared the hydroxylation efficiencies on our plates of ammonium hydroxide and HF. Chapter 5 reports a series of improvements in TLC plate preparation. The first is the low-pressure chemical vapor deposition (LPCVD) of silicon nitride onto CNTs, which can be used to make very robust TLC plates that have the necessary SiO2 surfaces. These TLC plates are the best we have prepared to date. We also describe here the ALD deposition of ZnO into these devices, which can make them fluorescent. Chapters 6 – 10 consist of contributions to Surface Science Spectra (SSS) of ToF-SIMS spectra of the materials used in our microfabrication process. SSS is a peer-reviewed database that has been useful to many in the surface community. The ToF-SIMS spectra archived include those of (i) Si/SiO2, (ii) Si/SiO2/Al2O3, (iii) Si/SiO2/Al2O3/Fe, (iv) Si/SiO2/Fe (annealed at 750 °C in H2), and (v) Si/SiO2/Al2O3/Fe(annealed)/CNTs. Both positive and negative ion spectra have been submitted. In summary, the present work is a description of advances in the development, thorough characterization, optimization, and application development of microfabricated thin layer chromatography plates that are superior to their commercial counterparts.

Thin-layer chromatography (TLC) is of great importance for the pharmaceutical industry as a simple, quick, and low cost analytical method. Considerable effort has been made over the past decades to combine the simplicity of TLC with the selectivity and sensitivity of mass spectrometry (MS) detection. In the pharmaceutical industry sensitivity is an especially important factor, since the allowed impurity level of most drugs is under 0.1%.The aim of the present thesis was to develop methods for the direct examination of pharmaceutical compounds from TLC plates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS). The study was started by comparing several approaches for the application of the matrix for direct TLC-MALDI including a newly developed electrospray matrix deposition method. This new method was found to be superior to the other techniques studied. It produced a stable signal, minimised analyte spreading, and hence allowed the scanning of a TLC plate to obtain chromatographic as well as mass spectral data. The plotted mass chromatograms assisted in spot location, and allowed the calculation of Rf-values. These showed good agreement with the Rf -values determined by UV detection. The decrease in mass resolution and mass accuracy commonly observed in TLC-MALDI TOF MS due to the uneven nature of the silica gel layer was corrected by internal recalibration on selected matrix ions during the scanning of the TLC plate. To enhance the signals recorded directly from a TLC plate the use of an extraction solvent prior the matrix application was explored. Further improvements in sensitivity were obtained by modifying a robotic x-y-z axis motion system to act as an electrospray deposition device and by use of special Si 60 F[254] HPTLC-MALDI targets. Using both approaches sensitivities in the high fmol range were obtained. To minimise matrix interference, which can suppress analyte signals, the application of suspensions of particles of different materials and sizes (Co-UFP, TiN, TiO[2], graphite and silicon) onto eluted TLC plates were investigated. The structural analysis of pharmaceutical compounds was achieved by post-source decay - matrix-assisted laser desorption/ionisation (PSD-MALDI) mass spectrometry performed directly on the separated spots. TLC-MALDI MS is not only applicable to the qualitative analysis of pharmaceutical compounds. The generation of quantitative data by using a structural analogue as an internal standard is also described. Different approaches to the incorporation of the internal standard into the TLC plate were tested. The most successful approach was to develop the TLC plate in the mobile phase to which the internal standard was added. Good accuracy, precision, linearity and sensitivity was obtained using this approach.

The purpose is to isolate a natural compound, which shows a high activity against Candida albicans, from plant, Vincetoxicum stocksii. Bio-Assay, Thin layer Chromatography, Column Chromatography, TLC bio Assay, and other extraction techniques are used in order to isolate the active compound. First, bio assay technique is carried out on the crude gum. Next, several flash chromatography columns are carried out in order to isolate the target compound, which has a Rf value of ~0.53 in 10:1 DCM/methanol solvent mixture. The TLC bioassay technique is also carried out in order to confirm the hypothesis that the target compound is indeed active.

This dissertation contains the following sections. Chapter 1 contains a detailed description of the theory of thin layer chromatography (TLC). Chapter 2 describes the benefits and practical considerations of elevated temperatures in liquid chromatography (LC). The porous graphitic carbon (PGC) I modified as part of my work is often used in elevated temperature LC. Chapter 3 shows a thermodynamic analysis of chromatographic retention at elevated temperature, and Chapter 4 contains a closer look at the van 't Hoff equation in LC and how it can be used in retention modeling. In Chapter 5, I describe a new procedure for microfabricating TLC plates that avoids the volume/feature distortions that occurred in our first microfabrication. The primary advance of this work was the priming of the carbon nanotube (CNT) forests with chemical vapor deposition (CVD) carbon and atomic layer deposition (ALD) alumina, which permitted effective ALD-like deposition of SiO2. Chapter 6 describes advancements in the microfabrication process of TLC, which excluded the use of the CVD carbon and Al2O3 coating as described in Chapter 5. The use of ozone, to lightly oxidize the CNT surface, primed the material for direct ALD deposition. Chapter 7 gives a detailed surface analysis of the microfabrication process up to and including the CNT forest. It was noticed that a channeling effect was present during Rutherford backscattering analysis of the CNTs. Additionally, characterization of CNTs using time-of-flight secondary ion mass spectrometry in the negative ion mode showed an odd-even effect for a homologous series of carbon, where the even moieties had a stronger signal. Chapter 8 describes the functionalization of PGC with di-tert-amyl peroxide (DTAP) and its effect on increasing the chromatographic performance as seen by a reduction in the tailing factors of test analytes. Chapter 9 -- 13 are detailed X-ray photoelectron analyses of the thin films and CNTs used in producing microfabricated TLC plates.

A sol-gel stationary phase was developed for in-situ infrared (IR) detection of analytes on thin-layer chromatography (TLC) plates. These sol-gel-based TLC plates have improved optical properties compared with conventional TLC plates in IR spectroscopic analysis. Samples can be analyzed in transmission geometry, requiring no special attachments. The sol-gel-based TLC plates demonstrate significantly better light throughput and a wider spectral range than conventional TLC plates analyzed in diffuse reflectance geometries.The sol-gel precursor, methyltrimethoxyorthosilicate (MTES), was templated with cetyltrimethylammonium bromide (CTAB) and urea in order to form a porous sol-gel. Aerosol deposition was used to apply the sol-gel solution onto either glass slides or silicon wafers within an enclosed chamber. Many variables were studied to determine their effect on the quality of the sol-gel stationary phases, including the ratio of MTES:methanol:water:CTAB:urea:HCl:, gelation times and temperatures, and deposition rate. Sol-gel films prepared using MTES/methanol/water/CTAB at ratios of 1 : 20 : 7 : 0.2 containing 5 wt% urea (relative to MTES) and pH 1.5 were crack-free, mechanically stable, and uniform in appearance. The films were tens of microns thick with a highly interconnected porous structure.For chromatographic separations, the films exhibited good solvent migration velocity and could be repeatedly washed and reused for TLC separations without showing degradation in the separation. Several different classes of compounds, including polyaromatic hydrocarbons and dyes, were successfully separated. Theoretical plate values measured on the MTES-based sol-gel films were comparable to those obtained on commercially available TLC plates.

Quality control of herbals has its roots in the study of morphoanatomic and organoleptic characters. Nevertheless, in the last century, with the evolution of analytical chemistry, the quality control rapidly evolved from elementary tests to the use of sophisticated instruments combined with software for data management. In the current days, many authorities and organizations recommend a suite of tests, featuring many of these instruments, to evaluate quality of herbal products. HPTLC offers a comprehensive set of data that can be used not only for identification but also to evaluate the purity and content of herbal drugs, herbal preparations, and herbal products. The objective of this doctoral thesis was to explore in-depth the capacities of HPTLC and develop applications for quality control of herbals, far beyond simple identification of the herbal drugs, preparations, and products. For that, five studies were developed. In the first study, the quality of herbal drugs, preparations, and products from milk thistle fruit, coneflower root and aerial parts and black cohosh root, regulated under food supplements or medicines were evaluated with existing HPTLC methods. The suitability of these methods, using the entire fingerprint and several detection modes, as a tool for detecting quality problems, mainly adulterations, was confirmed. In the second study, the comprehensive HPTLC fingerprinting concept was developed with the goal of simplifying the quality control process. This concept combines the qualitative and quantitative information of HPTLC images, obtained in a single analysis, to evaluate the identity, purity and content of herbals. The possibilities of applying it to identify an herbal drug, detect mixtures with re¬lated species (purity), and develop a minimum content test of an analytical marker were demonstrated in Angelica gigas root. In the third study, the application of comprehensive HPTLC fingerprinting aimed to go one step beyond in the test for adulterants and to evaluate the use of the HPTLC for purity limit tests. This approach was evaluated with sam¬ples of ginkgo leaf and extracts, commercialized as food supplements in different countries. This study demonstrated that the information contained in the HPTLC finger¬prints was suitable for verifying levels of rutin and quercetin, providing results similar to that of the HPLC limit test. It was also useful for detecting mixtures of ginkgo products not only with rutin and quercetin but also with buckwheat herb and sophora (flower bud or fruit). In the fourth study, it was evaluated the use of comprehensive HPTLC fingerprinting as an alternative method to the current HPLC assay of markers of TCM drugs in the Ph. Eur. The goal of this project was to simplify the determination of content and thus reducing the number of tests to be performed during quality control. For this evaluation, two TCM herbal drugs were chosen by the experts of the TCM working party of the Ph. Eur.: Fritillaria thunbergii bulbs and corydalis rhizome. In both cases, comprehensive HPTLC fingerprinting was proven useful for identification and minimum content testing in one single analysis. The fifth study goes one step beyond in the content determination. While the previous studies focused in the quantification of single markers, this study aimed to apply comprehensive HPTLC fingerprinting to quantify a group of constituents in an herbal drug, as an example of a more holistic assessment of quality. This determination was combined with the tests for purity and identity. To illustrate this concept, Ganoderma lucidum fruiting body was chosen. In this work, HPTLC proved to be a useful technique for routine quality control of herbal drugs, preparations and products. As demonstrated, it can simplify this process by applying the concept of comprehensive HPTLC fingerprinting. A detailed guideline of how to develop, validate and apply comprehensive HPTLC fingerprinting methods for routine quality control of herbals has been elaborated and is also included in the thesis.
El control de qualitat dels productes a base de plantes té les seves arrels en l'estudi dels caràcters morfoanatòmics i organolèptics. No obstant això, al segle passat, amb l'evolució de la química analítica, el control de qualitat va evolucionar ràpidament des de proves elementals a l'ús d'instruments sofisticats combinats amb programari per a la gestió de dades. Actualment, moltes autoritats i organitzacions recomanen un conjunt de proves, amb molts d'aquests instruments, per avaluar la qualitat dels productes a base de plantes. La HPTLC ofereix un conjunt complet de dades que poden usar-se no només per a la identificació, sinó també per avaluar la puresa i el contingut de drogues i preparats vegetals i productes a base de plantes. L'objectiu d'aquesta tesi doctoral va ser explorar en profunditat les capacitats de la HPTLC i desenvolupar aplicacions per al control de qualitat dels productes de plantes medicinals, molt més enllà de la simple identificació de drogues i preparats vegetals i productes acabats comercialitzats. Per això, es van desenvolupar cinc estudis. En el primer estudi, es va avaluar la qualitat de les drogues vegetals, preparats vegetals i productes a base de plantes del fruit de card marià, l'arrel i la part aèria de equinàcia i l'arrel de cimicífuga, regulats com complements alimentosos o medicaments, amb els mètodes existents de HPTLC. Es va confirmar la idoneïtat d'aquests mètodes, utilitzant l'empremta dactilar completa i diversos formes de detecció, com una eina per a detectar problemes de qualitat, principalment adulteracions. En el segon estudi, es va desenvolupar el concepte d'anàlisi integral de l'empremta dactilar per HPTLC (comprehensive HPTLC fingerprinting) amb l'objectiu de simplificar el procés de control de qualitat. Aquest concepte combina la informació qualitativa i quantitativa de les imatges d’HPTLC, obtingudes en una única anàlisi, per avaluar la identitat, la puresa i el contingut dels productes a base de plantes. La seva aplicabilitat per identificar una droga vegetal, detectar mescles amb espècies relacionades (puresa) i desenvolupar un assaig de contingut mínim d'un marcador analític es van demostrar en l'arrel d'Angelica gigas. En el tercer estudi, l'aplicació de l'anàlisi integral de l'empremta dactilar per HPTLC va tenir com a objectiu anar un pas més enllà en l'assaig de adulterants i avaluar l'ús de l’HPTLC per a l'assaig límit de puresa. Aquest enfocament es va avaluar amb mostres de fulla i extracte de ginkgo, comercialitzats com a complements alimentosos en diferents països. Aquest estudi va demostrar que la informació continguda en les empremtes dactilars per HPTLC era adequada per verificar els nivells de rutina i quercetina, proporcionant resultats similars als de l'assaig límit per HPLC. També va ser útil per detectar mescles de productes de ginkgo no només amb rutina i quercetina, sinó també amb part aèria de blat sarraí i sòfora (botó floral i fruit). En el quart estudi, es va avaluar l'ús de l'anàlisi integral de l'empremta dactilar per HPTLC com un mètode alternatiu a l'actual valoració de marcadors per HPLC en drogues vegetals de la medicina tradicional xinesa (MTC) a la Ph. Eur. L'objectiu d'aquest projecte era simplificar la determinació del contingut i, per tant, reduir el nombre de proves a realitzar durant el control de qualitat. Per a aquesta avaluació, dues drogues vegetals de la MTC van ser elegides pels experts del grup de treball TCM de la Ph. Eur.: bulb de Fritillaria thunbergii i rizoma de coridalis. En tots dos casos, es va demostrar que l'empremta dactilar completa per HPTLC era útil per a la identificació i l'assaig de contingut mínim en una sola anàlisi. El cinquè estudi va un pas més enllà en la determinació del contingut. Si bé els estudis anteriors es van centrar en la quantificació de marcadors individuals, aquest estudi va tenir com a objectiu aplicar l'anàlisi integral de l'empremta dactilar per HPTLC a la quantificació d'un grup de components en una droga vegetal, com un exemple d'una avaluació més holística de la qualitat. Aquesta determinació es va combinar amb els assajos d'identitat i puresa. Per il·lustrar aquest concepte, es va triar el carpòfor de Ganoderma lucidum. En aquest treball, s'ha demostrat que la HPTLC és una tècnica útil per al control de qualitat rutinari de drogues i preparats vegetals i productes a base de plantes, i que es pot simplificar aquest procés aplicant el concepte d'anàlisi integral de l'empremta dactilar per HPTLC. S'ha elaborat una guia detallada (inclosa a la tesi) sobre com desenvolupar, validar i aplicar mètodes d'anàlisi integral de l'empremta dactilar per HPTLC per al control de qualitat rutinari de productes a base de plantes.
El control de calidad de los productos a base de plantas tiene sus raíces en el estudio de los caracteres morfoanatómicos y organolépticos. Sin embargo, en el siglo pasado, con la evolución de la química analítica, el control de calidad evolucionó rápidamente de las pruebas elementales al uso de instrumentos sofisticados combinados con software para la gestión de datos. Actualmente, muchas autoridades y organizaciones recomiendan un conjunto de pruebas, con muchos de estos instrumentos, para evaluar la calidad de los productos a base de plantas. La HPTLC ofrece un conjunto completo de datos que pueden usarse no sólo para la identificación, sino también para evaluar la pureza y el contenido de drogas y preparados vegetales y productos a base de plantas. El objetivo de esta tesis doctoral fue explorar en profundidad las capacidades de HPTLC y desarrollar aplicaciones para el control de calidad de los productos de plantas medicinales, mucho más allá de la simple identificación de drogas vegetales, preparados vegetales y productos finales comercializados. Para eso, se desarrollaron cinco estudios. En el primer estudio, se evaluó la calidad de las drogas vegetales, preparados vegetales y productos a base de plantas del fruto del cardo mariano, la raíz y la parte aérea de equinácea y la raíz de cimicífuga, regulados como complementos alimenticios o medicamentos, con los métodos existentes de HPTLC. Se confirmó la idoneidad de estos métodos, utilizando la huella digital completa y varios modos de detección, como una herramienta para detectar problemas de calidad, principalmente adulteraciones. En el segundo estudio, se desarrolló el concepto de análisis integral de la huella dactilar por HPTLC (comprehensive HPTLC fingerprinting) con el objetivo de simplificar el proceso de control de calidad. Este concepto combina la información cualitativa y cuantitativa de las imágenes de HPTLC, obtenidas en un único análisis, para evaluar la identidad, la pureza y el contenido de los productos a base de plantas. Su aplicabilidad para identificar una droga vegetal, detectar mezclas con especies relacionadas (pureza) y desarrollar un ensayo de contenido mínimo de un marcador analítico se demostraron en la raíz de Angelica gigas. En el tercer estudio, la aplicación del análisis integral de la huella dactilar por HPTLC tuvo como objetivo ir un paso más allá en el ensayo de adulterantes y evaluar el uso de la HPTLC para el ensayo límite de pureza. Este enfoque se evaluó con muestras de hoja y extracto de ginkgo, comercializados como complementos alimenticios en diferentes países. Este estudio demostró que la información contenida en las huellas dactilares por HPTLC era adecuada para verificar los niveles de rutina y quercetina, proporcionando resultados similares a los del ensayo límite por HPLC. También fue útil para detectar mezclas de productos de ginkgo no sólo con rutina y quercetina, sino también con parte aérea de trigo sarraceno y sófora (botón floral y fruto). En el cuarto estudio, se evaluó el uso del análisis integral de la huella dactilar por HPTLC como un método alternativo a la actual valoración de marcadores por HPLC en drogas vegetales de la medicina tradicional china (MTC) en la Ph. Eur. El objetivo de este proyecto era simplificar la determinación del contenido y, por lo tanto, reducir el número de pruebas a realizar durante el control de calidad. Para esta evaluación, dos drogas vegetales de la MTC fueron elegidas por los expertos del grupo de trabajo TCM de la Ph. Eur.: bulbo de Fritillaria thunbergii y rizoma coridalis. En ambos casos, se demostró que la huella digital completa de HPTLC era útil para la identificación y el ensayo de contenido mínimo en un solo análisis. El quinto estudio va un paso más allá en la determinación del contenido. Si bien los estudios anteriores se centraron en la cuantificación de marcadores individuales, este estudio tuvo como objetivo aplicar el análisis integral de la huella dactilar por HPTLC a la cuantificación de un grupo de componentes en una droga vegetal, como un ejemplo de una evaluación más holística de la calidad. Esta determinación se combinó con los ensayos de identidad y pureza. Para ilustrar este concepto, se eligió el carpóforo de Ganoderma lucidum. En este trabajo, se ha demostrado que la HPTLC es una técnica útil para el control de calidad rutinario de drogas y preparados vegetales y productos a base de plantas, y que se puede simplificar este proceso aplicando el concepto de análisis integral de la huella dactilar por HPTLC. Se ha elaborado una guía detallada (incluida en la tesis) sobre cómo desarrollar, validar y aplicar métodos de análisis integral de la huella dactilar por HPTLC para el control de calidad rutinario de productos a base de plantas.

This research contributes to worldwide efforts to miniaturize one of the most powerful and versatile analytical tools, gas chromatography (GC). If a rapid, sensitive and selective hand-held GC system is realized, it would have a wide range of applications in many industries and research areas. As a part of developing a hand-held GC system, this research focuses on the separation column, which is the most important component of a GC system. This thesis describes the development of a miniature separation column that has low thermal mass and an embedded heating element for rapid thermal cycling. The worlds first thin polymer film (parylene) GC column has been successfully developed. This thesis includes: first, a study of theoretical column performance of rectangular GC column; second, the design optimization of parylene column and embedded heating element; third, the development of new processes such as parylene micromolding and stationary phase coating technique for parylene column; fourth, the fabrication of parylene GC column with an embedded heating element; and lastly, the testing and evaluation of parylene GC column through GC analysis.

Ionic self-assembled multilayer (ISAM) thin film nanostructures, including highly porous and conductive gold nanoparticles (GNP), and highly porous and thermally stable silica nanoparticles (SNP), were fabricated via the layer-by-layer (LbL) self-assembly technique. Their application in ionic polymer-metal composite (IPMC) electromechanical bending actuators and microfabricated gas chromatography (microGC) devices were investigated and significant performance improvements of these devices were achieved. IPMC bending actuators, consisting of an ionic electroactive polymer (iEAP) membrane as backbone, ionic liquids (IL) as electrolyte, and ISAM GNP thin film as porous electrode, were fabricated and investigated. The influences of humidity, conductive network composite (CNC), and IL uptake on the bending performance were examined and discussed. An equivalent circuit model to simulate both the electrical and mechanical responses was also proposed and experimentally verified. Moreover, IPMC actuators made from other newly synthesized iEAP membranes were fabricated and tested. Some of them showed promising performance that was comparable or even better as compared to the ones made from Nafion. LbL fabricated ISAM SNPs thin film coatings were also applied in the microGC devices including micro fabricated thermal preconcentrators (microTPC) and separation columns (microSC) as adsorbent and stationary phase materials, respectively. New fabrication approaches were developed to selectively coat uniform conformal ISAM SNP coatings in these devices with different 3D microstructures. Thus, functionalized microTPCs and microSCs showed good performance, which can be further improved by using the ISAM SNPs coating as a nanotemplate for modifying additional polymer adsorbents or as the anchor sites for incorporating functional molecules for targeting detection.
Ph. D.

Mestrado em Engenharia Alimentar - Processamento de Alimentos - Instituto Superior de Agronomia
This work investigates the effect of a drastic cooking treatment in chemical composition, particularly, on lipid profile of two species of farmed fish, salmon and meagre, and quantifies the percentage of each fatty acid bioacessibility on these two species in each culinary treatment. To this end, are used methodologies for extracting total lipids (TL), based on the techniques of Bligh & Dyer (1959), for thin layer chromatography and the transesterification of fatty acid methyl esters (FAME - Fatty Acid Methyl Esters). It is verified that the culinary treatment promotes the moisture loss and, consequent, concentration of the fatty acids from the food, although this effect is not uniform for all fatty acids or to both species concerned. Regarding the bioaccessibility it turns out that the fatty acids more bioaccessible on the salmon, are the polyunsaturated fatty acids with a percentage of 67.40% to 60.72% in raw and grilled and within these, docosahexanoic acid is the most bioaccessíble with a percentage of 92.30% in raw salmon and 60.22% on grilled salmon. In the meagre, the fatty acids more bioaccessible are the saturated, with 86.51% in raw and 56.28% in raw and grilled, thus revealing the consumption of salmon is more healthier

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

The chemical profiling, characterization of Aloe products and phytochemical properties of Aloe vera were studied. The adulteration of commercial products derived from medicinal plants has been a major muddle for both the society and the pharmaceuticalindustry. Economically motivated adulteration includes the potential for contaminated, sub‐potent or counterfeit medication to enter the supply chain at several levels, from the production of raw ingredients through to the point of retail sale. Darwin’s theory of evolution states that, species undergo genetic variation with time to adapt to environmental changes. Therefore, the same species growing in widely different habitats may drift from the original genetic makeup as a mechanism of adaptation and that may result in them having different chemical profiles. Therefore this study aimed at investigating the phytochemical properties of Aloe vera growing in South Africa. Also, this study aims to utilize Thin Layer Chromatography to profile this plant, as well as use Infra Red spectroscopy to characterize commercial Aloe vera products. A large quantity of Aloe vera plant was collected from AloeWay, Iphofolo Game Farm, Polokwane in the Limpopo province of South Africa. The identity of the plant was confirmedrom literature and authenticated by Professor DS Grierson of Botany Department, University of Fort Hare, Alice. The plant leaves were divided into two portions. One portion was extracted fresh while the other was cut into pieces and oven dried at 400C then and milled to a homogenous powder once dried completely. The phytochemical composition of the gel and leaf extracts revealed the presence of alkaloids, flavonoids, saponins, tannins and phenols at different concentrations. Results showed that the dry plant material yielded more phytochemicals than the fresh plant material. In particular, it was found that the acetone extract showed much more amounts ofphychemicals than the dichloromethane and aqueous extracts. The percentage compositions of phenols (71.86), flavonols (36.61), proanthocyanidins (82.71), saponins (37.73) and alkaloids (13.29) were significantly high in the acetone extract, followed by the dichloromthane extract with values of 46.85, 37.73, 49.51, 89.0 and 11.11 respectively, while the least composition was found in the aqueous extract. Furthermore, flavonoids were somewhat high in composition in both the aqueous extract of the dried and of the fresh plant material while others were very low. Tannins levels were significantly very low in all the solvent extracts. It was found that the acetone extract showed great amounts of phytochemicals than dichloromethane and aqueous extracts. Since A. vera is used in the treatment of different ailments such as skin wounds and abrasions, eczema, constipation, rheumatoid arthritis etc, the medicinal uses of this plant could be associated to such analysed bioactive compounds. Acetone, hexane, ethanol, water and dichloromethane were used to extract the Aloe vera leaf and the best solvent extract was determined. Thin layer chromatography was used to profile the leaf extracts with the aim of documenting the main phytochemicals present in the Aloe vera growing in South Africa. The best spraying reagent was determined. Fourier transform infrared spectrophotometer was used to validate the presence of Aloe vera ingredients in commercial products. The yield extraction ability of the solvent was the order: water>ethanol> hexane >dichloromethane and acetone for the dry portion. However, for the plant extracted fresh, the order of yield produced was ethanol-acetone-dichloromethane > and water. The different solvent systems separated the compounds differently. Hexane: acetone: ethanol (20 : 5: 2) and Benzene: ethanol: ammonium (80): ethanol (10): ammonium solvent systems were noted to be the best mobile phase as they gave the best separation compared to other systems.EMW [ethyl acetate (81): methanol (11): water (8)] showed better separation than the other two separating solvent systems. Vanillin- sulphuric acid spray was seen to be the best spraying reagent as compared to vanillin- phosphoric acid. Fourier transform infrared spectrophotometer validated the presence aloe ingredients in aloe vera commercial products.

Amongst the polar organic compounds occurring in unrefined and refined crude oils and the associated polluted production waters, complex mixtures of acids, known historically as naphthenic acids (NAs), have achieved prominence. This is particularly because NAs have been designated a toxicant class of concern in the oil sands process-affected water (OSPW) that has accumulated in vast quantities following exploitation of the oil sands of Northern Alberta, Canada in recent years. However, though there have been calls for NAs to be added to pollutant inventories, at the initiation of the current study, little knowledge existed of the exact composition of refined or unrefined NAs. The overall aim of the current study was therefore to identify individual NAs in refined (commercial) and unrefined (e.g. oil sands process-derived) complex mixtures of acids and then to assess the toxicity of any identified NAs. Individual NAs were tentatively identified by interpretation of the electron ionisation mass spectra of methyl ester derivatives, following comprehensive multidimensional gas chromatography-mass spectrometry (GCxGC-MS). Reference acids were then either purchased, or more commonly, where they were not commercially-available, synthesised, mainly by micro-hydrogenation methods, for co-chromatography and comparison of mass spectra of methyl esters with those of unknowns. The synthetic NAs, purified to >97% were then subjected to toxicological assessments using the Microtox™ assay. In all, 34 compounds were obtained pure enough for testing. Microtox results revealed that the toxicity endpoint (50% Inhibition Concentration, IC50) was between 0.004 and 0.7 mM. Exponential and other correlations were noted between carbon number and toxicity in several of the structural groups of acids assayed, which may be beneficial for predictions of toxicity of non-synthesised acids. Although n-hexanoic acid (IC50 0.7 mM) had the lowest toxicity, adamantane-type acids were the least toxic as a group overall. Conversely, the decahydronaphthalene (decalin)-type acids had the largest range of toxicities (IC50 0.004 to 0.3 mM) and the most toxic acid assayed was 3-decalin-1-yl-propanoic acid. According to USEPA guidelines many individual acids can be said to show low to medium toxicity. Since the acids in commercial and unrefined NAs occur in complex mixtures, an attempt was also made to assess mixture toxicity. Mixtures of individual structural groups of acids (e.g. acyclic isoprenoid acids, n-acids) and a mixture of all 34 acids were assessed. Apart from the adamantane sub-group of acids, all of the mixtures showed toxicities lower than the sum of the parts when calculated using equations for Concentration Addition and Model Deviation Ratios (simply the predicted IC50/Observed IC50). A hypothesis that achievement of a critical micelle concentration is required to produce toxicity was proposed to explain the lower than expected results. Some of the mass spectra of NA present in the commercial and unrefined mixtures were inconsistent with those of any of the alicyclic acids synthesised or purchased. These were hypothesised to be aromatic acids. Fractionation experiments of the NA mixtures using silver ion thin layer chromatography and solid phase extraction (Ag+TLC and Ag+SPE) were carried out in order to provide further evidence for aromatic acids. Ag+TLC allowed separation of a methylated NA mixture from OSPW into three distinct fractions; Ag+SPE resulted in eleven fractions, through the use of a wider range of solvents and differential solvent ratios. Analysis of the fractions by GC-MS revealed that each fraction was largely still made up of unresolved acids (as esters), although one or two fractions revealed some resolved acids. Use of averaged mass spectra and mass chromatography on each fraction revealed further resolved chromatographic peaks and associated interpretable mass spectra. Each of eight of the eleven sub-fractions were examined by GC-MS, in some cases by GCxGC-MS, and all by infrared spectroscopy, ultraviolet visible spectrophotometry and elemental analysis. A number of structures were proposed for the aromatic acids, including those with sulphur-containing moieties. It was noted that far from being minor components, aromatic acids comprised ca.25-40% of the OSPW acid extracts.

Surfaces that exhibit a gradual change in their chemical and/or physical properties are termed as surface gradients. Based on the changes in properties they are classified either as physical or chemical gradients. Chemical gradients show variations in properties like polarity, charge, functionality concentration and have found potential applications in fields of biology, physics, biosensing, catalysis and separation science. In this dissertation, surface gradients have been prepared using controlled rate infusion (CRI). CRI is a simple method in which a surface gradient is formed by carrying out the infusion of organoalkoxysilane in a time-dependent fashion using a set infusion rate. Depending on concentration of silane, rate of infusion and time of infusion, the gradient profiles on surfaces can be varied and the surface chemistry of the substrate can be altered. Initial work in the dissertation focuses on demonstrating different gradient profiles and selectivity obtained using amine and/ or phenyl functionalized gradient stationary phases on thin layer chromatography (TLC) plates prepared by CRI. The presence of amine and phenyl on the surfaces were confirmed by X-ray Photoelectron Spectroscopy (XPS) and diffuse reflectance spectroscopy, respectively. The change in surface chemistry was demonstrated by changes in the selectivities of water and fat soluble vitamins. After successful preparation and characterization of single and multi-component stationary phase gradients for planar chromatography, single-component gradients were prepared for column chromatography (Silica monolithic columns). Similar to that observed for planar chromatography, the selectivity was evaluated from retention factors and was found to be different for a weak acid/weak base mixture. The results obtained showed the promising approach of using gradient stationary phases in column chromatography. This work was further extended to prepare amine and phenyl multi-component gradients on silica monolithic columns to investigate mixed-mode and synergistic effects. Finally, amine, phenyl and thiol gradients were also prepared on cellulose substrates, particularly water color paper, The goal was to study the formation of functionality gradients on cellulose substrates particularly the interaction between hydroxyl groups on cellulose and silanols and to study the stability of the silanes on the cellulose surface.

Vitamin C is industrially produced by the Reichstein method, which uses gluconobacters to oxidize sorbitol to sorbose then a chemical process to convert sorbose to 2-keto-L-gulonic acid (2-KLG). The establishment of a more extensive microbial process for 2-KLG production translates into a less expensive and more efficient production of vitamin C. I examined pure strains and mixed cultures for their ability to produce 2-KLG using thin layer and high performance liquid chromatography. The DSM 4027 mixed culture produced the highest yield, 25 g/L, of 2-KLG from 100 g/L of sorbose, while the gram-negative rods isolated from DSM 4027 produced 8.8 g/L, and B. megaterium isolated from DSM 4027 produced 1.4 g/L. Thus, the gram-negative rods in the mixed culture were the primary 2-KLG producer, but B. megaterium in the DSM 4027 mixture enhanced this synthesis. Authentic pure cultures of Gluconobacter oxydans IFO strain 3293 and ATCC strain 621 produced 3.4 g/L and 5.7 g/L, respectively. Attempts to co-culture the isolated B. megaterium with the isolated gram-negative rods and authentic Gluconobacter strains did not increase 2-KLG production, nor did growing the cultures on B. megaterium spent media. Bacillus megaterium produced an unidentified keto-compound detected on the TLC chromatograms, which suggested that B. megaterium converted sorbose to an intermediate that may then be converted by the gram-negative rods in DSM 4027 to 2-KLG. Limited phenotypic tests suggested that the gram-negative rods in the DSM 4027 mixture are not gluconobacters.
Master of Science

Gradient surfaces exhibit a variation in functionality along the length of the surface. One method for preparing gradients is controlled-rate infusion (CRI). In Part 1 of this work, CRI was used to prepare gradients for the purpose of separating transition and heavy metals. Initial work on this project was focused on controlling the retention of the metal ions by varying the number of amine groups, aminoalkoxysilane concentration, and the infusion time. The retention factors of four metal ions varied predictably with increasing number of amine groups, increasing aminoalkoxysilane concentration, and increasing infusion time, producing small but useful changes in the retention factors. The continuation of this project involved the preparation of two-dimensional multi-component gradients on TLC plates, which were used to separate six transition and heavy metals. The retention, and thus the separation, was affected by the presence or absence of a gradient and the direction of the gradient. Part 2 of this work focused on understanding the factors that motivated instructors in the early and late stages in the process of change. Instructors who attended the POGIL-PCL (Process-Oriented Guided Inquiry Learning in the Physical Chemistry Laboratory) workshops were asked to complete online surveys. The goals of the first survey were to understand the factors that initially interested instructors in POGIL-PCL, to determine if instructors enter the implementation stage, and to understand the factors that affect how instructors implement POGIL-PCL. Later surveys were designed to explore the development of the POGIL-PCL network and assess whether implementation is sustained over time.

Tyrimo objektas ir metodai: tiriamieji antidepresantai: bušpirono hidrochloridas; sertralino hidrochloridas; amitriptilino hidrochloridas, fluvoksamino maleatas ir paroksetino hidrochloridas. Šių antidepresantų mišinio skirstymui ir komponentų identifikavimui plonasluoksnės chromatografijos metodu naudoti tirpikliai ir ryškinimo reagentai: metanolis, 25 proc. amonio hidroksidas, trichlormetanas, etanolis, etilacetatas, cikloheksanas, etano rūgštis, dimetilacetonas, acetonitrilas, propanolis, trifluoracto rūgštis, dichlormetanas, 1,4–dioksanas, benzenas, petrolio eteris, izoamilo spiritas, dietileteris, oktanas, metano rūgštis, N,N –dimetilanilinas, nitrobenzenas ir heksanas; Dragendorfo reagentas (modifikuotas pagal Munjė), ninhidrinas, Mandelino reagentas ir UV spinduliuotė (254 nm; 365 nm). Darbo tikslas: sukurti ir validuoti plonasluoksnės chromatografijos metodiką, tinkamą išskirstyti antidepresantų mišinio (amitriptilino hidrochloridas, paroksetino hidrochloridas, sertralino hidrochloridas, fluvoksamino maleatas ir bušpirono hidrochloridas) komponentus ir juos identifikuoti. Darbo uždaviniai: atlikti mokslinės literatūros šaltinių analizę įvertinant amitriptilino, paroksetino, sertralino, fluvoksamino ir bušpirono fizines, chemines, farmakologines savybes ir analizės metodus naudojamus jiems identifikuoti. Aprašyti skirtingas plonasluoksnės chromatografijos metodikas, tinkamas amitriptilino, paroksetino, sertralino, fluvoksamino ir bušpirono mišinio skirstymui ir... [toliau žr. visą tekstą]
Object and methods: research antidepressants: buspirone hydrochloride, sertraline hydrochloride, amitriptyline hydrochloride, fluvoxamine maleate and paroxetine hydrochloride. Used solvents and visualization reagents for those antidepressant mixture distribution and component identification by thin-layer chromatography: methanol, 25 percent. ammonium hydroxide, trichloromethane, ethanol, ethyl acetate, cyclohexane, ethane acid, dimethylketone, acetonitrile, propanol, trifluoroacetic acid, dichloromethane, 1,4-dioxane, benzene, petroleum ether, isoamyl alcohol, diethyl ether, octane, methane acid, N, N - dimethylaniline, nitrobenzene and hexane; Dragendorff reagent (modified by Munjė), ninhydrin, Mandelina reagent and UV radiation (254 nm; 365 nm). Aim: to develop and validate a thin-layer chromatographic method suitable for separating of antidepressants mixture components (amitriptyline hydrochloride, paroxetine hydrochloride, sertraline hydrochloride, fluvoxamine maleate and buspirone hydrochloride) and identify them. Objective: to perform analysis of scientific literature suporting amitriptyline, paroxetine, sertraline, fluvoxamine and buspirone physical, chemical, pharmacological properties, and analytical methods used to identify them. Describe the different thin-layer chromatographic techniques suitable for amitriptyline, paroxetine, sertraline, fluvoxamine and buspirone mixture distribution and component identification. Create a suitable thin-layer chromatographic... [to full text]

A presença de aminas biogénicas nos alimentos é um ponto crítico da segurança alimentar pela sua ligação a casos de intoxicações alimentares, sendo por isso importante testar e otimizar novas metodologias analíticas para a deteção e quantificação destes compostos. Neste trabalho, pretendeu-se otimizar uma metodologia baseada na técnica de cromatografia em camada fina para analisar 7 aminas biogénicas: cadaverina, espermina, espermidina, putrescina, tiramina, 2-feniletilamina e histamina. Numa primeira fase, testou-se a eficácia de separação de três eluentes distintos, tendo-se selecionando o eluente clorofórmio: éter diétilico: trietilamina (40:10:10) porque permitiu melhor resolução entre os picos das aminas biogénicas. Posteriormente, avaliou-se o desempenho da metodologia analítica relativamente à calibração de cada amina biogénica para a análise quantitativa. Os coeficientes de correlação (R) obtidos para a maioria das aminas biogénicas foi superior a 0,99, exceto na calibração da 2-fenileilamina cujo R=0,977. Os limites de deteção situaram-se entre 2,6 mg/L (tiramina) e 5,8 mg/L (2-feniletilamina), enquanto os limites de quantificação se situam entre 7,9 mg/L (tiramina) e 17,6mg/L (2-feniletilamina). Finalmente, aplicou-se o método otimizado em amostras comerciais do cogumelo de cultivo Pleurotus ostreatus, em fresco. Estabeleceu-se um procedimento apropriado para a extração das aminas biogénicas nesta matriz e avaliou-se a presença de aminas biogénicas nas amostras de cogumelos conservados à temperatura ambiente ao longo de 8 dias. Neste estudo foram encontrados 4 compostos, três dos quais desconhecidos suspeitando tratar-se de aminas biogénicas. A amina biogénica que foi detetada e quantificada nas amostras de cogumelos foi a espermidina.The presence of biogenic amines in foods is a critical issue of food safety due to its links with cases of food poisoning, being important to test and optimize new analytical methods for detection and quantification of these compounds. In this work it was intended to optimize a methodology based on the technique of thin layer chromatography to analyze seven biogenic amines: cadaverine, spermine, spermidine, putrescine, tyramine, 2-phenylethylamine and histamine. Initially, it was tested the efficacy of separation of three different eluents, being selected the chloroform: diethyl ether: triethylamine (40:10:10) eluent because it allowed better resolution between the biogenic amines peaks. Subsequently, it was evaluated the analytical methodology performance of calibration of each biogenic amine for quantitative analysis. The correlation coefficients (R) obtained for most of the biogenic amines was higher than 0.99, except in the calibration of 2-fenileilamine, with R = 0.977. The limits of detection ranged from 2.6 mg/L (tyramine) and 5.8 mg/L (2-phenylethylamine), while the quantification limits are between 7.9 mg/L (tyramine) to 17.6 mg/L (2-phenylethylamine). Finally, the optimized method was applied in fresh commercial samples of Pleurotus ostreatus mushrooms. It was established a suitable procedure for the extraction of biogenic amines in this matrix and carried out the analysis of the biogenic amines in mushrooms samples stored at room temperature over 8 days. In this study, four compounds were detected, three of which are unknown although suspected to be biogenic amines. The biogenic amine that was detected and quantified in the mushroom samples was the spermidine

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:11/03138-8

A process, called carbon nanotube templated microfabrication (CNT-M) makes high aspect ratio microstructures out of a wide variety of materials by growing patterned vertically aligned carbon nanotubes (VACNTs) as a framework and then infiltrating various materials into the frameworks by chemical vapor deposition (CVD). By using the CNT-M procedure, a partial Si infiltration of carbon nanotube frameworks results in porous three dimensional microscale shapes consisting of silicon-carbon nanotube composites. The addition of thin silicon shells to the vertically aligned CNTs (VACNTs) enables the fabrication of robust silicon nanostructures with edibility to design a wide range of geometries. Nanoscale dimensions are determined by the diameter and spacing of the resulting silicon/carbon nanotubes while microscale dimensions are controlled by the lithographic patterning of CNT growth catalyst. The characterization and application of the new silicon nanomaterial, silicon-carbon core-shell nanotube (Si/CNT) composite, is investigated thoroughly in the dissertation.The Si/CNT composite is used as thin layer chromatography (TLC) separations media with precise microscale channels for fluid flow control and nanoscale porosity for high analyte capacity. Chemical separations done on the CNT-M structured media outperform commercial high performance TLC media resulting from separation efficiency and retention factor. The Si/CNT composite is also used as an anode material for lithium ion batteries. The composite is assembled into cells and tested by cycling against a lithium counter electrode. This CNT-M structured composite provides an effective test bed for studying the effects of geometry (e.g. electrode thickness, porosity, and surface area) on capacity and cycling performance. A combination of high gravimetric, volumetric, and areal capacity makes the composite an enabling materials system for high performance Li-ion batteries.Last, a thermal annealing to the Si/CNT composite results in the formation of silicon carbide nanowires (SiCNWs). This combination of annealing and Si/CNTs yields a unique fabrication approach resulting in porous three dimensional silicon carbide structures with precise control over shape and porosity.

A aflatoxina M1 (AFM1) é um metabólito tóxico resultante da biotransformação da aflatoxina B1 e pode ser secretada no leite de animais que ingerem alimentos contaminados com esta última. Considerando os efeitos adversos que podem ocorrer devido à ingestão do produto contaminado e visto que as crianças, maiores consumidoras deste alimento, são potencialmente mais sensíveis que os adultos aos efeitos desta micotoxina, a avaliação da presença de AFM1 no leite se faz necessária. Durante o período de março a novembro de 2006 foram analisadas 48 amostras de leite cru provenientes de 8 propriedades fornecedoras de leite para uma Cooperativa de Leite da Serra Gaúcha e 80 amostras de leite UHT, provenientes de 7 marcas distintas, comercializadas em Porto Alegre (RS). A metodologia empregada na análise de aflatoxina M1 envolveu partição líquido-líquido na etapa de extração, uso de coluna de sílica gel na etapa de purificação e Cromatografia em Camada Delgada para a detecção. O limite de detecção foi de 10 ng e a avaliação da eficiência do método apresentou valor de 86% no teste de recuperação. Nas condições de trabalho e pelo método utilizado nenhuma das amostras analisadas foi positiva para a presença de AFM1, sugerindo que as mesmas encontram-se dentro das conformidades legais.
Aflatoxin M1 (AFM1) is a toxic metabolite resulting of the biotransformation of aflatoxin B1, and may be sectreted in milk of animals that consume foods contaminated with aflatoxin B1. Considering the adverse effects that can occur when foods contaminated are consumed, and since children, the greatest milk consumer are potentially more susceptible than adults to the effects of this mycotoxin, the evaluation of the presence of AFM1 in milk is necessary. From March to November of 2006 48 samples of raw milk from 8 dairy farms that integrate a Milk Cooperative of mountain region of Rio Grande do Sul and 80 samples of UHT milk from 7 different brands commercialized in Porto Alegre were analized. The mehodology employed for the analysis of aflatoxin M1 involved liquid-liquid partition on the extraction step, use of silic gel column for the purification step and Thin Layer Chromatography for the detection. The evaluation of the method efficiency present a value of 86% in the recovery test and the detection level was 10ng. Following analysis conditions and the method employed none of the samples analyzed were positive for the presence of aflatoxin M1, suggesting that samples analysed attend the legal conformities.

As plantas medicinais são conhecidas há muitos séculos pelos homens, e ainda hoje são amplamente utilizadas no tratamento de enfermidades. A população acredita que é uma alternativa segura e de baixo custo em relação aos produtos farmacêuticos industrializados, e faz seu uso indiscriminado e sem acompanhamento médico. A automedicação e a falta de controle de qualidade das drogas vegetais (DVs) representam um sério risco à saúde de seus usuários. A diversidade de espécies conhecidas como plantas medicinais, e o uso de nomes populares regionais podem ocasionar erros de identificação taxonômica ou mesmo adulteração do produto. Outro fator que pode alterar a qualidade das DVs é a contaminação de origem química ou biológica, proveniente das diversas fases de seu preparo. Com o uso disseminado de DVs e fitoterápicos, cresceu a preocupação com os efeitos adversos e a interação com medicamentos convencionais. Esse panorama evidencia a interdisciplinaridade na área de plantas medicinais. Assim, o projeto, do qual derivou este de mestrado, é interdisciplinar, com a participação de pesquisadores da etnofarmacologia, microbiologia, farmacovigilância e farmacognosia. O objetivo específico desta dissertação de mestrado é avaliar a qualidade das DVs com possíveis ações psicoativas, adquiridas em barracas de rua na cidade de Diadema. As amostras selecionadas e adquiridas pelo grupo da etnofarmacologia foram confrontadas a monografias farmacopêicas, literatura especializada e/ou amostra autêntica, avaliando-se os caracteres macroscópicos, microscópicos, perfis cromatográficos e pureza das DVs. Cortes histológicos foram preparados conforme as técnicas usuais, cromatografias em camada delgada foram realizadas de acordo com literatura e fotografias documentam as análises. De um total de 35 lotes analisados, 88,6% confirmaram sua autenticidade, e apenas 57,1% estavam em conformidade com os valores máximos permitidos para materiais estranhos. Também ficou evidente a baixa qualidade de armazenamento do material e problemas com embalagens e rótulos, em total desacordo com a legislação vigente. Concluiu-se então que as DVs adquiridas demandam atenção e melhorias quanto à sua qualidade, fato que demonstra a necessidade de maior orientação e conscientização dos vendedores quanto aos cuidados para sua comercialização à população.
Medicinal plants have been known for centuries by humanity and are still widely used for the treatment of illnesses. People believe that it is a safer and low cost alternative in relation to manufactured pharmaceutical products and makes indiscriminate use of them, without medical supervision. Self-medication and the lack of quality control of herbal drugs (HDs) represent a serious health risk to users. The diversity of species known as medicinal plants and the use of popular regional names can cause errors in taxonomic identification or even product adulteration. Another factor that may alter the quality of HDs is contamination from biological or chemical origin, from various stages of their preparation. With the widespread use of herbal remedies and HDs, the concern about adverse effects and interactions with conventional medicines has increased. This scenario highlights the interdisciplinarity of the medicinal plants field. Thus, the project, which this master`s program was derived from, is interdisciplinary, involving researchers from ethnopharmacology, microbiology, pharmacovigilance and pharmacognosy. The specific aim of this master\'s project is to evaluate the quality of HDs with possible psychoactive action, acquired in street stalls in the city of Diadema. The samples selected and acquired by ethnopharmacology were confronted with pharmacopoeial monographs, literature and/or authentic samples and the macroscopic and microscopic characters, chromatographic profiles and purity of HDs were evaluated. Histological sections were prepared according to the usual techniques, thin layer chromatographys were performed according to the literature, and photographs document the analysis. From a total of 35 lots, 88.6% confirmed their authenticity, and only 57.1% were in compliance with the maximum limit for the amount of foreign material. The poor quality of material storage and problems with packaging and labels, in total disagreement with the law, was also made evident. We concluded that the acquired HDs require caution and improvement of their quality, which demonstrates the need for more guidance and awareness of vendors about the care involved in the commercialization of HDs to the population.

Vanillin (4-hydroxy-3methoxybenzaldehyde) is one of the most employed aromatic and flavoring additives in food and cosmetic industry. The industrial interest in vanillin could also apply to its biodegradation products. The microbial transformation of vanillin can open the possibility of new products with new areas of application for products related to vanillin. For example, vanillyl alcohol, vanillic acid and ferulic acid are currently used in the pharmaceutical or food industry. Some species reported to biodegrade vanillin into the related products vanillyl alcohol and vanillic acid, are: Brettanomyces anomalus and Saccharomyces cerevisiae. Moreover, certain microorganisms possess the ability to accumulate lipids when cultivated on different carbon sources, opening the possibility of microbial lipid production as another industrial application. The present investigation focuses on the optimization of extraction methods for vanillin biodegradation products, as well as identifying the isolates of a collection of microorganisms originating from the Faroe Islands that are amenable to being cultivated on a lignin-based media. Finally, the potential for microbial lipid accumulation was also studied. Two analytical methods, Thin-Layer Chromatography (TLC) and Gas Chromatography (GC) were employed for characterizing the biodegradation products obtained after 24 hours and 72 hours of culture in growth medium supplemented with 1 mM of vanillin. The results showed that after 24 hours of incubation, the model microorganism, strain FMYD002, had consumed some of the vanillin and transformed it into biodegradation products. TLC retention factors and GC chromatograms revealed that the main biodegradation product after 24 hours - when compared to a standard – is likely to be to vanillyl alcohol. Furthermore, vanillin and its biodegradation products were relatively temperature-stable based on a temperature test of supernatant from a 24-hour culture, however, when the 72-hour culture had been subjected to the highest temperature (60 °C) some spontaneous decomposition occurred. The biodegradation pattern of the 72-hour culture evidenced by TLC revealed two additional biodegradation products, one of which migrates in a similar fashion to vanillic acid. After 72 hours of incubation, the biodegradation product presumed to be vanillyl alcohol was no longer observed. Acidification tests showed that the best route for extraction of the product believed to be vanillyl alcohol is to adjust the extracted sample to a pH of 9. The cultivation test of the isolates in media prepared from different lignin-based residual products showed that 26 out of 60 initial strains grew regardless of the concentration of lignosulfonates and vanillin. Moreover, 17 strains grew in nitrogen-limited medium. Eight of the strains accumulated lipids. A preliminary categorization of isolates based on their colony morphology and capacity of growth on different substrates showed that to some extent, their morphology can predict the ability to grow on lignin- and vanillin-based media. This could help future scientists to easily screen for and select isolates with interesting activity for the ligno-cellulose industry.

In many developing countries, farmers and pastoralists still rely on their indigenous knowledge, practices and locally available plants to control nematode parasitic infections, both in livestock and humans. The overall aim of my thesis was to undertake bioassay-guided phyto-chemical study of extracts and their constituents from Ethiopian anti-parasitic plants used by healers to control gastrointestinal nematode parasites in livestock to validate their ethno-medicinal use and to characterise and identify their active ingredients. As a first experiment (Chapter Three), four types of crude extracts (water, 70% methyl-alcohol, absolute methanol and acetone) of four indigenous Ethiopian medicinal plants (Adenia species, Cissus ruspolii, Ipomoea eriocarpa and Euphorbia thymifolia) were screened against Teladorsagia circumcincta egg hatching in vitro, not only as a first step to validate the traditional healers claim but also to choose the most promising plant extract(s) for further phyto-chemical studies. The egg hatching inhibition (EHI) test results revealed that the anti-parasitic properties of these plants depended on plant species, dose, and solvent polarity. The water extracts of both C. ruspolii and Adenia sp. exhibited largest, up to 100% EHI but also larval migration inhibition activities, and were selected for further studies. The second experiment (Chapter Four) assessed the nature of active constituents in these extracts by physico-chemical methods. It was observed that the major constituents of both plant extracts responsible for the EHI activities are likely highly polar, water-soluble, small and moderately heat-labile molecules. The third and fourth experiments (Chapters Five and Six) consisted of separating Cissus ruspolii and Adenia sp. water extracts into discrete fractions by gel-permeation chromatography, EHI tests of Bio-Gel P-2 fractions followed by thin layer chromatography (TLC) profiling of these fractions to detect separated spots (in day light, under UV-light or after staining with various staining reagents) and also to see how elution patterns of separated spots affected by column parameters. The EHI tests on the fractions obtained revealed that the active constituents of C. ruspolii and Adenia sp. water crude extracts were eluted into few fractions based on their molecular sizes. The TLC profilings of these fractions identified spot patterns of active and inactive fractions, which allowed pooling of active constituents based on their EHI and TLC profiling into three pools for each plant. The fifth experiment (Chapter Seven) was to isolate and purify compounds from these pools using various preparative planar and column chromatographic methods. Sequential applications of column chromatography followed by preparative thin layer chromatography isolated and purified five active compounds from C. ruspolii and two active compounds from Adenia sp. The sixth experiment (Chapter Eight) was to characterize and propose/elucidate structures of compounds from the active fractions using chromatographic, analytical and spectroscopic methods. In this regard, the structures of two oleanane type triterpenoid saponins isolated from one of active fractions of Adenia sp. were proposed based on their mass spectrometry (MS) and nuclear magnetic resonance (NMR) data with support of compounds property, TLC and literature. Similar outcomes for C. ruspolii were not achieved due to lack of sufficient sample to run 13C-nuclear magnetic resonance spectroscopy and distortionless enhancement by polarization transfer (DEPT), contamination of some purified compounds with ill-characterised substance from the preparative TLC matrix and in some cases mass spectrometry (MS) and nuclear magnetic resonance (NMR) data did not support each other. The last experiment (Chapter Nine) was to assess anthelmintic efficacy and safety of C. ruspolii and Adenia sp. crude water extracts in Heligmosomoides bakeri infected mice. This in vivo test revealed that both plant extracts exhibited significant reduction in worm burdens and worm egg excretion, with moderate effects on haematology and organ weights at tolerated dosages. In conclusion, both in vitro and in vivo data revealed that Adenia sp. and C. ruspolii have anthelmintic properties, thus validating traditional healer claims and supporting ethno-medicinal use. The bioassay-guided phytochemical study resulted in the isolation of a number of active compounds from these plants, for some of which a structure has been proposed.

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The aim of this study was to evaluate the fertilizing capacity, acrosomal integrity, and qualify and quantify by chromatographic techniques the incorporation of cholesterol to the sperm membrane by cyclodextrin in different diluents on cryopreservation of goat sperm. Four males, Saanen (2) and Parda Alpine (2) breeds were used. It was performed a completely randomized design, with each semen sample divided into the following treatments: TG - Tris-glycerol diluent; TGCCC - Cyclodextrin-cholesterol complex (CCC) + Tris-glycerol diluent; TG15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the Tris-glycerol diluent; EE - egg yolk + ethylene glycol diluent; EECCC - CCC + egg yolk + ethylene glycol diluent; EE15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the egg yolk + ethylene glycol diluent. Fresh semen, after its examination, was submitted to the cryopreservation process and stored in a cryogenic cylinder for 10 days. After thawing, the following analysis were performed: acrosomal integrity, sperm fertilizing capacity through perivitelline membrane of hen egg yolk binding test (MPEY) and analysis of sperm motility and vigor. Besides, techniques of gas and thin layer chromatography were conducted to evaluate the incorporation of cholesterol to the sperm membrane. Data were submitted to Lilliefors and Cochran and Bartlett tests to verify normality and homogeneity of variances, respectively. The characteristics that met the assumptions of these tests were submitted to ANOVA and means were compared by Duncan s test at 5% of probability. When data did not meet the assumptions of normality and homogeneity of variances, the means were compared by Kruskal-Wallis test. Pearson s correlation coefficient was calculated in all features.Addition of cyclodextrin-cholesterol complex did not increase binding of sperm to MPEY (P > 0.05). The EE15CCC treatment was superior to EECCC treatment in maintaining the acrosomal integrity but did not differ from control (EE; P > 0.05). In Tris-glycerol diluent, the values observed in the control treatment were higher (P < 0.05) than values of the other treatments (TGCCC and TG15CCC) in maintaining the acrosomal integrity. There was a negative correlation between the binding assay and acrosomal integrity (r = -0.25) and positive correlation between the binding assay and sperm motility (r = 0.20). The sperm motility and acrosomal integrity were negatively correlated (r = -0.26). In quantitative and qualitative evaluation of cholesterol by chromatographic techniques (gas and thin layer) there was no difference between the samples of semen in different treatments (P > 0.05). Both techniques showed no incorporation of cholesterol to spermatozoa. It was concluding that addition of cholesterol-cyclodextrin complex to the medium did not improve the physical aspects of goat semen, or the sperm binding capacity. Pre-incubation of semen with CCC for 15 minutes before addition of ethylene + egg yolk diluent (EE15CCC) did not enhance the integrity of the acrosome in relation to control (EE). The gas and thin layer chromatography showed, respectively, an efficient method for quantitatively and qualitatively determining the cholesterol present in the cryopreserved goat sperm. The concentration of 1 mg of cholesterol-cyclodextrin complex added to the goat semen was not effective for increasing the concentration of cholesterol in sperm.
O objetivo do presente estudo foi avaliar a capacidade fecundante, integridade acrossomal, além de qualificar e quantificar por técnicas cromatográficas a incorporação do colesterol à membrana plasmática do espermatozoide pela ciclodextrina em diferentes diluentes na criopreservação de espermatozoides caprinos. Foram utilizados quatro machos caprinos das raças Saanen (2) e Parda Alpina (2) seguindo um delineamento inteiramente casualizado, dividido nos seguintes tratamentos: TGcontrole negativo para o diluente a base de Tris-glicerol; TGCCC- Complexo ciclodextrina-colesterol (CCC) + diluente Tris glicerol; TG15CCC- CCC diluído em solução isosmótica ao sêmen (soro fisiológico) com 15 minutos de incubação antes da adição do diluente Tris glicerol; EE- controle negativo para o diluente a base de Gema de ovo + etilenoglicol; EECCC- CCC + diluente Gema de ovo + etilenoglicol; EE15CCC- CCC diluído em solução isosmótica ao sêmen (soro fisiológico) com 15 minutos de incubação antes da adição do diluente Gema de ovo + etilenoglicol. O sêmen fresco, após realização de sua análise física foi submetido ao processo de criopreservação e estocado em botijão de nitrogênio por 10 dias. Após o descongelamento realizou-se análise da integridade acrossomal, capacidade fecundante do espermatozoide por meio do teste de ligação à membrana perivitelina da gema do ovo de galinha (MPGV) e as análises de motilidade progressiva e vigor espermático. Além destes testes foi realizada a avaliação da incorporação do colesterol à membrana plasmática dos espermatozoides pelas técnicas de cromatografia gasosa e de camada delgada. Para verificação da normalidade e homogeneidade dos dados foi empregado, respectivamente, o teste de Lilliefors e Cochran e Bartlett. As características que atenderam as premissas destes testes foram submetidas à ANOVA e as médias foram comparadas pelo teste de Duncan com 5% de probabilidade de erro. Quando as distribuições não atenderam as premissas de normalidade e homogeneidade, as médias foram comparadas pelo teste de Kruskal-Wallis. Realizou-se a correlação simples de Pearson entre todas as características. A adição do complexo ciclodextrina-colesterol não aumentou a ligação dos espermatozoides a MPGV (P>0,05). O tratamento empregando o complexo ciclodextrina-colesterol (CCC) diluído em solução isosmótica ao sêmen com 15 minutos de incubação antes da adição do diluente a base gema de ovo + etilenoglicol (EE15CCC) foi superior aos valores médios do tratamento EECCC na manutenção da integridade acrossomal, porém não diferiu dos valores do tratamento controle para este diluente (EE; P >0,05). No diluente Tris-Glicerol, os valores observados no tratamento controle foi superior (P<0,05) aos valores médios dos demais tratamentos (TGCCC e TG15CCC) na manutenção da integridade acrossomal. Houve correlação negativa entre o teste de ligação e a integridade do acrossoma (r= -0,25) e positiva entre o teste de ligação e a motilidade espermática progressiva (r= 0,20). A motilidade espermática progressiva e a integridade do acrossoma apresentaram correlação negativa (r= -0,26). Na avaliação quantitativa e qualitativa do colesterol pelas técnicas cromatográficas (gasosa e camada delgada) não se verificou diferença entre as amostras do sêmen nos diferentes tratamentos (P>0,05). Ambas as técnicas demonstraram que não houve incorporação do colesterol aos espermatozoides. Conclui-se que o complexo ciclodextrina-colesterol no meio diluidor não melhorou os aspectos físicos do sêmen caprino pós-descongelamento e a capacidade de ligação à membrana perivitelina da gema do ovo de galinha. A pré-incubação do sêmen com o CCC por 15 minutos antes da adição do diluente a base de Etilenoglicol+ gema de ovo (EE15CCC) não proporcionou um aumento na integridade do acrossoma a ponto de diferir dos valores do tratamento controle (EE). A cromatografia gasosa e de camada delgada demonstraram, respectivamente, um eficiente método quantitativo e qualitativo para determinar o colesterol presente no espermatozoide caprino criopreservados. A concentração de 1 mg do complexo ciclodextrina-colesterol adicionada ao sêmen caprino não foi eficaz em aumentar a concentração de colesterol presente no espermatozoide.

Orientadores: Pedro Eduardo de Felicio, Jana Pickova
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Em função da alta perecibilidade do pescado, a avaliação do frescor e qualidade deste alimento deve ser precisa e baseada em métodos consolidados. Bases nitrogenadas voláteis (BNV), trimetilamina (TMA), aminas biogênicas, nucleotídeos, uréia e triptofano livre têm sido propostos como índices de frescor para pescado e podem ser efetivos ou não dependendo da espécie de pescado, microbiota contaminante e condições de armazenamento. A quantificação dos teores de aminas biogênicas é também importante devido ao seu potencial tóxico. Os objetivos deste estudo foram avaliar a utilidade das aminas biogênicas, BNV, TMA, triptofano livre e uréia como índices de qualidade e frescor em peixes e moluscos cefalópodes, desenvolver e avaliar sistemas de solventes para separação de aminas biogênicas por cromatografia de camada delgada e avaliar o efeito do tempo e temperatura de derivatização sobre a conversão das aminas biogênicas em seus derivados dansilados. Derivados dansilados de agmatina (AGM), putrescina (PUT), triptamina (TRY), cadaverina (CAD), histamina (HIS), espermidina (SPD), espermina (SPM), tiramina (TYR) e feniletilamina (PHE) foram separados utilizando-se o sistema de solventes clorofórmio: éter dietílico: trietilamina (6:4:1 - v/v) seguido de clorofórmio: trietilamina (6:1 ¿ v/v). A melhor condição para derivatização das aminas biogênicas com cloreto de dansila foi 1h a 40oC. AGM permaneceu no local de aplicação (RF=0,0), o que indica que a metodologia deve ser utilizada com cautela para a determinação desta amina. As percentagens de recuperação de TRY, SPM, SPD e TYR foram baixas, indicando que a metodologia para extração destas aminas deve ser aperfeiçoada. BNV e TMA foram consideradas índices de frescor inadequados para bacalhau (Gadus morhua) e hadoque (Melanogrammus aeglefinus). O padrão de produção mais acentuado a partir da segunda semana de armazenamento, caracterizou as aminas voláteis e as aminas biogênicas CAD e PUT como índices de deterioração para bacalhau e hadoque. Uréia se mostrou inadequada como índice de qualidade para as duas espécies de pescado estudadas. Modelos de regressão linear e quadrática indicaram um aumento progressivo dos teores de AGM em lula (Illex coindetii), de uréia em sépia (Sepia officinalis), e de BNV e triptofano livre nas duas espécies de cefalópodes desde o início do armazenamento. BNV e triptofano livre foram considerados bons índices de frescor para I. coindetii e S. officinalis, pois seus teores aumentaram significativamente (p<0,05) desde o início do armazenamento. AGM e uréia foram consideradas índices de deterioração para I. coindetii e S. officinalis, respectivamente. Os teores de TMA (% NNP) aumentaram significativamente (p<0,05) durante a primeira semana de armazenamento em I. coindetii o que sugere sua utilidade como índice de frescor para lula. Para S. officinalis TMA é mais adequada como índice de deterioração
Abstract: Fish and shellfish muscle is highly susceptible to spoilage during storage. Because quality quickly decreases during storage reliable chemical indices for quality and freshness evaluation are greatly needed. Total volatile bases-nitrogen (TVB-N), trimethylamine (TMA), biogenic amines, nucleotides, urea and free tryptophan have been suggested as freshness indices for fish and shellfish. Such indices are useful depending on the species, microbial flora and storage conditions. Biogenic amines levels are also cause of concernment due to their toxicological effects. The objectives of this study were to evaluate the usefulness of biogenic amines, TVB-N, TMA, free tryptophan and urea as freshness and quality indices for fish and cephalopods; to develop and evaluate solvent systems for biogenic amines separation by thin-layer chromatography; and to evaluate the effects of time and temperature of derivatisation on the conversion of biogenic amines to their dansyl derivatives. Dansyl derivatives of agmatine (AGM), putrescine (PUT), tryptamine (TRY), cadaverine (CAD), histamine (HIS), spermidine (SPD), spermine (SPM), tiramine (TYR) and phenylethylamine (PHE) were separated using the solvent system chloroform: diethyl ether: triethylamine (6:4:1 ¿ v/v), followed by chloroform: triethylamine (6:1 ¿ v/v). The best dansylation condition was 1h at 40oC. AGM remained at the start position, indicating the determination of AGM by this method should be considered with caution. The percentages of recovery of TRY, SPM, SPD and TYR were low, indicating that the extraction methodology must be improved. TVB-N and TMA were considered inappropriate as freshness indices for cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). CAD and PUT showed the greatest increases during the storage, but their levels did not increase significantly (p<0.05) during the first week. Volatile amines and biogenic amines were characterized as spoilage indices for G. morhua and M. aeglefinus due to the intense increasing of their amounts from the second week of storage. Urea was not useful as a quality index for cod and haddock. Linear and quadratic models indicated progressive increasing of AGM levels in squid (Illex coindetii), urea in cuttlefish (Sepia officinalis) and also increasing of TVB-N and free tryptophan in both cephalopods, since the beginning of the storage. TVB-N and free tryptophan were considered good freshness indices for both cephalopod species, because their levels significantly increased (p<0.05) during the first week of storage. AGM and urea were useful as spoilage indices for I. coindetii and S. officinalis, respectively. It was observed a significant increase of TMA (% of NNP) during the first week of storage in I. coindetii indicating the TMA usefulness as freshness index for squid. For S. officinalis TMA was a good spoilage index
Doutorado
Doutor em Tecnologia de Alimentos

As caprilinas são acilgliceróis de cadeia média com diversas aplicações nos campos alimentício, farmacêutico e cosmético. A esterificação direta é um processo importante para a síntese de caprilinas, dado que o glicerol é subproduto da produção de biodiesel e que a tricaprilina, material de partida para a glicerólise e hidrólise, não é um composto abundante naturalmente. As reações existentes para a síntese de acilgliceróis podem ser catalisadas por via química ou enzimática, sendo esta última superior em diversos aspectos. Dois dos principais contrapontos à utilização de enzimas são seu alto custo e dificuldade de recuperação. A imobilização de enzimas é uma das maneiras existentes para contornar estas questões. A maioria dos estudos científicos relativos à obtenção destes compostos refere-se aos acilgliceróis de cadeia longa. Portanto, é necessário aprimorar a esterificação direta entre o glicerol e ácido caprílico catalisada por lipase, através da compreensão da cinética reacional e da influência dos parâmetros reacionais. A existência de uma metodologia de separação, identificação e quantificação simples e eficaz também é importante, tornando as pesquisas relacionadas às caprilinas mais rápidas e menos complicadas. Desta maneira, este trabalho consistiu de três etapas principais: (1) a imobilização de lipases em resinas de troca aniônica, visando reduzir os custos em relação à lipase imobilizada comercial; (2) o desenvolvimento de um método de cromatografia em camada delgada capaz de quantificar as caprilinas formadas; (3) o estudo da reação de esterificação para a obtenção de caprilinas em regime descontínuo e descontínuo-alimentado. A resina DOWEX® 1X2-400 foi a que apresentou a melhor eficiência de imobilização (EI) da Palatase®. Sua atividade lipolítica foi apenas 12,7% inferior à versão imobilizada comercial da lipase, a Lipozyme®RM IM, indicando a viabilidade de substituição desta última. O desenvolvimento de método de cromatografia em camada delgada resultou em relações lineares para a detecção e quantificação de ácido caprílico, monocaprilina, dicaprilina e tricaprilina com R2 e R2pred de 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectivamente, e com valor-p de 0,000. As reações em regime descontínuo foram realizadas seguindo planejamento fatorial completo, no qual foram variadas a temperatura (30 ou 70°C), a proporção molar entre os reagentes (1:1 ou 3:1 - ácido caprílico : glicerol) e o tempo reacional (6 ou 10h). A análise de regressão do rendimento reacional expresso em porcentagem de consumo de ácido caprílico indicou que, nestas condições, os fatores relevantes para o resultado final são a temperatura e a proporção molar, bem como a interação entre eles. As melhores condições para a obtenção de mono e dicaprilinas foram, portanto, de 30ºC e razão molar de 1:1. O estudo da cinética da reação indica que 40,4% do ácido caprílico são consumidos na primeira hora e que o consumo máximo ocorre na terceira hora de reação. Houve uma queda drástica no rendimento reacional, quando a reação foi conduzida em regime descontínuo-alimentado, provavelmente devido à menor eficiência catalítica da lipase devido ao baixo teor de água na primeira hora de reação.
Caprylins are medium chain acylglycerols that find several applications in food, pharmaceutical and cosmetic fields. Direct esterification is an important route for the synthesis of caprylins, since glycerol is a byproduct of biodiesel production and tricaprylin, the starting material of glycerolysis and hydrolysis, is not a naturally abundant compound. The glycerides synthesis can be catalyzed by either chemical or enzymatic routes, the latter being superior in several aspects. The biggest issues regarding the use of enzymes are their high cost and difficult recovery. Using immobilized enzymes can minimize these concerns. Most of scientific studies concerning the obtainment of these compounds address to long chain acyglycerols. Thus, the improvement of lipase catalyzed esterification of glycerol and caprylic acid is necessary, by understanding the reaction kinetics and the role of reaction parameters. A simple and effective method of separation, identification and quantification of caprylins is also a relevant contributing factor for easier and faster researches. Therefore, this work consisted of three main steps: (1) immobilization of lipases on anion exchange resins in order to produce a cheaper alternative to imported commercial immobilized lipases; (2) development of a thin layer chromatographic method capable of quantifying caprylins; (3) studying the obtainment of caprylins by direct esterification in batch and fed batch systems. DOWEX® 1X2-400 resin presented the best immobilization efficiency (IE) results. Additionally, its lipolytic activity was only 12.7% lower than the commercial Lipozyme® RM IM, suggesting that Lipozyme\'s substitution is feasible. Regarding the developing of a thin layer chromatography method, linear correlations were obtained for detection and quantification of caprylic acid, monocaprylin, dicaprylin and tricaprylin, with a R2 and R2pred of 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectively, with a p-value of 0,000. The batch reactions were carried out according to a complete factorial design, in which the temperature (30 or 70ºC), the molar ratio between the reagents (1:1 or 3:1) and reaction time (6h or 10h) were varied. The regression analysis of reactional yield expressed in terms of percentage of caprylic acid consumption indicated that only the temperature, molar-ratio and their interaction were important for reaction yields, within the studied conditions. The best conditions related to the obtainment of mono and dicaprylins were 30ºC and molar ratio of 1:1. The reaction kinetics indicates that 40.4% of the caprylic acid is consumed within one hour of reaction and that the maximal consumption was achieved in the third hour. A drastic drop in reaction yield was observed when the fed batch mode was adopted, probably due a lower catalytic efficiency presented by lipase as a result of the lower water content in the first hour of reaction.

A atual Política Nacional de Plantas Medicinais e Fitoterápicos prevê investimentos em pesquisa e desenvolvimento para suprir a necessidade da indústria nacional em adquirir a matéria prima para a produção de novos fitoterápicos de maneira segura e controlada. As espécies do gênero Smilax, conhecidas popularmente como salsaparrilha, são empregadas na medicina popular como fortificante, antirreumático e antissifilítico e são vendidas em farmácias, casa de ervas e mercados públicos sem que exista um controle de qualidade de sua origem e eficácia. Além disso, a procedência do material é baseada principalmente no extrativismo. O controle de qualidade das drogas vegetais deveria ter uma base mais segura de identificação das drogas através da caracterização e definição de particularidades anatômicas e químicas. Este estudo teve como objetivo apresentar a quantidade, o valor, o modo de preparo, utilização e a procedência da salsaparrilha comercializada no Estado de São Paulo, bem como analisar a anatomia e o perfil químico de 44 amostras de salsaparrilha comercializada. Amostras de raiz foram submetidas às técnicas convencionais de microscopia de luz e eletrônica de varredura. Também foram realizados testes histoquímicos. Para determinar o perfil químico por Cromatografia em Camada delgada (CCD), foram realizados extratos etanólicos de amostras de raízes. O perfil químico do material comercializado foi comparado com o perfil das espécies de Smilax previamente identificadas (S. goyazana A. De Candolle, S. rufescens Grisebach, S. brasiliensis Sprengel, S. campestris Grisebach, S. cissoides Martius ex Grisebach, S. fluminensis Steudel, S. oblongifolia Pohl ex Grisebach e S. polyantha Grisebach). A quantidade média de salsaparrilha comercializada nas farmácias (400g) e nas casas de ervas (20Kg) é elevada se for considerado o fato de que as raízes não são procedentes de cultivo. Embora tenha sido observada grande semelhança entre a estrutura anatômica das amostras de salsaparrilha comercializadas e a estrutura já descrita na literatura para as espécies de Smilax, houve diferenças em relação à organização do floema, à presença de séries de idioblastos contendo ráfides na medula, à ausência de idioblastos fenólicos na medula e presença de metaxilema no centro da estrutura. O teste histoquímico confirmou a presença de amido em todas as amostras comerciais. As análises em CCD dos extratos etanólicos das amostras comerciais evidenciaram diversas manchas com tonalidades variando de amarelo a verde. Além disso, as manchas apresentaram componentes de mesmo Fator de retenção (Rf), indicando semelhança química entre as diversas amostras. No entanto, o padrão de distribuição de manchas, bem como o Rf das amostras comerciais diferiu daqueles obtidos para as oito espécies de Smilax, os quais foram muito similares entre si.
The current National Policy on Herbal Medicine provides investments in drug research and development to supply the need of national industry to acquire the raw material for production new herbal medicines in a safe and controlled way. The species of Smilax, popularly known as sarsaparilla, are used in folk medicine to be effective as tonic, antirheumatic and antisyphilitic properties and they are commercialized in pharmaceutical stores, natural products stores and public markets in Brazil with no quality control of its origin and efficacy. In addition, these plants are still harvested in extractive way. The quality control of herbal drugs should have more secure identification of drugs through the characterization and definition of anatomical and chemical peculiarities. This study aims to present the quantity, value, method of preparation, use and origin of the commercialized sarsaparilla in cities from the São Paulo state and to investigate the anatomy and chemical profile of 44 samples of the commercialized sarsaparilla. Root samples were processed and analyzed using conventional light and scanning electron microscopy techniques. Usual histochemical tests were also performed. Chemical profiles of samples were analyzed by thin layer chromatography (TLC) using ethanolic extracts of the roots. The chemical profile of the commercialized material was compared with profiles of previously identified species of Smilax (S. goyazana A. De Candolle, S. rufescens Grisebach, S. brasiliensis Sprengel, S. campestris Grisebach, S. cissoides Martius ex Grisebach, S. fluminensis Steudel, S. oblongifolia Pohl ex Grisebach and S. polyantha Grisebach). The average amount of sarsaparilla sold in pharmaceutical stores (400g) and natural products stores (20kg) is high if one considers the fact that the roots are not found under cultivation. Although there was a great similarity between the anatomical structure of commercialized sarsaparilla and the structure already described in literature for the Smilax species, there were found some differences in the organization of the phloem, the occurrence of series of idioblasts containing raphides in the pith, the absence of phenolic idioblasts in the pith and the presence of metaxylem in the center of the organ. The histochemical tests confirmed the presence of starch in all commercialized samples. Chemical profile showed several spots with colors ranging from yellow to green. Moreover, the spots showed the same components retention factor (Rf), indicating chemical similarity between the different samples. However, the distribution pattern of spots, Rf value of commercial samples differed from the eight species of Smilax, which were very similar to each other.

This diploma thesis deals with the microbial production of biosurfactants of selected bacterial strains. In order to test the biosurfactant production ability, screening methods were chosen to be able to review the potential of the selected strains to produce biosurfactants. With the scope of the work, 11 bacterial strains, which are used as polyhydroxyalkanoate (PHA) producers, have been tested. The ability to produce biosurfactants was tested in all strains both in complex inoculation and mineral production media. The presence of biosurfactants in Pseudomonas putida was detected on the basis of the results obtained after cultivation in inoculation and production media. The bacteria Pseudomonas fulva was put under more deep study to support their production by cultivation in different types of production media supplemented by different sources of carbon and nitrogen, and the effect of cultivation time was tested as well. Biosurfactants produced by these bacteria were subsequently identified by Fourier transform infrared spectroscopy (FTIR) on the basis of which the substances were identified as rhamnolipids. According to thin-layer chromatography result (TLC), Pseudomonas putida produces a mixture of mono- and dirhamnolipids, with monorhamnolipids being more dominant in our samples.