Dissertations / Theses on the topic 'Chemical activity'

Propolis or bee glue, is collected by bees and contains secondary metabolites largely derived from trees or shrubs; it has been used traditionally as a natural remedy with a wide range of biological activities. Its chemical composition is highly complex and variable, and it has been studied in detail worldwide except in Africa. This study investigated the chemical composition and activity of African propolis against blood stream form of Trypanosoma brucei, the causative agent for sleeping sickness that threatens a large population of both humans and animals in sub-Saharan Africa. Extracts of propolis samples (n=12) collected from different regions in Nigeria and one sample collected from South Africa were chemically profiled by using various analytical techniques. These included high performance liquid chromatography (HPLC), coupled with different detection systems including evaporative light scattering detection (ELSD), ultraviolet detection (UV), and high resolution mass spectrometry (HRMS), along with gas chromatography- mass spectrometry (GC-MS) and proton-nuclear magnetic resonance (1H-NMR).Principal components analysis (PCA) of the processed LC-MS data collected was used in order to characterize samples according to their chemical composition. PCA demonstrated the uniqueness in chemical composition of some samples that were also active against Trypanosoma brucei. Therefore, the study proceeded to investigate in detail four samples collected mainly from the southern part of Nigeria. An optimized medium pressure chromatographic technique was used to isolate some of the component(s) responsible for the anti-trypanosomal activity. Two samples collected from Rivers State Nigeria had a different appearance from the rest of the propolis samples, being red in colour and had the highest trypanocidal activity (EC50=4.2 and 6.9 μg/mL) respectively. Their chemical composition was comparable to that of Brazilian red propolis. Fractionation work led to the isolation of ten phenolic compounds including calycosin, liquiritigenen, pinocembrin, vestitol, medicarpin, 8-prenylnaringenin, 6-prenylnaringenin, propolin D, macarangin and a new benzofuran. All compounds structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy and LC-MSn. Some compounds showed strong inhibitory activity against trypanosomes such as medicarpin (MIC=11.5 μM) and propolin D (MIC=7.4 μM), macarangin (EC50 =18.5 μM), 8-prenylnaringenin (EC50= 17.9 μM), and vestitol (EC50= 30.5 μM). The new benzofuran was moderately active with (EC50=58.01 μM). Fractionation of the propolis sample collected from the Ugelli/Delta sample led to isolation of three compounds 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl) xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl) xanthone and a new xanthone. These compounds were tested against T. brucei and presented high activities of EC50= 3.9, 11.04, 14.7 μM respectively. Triterpenes were the main fingerprint compounds in a sample collected from Ijebu-Ode/Ogun; three compounds were isolated and elucidated as ambonic acid, mangiferonic acid and α-amyrin. These compounds had EC50 values against T. brucei of 39.5, 25.5 and 20.9 μM respectively. Finally, sample D46SA from South Africa was found to contain mainly flavonols and diterpenic acids; three compounds pinocembrin, acetylimbricatolic acid and (-)-pimara-8 (14), 15-dien-19-oic acid were isolated and tested. All were moderately active against T. brucei.with MIC ranging from 41.4-137.3 μMIn conclusion, this work has proved the variabiliy of propolis collected even from the same region and the widespread activity of propolis against (blood stream form) T. brucei. It is likely that some of the propolis samples contain compounds with even higher activity that have not yet been isolated.

Biological screening of extracts of various bryophytes showed that the species Dicranum fulvum gave extracts with activity in both in vitro and in vivo bioassays. This plant was thus selected for extraction and fractionation, monitored by iin vitro bioassays.

Isolation was guided by a combination of bioassay and chemical methods, and led to the isolation of three compounds, betulin, 9,l9- cyclolanostâ 23â eneâ 3,25â diol, and B-sitosterol. Purification was achieved by open column, flash column, gel filtration, thin layer chromatography, the chromatotron and crystallization.

The isolated compounds were identified by comparisons of spectroscopic data with those of authentic samples and the matching of experimental and literature melting points and optical rotations.

Master of Science

Sphingolipids (SLs) are one of the major classes of lipids in eukaryotes. In addition to being essential structural components of cell membranes, SLs also play capital roles as signalling molecules. Ceramides (Cer) are a family of bioactive SLs consisting of a long chain base (LCB), known as the sphingoid base, linked to a fatty acid (FA) of variable chain length via an amide bond. Due to their metabolic inter-relations with other SL species, Cer are considered key intermediates in the SL pathway. Cer are important second messengers in several cellular processes including apoptosis, autophagy, cell differentiation and sensescence. Ceramide synthases (CerS) are a group of enzymes, primarily localised at the endoplasmic reticulum, that catalyse the N–acylation of sphingoid bases such as sphingosine (So) and dihydrosphingosine (dhSo), using acyl CoA thioesters of variable chain lengths, to afford Cer and dihydroceramides (dhCer), respectively. Six isoforms of CerS (CerS1–6) have been identified in mammals. Each CerS isoform utilizes a small subset of FA-CoAs of defined chain lengths and, thus, each of them produces specific Cer populations. In the recent years, it has become apparent that Cer with different acyl chains vary in their biophysical properties and in the signalling pathways they participate. Furthermore, Cer with defined acyl chain lengths have been found to be implicated in the onset of a variety of human diseases, including cancer, type-2 diabetes mellitus, Alzheimer’s disease, multiple sclerosis and cardiomyopathy. In this context, the development of appropriate tools to study the activity of CerS enzymes, which is crucial to decipher the molecular mechanisms by which Cer elicit their effects, was the ultimate goal of the present doctoral thesis. The first part of this thesis was devoted to the development of a new CerS activity assay based on the Förster Resonance Energy Transfer (FRET) phenomenon. To that end, we designed and synthesized a series of fluorescently labelled (or labelable) 1-deoxy sphingoid probes derived from spisulosine, a small library of clickable FA analogues of different chain lengths, and a collection of bicyclo[6.1.0]nonyne (BCN) or 1,2,4,5-tetrazine (Tz) based fluorescent reagents. The absorption and fluorescence emission properties of these compounds was thoroughly studied in various solvents by means of cuvette-based experiments. Based on these studies, we anticipated that a highly efficient FRET process would take place between the donor-acceptor fluorophore pairs that had been selected, namely MCC/NBD and NBD/NR. Next, the metabolic incorporation of the different spisulosin-based probes and the FA analogues was evaluated in various biological contexts. Mass spectrometry analysis evidenced an extensive metabolization of the synthetic LCB probes and the FA analogues by CerS enzymes to form the corresponding Cer metabolites. Unfortunately, the FA analogues were also incorporated into other lipidic metabolic pathways, resulting in the generation of a strong fluorescence background after the fluorescent labelling reactions. Our different attempts to solve this issue were unfruitful and, thus, the development of the FRET based fluorescence assay to determine the CerS activity could not be achieved. The second part of this thesis was aimed at the development of new click-formed proteolysis targeting chimeras (CLIPTACs) targeting the ubiquitination and proteasomal degradation of CerS, as an alternative to small molecule inhibitors for the modulation of the CerS activity. To this end, we designed and synthesized four BCN derivatives containing known ligands for recruiting different E3 ubiquitin ligases. These BCN-tagged E3 ligase recruiters will be used in future studies in combination with an azido-functionalized analogue of the CerS substrate Jaspine B to obtain the desired CLIPTACs.
Los esfingolípidos (SLs) son una de las principales categorías de lípidos presentes en los organismos eucariotas. Los SL no sólo son componentes estructurales esenciales de las membranas celulares, sino que también actúan como moléculas señalizadoras. Las ceramidas (Cer) son una tipología de SL que están formadas por una base esfingoide y una cadena de ácido graso de longitud variable unidos a través de un enlace amida. Las Cer participan como segundos mensajeros en procesos celulares como la apoptosis, la autofagia, la diferenciación celular y la senescencia. Las ceramida sintasas (CerS) son un grupo de enzimas del retículo endoplasmático que catalizan la N-acilación de bases esfingoides, como la esfingosina, utilizando acil-CoAs de distintas longitudes, para dar Cer. En los mamíferos se han descrito seis isoformas de la CerS y cada una de ellas tiene preferencia por un pequeño grupo de ácidos grasos de longitud de cadena definida, por lo que cada una produce perfiles de Cer característicos. En los últimos años se ha visto que la longitud de la cadena acilo de las Cer influye en sus propiedades biofísicas y en las cascadas de señalización en las que participan. Además, se sabe que ciertas Cer están involucradas en el desarrollo de distintas enfermedades como el cáncer, la diabetes, el Alzheimer o la esclerosis múltiple. En este sentido, el desarrollo de herramientas adecuadas para el estudio de la actividad de CerS es fundamental para descifrar los mecanismos moleculares a través de los cuáles actúan las Cer, siendo este el objetivo principal que se persiguió en la presente tesis doctoral. La primera parte de la tesis se centró en el desarrollo de un nuevo ensayo para determinar la actividad CerS por medio del fenómeno de FRET. Para ello, se diseñaron y sintetizaron una serie de sondas esfingoides derivadas de la espisulosina, una pequeña quimioteca de análogos de ácidos grasos “clicables” de distintas longitudes de cadena y una colección de reactivos fluorescentes marcados con un grupo biciclo[6.1.0]nonino (BCN) o 1,2,4,5-tetrazina (Tz). Las propiedades de absorción y emisión de fluorescencia de estos compuestos fueron estudiadas en varios disolventes a través de experimentos “en cubeta”. En base a estos experimentos anticipamos que las parejas de fluoróforos seleccionadas eran adecuadas para su uso en experimentos de FRET. A continuación, se evaluó la incorporación metabólica de las distintas sondas esfingoides y de los varios análogos de ácidos grasos en medios biológicos. Los estudios de lipidómica mostraron que tanto las sondas como los análogos de ácidos grasos eran procesados por las CerS para dar las Cer correspondientes. Sin embargo, los análogos de ácidos grasos también entraron en otras rutas metabólicas de los lípidos dando lugar a un elevado ruido de fondo tras la reacción de marcaje de fluorescencia. Nuestros intentos para solventar este problema fueron en vano y, por tanto, finalmente no fue posible la implementación del ensayo de fluorescencia para medir la actividad CerS. En la segunda parte de la tesis se propuso el desarrollo de nuevos CLIPTACs dirigidos a la degradación de CerS, como alternativa a los inhibidores clásicos para la modulación de la actividad CerS. Para ello se diseñaron y sintetizaron cuatro derivados de BCN que contuvieran un ligando para reclutar distintos enzimas E3 ligasas de ubiquitina. Estos reclutadores de E3 ligasa serán utilizados en un futuro en combinación con un derivado de la Jaspina B, un análogo del sustrato de las CerS, para obtener los CLIPTACs deseados.

The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atoms and substitution patterns. These compounds tend to be biomagnified in foodwebs and have been shown to induce an array of effects in exposed organisms. The structural characteristics of the PCBs influence their potency as well as mechanism of action. In order to assess the biological potency of these compounds a multi-step quantitative structure-activity relationship (QSAR) procedure was used in the project described in this thesis.

The ultraviolet absorption (UV) spectra were measured for all 209 PCBs, and digitised for use as physico-chemical descriptors. Interpretations of the spectra using principal component analysis (PCA) showed the number of ortho chlorine atoms and para-para substitution patterns to be significant. Additional physico-chemical descriptors were derived from semi-empirical calculations. These included various molecular energies, the ionisation potential, electron affinity, dipole moments, and the internal barrier of rotation. The internal barrier of rotation was especially useful for describing the conformation of the PCBs on a continuous scale.

In total 52 physico-chemical descriptors were compiled and analysed by PCA for the tetra- to hepta-chlorinated congeners. The structural variation within these compounds was condensed into four principal properties derived from a PCA for use as design variables in a statistical design to select congeners representative for these homologue-groups. The 20 selected PCBs have been applied to study structure-specific biochemical responses in a number of bioassays, and to study the biomagnification of the PCBs in various fish species.

QSARs were established using partial least squares projections to latent structures (PLS) for the PCBs potency to inhibit intercellular communication, activate respiratory burst, inhibit dopamine uptake in synaptic vesicles, compete with estradiol for binding to estrogen receptors, and induce cytochrome P4501A (CYP1A) related activities. By the systematic use of the designed set of PCBs the biological potency was screened over the chemical domain of the class of compounds. Further, sub-regions of highly potent PCBs were identified for each response measured. For risk assessment of the PCBs potency to induce dioxin-like activities the predicted induction potencies (PIPs) were calculated. In addition, two sets of PCBs were presented that specifically represent congeners of environmental relevance in combination with predicted potency to induce estrogenic and CYP1A related activities.

Bethoxazin is a broad spectrum industrial biocide with commercial applications as a material preservative; however its mechanism of action has not been investigated. In this study, the chemical reactivity of bethoxazin towards biologically important nucleophiles was assessed with UV-Vis spectroscopy. Bethoxazin reacted with molecules containing free sulfhydryl groups such as glutathione and human serum albumin but not with amino, acetate or phenol containing compounds. Bethoxazin was shown to potently inhibit the growth of the K562 human cancer cell line with an IC50 value in the micromolar range. The sulfydryl fluorescent label ThioGlo-1 was used to investigate the biological effects of bethoxazin in K562 cells and explore its mechanism of action. Bethoxazin inhibited the formation of covalent adducts in K562 cells between the free sulfhydryl group of biomolecules and ThioGlo-1, implying that bethoxazin reacts with molecules containing free sulfhydryl groups. Likewise, when glutathione was depleted in K562 cells, by buthionine sulfoximine, high concentrations of bethoxazin were able to inhibit the formation of covalent adducts between sulfhydryl biomolecules and ThioGlo-1. The growth inhibition assay (MTS) was used to investigate the effect of continuous bethoxazin treatment versus wash out in K562 cells. The MTS assay revealed a reduction in the potency of bethoxazin due to the wash out effect, suggesting that the growth inhibition effects of bethoxazin are likely not due to glutathione depletion. A two-colour flow cytometry analysis of bethoxazin treated K562 cells for eight hours demonstrated that bethoxazin provokes necrosis induced cell death in K562 cells. Taken together, these experimental results demonstrate that the reaction of bethoxazin with proteins containing an accessible sulfhydryl group is more likely to be the mechanism of action of the cell growth inhibition effects rather than glutathione depletion.

Quantitative structure-activity relationships (QSAR) rationalize interrelation between molecular structure and biological response in terms of either physicochemical parameters, as in linear free energy relationships (LFER), or via purely empirical parameters, as is the case for De Novo schemes. In LFER the leading process is often the partitioning of a compound between two solvent phases, taken to represent the transfer of a drug molecule across a biological membrane. This study has investigated the partitioning behaviour of three series of hydroxybenzoate esters, viz. o-, m- and predominantly p-esters, the latter being preservatives in pharmaceutical formulations. The thermodynamic parameters AH, AG and AS for the transfer process were derived in an attempt to establish a QSAR. on a fundamental thermodynamic basis. Such parameters have identifiable physicochemical meaning and lend themselves more readily to interpretation. This facilitates application to alternative systems. A new Gibbs function factor analysis was developed and utilized to obtain thermodynamic contributions for parent and incremental methylene group portions of thestudy molecules. The empirical Collander equation for interrelation of various solute/solvent systems was also rationalized on a thermodynamic basis. Further extension of the Gibbs function factor analysis allowed scaling of "solvent" systems including chromatographic packings, solvents and liposomes. The scheme indicated capacity for optimized selection of bulk solvent systems to mimic biological membranes. A novel analytical procedure for direct measurement of biological response was developed. The bioassay appeared capable of discrimination i) between the closely related structural homologues, ii) between gram-negative and gram-positive bacteria, and further, iii) between certain cell batches of the same bacteria type. Also, the bioassay demonstrated a Collander interrelation between the two bacteria types. Flow microcalorimetry was the technique employed to measure thermal response of respiring E. coli and Staph, aur. bacteria. The modification of biological response with drug concentration was quantitated and a log dose max term was derived for each homologue. The results indicated potential for a predictive, additive structure-activity scheme based on assessment of biological response (BR) direct rather than through f(BR) via physicochemical or empirical parameters.

Adverse drug reactions (ADRs) are responsible for an increasing number of hospitalised patients, with the large majority of these ADRs classed as either type A or type B. Drug hypersensitivity reactions fall within the type B category and one such drug responsible for this form of ADR is abacavir (ABC). ABC, a nucleoside reverse transcriptase inhibitor, is used to treat the HIV-1 virus. It is responsible for a potentially life-threatening type IV hypersensitivity reaction which occurs in patients bearing the HLA-B*57:01 allele. Although many mechanisms have been proposed, it was the objective of this research to examine all these previous proposals to further extend and develop the mechanism of ABC toxicity. In Chapter 2, deuterated-ABC (D2-ABC) was designed and synthesised where the two 5'-H atoms were replaced with two 5'-D atoms. The design of this analogue was intended to retard the oxidative metabolism of ABC to its aldehyde and carboxylic acid metabolites. The synthesis of this compound was paramount to investigating this metabolism and through a series of metabolic experiments, described in Chapter 3, a kinetic isotope effect between ABC and D2-ABC was determined, ultimately showing an altered metabolism between the two compounds. To investigate binding of ABC within the HLA-B*57:01 protein, analogues of ABC, with alterations at varying positions within the molecule, were required. Using a racemic method, ABC enantiomers were synthesised and ABC’s enantiomer failed to stimulate T-cells in vitro. The creation of further analogues required the development of an asymmetric synthetic route. A total synthetic method was desired to synthesise intermediates to be used in future analogue synthesis. Finally, as described in Chapter 5, a range of 6-position analogues were designed, using a structure-activity relationship method, and synthesised, to further investigate the altered repertoire mechanism. These analogues, consisting of primary and secondary amine and oxy moieties, were subjected to in vitro immunological assays to determine their stimulation of T-cells. Additionally, the synthesised analogues were modelled in silico using molecular docking within the HLA protein. The in silico results assisted in explaining the basis of such T-cell activation/inactivation and will direct future analogue design. IC50 and EC50 values were determined for analogues that presented a negative T-cell response and a compound showing positive values was subjected to further pharmacokinetic testing. The oxidative metabolism of ABC was affected by isotopic substitution, but initial results have shown no altered T-cell stimulation of D2-ABC compared to ABC. This mechanism cannot be discarded, with further investigational work required. However, the synthesised 6-position analogues have assisted in further examining the altered repertoire mechanism and initial findings have enabled further understanding of the binding of ABC within HLA-B*57:01. This mechanism of ABC toxicity appears paramount to others proposed and the results presented in this thesis support this. Additional analogue synthesis and in vivo experiments will assist in confirming this further.

There are difficulties in the standardization of traditional Chinese medicines (TCM) because of the complexity of TCM preparations. This leads to regulatory problems. In the present work, several Chinese medicinal plants were investigated including Danshen (Salvia miltiorrhiza Bunge), Sanqi (Panax notoginseng Burk.), compound Danshen dripping pill, and several species of the genus Panax. Plant materials and commercial products containing these taxa were analysed by a range of techniques, including chemical- and activity-based standardization methods: the chemical techniques used include high performance liquid chromatographic (HPLC), nuclear magnetic resonance (NMR) spectroscopic and infrared (IR) spectroscopic methods, followed by chemometric analysis. Different brands of Danshen finished products, Danshen plant root materials of different origin, and Sanqi plant root materials with different production dates were successfully discriminated from each other. Asian and American ginseng were successfully discriminated, and arginine was found for the first time as a main difference, together with other primary and secondary metabolites, using 1H-NMR-PCA. For wild and cultivated American ginseng which had reputed activity in diabetes, an activity-based standardization method based on a cell viability test and proteomic analysis (using two-dimensional difference gel electrophoresis (2D-DIGE) analysis) of mouse insulinoma (MIN6) cells was performed. 83 proteins were found to be significantly up or down-regulated. This work shows that HPLC, and NMR and IR spectroscopy coupled with PCA methods are applicable to standardization methodologies. The advantages and disadvantages of HPLC, and NMR and IR spectroscopic technology are discussed and compared. The 2D-DIGE protein profile can be correlated with TCM treatments. This work contributes to the current problem of regulatory control of TCM preparation as traditional medicines and may suggest a practical way forward.

The aim of this project was to study the effect of pigment volume concentration on enzyme activity by using two different kinds of pigments (kaolin clay and calcium carbonate) in various concentrations. The coating colors were coated on various kinds of substrates for the different methods including flexible paper, board and plastic film. All coating colors contained a latex of standard grade, starch, pigment and the chemical compound used by enzyme, in this case β-D-glucose. The paper substrate used was a white top kraft liner with a grammage of 140 g/m2, while the board had a grammage of 244 g/m2. The plastic film used was a standard Mylar film. All coating colors with various amounts of the two pigments (without enzyme) were coated on the paper and board substrates. The resulting coat weight and the thermal conductivity were measured. The Cobb-Unger oil absorbency was evaluated using double coated plastic sheets for different pigment volume concentrations of the two pigments. The oxygen scavenging ability of the enzyme in the various coating colors coated on the barrier board was also evaluated. For the determination of enzyme activity, glucose oxidase, catalase and glucose were added to latex dispersions in the preparation of the coating colors. The enzymes were entrapped in the coating layers after coating and drying. The activity of enzyme incorporated in the coating dispersions was compared for the two different pigments. The results indicated that the pigment concentration had a significant effect on the enzyme activity.

Urease was microencapsulated via interfacial polymerization within polyamide membranes with high yields of protein (83.1%) and specific activity (92.5%) observed. Intracapsular urease concentration was determined spectrophotometrically and represented 6.2 mg/ml of microcapsule volume. The activity distribution analysis of urease microcapsules indicated that 94% of total intracapsular urease was in a soluble form. The balance (6%) was observed to be incorporated into the encapsulating membrane. The specific activities of microcapsule homogenate (131.7 IU/mg) were similar to that of the intact microcapsules (123.2 IU/mg) suggesting minimal mass transfer and diffusional limitations on the overall rate of urea conversion.
Urease was co-encapsulated with hemoglobin with high yields of mass (91.5%) and specific activity (84%). The specific activity yield was 10% lover than that observed when pure urease was microencapsulated. Thus, the protective role of hemoglobin against urease interfacial denaturation did not appear to be significant.
The pH activity profiles of both free and intracapsular urease were similar with pH optima of 6.0. Thus, effects of charge interactions between ammonia product and the encapsulating nylon membrane were not observed. Similar K$ sb{ rm M}$ values for free (7.6 mM) and intracapsular urease (8.4 mM) suggested that substrate diffusion through the encapsulating membrane did not affect initial rates of catalysis. This was further confirmed by application of the data to a model (Sundaram, 1973) considering the effects of diffusion and mass transfer on the overall reaction rate of intracapsular urease. Under the conditions examined, kinetic rates were slower than the mass transfer rate and thus rate limiting.

In order to establish the viability of using a biological system to decaffeinate coffee, a bacterium (Pseudomonas putida IF-3) capable of living on caffeine as its only source of carbon and nitrogen was studied. Resting cell suspensions and cell-free extracts of P. putida IF-3 were tested to assess their ability to degrade caffeine, and to determine their capacity to retain activity at different temperatures. A method to quantify cell lysis was developed; this method allowed comparison of cell free extract and resting cell caffeine degradation rates. Caffeine degradation rates for cell free extracts were found to be 55 times faster than previously reported P. putida data. Resting cells degraded caffeine 12 times faster than cell free extracts, at 22°C. However, both systems were equivalently active at 50°C. Finally, resting cells were found to be more stable than cell free extracts; this was significantly more evident at 50°C than at 22°C.

In the synthesis of zeolites optimization of factors such as crystallinity, which encapsulates morphology, and acidity can result in optimal activity. Optimal activity is vital in chemical industries due to the fact that it results in higher yields, better selectivities and lower operational costs. In this project the effect of crystallinity on activity is investigated to get a better understanding of performance in terms of activity of amorphous, partially crystalline and highly crystalline materials using isomerisation of para-xylene as the probe reaction.

Mestrado em Biotecnologia - Biotecnologia Alimentar
Atualmente, existe um elevado interesse na valorização de recursos naturais como fontes de compostos bioativos com potenciais efeitos benéficos para a saúde. A salicórnia é uma planta halófita que tem sido usada na alimentação e na medicina tradicional e mais recentemente no desenvolvimento de novos produtos alimentares, ilustrando o interesse da sua caracterização e avaliação. Esta planta encontra-se dispersa mundialmente, estando presente em algumas regiões em Portugal, nomeadamente na Ria de Aveiro e na Ria da Formosa, no Algarve. Esta planta cresce espontaneamente em ambientes salinos, estando inserida num ambiente de elevado stress. O género Salicornia compreende cerca de 25-30 espécies sendo a Salicornia ramosissima uma das menos estudadas atendendo à sua composição química. Alguns compostos bioativos são reportados nesta espécie, nomeadamente ácidos gordos, esteróis e compostos fenólicos, no entanto a informação encontra-se muito dispersa atendendo aos seus efeitos biológicos e composição. Neste sentido, o conhecimento da composição química e dos potenciais efeitos biológicos da S. ramosissima é extremamente importante para introduzir novas aplicações. Assim, o objetivo principal desta tese foi a caracterização química e avaliação in vitro da atividade antioxidante e anti-inflamatória extratos da S. ramosissima, recolhida na ria de Aveiro. Foram recolhidas quatro amostras, no estado de frutificação, em três locais da ria de Aveiro, estas amostras foram tratadas e armazenadas para posterior caracterização. Inicialmente foi estudado a fração lipofílica (extratos de diclorometano) da planta por GC-qMS, seguida do estudo da fração polar (extratos de metanol seguidos de extração com éter de petróleo/água) por LC-QqQ-MS. Posteriormente foram analisados minerais essenciais por ICP-OES e componentes potencialmente tóxicos por ICP-MS. Na fase final foi avaliada ainda a atividade antioxidante e anti-inflamatória dos extratos da fração polar da planta. De um total de 35 compostos da fração lipofílica, o ácido linolénico, o ácido linoleico, o stigmasterol, o β-sitosterol e o ácido palmítico foram os compostos maioritários. O conteúdo total de lipofílicos variou entre 541 e 5412 mg/100g peso seco. Na fração polar, o conteúdo em fenóis totais variou entre 1391 e 3398 mg de equivalentes de ácido gálico por 100g. A amostra vermelha da Marinha dos peixinhos (MPR) apresentou o maior conteúdo de fenóis. Da análise detalhada dos compostos fenólicos, foram identificados 32, sendo que 22 são reportados pela primeira vez nesta espécie. Isorhamnetina é composto maioritário presente nesta espécie. MPR apresentou um maior número de compostos identificados assim como um maior conteúdo estimado (1676.6 μg/g de extrato peso seco). O estudo dos minerais revelou que o sódio é o mineral mais abundante em todas as amostras, no entanto o consumo de 5g desta planta fresca numa salada corresponde apenas a 6.0-7.1% da dose diária recomendada (DDR) para este mineral. Relativamente ao selénio, magnésio e potássio pode contribuir para a DDR dos mesmos com 1.9-2.6%, 1.3-2.1% e 0.2-0.3%, respetivamente. Os estudos in vitro da atividade antioxidante foram expressos em valores de EC50. Os diferentes estudos permitiram avaliar o potencial da S. ramosissima como fonte de antioxidantes, estando os compostos fenólicos relacionados com esta atividade (r2>0.77). A atividade anti-inflamatória foi avaliada através da inibição da produção de dois metabolitos do metabolismo do ácido araquidónico (TXA2 e PGE2). Apesar de nenhum dos extratos inibir a produção de PGE2, o extrato da Marinha dos Peixinhos (MP) e o extrato do Rio Boco (BC) inibiram a produção de TXA2 em 33.2% e 18.1%. A aspirina, conhecida pelos seus efeitos em processos anti-inflamatórios foi usada na mesma metodologia, inibindo o PGE2 e o TXA2 em 18% e 69.3%, respetivamente. Sendo que algumas reações neste metabolismo envolvem radicais, os compostos previamente identificados nos extratos podem ser fundamentais para a atividade reportada. Além disso sendo que a aspirina inibe preferencialmente TXA2, pode-se inferir que os extratos de S. ramosissima apresentam um comportamento similar. Em conclusão os resultados obtidos permitiram a caracterização química sumária da S. ramosissima da Ria de Aveiro, com especial destaque para os compostos lipofílicos e fenólicos e ainda a presença de minerais essenciais. A presença de compostos com potenciais efeitos benéficos para a saúde humana (flavonoides, fitoesteróis e ácidos gordos ω-3 e ω-6) pode ser um fator determinante para a valorização desta planta assim como a presença de baixo teor de sal, podendo ser usado como coadjuvante na dieta de forma a diminuir o risco de doenças cardiovasculares. O efeito anti-inflamatório dos extratos da S. ramosissima revelou o seu potencial impacto na produção de TXA2.
Relativamente ao selénio, magnésio e potássio pode contribuir para a DDR dos mesmos com 1.9-2.6%, 1.3-2.1% e 0.2-0.3%, respetivamente. Os estudos in vitro da atividade antioxidante foram expressos em valores de EC50. Os diferentes estudos permitiram avaliar o potencial da S. ramosissima como fonte de antioxidantes, estando os compostos fenólicos relacionados com esta atividade (r2>0.77). A atividade anti-inflamatória foi avaliada através da inibição da produção de dois metabolitos do metabolismo do ácido araquidónico (TXA2 e PGE2). Apesar de nenhum dos extratos inibir a produção de PGE2, o extrato da Marinha dos Peixinhos (MP) e o extrato do Rio Boco (BC) inibiram a produção de TXA2 em 33.2% e 18.1%. A aspirina, conhecida pelos seus efeitos em processos anti-inflamatórios foi usada na mesma metodologia, inibindo o PGE2 e o TXA2 em 18% e 69.3%, respetivamente. Sendo que algumas reações neste metabolismo envolvem radicais, os compostos previamente identificados nos extratos podem ser fundamentais para a atividade reportada. Além disso sendo que a aspirina inibe preferencialmente TXA2, pode-se inferir que os extratos de S. ramosissima apresentam um comportamento similar. Em conclusão os resultados obtidos permitiram a caracterização química sumária da S. ramosissima da Ria de Aveiro, com especial destaque para os compostos lipofílicos e fenólicos e ainda a presença de minerais essenciais. A presença de compostos com potenciais efeitos benéficos para a saúde humana (flavonoides, fitoesteróis e ácidos gordos ω-3 e ω-6) pode ser um fator determinante para a valorização desta planta assim como a presença de baixo teor de sal, podendo ser usado como coadjuvante na dieta de forma a diminuir o risco de doenças cardiovasculares. O efeito anti-inflamatório dos extratos da S. ramosissima revelou o seu potencial impacto na produção de TXA2.

Doutoramento em Química
The Mediterranean species Cynara cardunculus L. is recognized in the traditional medicine, for their hepatoprotective and choleretic effects. Biomass of C. cardunculus L. var. altilis (DC), or cultivated cardoon, may be explored not only for the production of energy and pulp fibers, but also for the extraction of bioactive compounds. The chemical characterization of extractable components, namely terpenic and phenolic compounds, may valorize the cultivated cardoon plantation, due to their antioxidant, antitumoral and antimicrobial activities. In this study, the chemical composition of lipophilic and phenolic fractions of C. cardunculus L. var. altilis (DC), cultivated in the south of Portugal (Baixo Alentejo region) was characterized in detail, intending the integral valorization of its biomass. The biological activity of cultivated cardoon extracts was evaluated in terms of antioxidant, human tumor cell antiproliferative and antibacterial effects. Gas chromatography-mass spectrometry (GC-MS) was used for the chemical analysis of lipophilic compounds. Sixty-five lipophilic compounds were identified, from which 1 sesquiterpene lactone and 4 pentacyclic triterpenes were described, for the first time, as cultivated cardoon components, such as: deacylcynaropicrin, acetates of β- and α-amyrin, lupenyl acetate and ψ-taraxasteryl acetate. Sesquiterpene lactones were the major family of lipophilic components of leaves (≈94.5 g/kg), mostly represented by cynaropicrin (≈87.4 g/kg). Pentacyclic triterpenes were also detected, in considerably high contents, in the remaining parts of cultivated cardoon, especially in the florets (≈27.5 g/kg). Taraxasteryl acetate was the main pentacyclic triterpene (≈8.9 g/kg in florets). High pressure liquid chromatography-mass spectrometry (HPLC-MS) was utilized for the chemical analysis of phenolic compounds. Among the identified 28 phenolic compounds, eriodictyol hexoside was reported for the first time as C. cardunculus L. component, and 6 as cultivated cardoon components, namely 1,4-di-O-caffeoylquinic acid, naringenin 7-O-glucoside, naringenin rutinoside, naringenin, luteolin acetylhexoside and apigenin acetylhexoside. The highest content of the identified phenolic compounds was observed in the florets (≈12.6 g/kg). Stalks outer part contained the highest hydroxycinnamic acids abundance (≈10.3 g/kg), and florets presented the highest flavonoids content (≈10.3 g/kg). The antioxidant activity of phenolic fraction was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stalks outer part, and receptacles and bracts extracts demonstrated the highest antioxidant effect on DPPH (IC50 of 34.35 μg/mL and 35.25 μg/mL, respectively). (cont.) abstract (cont.) The DPPH scavenging effect was linearly correlated with the total contents of hydroxycinnamic acids (r = -0.990). The in vitro antiproliferative activity of cultivated cardoon lipophilic and phenolic extracts was evaluated on a human tumor cells line of triple-negative breast cancer (MDA-MB-231), one of the most refractory human cancers to conventional therapeutics. After 48 h of exposition, leaves lipophilic extract showed higher inhibitory effect (IC50 = 10.39 μg/mL) than florets lipophilic extract (IC50 = 315.22 μg/mL), upon MDA-MB-231 cellular viability. Pure compound of cynaropicrin, representative of the main compound identified in leaves lipophilic extract, also prevented the cell proliferation of MDA-MB-231 (IC50 = 17.86 μM). MDA-MB-231 cells were much more resistant to the 48 h- treatment with phenolic extracts of stalks outer part (IC50 = 3341.20 μg/mL) and florets (IC50 > 4500 μg/mL), and also with the pure compound of 1,5-di-O-caffeoylquinic acid (IC50 = 1741.69 μM). MDA-MB-231 cells were exposed, for 48 h, to the respective IC50 concentrations of leaves lipophilic extract and pure compound of cynaropicrin, in order to understand their ability in modelling cellular responses, and consequently important potentially signaling pathways for the cellular viability decrease. Leaves lipophilic extract increased the caspase-3 enzymatic activity, contrarily to pure compound of cynaropicrin. Additionally, leaves lipophilic extract and pure compound of cynaropicrin caused G2 cell cycle arrest, possibly by upregulating the p21Waf1/Cip1 and the accumulation of phospho-Tyr15-CDK1 and cyclin B1. The inhibitory effects of leaves lipophilic extract and cynaropicrin pure compound, against the MDA-MB-231 cell proliferation, may also be related to the downregulation of phospho-Ser473-Akt. The antibacterial activity of cultivated cardoon lipophilic and phenolic extracts was assessed, for the first time, on two multidrug-resistant bacteria, such as the Gram-negative Pseudomonas aeruginosa PAO1 and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), two of the main bacteria responsible for health care-associated infections. Accordingly, the minimum inhibitory concentrations (MIC) were determined. Lipophilic and phenolic extracts of florets did not have antibacterial activity on P. aeruginosa PAO1 and MRSA (MIC > 2048 μg/mL). Leaves lipophilic extract did not prevent the P. aeruginosa PAO1 growth, but pure compound of cynaropicrin was slightly active (MIC = 2048 μg/mL). Leaves lipophilic extract and pure compound of cynaropicrin blocked MRSA growth (MIC of 1024 and 256 μg/mL, respectively). The scientific knowledge revealed in this thesis, either by the chemical viewpoint, or by the biological viewpoint, contributes for the valorization of C. cardunculus L. var. altilis (DC) biomass. Cultivated cardoon has potential to be exploited as source of bioactive compounds, in conciliation with other valorization pathways, and Portuguese traditional cheeses manufacturing.
A espécie mediterrânica Cynara cardunculus L. é reconhecida na medicina tradicional, pelos seus efeitos hepatoprotetor e colerético. A biomassa de C. cardunculus L. var. altilis (DC), ou cardo cultivado, poderá ser explorada não só para a produção de energia e fibras de pasta de papel, mas também para a extração de compostos bioativos. A caracterização química dos componentes extratáveis, nomeadamente os compostos terpénicos e fenólicos, poderá valorizar a plantação de cardo cultivado, dadas as suas atividades antioxidante, antitumoral e antimicrobiana. Neste estudo, a composição química das frações lipofílica e fenólica de C. cardunculus L. var. altilis (DC), cultivado no sul de Portugal (região do Baixo Alentejo), foi caracterizada em detalhe, com vista à valorização integral da sua biomassa. A atividade biológica dos extratos de cardo cultivado foi avaliada em termos de efeitos antioxidante, antiproliferativa em células humanas tumorais, e antibacteriano. A análise química dos compostos lipofílicos foi realizada por cromatografia em fase gasosa acoplada à espectrometria de massa (GC-MS). Identificaram-se 65 compostos lipofílicos, dos quais 1 lactona sesquiterpénica e 4 triterpenos pentacíclicos foram descritos, pela primeira vez, como componentes do cardo cultivado, tais como: desacilcinaropicrina, acetatos de β- e α-amirina, acetato de lupenilo e acetato de ψ-taraxasterilo. As lactonas sesquiterpénicas foram a principal família de compostos lipofílicos das folhas (≈94,5 g/kg), maioritariamente representadas pela cinaropicrina (≈87,4 g/kg). Os triterpenos pentacíclicos foram também detetados, em teores consideravelmente elevados, nas restantes partes do cardo cultivado, em especial nos floretos (≈27,5 g/kg). O acetato de taraxasterilo foi o triterpeno pentacíclico maioritário (≈8,9 g/kg nos floretos). Para a análise química dos compostos fenólicos foi utilizada a cromatografia líquida de alta resolução acoplada à espectrometria de massa (HPLC-MS). Entre os 28 compostos fenólicos identificados, o erioditiol hexósido foi descrito pela primeira vez como componente de C. cardunculus L., e 6 como componentes do cardo cultivado, nomeadamente ácido 1,4-di-O-cafeoilquínico, naringenina 7-O-glucósido, naringenina rutinósido, naringenina, luteolina acetil-hexósido e apigenina acetil-hexósido. A concentração mais alta de compostos fenólicos identificados foi observada nos floretos (≈12,6 g/kg). A parte externa dos caules continha o maior teor em ácidos hidroxicinâmicos (≈10,3 g/kg), e os floretos apresentaram o maior teor em flavonoides (≈10,3 g/kg). A atividade antioxidante da fração fenólica foi examinada face ao radical livre 2,2-difenil-1-picril-hidrazilo (DPPH). Os extratos da parte externa do caule, e dos recetáculos e brácteas demonstraram o maior efeito antioxidante, face ao DPPH (IC50 de 34,35 μg/mL e 35,25 μg/mL, respetivamente). (cont.) resumo (cont.) A atividade antioxidante face ao DPPH foi correlacionada linearmente com a concentração total de ácidos hidroxicinâmicos (r = -0.990). A atividade antiproliferativa in vitro dos extratos lipofílicos e fenólicos de cardo cultivado foi avaliada numa linha de células tumorais humanas de cancro da mama de fenótipo triplo-negativo (MDA-MB-231), um dos tipos de cancro humano mais refratários às terapêuticas convencionais. Após 48 h de exposição, o efeito inibitório do extrato lipofílico das folhas (IC50 = 10,39 μg/mL) foi superior ao do extrato lipofílico dos floretos (IC50 = 315,22 μg/mL), sobre a viabilidade celular de MDA-MB-231. O composto puro da cinaropicrina, representativo do composto maioritário identificado no extrato lipofílico das folhas, também inibiu a proliferação das células MDA-MB-231 (IC50 = 17,86 μM). As células MDA-MB-231 foram muito mais resistentes, ao tratamento de 48 h, com os extratos fenólicos da parte externa dos caules (IC50 = 3341,20 μg/mL) e dos floretos (IC50 > 4500 μg/mL), e também com o composto puro do ácido 1,5-di-O-cafeoilquínico (IC50 = 1741,69 μM). As células MDA-MB-231 foram expostas, durante 48 h, às respetivas concentrações de IC50 do extrato lipofílico das folhas e do composto puro da cinaropicrina, de modo a perceber a sua capacidade em modelar respostas celulares, e consequentemente potenciais vias de sinalização importantes para o decréscimo da viabilidade celular. O extrato lipofílico das folhas aumentou a atividade enzimática da caspase-3, ao contrário do composto puro da cinaropicrina. Além disso, o extrato lipofílico das folhas e o composto puro da cinaropicrina causaram paragem do ciclo celular na fase G2, possivelmente através do aumento da expressão proteica de p21Waf1/Cip1 e da acumulação das proteínas fosfo-Tyr15-CDK1 e ciclina B1. Os efeitos inibitórios do extrato lipofílico das folhas e do composto puro da cinaropicrina, contra a proliferação das células MDA-MB-231, poderão também estar relacionados com a diminuição da expressão proteica da fosfo-Ser473-Akt. A atividade antibacteriana dos extratos lipofílicos e fenólicos de cardo cultivado foi avaliada, pela primeira vez, sobre duas bactérias multirresistentes, tais como a bactéria Gram-negativa Pseudomonas aeruginosa PAO1 e a bactéria Gram-positiva Staphylococcus aureus resistente à meticilina (MRSA), duas das principais bactérias responsáveis pelas infeções associadas aos cuidados de saúde. Para tal, determinaram-se as concentrações inibitórias mínimas (MIC). Os extratos lipofílicos e fenólicos dos floretos não revelaram atividade antibacteriana contra P. aeruginosa PAO1 e MRSA (MIC > 2048 μg/mL). O extrato lipofílico das folhas não inibiu o crescimento de P. aeruginosa PAO1, mas o composto puro da cinaropicrina foi ligeiramente ativo (CIM = 2048 μg/mL). O extrato lipofílico das folhas e o padrão puro da cinaropicrina bloquearam o crescimento de MRSA (MIC de 1024 e 256 μg/mL, respetivamente). O conhecimento científico revelado nesta tese, quer do ponto de vista químico, quer do ponto de vista biológico, contribui para a valorização da biomassa de C. cardunculus L. var. altilis (DC). O cardo cultivado tem potencial para ser explorado como fonte de compostos bioativos, em conciliação com outras vias de valorização, e a produção de queijos tradicionais portugueses.

The activity coefficients of glycylglycine and DL, alpha-aminobutyric acid in four aqueous electrolyte solutions (+NaCI, +NaBr, +KCI and +KBr) were obtained at 298.2K. The mean ionic activity coefficient of the electrolyte in aqueous solutions containing these biomolecules was determined from measurements of the potential differences of a cation and an anion ion-selective-electrodes, each measured versus a double junction reference electrode.
For systems containing glycylglycine, the results show that the nature of the anion has a major effect on the activity coefficients of glycylglycine. Comparison of activity coefficient data for glycylglycine with literature data for glycine, both in aqueous NaCl solutions, indicates that the effect of the electrolyte is larger for the peptide than for the amino acid. For the peptide, in all cases, the effect of the electrolyte is more important at low molalities of the electrolyte. The Wilson equation was used to correlate the activity coefficient data obtained. The correlation results were satisfactory for the region of concentrated electrolyte. For systems of DL, alpha-aminobutyric acid in electrolyte solutions, the cation was found to have a significant effect on the activity coefficients of DL, alpha-aminobutyric acid. There exists a distinctively different tendency of the activity coefficients in the dilute and in the concentrated regions of electrolyte in the presence of DL, alpha-aminobutyric acid.

Thesis advisor: Eranthie Weerapana
Functional amino acids that play critical roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chemical-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chemical probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid specificity of these groups. The sulfonyl fluoride activity-based probe (ABP) DAS1 was discovered to label and inhibit both serine proteases and glutathione S-transferases (GSTs). In the case of GSTs, DAS1 covalently bound to a tyrosine residue, despite predicted serine reactivity. Investigation of potential aryl halide electrophiles for ABPP found that chloronitrobenzene RB2 and dichlorotriazine RB7 covalently modify cysteine and lysine residues in target proteins. Applying an existing ABP, iodoacetamide alkyne (IA-alkyne), demonstrated the ability of ABPP to discover novel reactive residues in short open reading frame (sORF)-encoded peptides, as well as previously unannotated cysteine residues on glycolysis enzymes. These studies illustrate the development and characterization of novel electrophiles and demonstrate the application of ABPs to interrogate biological systems. Looking further ahead, the novel electrophiles also provide new tools for the development of covalent inhibitors for treatment of disease
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

This thesis describes efforts made towards the synthesis of a biologically stable, luminescent molecular probe, which could be used to investigate in vivo the processing of sugars by β-galactosidases. To this end, a lactose-based probe was designed, featuring a Lanthanide held within a chelate and appended to the glucosyl unit, and a proximal naphthyl moiety, attached to the galactose unit, which would function as a sensitiser for luminescence. A β-galactosidase enzyme from B. circulans was chosen to carry out the investigation. A number of novel methyl glucosides, functionalised with a naphthyl moiety at C6 of the sugar, were prepared. These were then used as glycosyl acceptors to make disaccharides (lactose analogues), with the enzyme (functioning in reverse) catalysing the glycosylation. The enzymatic reaction was optimised by varying the amount of enzyme, the reaction pH, the ratio of glycosyl acceptor to donor, the reaction temperature, concentration and solvent mixture. The optimal conditions were found to be a 0.4 M reaction solution at pH 7.0 with 20% acetonitrile, a 7:1 ratio of glycosyl acceptor to donor, 19.6 U of enzyme per mmol of acceptor, and a reaction temperature of 30°C. The resulting disaccharide products exhibited unusual regioselectivity for the β-galactosidase from B. circulans, with unexpected β(1→3) and β(1→2) glycosidic linkages being formed. In an effort to increase the efficiency of the process of identifying suitable substrates for the enzyme, a dynamic combinatorial chemistry approach was also explored. This used disulfide bonds to attach the naphthyl moiety to the methyl glucoside using linkers of different lengths. From this library, the enzyme successfully processed the novel disulfide GlcOMe-S-S-CH\(_2\)Np as a glycosyl acceptor with p-nitrophenyl galactose as the glycosyl donor. This resulted in a novel disaccharide featuring a naphthyl group attached via a disulfide bond to the glucosidic residue.

Includes bibliographical references (leaves 67-72).
Liver dysfunction is common, often unrecognised and likely to increase in incidence in the population in parallel with the obesity and attendant type 2 diabetes mellitus epidemics. Liver fibrosis is a significant finding in liver pathology as it imparts important clinical staging and prognostic information, is a risk marker of adverse clinical outcome yet, even if advanced, is capable of reversal. Histological examination of liver biopsy material is the reference standard in the assessment of liver fibrosis, but it is impractical to biopsy all patients suspected of having liver disease. Serumprolidase is among novel biomarkers that have been described in diagnosing and/or staging liver fibrosis. This study evaluated the measurement and the diagnostic accuracy of serumprolidase in determining the potential presence and degree of liver fibrosis compared with liver biopsy.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
This present work reports the chemical and biological analysis of the stem and leaves from Bauhinia pulchella. In this study, the ethanol extract from stems was obtained by maceration, subjected to chromatographic fractionation, leading to isolation of three flavonoids: (+)-3â,4â-dihydroxyphenyl-chroman-7-ol (BP-2), (-)-fisetinidol (BP-3) and (+)-epicatechin (BP-4); a mixture of triterpenes taraxerone and β-amyrenone (BP-1); a mixture of steroids sitosterol and stigmasterol (BP-5); and a bibenzyl named 2-hydroxy-3â,5â-dimethoxybibenzyl (BP-6). It is notewhorthy to mention that BP-1 and BP-4 substances are unprecedented in the genus, while BP-2 is unpublished. Chemical structures of secondary metabolites obtained were elucidated by 1H and 13C NMR; IR and MS associated with comparison of data described in the literature. Chemical composition of the essential oil from leaves of B. pulchella, obtained by hydrodistillation, was determined and quantified by gas chromatography-mass spectroscopy (GC/MS) and gas chromatography-flame ionization detector (GC/FID), which identified 95,68% of all constituents. α-pinene (23.89%); caryophyllene oxide (22.43%) and β-pinene (12.19%) were the major components. The essential oil was tested against Aedes aegypti larvae and showed LC50 value of 105.93 Â 1.48 μg/mL. The cytotoxic activity of essential oil was evaluated on human tumor cell lines (HL-60; MCF-7; NCI-H292 and HEP-2) was evaluated, showing IC50 values with confidence intervals of 9.941 (8.238 to 12.00), 53.05 (41.39 to 67.99), 48.98 (44.22 to 54.25) and 50.42 (42.47 to 59.87) μg/mL, respectively and the cell line HL-60 the most sensitive among the cells tested. This is the first report of the chemical study of Bauhinia pulchella, as well the investigation of larvicidal activity and cytotoxicity of the essential oil from its leaves.
O presente trabalho relata o estudo quÃmico e biolÃgico do caule e das folhas de Bauhinia pulchella. Nesse estudo, o extrato etanÃlico do caule, obtido por maceraÃÃo, foi submetido a fracionamento cromatogrÃfico levando ao isolamento de trÃs flavonoides (+)-3â-4âdiidroxifenil-cromano-7-ol (BP-2), (-)-fisetinidol (BP-3) e (+)-epicatequina (BP-4); da mistura de triterpenos taraxerona e β-amirenona (BP-1); da mistura de esteroides sitosterol e estigmasterol (BP-5) e de um bibenzil denominado 2-hidrÃxi-3â-5â-dimetoxibibenzila (BP-6). Cabe ressaltar que as substÃncias BP-1 e BP-4 sÃo inÃditas no gÃnero, enquanto BP-2 à inÃdita na literatura. As estruturas dos metabÃlitos secundÃrios isolados foram elucidadas por RMN 1H e 13C; IV e EM, juntamente com a comparaÃÃo com os dados descritos na literatura. A composiÃÃo quÃmica do Ãleo essencial das folhas de B. pulchella, obtido por hidrodestilaÃÃo, foi determinada e quantificada por cromatografia gasosa acoplada à espectrometria de massas (CG-EM) e detector de ionizaÃÃo por chama (CG-DIC), sendo, portanto, identificados 95,68% dos seus constituintes: α-pineno (23,89%), Ãxido de cariofileno (22,43%) e β-pineno (12,19%) foram os constituintes majoritÃrios. O Ãleo essencial teve sua atividade larvicida sobre Aedes aegypti avaliada, sendo obtido um valor de CL50 igual a 105,93  1,48 μg/mL. O poder citotÃxico do Ãleo essencial foi avaliado sobre as linhagens tumorais humanas HL-60, MCF-7, NCI-H292 e HEP-2, sendo obtidos valores de CI50 e intervalos de confianÃa iguais a 9,941 (8,238 a 12,00); 53,05 (41,39 a 67,99); 48,98 (44,22 a 54,25) e 50,42 (42,47 a 59,87) μg/mL, respectivamente, sendo a linhagem celular HL-60 a mais sensÃvel dentre as cÃlulas testadas. Este à o primeiro relato do estudo quÃmico de Bauhinia pulchella, bem como da investigaÃÃo da atividade larvicida e citotÃxica do Ãleo essencial de suas folhas.

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2)).

Thesis advisor: Eranthie Weerapana
Cysteine residues on proteins play important catalytic and regulatory roles in complex proteomes. These functional residues can be modified under physiological conditions by posttranslational modifications (PTMs) to regulate protein activities and modulate cysteine reactivity. Many PTMs are highly labile and dynamic, rendering it difficult to detect modified proteins within complex systems. To contribute to the chemical-proteomic methods currently available, chemical probe-Mass Spectrometry (MS) platforms were developed to study oxidative cysteine modifications. A MS platform for the assessment of S-nitrosation in vitro identified Cys329 of Cathepsin D (CTSD) as highly sensitive to S-nitrosothiol formation. To achieve a more physiological relevant representation of S-nitrosation, this platform was later adapted for study in live cells using a caged electrophile, Caged BK. Additionally, oscillation of cysteine oxidation as a function of circadian rhythm in Drosophila melanogaster and human samples was explored. As a compliment to these MS platforms, a 4-aminopiperidine-based cysteine-reactive probe library was developed. These probes have been used to target specific reactive cysteines as an alternate way to regulate protein function and can be used as tools to provide insight into the roles of these residues in protein activities
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Both an electrochemical and an isopiestic methods were used to measure the mean activity coefficients of amino acids in water-electrolyte-amino acid systems. The electrochemical method used an electrochemical cell with an anion and a cation ion selective electrodes, each measured versus a double junction reference electrode. The activity coefficients of the electrolyte were obtained from the e.m.f measurements for four systems at 298.15K. The amino acids were glycine, DL-serine and DL-valine and the electrolyte were KNO3 and NaNO3. The isopiestic method was applied to measure the activity of water for the system water + DL-serine + KNO3 at 298.15K and the results were compared with those from electrochemical method. The activity coefficients of the amino acids in the ternary systems were obtained from those of the electrolyte measured by the electrochemical method or from those of the water measured by isopiestic method, according to the cross differential relation.
Two models were used to correlate the measured data. First the excess Gibbs free energy model proposed by Khoshkbarchi and Vera (1996a) with the NRTL equation was used to correlate the activity coefficient data. A simple model derived from a modification of a more complex model based on the perturbation theory (Khoshkbarchi and Vera, 1996c) was proposed. The model was applied to correlate the activity coefficients and solubilities of amino acids in binary aqueous solutions and the activity coefficients in ternary systems with satisfactory results.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 8, 2009) Includes bibliographical references.

The chemical oxygen-iodine laser (COIL) has several important military and industrial applications. The concern of this work is do develop a partial differential equation model describing optical behavior in the COIL. Optical behavior of the COIL has traditionally been investigated via a ray tracing method. Photons are represented as discrete particles, and their behavior is described by the geometry of the system. We develop an optical model wherein photons have a wave description. In order to construct the mathematical model, we utilize the theory of paraxial wave optics and Gaussian beams. Doing so allows us to incorporate physical effects such as diffusion/diffraction and refraction into the model. After describing the optical model, we present numerical methods for obtaining approximate solutions to the model in the cases of one and two transverse directions. Results are presented illustrating the efficacy of the numerical methods.
Ph. D.

Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency.
Chemistry and Chemical Biology

This study was conducted to investigate the physico-chemical properties and antioxidant activity of five pomegranates fruit (Punica granatum L.) cultivars grown in Iran. Significant differences were found among the pomegranate cultivars for many of the properties studied. Results showed that, in particular, fruit diameter ranged from 63.63 mm (Syah) to 79.29 mm (Rabab), fruit volume from 153.3 cm3 (Syah) to 293.3 cm3 (Rabab), fruit density from 0.93 g cm-3 (Rabab) to 1.13 g cm-3 (Torsh Sefeed). Although Syah showed the lowest fruit weight (144.8 g), fruit yield (8.28 ton ha–1) and fruit skin thickness (1.55 mm), Rabab had the highest fruit yield (27.1 ton ha–1) and fruit skin thickness (2.32 mm). Juice volume was between 61.1 and 67.0 cm3. Percent of aril ranged from 59.64% (Rabab) to 75.3% (Syah) and weight of aril was between 108.9 and 199.8 g. Also, results indicated that titratable acidity content varied from 0.39% (Syah) to 1.13% (Torsh Sefeed). The total soluble solids content varied from 12.67 ◦Brix (Torsh Sefeed) to 15.67 ◦Brix (Zardeh Anar), pH values from 3.05 to 3.77, Electrical conductivity values from 2.8 to 3.14 dSm-1 and vitamin C content from 59.25 to 69.52 mg 100g1. The anthocyanins content was observed between 80.36 (Syah) and 216.97 (Zardeh Anar). The antioxidant activity of pomegranate cultivars ranged from 27.24% (Syah) to 84.04% (Torsh Sefeed). These results demonstrated that the cultivar was the major factor which influences the morpho-pomological and chemical (especially, antioxidant activity), properties in pomegranates.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2018
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 132-143).
Since its discovery by Banting and Best, the administration of exogenous insulin to control blood glucose levels has been a primary method of treatment for severe cases of diabetes mellitus. Several decades of insulin engineering and development has led to the clinical introduction of two broadly classified categories of protein therapies: prandial and basal insulins. Although these developments have had profound effects on disease management with respect to insulin-dependent diabetes, the overall strategy for development has historically been restricted to rational design criteria based on static pharmacodynamic profiles, profiles that are inherently naive to physiological changes in the diabetic patient. As a result, stringent patient-dependent regimens are the standard of care with regard to glycemic monitoring and management.
When coupled with issues such as patient noncompliance, severe hypoglycemia, as well as the adverse health effects that result from chronic mismanagement of hyperglycemia, it is obvious that there are still major hurdles that must be overcome to properly treat the disease. Herein, we introduce innovative strategies aimed towards the advancement of novel insulin bioconjugate design and development for enhanced long-term efficacy and glucose-responsive activity. First, we develop a class of unimolecular, glucose-responsive insulin conjugates towards the design of a state-responsive, patient-dependent therapy.
Optimization of this system resulted in the identification of a lead candidate, lns-PL-4FPBA, capable of (1) glucose-mediated changes in solubility for long-term sequestration and intelligent depot formation, (2) superior safety in comparison to clinically used long-acting insulins, and (3) glucose-responsive pharmacokinetic and pharmacodynamic activity in both healthy and diabetic animal models. Next, we pioneer the first design and synthesis strategy of a novel class of sugar-responsive insulin conjugates, with the ultimate goal of developing an insulin bioconjugate capable of sugar-mediated receptor binding interactions. In this effort, we created dynamically cyclized insulin conjugates that were found to exhibit enhanced chemical stability and superior thermal stability relative to the native protein, as well as sugar-mediated destabilization, suggesting the potential to exploit the insulin receptor binding mechanism.
Lastly, we focus on improving basal activity of the insulin protein by utilizing a novel class of zwitterionic insulin polymer conjugates towards the generation of ultra long-acting insulin therapies. The resulting library is demonstrated to afford equivalent biological potency relative to native human insulin, augmented thermal and chemical stability capable of withstanding thermal aggregation for over 80 days, as well as ultra long-acting basal insulin potential.
by Abel Bryan Cortinas.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

Includes bibliographical references (p. 170-186).
Fischer-Tropsch catalysts tyically contain metals such as iron, cobalt and ruthenium. In this study a supported cobalt catalyst was used, in which cobalt was put on the support by colloidal impregnation. Some literature on CO hydrogenation indicates that molybdenum oxide and vanadium oxide used as a promoter may be beneficial for the activity and selectivity of a cobalt based catalyst. To eliminate the structural influence on the selectivity by using commercial MoO3 and V2O5 supports, and alumina support was modified with molybdenum and vanadium in an attempt to create molybdenum and vanadium oxide layer covering the alumina surface.

Bibliography: pages 128-140.
The isopropylation of benzene to produce cumene is an important petrochemical process since cumene is used as a chemical intermediate for the production of phenol and acetone. In recent years, the good performance of zeolite Beta for this particular reaction has often been reported in the literature (Reddy et al., 1993, Bellussi et al., 1995 and Perego et al., 1996). It is known, from the numerous studies that have been carried out on other zeolite types, that post synthesis modifications such as dealumination of the zeolite framework tend to enhance the activity of these zeolites for catalytic reactions. Moreover, the effect of synthesis parameters on the catalytic activity of zeolite Beta is an important issue that has not been thoroughly investigated in the literature. In this study, a commercial parent zeolite Beta catalyst (sample A) was modified by post synthesis modifications such as steaming, acid washing or steaming followed by acid washing. A series of zeolite Beta catalysts were also synthesised by different techniques. The samples were characterised with respect to structure, morphology, particle size, number of acid sites, co-ordination state of aluminium and the environment of silicon atoms. The isopropylation of benzene to cumene was used as a test reaction to evaluate the effect of post-synthesis modifications and synthesis procedure on the catalytic activity of the zeolite Beta catalysts.

In the first part of this thesis (chapters 1-8) the structure/activity (S/A) correlation studies on a class of anti-cancer drugs based on flavone acetic acid (FAA) by means of computer modelling techniques are reported. In particular, semiempirical and ab-initio quantum mechanical calculations have been performed on ten FAAs whose expeirmental anti-cancer activity was known. The results show that some calculated properties such as bond lengths, atomic charges, energies of the HOMO and the atomic orbitals involved in its formation, correlate with the anti-tumour activity. The correlations found were then used on another 38 molecules analogous to FAA whose anti-cancer activity had also been measured and of the 21 active molecules, 20 were predicted to be active by these SA correlations (95% success rate). From this study it also emerged that the pyrone ring may be directly involved in the anti-tumour mode of action of the FAA and it is suggested that vitamin-K may also play a role. The second part (chapter 9) is a study of the dependence of the molecular electrostatic potential on the basis set. From this study it emerged that GEOSMALL and MINI-1 minimal basis sets produce MEPs that are more similar to those obtained with the 6-31G** basis set than the MEP obtained with the STO-NG basis set. GEOSMALL and MINI-1 also give better energies and better properties than STO-NG and their use is recommended when properties of large organic molecules are of interest. Also, from this study it emerged that the use of Mulliken charges for the calculation of the MEP with the point charge approximation is not advisable for it may lead to very different pictures of the electrostatic potential calculated directly from the wave function.

The phenomena of hydrogen spillover on heterogeneous catalysis has been studied extensively. However, questions involving the nature of the spiltover species; the possibility of activated sites on an inert support; the magnitude and importance of spillover in terms of industrial catalysis and kinetics, etc. are still unresolved. Using Proton NMR Spectrometry, Mass Spectrometry, MacBain Balance, and Isotopic studies, these questions are addressed for the system of hydrogen spillover on silica aerosil. The proton NMR studied indicate that spiltover hydrogen has a noticeable effect on the resonance spectra of hydrogen on aerosil. Three peaks are present, one upfield and one downfield from a central H(,2)-OH resonance peak. The experimental results can be explained most simply by the existance of a radical hydrogen species involved in the formation of a surface complex. Also, a resonance peak associated with spillover-induced active sites is observed. The active sites seem to have a high electron density and are thus electron donor or "metallic" in nature. Depending on the activation conditions, one or two types of sites seem to be created. The mechanism of site activation can involve vicinal and/or geminal hydroxyls at specific surface sites on the oxide interacting with spiltover hydrogen to form an F center-type defect and gaseous water. Stressed siloxane bonds could also be involved. The rate of this zero order reaction is approximately 3.3 x 10('11) sites formed per hour with an apparent activation energy of 28 kcal/gmol. This induced activity is independent of the metal and quite different. The mechanism of ethylene hydrogenation on activated aerosil is similar to that on metal oxides in the sense that the molecular identity of the reacting deuterium is retained and dideuteroethane is the primary product. Differences in activity from both metals and metal oxides are apparent such as partial ethylene exchange and H(,2)-D(,2) exchange. It is not clear whether these differences are due to unique catalytic properties of the activated silica or to the higher reaction temperatures (200(DEGREES)C) involved.

碩士
高雄醫學大學
天然藥物研究所
96
Fatoua pilosa Gaud. (Moraceae) is a small perennial herb, which distributed in Taiwan, Philippine, Moluccas and New Guinea. In a series of studies on the active constituents of Formosan plants, the methanolic extract of the whole plant of this species showed antitubercular activity against Mycobacterium tuberculosis H37Rv in vitro. There are only three species of Fatoua in the world. The chemical constituents and bioactivities of Fatoua plants have never been conducted. The methanolic extract of whole plant of this species was partitioned into EtOAc and H2O-soluble fractions. Investigation of EtOAc-soluble fraction led to the isolation of six new coumarins: (+)-fatouain A (1), (-)-fatouain B (2), (+)-fatouain C (3), (-)-fatouain D (4), (+)-fatouain E (5), (-)-fatouain F (6) and four new bis-coumarins: (+)-fatouain G (7), (+)-fatouain H (8), fatouain I (9), fatouapilosin (10), along with eighteen known compounds, including three simple coumarins: umbelliferone (11), scopoletin (12), phellodenol-A (13), six furanocoumarins: psoralen (14), bergapten (15), S-(+)-marmesin (16), S-(+)-rutaretin methy1 ether (17), S-(+)-rutaretin (18), 2’,3’-dehydromarmesin (19), one pyranocoumarin: xanthyletin (20), three chaclones: isobavachalcone (21), kanzonol C (22), (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(2,2-di- methyl-8-hydroxy-2H-benzopyran-6-yl)-2-propen-1-one (23), one benzenoid: syringaldehyde (24), one quinone: α-tocopheryl quinone (25), one triterpenoid: lupeol (26), two steroids: a mixture of β-sitosterol (27) and stigamasterol (28). The structures of these compounds were elucidated by spectral analysis or by chemical derivation. Scopoletin (12), isobavachalcone (21) and (E)-1-[2,4-dihydroxy-3-(3-methyl- 2-butenyl)-phenyl]-3-(2,2-dimethyl-8-hydroxy-2H-benzopyran-6-yl)-2-propen-1- one (23) showed antituburcular acivity aganist Mycobacterium tuberculosis H37Rv in vitro with MIC values: 42.1, 17.6 and 30.0 μg/ mL, respectively.

碩士
靜宜大學
應用化學系
82
1-芳香基-2,2-二氯環丙烷化合物具高度環張力及電子密度，在進行硝化 反應後，可得異▌▌之開環產物﹔對含其他取代基之苯基環丙烷，如甲基 、氯甲基、苯環或是以 基取代苯基，進行硝化反應，來探討溫度及NO2+ 與反應物之比例對產物分佈之影響，除了1-甲基-1-苯基-2,2-二鹵環丙烷 形成開環主產物 5-甲基-5-(4'-硝基苯)-3-鹵異▌▌▌外，其他六個環丙 烷化合物的產物皆以苯環上硝基取代為主之原因。另外，利用電腦輔助分 子模擬之理論計算—半經驗法，來探討一系列1-芳香基-2-鹵環丙烷的環 丙烷上三個碳原子之13C NMR SCS值、Hammett值與三種方法(MNDO,AM1, PM3)之電荷密度的關係，並得到三種方法之電荷密度值皆可與1-芳香基環 丙烷之13C NMR SCS值、Hammett值相呼應，但 順-1-芳香基-2-溴環丙烷 之Cβ，1-芳香基-2,2-二氯環丙烷之Cβ、Cγ及1-芳香基-2,2-二溴環丙 烷之Cβ的13C NMR SCS值、 Hammett值，分別與 MNDO,AM1,PM3之電荷密 度值有較一致性的關係。 The high ring strain and high electron density of cyclopropanes result in formation of isoxazoles from the ring opening during the nitration of 1-aryl-2,2-dichlorocyclopropanes. The study of phenylcyclopropanes contain an additional of substituents,such as methyl, chloromethyl, phenyl or naphthyl group instead of phenyl, were nitrated under various temperature and the ratio between cyclopropane and HNO3 to investigate the effect on the resultants. The major products from six cyclopropane are the nitro substitution on phenyl, except for 2,2-dihalogeno-1- methyl-1-phenylcyclopropane which yields 3-halogeno-5- methyl-5-(4'-nitrophenyl)isoxazoline. In addition, the Semi- Empirical method (MNDO,AM1,PM3) is applied for the calculation of the charge densities of three carbons for the cyclopropanes of 1-aryl-2-halogenocyclopropanes to study of the correlation between SCS from 13C NMR chemical shift, Hammett constant and charge densities from calculation. The charge densities from three calculative methods are correlated to SCS from 13C NMR and Hammett constant of 1-arylcyclopropanes. But SCS from 13C NMR and Hammett constant of Cβ of cis-1-aryl-2- bromocyclopropane, Cβ,Cγ of 1-aryl-2,2-dichlorocyclopropane, C βof 1-aryl-dibromocyclopropane only are well correlated to the charge densities from MNDO,AM1, PM3, respectively.

博士
國立屏東科技大學
食品科學系所
102
Longan seed extract was found to contain high levels of effective plant polyphenolic compounds, such as ellagic acid and gallic acid. Plant polyphenols have been published to show significant antioxidant activity and free radical scavenging activity. Prior research about longan seeds are mostly related to anti-inflammatory and component analysis, but no more in-depth research about antibacterial activity. This study researches and analyses the antibacterial activity by longan seed extracts in hierarchical objects layers, and depth probe their qualitative and quantitative analysis of effective antimicrobial activity, in order to clarify its antibacterial scope and application areas of antimicrobial. Methicillin-resistant Staphylococcus aureus (MRSA), a species of multidrug-resistant Staphylococcus aureus, has 60 to 80 percent detected rate in hospitals. It’s the most common nosocomial bacteria in hospitals and can cause human opportunistic infections, including endocarditis, wound infection and cause menstrual toxic shock syndrome among women. The strain of bacteria is as an indicator of nosocomial infections. Another strain, referred to as AB(Acinetobacter baumannii) strain, is a gram-negative bacilli, can cause infections to many body parts, including respiratory infections, urinary tract infections, surgical wound infections, bacteremia, etc. It’s also an important pathogenic bacteria for human being. The mid-1980s began, the infections caused by AB bacteria in clinical are more and more common and the clinical medicines have also started to be increased. The AB bacteria has gradually become resistant to many antibiotics, including broad-spectrum penicillin, the third-generation cephalosporin sub streptozotocin, amino- glycosides, etc. With the abuse of human antibiotics and the necessary using of livestock and aquaculture industry, emerge in endlessly of resistant the existing antibiotics. After screening from several kinds of natural extracts, we found the longan seed, the general discard unused, have antibiotic effects for this two kinds of human health menacing strains. This experiment researches and analyses the antibacterial activity of the longan seed extracts by various layered materials, and depth probe the antimicrobial activity by qualitative and quantitative analysis. In the use of 1mg/disc sample volume, the DL-extracted material P01-SI01 in the third sub-layer extract’s inhibition ring diameter can be up to 1.5cm and the MIC concentration is 64μg/mL. It’s generally rare effect of natural product extractions. In the future, we will further evaluate its antibacterial effect in practical application.

碩士
高雄醫學大學
天然藥物研究所
98
Abstract Aristolochia manshuriensis (Guanmuton) (Aristolochiaceae) is a perennial shrub plant, and distributed in the northeastern of the China and Korea. Aristolochia species were used as antibacterial, antiinflammatory, antiasthmatic agents, etc. in the Chinese medicine. In previous reserches, aristolochic acids had been proved to be the most potent constituents of Aristolochia species and were reported to be nephrotoxins and carcinogens with long duration for body metabolism. The MeOH extract of the stem was partitioned into CH2Cl2 and H2O layers. The CH2Cl2 layer was subjected to Celite column chromatography into n-Hexane, n-Hexane:EA (1:1), EA, MeOH, n-BuOH and H2O-soluble layers. Among them, which was based on the anti-inflammatory assay, the n-Hexane, n-Hexane:EA (1:1), EA and MeOH-soluble layers showed inhibitory effects on superoxide anion generation and elastase release by human neutrophils which induced by fMLP/CB. In the current phytochemical investigation, the CH2Cl2-soluble layer was subjected to repeated column chromatography. The structures were elucidated by spectroscopic methods that led to the identification of twenty-five compounds, including four aristolochic acids︰ aristolochic acid I (4), aristolochic acid-IIIa (7), aristolochic acid-IVa (6),and aristolochic acid II (5), one sodium salt of aristolochic acids︰sodium aristolochate I (21), five aristolactams ︰aristolatams IIIa (8), aristolactam III (11), aristolactam I (9), aristolactam II (12), and aristolactam Ia (10), three denitroaristolochic acids ︰aristolamide (2), aristolamide II (3), and demethylaristofolin E (16), four aristolochic acid alkyl esters ︰aristolic acid methyl ester (17), 6-methoxyaristolic acid methyl ester (18), aristolochic acid-I methyl ester (13), aristolochic acid-IV methyl ester (14), and aristolochic acid-IVa methyl ester (15), two amides N-trans-feruloylmethoxytyramine (19) and aristopyridinone (25), one benzenoid︰trans-p-Coumaric acid (20), four steroids︰??-sitosteryl-3-O-??-D-glucoside & Stigmasteryl-3-O-??-D-glucoside (22), β-stigmasterol (23), 6β-hydroxystigmast-4-en-3-one (24) and β-sitosterone (25)。 In the anti-inflammatory assay, aristolactam IIIa (8) showed better inhibitory effect on superoxide anion generation and elastase release than genistein, and aristolamide II (3) showed better inhibitory effect on elastase release than genistein.

碩士
高雄醫學大學
天然藥物研究所
100
Clinacanthus nutans (Acanthaceae) is a small shrub native to tropical Asia. The species has long been used in Thailand as a traditional medicine for the treatment of skin rashes, insect and snake bite, herpes simplex virus (HSV), and varicella-zoster virus (VZV) lesions. In this study, the cultivated materials were collected in Taichung, Taiwan. The aerial parts of C. nutans (2.3 kg) were extracted by 95% EtOH aq.. The extract was partitioned to yield four layers (n-hexane, 80% EtOH aq., n-BuOH, and H2O layers). Among the four layers, only the 80% EtOH aq. layer showed immune-modulating activity. Chromatographic separation of the active layer yielded twenty-seven compounds. Among them, sixteen compounds were identified, including seven sulfur-containing compounds: entadamide A (1)、2-cis-entadamide A (2)、trans-N-(2-(3-(2-hydroxyethylamino)-1-(methylthio)-3-oxopropoxy)ethyl)-3-methylthiopropenamide (3)、trans-N-(2-hydroxyethyl)-3-methylsulfonylpropenamide (4)、trans-2-(3-(methylsulfinyl)acrylamido)ethyl acetate (5)、entadamide C (6)、trans-3-methylsulfinyl-2-propenol (7), three megastigmanes: 3β-hydroxy-5α,6α-epoxy-7-megastimen-9-one (8)、loliolide (9)、3α-hydroxy-4,7-megastigmadien-9-one (10), five benzenoids: ethyl-trans-3-(4-hydroxyphenyl)prop-2-enoate (11)、ethyl-cis-3-(4-hydroxyphenyl)prop-2-enoate (12)、4-hydroxyacetophenone (13)、4-hydroxybenzaldehyde (14)、4-hydroxybenzoic acid (15), one alkaloid: indole-3-aldehyde (16). The structures of the isolates were identified by spectral analysis. In addition, eleven unknown compounds are still under elucidating. Compounds 2-5 and 7-16 were isolated for the first time from Clinacanthus genus. Among these ingredients, 2-5 are new compounds. The biological activities of isolates are currently under investigation.

碩士
高雄醫學大學
天然藥物研究所
96
Coptosapelta diffusa Steenis (Thysanospermum diffusum Champ., Rubiaceae) is a woody lianas, distributed in Southern China, Ryukyu, and Taiwan in broad-leaved forests. The MeOH extract of its whole plant showed significant antitubercular activity against Mycobacterium tuberculosis H37Rv. and was partitioned into EtOAc, n-BuOH and H2O-soluble parts. Investigation of the EtOAc-soluble part by repeated column chromatography led to the isolation of thirty-four compounds, including fourteen triterpenoids: thysanolactone (1), coptosalactone A (2), coptosalactone B (3), coptosalactone C (4), coptosalactone D (5), coptosalactone E (6), coptosalactone F (7), lupeol (8), lupenone (9), lupane (10), friedelin (11), β-amyrin (12), 3-acetyl ursolic acid (13), squalene (14); one naphthalene: methyl 1,4-dimethoxy-2-naphthoate (15); one benzophenone: 2-carbomethoxy-2''-hydroxy-5''-methyl-benzophenone (16); seven anthraquinones: 2-dimethoxymethylanthraquinone (17), 1-hydroxy-6-methylanthraquinone (18), 2-methylanthraquinone (19), 1-hydroxy-2-methylanthraquinone (20), 2-carbomethoxyanthraquinone (21), 2-carbomethoxy-1-hydroxyanthraquinone (22), 2-formylanthraquinone (23); three naphthoquinones: hydroxyhydrolapachol (24), lapachol (25), dehydro-α-lapachone (26); four steroids: β-sitostenone (27), ergosta- 4,6,8(14),22-tetraen-3-one (28), a mixture of β-sitosterol (29) and stigmasterol (30); one benzenoid: octadecyl ferulate (31); one quinone: α-tocopheryl quinone (32); one diterpenoid: trans-phytol (33); one fatty acid ester: methyl linoleate (34). The structures of these compounds were determined through spectral analysis. Among these isolates, 2, 4, 5, 6, 7 and 16 are new compounds, and compounds 3, 15, 17, 18 and 24 were first isolated from natural source. Compounds 23, 24, 25 and 26 showed antitubercular activity in vitro.