published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

Background: Cassia angustifolia Vahl. (commonly known as senna makkai or cassia senna), native to Saudi Arabia, Egypt, Yemen and also extensively cultivated in Pakistan, is a medicinal herb used traditionally to cure number of diseases like liver diseases, constipation, typhoid, cholera etc. This study was conducted to evaluate the in-vitro antimicrobial, antioxidant and anticancer assays and phytochemical constituents of aqueous and organic extracts of C. angustifolia leaves. Methods: The antimicrobial activities of C. angustifolia aqueous and organic (methanol, ethanol, acetone, ethyl acetate) extracts were investigated by the disk diffusion method. These extracts were further evaluated for antioxidant potential by the DPPH radical scavenging assay. Anticancer activities of the extracts were determined by the MTT colorimetric assay. The total phenolic and flavonoid contents of C. angustifolia extracts were evaluated by the Folin-Ciocalteu method and aluminum chloride colorimetric assay, respectively. The structures of the bioactive compounds were elucidated by NMR and ESI-MS spectrometry. Results: Bioactivity-guided screening of C. angustifolia extracts, led to the isolation and identification of three flavonoids quercimeritrin (1), scutellarein (2), and rutin (3) reported for the first time from this plant, showed significant anticancer activity against MCF-7 (IC50, 4.0 mu g/mu L), HeLa (IC50, 5.45 mu g/mu L), Hep2 (IC50, 7.28 mu g/mu L) and low cytotoxicity against HCEC (IC50, 21.09 mu g/mu L). Significant antioxidant activity was observed with IC50 2.41 mu g/mL against DPPH radical. Moreover, C. angustifolia extracts have the potential to inhibit microbial growth of E. cloacae, P. aeruginosa, S. mercescens and S. typhi. Conclusion: C. angustifolia extracts revealed the presence of quercimeritrin (1), scutellarein (2), and rutin (3), all known to have useful bioactivities including antimicrobial, antioxidant and anticancer activities.

Charles University Faculty of Pharmacy in Hradec Kralove Department of Farmacology & Toxikology Student: Martina Fišrová Supervisor: PharmDr. Marie Vopršalová, CSc. Title of diploma thesis: The effects of some flavonoids on isolated rat aortic rings Flavonoids are secondary plant metabolites that are profusely represented in nature. They are known for their wide range of effects and many of them have beneficial effects on the human body. Above all, they have anti-bacterial and anti-inflammatory properties and effects on cardiovascular system - they cause vasodilation. The aim of this diploma thesis was to investigate vasorelaxation effects of selected flavonoids from the group of flavonolignans - 2,3-dehydrosilybin A (DHS-A), 2,3-dehydrosilybin B (DHS-B) and metabolites, which are components of the silymarin complex found in the plant Silybum marianum (Asteraceae). The vasorelaxation potential of tested substances was verified in ex vivo conditions on isolated rat aortic rings. The effect of increasing doses of individual substances in precontracted aortic rings with intact endothelium was measured. From the measured values, the DRC curves were constructed and EC50 values were determined. The analysis of the results shows, that DHS-A (EC50 = 30,1 mol/l) had the most signifiant activity. Its...

Fabaceae é a terceira maior família botânica, apresentando grande importância ecológica, ornamental e econômica. Por sua grande diversidade de espécies está dividida em seis sub-famílias Caesalpinioideae, Cercidoideae, Detarioideae, Dialioideae, Duparquetioideae e Papilionoideae. Papilionoideae é considerada um grupo monofilético e nesta está inserida Glycine max (L.) Merr., espécie de importância econômica alimentícia (grãos, farelo, óleo) e medicinal. A soja tem seu centro de origem na China, sendo que a soja moderna foi modificada por cruzamentos entre duas espécies selvagens, por cientistas ainda na antiga China. É considerada um alimento completo, pois contém proteínas, gorduras, óleos, aminoácidos essenciais e metabólitos secundários, tais como isoflavonas e outras substâncias fenólicas, importantes antioxidantes naturais. A liberação do plantio da soja se deu com a Lei 11.105 de 24 de março de 2005, levando a um aumento exponencial de sua produção no país. Seu consumo ocorre em todo o mundo, e sua produção aumenta a cada ano, pois também vem sendo utilizada como fonte para a produção de biocombustível. Atualmente, sabemos que a poluição atmosférica e outras atividades humanas contribuem para o aumento de poluentes na atmosfera e consequente impacto na camada de ozônio. A radiação ultravioleta é dividida em três: ultravioleta A (315 a 400 nm), ultravioleta B (280 a 315 nm) e ultravioleta C (200 e 280 nm). Por conta disso, estudos que visam avaliar como a radiação ultravioleta pode interferir no metabolismo vegetal podem contribuir para o conhecimento mais aprofundado sobre como espécies cultivadas são afetadas por esse fator de estresse. Os metabólitos especiais são importantes estratégias químicas usadas pelas plantas na sua defesa contra fatores de estresse biótico ou abiótico. Em particular, as substâncias fenólicas como os flavonoides e isoflavonas são importantes metabólitos envolvidos nos mecanismos de defesa vegetal associados à fotoproteção. Este projeto teve por objetivo contribuir com conhecimentos quanto aos efeitos da radiação ultravioleta sobre a produção de metabólitos fenólicos durante o desenvolvimento vegetativo de dois cultivares de soja CD202RR e CD202, quando submetidos a três tratamentos com diferentes incidências de radiação ultravioleta (UV): 1. Ausência de radiação UV (UV-), 2. Alta incidência de radiação UV (UV+) e 3. Incidência natural de radiação UV solar (Controle). A hipótese deste estudo partiu do pressuposto que substâncias fenólicas são reconhecidos metabólitos vegetais com propriedades de absorção de luz nos comprimentos de onda do espectro UV-Visível, sendo assim, aumentariam em conteúdo em resposta a alta intensidade de radiação UV. Foram feitas análises por CLAE-DAD e CLAE-MS das substâncias fenólicas em folhas de G. max em três estádios vegetativos (V3, V4 e V5). As sementes de soja foram germinadas dentro das câmaras e 10 plantas foram coletadas por estádio, em cada câmara e em 3 repetições de experimentos (setembro de 2016 a março 2017). Foi verificado que o tratamento UV+ resultou em menores massas foliares quando comparado ao Controle e UV-. Há também diferença nas alturas das plantas de soja e danos nas folhas produzidos pela maior incidência de radiação UV. Foram identificados 28 constituintes, sendo 13 derivados do ácido cinâmico, 9 derivados dos flavonoides campferol e apigenina e 6 isoflavonas derivadas de genisteína e daidzeína. Quanto aos conteúdos fenólicos, pode-se observar que há um aumento da quantidade de isoflavonas na câmara UV+ em relação ao Controle para ambos os cultivares. Para os derivados cinâmicos e os flavonóis encontrados, observa-se um decréscimo em relação ao Controle para grande parte desses constituintes. Diminuição no conteúdo de flavonóis e aumento de isoflavonas pode ser devido a um possível desvio da via de produção dos flavonoides, uma vez que naringenina é o precursor para a síntese de ambas as classes. Além disso, isoflavonas apresentam espectro de absorção UV-Vis com banda máxima no comprimento de onda entre 260-280 nm, o que pode resultar em maior fotoproteção para plantas expostas a maiores intensidades de radiação UV / Fabaceae is the third largest botanical family, with great ecological, ornamental and economic importance. For its great diversity of species, the family is divided into six sub-families Caesalpinioideae, Cercidoideae, Detarioideae, Dialioideae, Duparquetioideae and Papilionoideae. Papilionoideae is a monophyletic group comprising Glycine max (L.) Merr., an important economic species for food (grains, bran, oil) and medicine. Glycine has its center of origin in China, and modern soybean has been modified by crosses between two wild species, by scientists still in ancient China. It is considered a complete food because it contains proteins, fats, oils, essential amino acids and secondary metabolites, such as isoflavones and other phenolic substances, important natural antioxidants. The liberation of soybean culture occurred in Brazil after the 11,105 law of March 24, 2005, leading to an exponential increase of its production in the country. Its consumption occurs all over the world, and its production increases every year, since it is also used as a source for the production of biofuel. We now know that atmospheric pollution and other human activities contribute to the increase of pollutants in the atmosphere and a consequent impact on the ozone layer. Ultraviolet radiation (UV) is divided into three: ultraviolet A (315 at 400 nm), ultraviolet B (280 to 315 nm), and ultraviolet C (200 and 280 nm). Because of this, studies aiming to evaluate how ultraviolet radiation can interfere on plant metabolism may contribute to better understand of how cultivated species are affected by this stress factor. Special metabolites are important chemical strategies used to plant defense against biotic or abiotic stress factors. In particular, phenolic substances such as flavonoids and isoflavones are important metabolites involved in the plant defense mechanisms associated to photoprotection. The objective of this project was to contribute with knowledge about the effects of ultraviolet radiation on the production of phenolic metabolites during the vegetative development of two soybean cultivars CD202RR and CD202, when exposed to three UV treatments: 1. Absence of UV radiation (UV-), 2. High incidence of UV radiation (UV +), and 3. Natural incidence of solar UV radiation (Control). The hypothesis of this study was based on the assumption that phenolic substances are recognized plant metabolites with properties of light absorption in the wavelengths of the UV-visible spectrum and thus could increase in content in response to high intensity of UV radiation. Analyzes were performed using HPLC-DAD and HPLC-MS of leaves of G. max at three vegetative stages (V3, V4 and V5). Soybean seeds were germinated within the chambers and 10 plants were collected per stage, in each chamber and in 3 experiment replicates (September of 2016 to March of 2017). It was observed that UV+ treatment resulted in smaller leaf biomass when compared to Control and UV-. There were also differences in soybean heights and leaf damage produced by the higher incidence of UV radiation. A total of 28 constituents were identified, of which 13 were cinnamic acid derivatives, 9 kaempferol and apigenin derivatives, and 6 genistein and daidzein derivatives (isoflavones). Was observed an increase in the contents of isoflavones in the UV+ comparing to Control, for both cultivars. Cinnamic acid derivatives and flavonoids showed decreased contents in relation to the Control. Decreases in flavonoid content together with increases in isoflavones might be explain by a possible flavonoid pathway deviation, since naringenin is precursor of both flavonoid classes. Isoflavones show UV-Vis absorption spectra with maximum absorption between 260-280 nm, which might result in higher capacity for UV photoprotection

\"Maracujá\" é o nome popular de várias espécies, dentre as quais Passiflora edulis e Passiflora alata (Passifloraceae). Esta última é uma planta medicinal amplamente utilizada no Brasil devido às suas propriedades ansiolíticas e sedativas, sendo também a espécie oficial da Farmacopéia Brasileira. Porém, freqüentemente ocorre a sua substituição indevida por Passiflora edulis, espécie usada para o preparo de sucos. Há também a confusão com a Passiflora incarnata, espécie usada na Europa, mas que não se adapta bem ao clima brasileiro. Neste trabalho foi desenvolvido um método espectrofotométrico por UV-visível para a quantificação dos flavonóides totais contidos nas folhas de Passiflora alata e de Passiflora edulis, a partir da adaptação das metodologias descritas nas Farmacopéias Francesa, Helvética e Européia para a espécie Passiflora incarnata. O procedimento proposto envolveu a complexação dos flavonóides com cloreto de alumínio e foi utilizada a rutina como padrão. Confrontou-se os dados obtidos pelo método espectrofotométrico desenvolvido neste trabalho com os obtidos por HPLC. Os melhores resultados foram obtidos com a metodologia adaptada da Farmacopéia Francesa. / \"Maracujá\" is the popular name of some species, among which are Passiflora edulis and Passiflora alata (Passifloraceae). The latter is a widely used medicinal plant in Brazil due to its ansiolytic and sedative properties, and also the official species of the Brazilian Pharmacopoeia. However, it is quite often improperly substituted for Passiflora edulis, which is used for the preparing juices. Another inadequate substitution occurs with Passiflora incarnata, wich is used in Europe, but does not adjust to the Brazilian climate. From adapting the methodologies described in the French, Helvetian and European Pharmacopoeia for the Passiflora incarnate species, a spectrophotometric method by UV-Visible has been developed to enable quantification of all flavonoids comprised in the leaves of both Passiflora edulis and Passiflora alata. The suggested procedure involved the complexation of the flavonoids with aluminum chloride, being rutin used as standard. Data obtained by the spectrophotometric method developed in the present work were compared with those obtained by HPLC. The French Pharmacopoeia methodology provided the most significant data in the study.

Orientador: Hideko Yamanaka / Banca: Maria Valnice Boldrin / Banca: Silvia Helena Pires Serrano / Banca: Ronaldo Censi Faria / Banca: Paulo Roberto Brasil de Oliveira Marques / Resumo: A molécula do DNA pode ser modificada por substâncias eletrofílicas, tanto de origem exógena quanto endógena. As lesões geradas podem ser mutagênicas e contribuir para o processo de carcinogênese. Assim, desvios significativos da estrutura da dupla hélice desempenham um papel importante no metabolismo do DNA. Um biossensor voltamétrico baseado na imobilização de DNA de fita dupla (dsDNA) sobre eletrodos descartáveis foi desenvolvido para aplicação nos estudos de interação entre corantes têxteis e a molécula do DNA, na ausência e na presença de flavonóides em solução. Os eletrodos modificados foram colocados em contato com soluções de corantes têxteis da classe dos dispersos, o Disperso Orange 1 (DO1) e o Disperso Red 1 (DR1), e seus produtos de eletrólise por oxidação e por redução. A variação dos sinais de oxidação das bases guanina e adenina presentes no dsDNA imobilizado, obtidos antes e após cada interação, foi utilizada como parâmetro de análise dos resultados. O tempo para que a interação DNA:corante ocorresse foi avaliado utilizando o biossensor e estabeleceu-se 180 s. A concentração dos corantes foi analisada na faixa de 1,0 x 10-8 a 1,0 x 10-4 mol L-1, sendo que a variação do sinal voltamétrico das bases foi mais intensa para DR1 (sinais da guanina e adenina decresceram 48% e 51% do seu valor original, respectivamente), enquanto que na presença de DO1, as intensidades de corrente da guanina e adenina diminuíram 30% e 10% de seu valor original, respectivamente, quando comparada a mesma concentração dos corantes de 1,0 x 10-6 mol L-1. Além da diminuição da intensidade de corrente das bases guanina e adenina, o aparecimento de novos picos e deslocamentos do potencial de pico das bases foram observados após as interações com produtos de eletrólises. Estudos de interação também... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: DNA molecule may be modified by electrophilic substances, either from endogenous or exogenous origin. Lesions generated may be mutagenic and contribute to the process of carcinogenesis. In this way, significant deviations on the double helix structure play an important role in the DNA metabolism. A biosensor based on double-stranded DNA (dsDNA) immobilization on disposable electrodes has been developed for application in interaction studies between textile dyes and DNA molecule in the absence and presence of flavonoids in solution. The modified electrodes were placed in solutions of disperse textile dyes, Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and their products of electrolyses by oxidation and reduction. The variation of the oxidation signals of guanine and adenine bases of the immobilized dsDNA, obtained before and after each interaction, was used as a parameter for analyzing the results. The time for DNA:dye interaction was evaluated using the biosensor and 180 s was established. The concentration of dyes was analyzed in the range from 1.0 x 10-8 to 1.0 x 10-4 mol L-1, and the bases voltammetric signal variation was more intense for DR1 (signals of guanine and adenine decreased 48% and 51% of its original value, respectively), whereas in the presence of DO1, the current intensities of guanine and adenine decreased by 30% and 10% of its original value, respectively, when compared the same dyes concentration of 1.0 x 10-6 mol L-1. Besides adenine and guanine current intensities decrease, the appearance of new peaks and peaks potential shifts were also observed after interactions with electrolysis products. Interaction studies were also performed by means of UV-Vis spectrophotometry in aqueous phase showed different effects of hypochromism and hiperchromism of DNA band after interactions... (Complete abstract click electronic access below) / Doutor

Croton é um dos maiores gêneros de Euphorbiaceae, amplamente distribuído nas regiões tropicais do mundo. No Brasil existem cerca de 300 espécies, muitas utilizadas como medicinais no tratamento de diversos males, tais como úlceras, inflamações, malária, diabetes, sífilis e câncer. A variedade de usos medicinais reflete a diversidade química inerente ao gênero, cuja taxonomia também é bastante complexa. O objetivo deste trabalho é a caracterização química de sete espécies nativas de Croton, a fim de contribuir para a ampliação do conhecimento de potenciais farmacológicos e quiçá fornecer ferramentas quimiotaxonômicas que auxiliem na compreensão das relações entre grupos infragenéricos. Foram analisados óleos voláteis, flavonóides e componentes de extratos etanólicos brutos das espécies. Óleos voláteis de folhas de C. betulaster e C. glutinosus, e folhas e caules de C. hemiargyreus, C. antisyphiliticus, C. grandivelum, C. cf pycnocephalus e C. cf montevidensis foram extraídos por hidrodestilação em Clevenger e analisados por CG/EM. Todas as amostras apresentaram componentes voláteis, exceto uma amostra (folhas e caule) de C. antisyphiliticus, a espécie com folhas e caules menos aromáticos, e caules de C. grandivelum, de uma amostra de C. cf pycnocephalus e uma de C. hemiargyreus. Monoterpenos e sesquiterpenos são os componentes mais abundantes e não foram detectados fenilpropanóides. Caules apresentam em geral menos componentes voláteis que folhas. Os sesquiterpenos β-cariofileno, germacreno D e espatulenol são os mais comuns, enquanto 8-isoproprenil-1,5-dimetil-ciclodeca-1,5-dieno (C. betulaster e C. hemiargyreus), elixeno (em todas as espécies exceto C. betulaster e C. glutinosus) e o sesquiterpeno ledol (abundante em C. cf pycnocephalus) nunca foram antes relatados para Cróton. Extratos hidroalcoólicos das amostras foram analisados por CLAE e quando possível CLAE/EM e CLAE/EM/EM para detecção de flavonóides. Apenas em C. betulaster e C. hemiargyreus houve predominância de flavonas, tais como isoorientina, orientina, vitexina e diglicosídeos de apigenina. Nas demais espécies são abundantes glicosídeos de quercetina, como rutina e quercitrina, assim como tilirosídeo (glicosídeo acilado de campferol), que só não foi detectado em folhas de C. betulaster, C. glutinosus e C. cf montevidensis. A análise de outras espécies das mesmas seções (Barhamia e Codonocalyx, respectivamente) pode confirmar a coerência da ausência do tilirosídeo nas folhas como característica. Flavonóides metoxilados, como a casticina (em amostras de C. cf pycnocephalus, C. betulaster e C. glutinosus), também foram encontrados. Os extratos etanólicos brutos de todas as espécies exceto C. glutinosus foram analisados por CG/EM. Triterpenos, esteróides, alcalóides e ésteres de ácidos graxos são os componentes mais abundantes. Diterpenos não foram detectados. Benzoato de benzila é o componente majoritário dos extratos etanólicos brutos de C. betulaster, no qual também foram detectados cinamato de benzila, lupeol e lupenona Esteróides como sitosterol, campesterol e estigmasterol ocorrem em diversas espécies. A casticina é o componente majoritário dos extratos obtidos de folhas de C. cf pycnocephalus. Muitos alcalóides foram detectados em C. grandivelum, principalmente N-metil-crotonosina. Somente glaucina foi detectada em C. hemiargyreus. Os diferentes componentes dos extratos etanólicos das espécies estudadas possuem diversas atividades biológicas. As variações encontradas entre amostras de diferentes populações das mesmas espécies podem ser influenciadas por fatores ambientais ou genéticos. Com base nos dados obtidos, o isolamento e caracterização de flavonóides por EM e RNM, ensaios farmacológicos de acordo com as propriedades de cada grupo de substâncias e a caracterização de demais espécies das mesmas seções podem ser um interessante enfoque para estudos futuros e contribuir ainda mais para a compreensão da química e da taxonomia de Croton. / Croton is one of the largest genera of Euphorbiaceae, widely distributed in tropical regions of the world. There are around 300 species in Brazil, many used in traditional medicine to treat gastric ulcers, inflammations, malaria, diabetes, syphilis and cancer. The variety of medicinal applications reflects the diversity of biologically active compounds and the complexity of taxonomic relationships within the genus. The present work focuses on the chemical screening of seven Brazilian species of Croton, contributing to increase the knowledge about pharmacological potentials and hopefully provide chemotaxonomic tools to help understanding ralationships at the infrageneric level. Essential oils, flavonoids and compounds of crude ethanolic extracts were analysed. Esssential oils of leaves of C. betulaster and C. glutinosus, leaves and stems of C. hemiargyreus C. antisyphiliticus, C. grandivelum, C. cf pycnocephalus and C. cf montevidensis were extracted by hydrodistillation in Clevenger and analysed by GC/MS. Volatile compounds were found in all samples, except one sample (leaves and stems) of C. antisyphiliticus (the less aromatic among the species), and in stems of C. grandivelum, C. cf pycnocephalus and C. hemiargyreus. Monoterpenes e sesquiterpenes are major compounds and no phenylpropanoids were found. In general, leaves are richer in volatile compounds than stems. Sesquiterpenes such as β- caryophyllene, germacrene D and spathulenol are common, while 8-isoproprenyl-1,5- dimethyl-cyclodeca-1,5-diene (C. betulaster and C. hemiargyreus), elixene (in all species except C. betulaster and C. glutinosus) and the oxigenated sesquiterpene ledol (in C. cf pycnocephalus) are reported for Cróton for the first time. Hydroalcoholic extracts were analysed by HPLC and whenever possible also by HPLC/MS and HPLC/MS/MS to detect flavonoids. Flavones, such as isoorientin, orientin, vitexin and apigenin diglycosides were predominant only C. betulaster and C. hemiargyreus. Other species showed a profile richer in flavonols, such as quercetin glycosides (rutin, quercitrin and others) and tiliroside (kaempferol acylglycoside), which was not detected only in leaves of C. betulaster, C. glutinosus and C. cf montevidensis. Analyses of other species from the same sections (Barhamia e Codonocalyx, respectively) might confirm the coherence of the absence of tiliroside in leaves as a taxonomic character. Methoxylated flavonoids, such as casticin (found in samples of C. cf pycnocephalus, C. betulaster and C. glutinosus), were found. Crude ethanolic extracts of all species except C. glutinosus were analysed by GC/MS. Triterpenes, steroids, alkaloids and fatty acid esters were the predominant compounds of those extracts. Diterpenes were not detected. Benzyl benzoate predominates in crude ethanolic extracts from leaves of C. betulaster, along with benzyl cinnamate, lupeol e lupenone. Steroids such as sitosterol, campesterol and stigmasterol were found in several species. The flavonoid casticin is the major compound in extracts obtainded from leaves of C. cf pycnocephalus. Several alkaloids were detected in C. grandivelum extracts, mainly N-methyl-crotonosine. Glaucine was the only alkaloid identified in C. hemiargyreus. Several biological activities are known for the different compounds found in the crude ethanolic extracts. The differences between populations of the same species may be accounted for environmental or genetic influences. The data obtained suggest isolation and total structural determination of flavonoids by MS and NMR, pharmacological assays according to the properties of each group of compounds and chemical screening of remaining species of the same sections as interesting focuses for future studies, with the expectation to enhance the contribution to the chemistry and pharmacology of Croton and provide chemical synapomorphies for infrageneric groups within the genus.

No presente trabalho estudou-se por métodos analíticos (CCD e CLAE/UV) a atividade antioxidante dos flavonóides presentes na cana-de-açúcar (Saccharum officinarum L.), visando sua possível utilização como alimento funcional. As técnicas CLAE/UV e microfracionamento por CLAE permitiram o fracionamento e o isolamento dos flavonóides, os quais foram analisados por CCD utilizando reagentes específicos para detecção de substâncias antioxidantes. Os resultados obtidos permitiram identificar os flavonóides da cana-de-açúcar (folhas e garapa) com atividade antioxidante, através da comparação dos espectros de UV/DAD e tempo de retenção: orientina-O-ramnosídeo nas folhas e schaftosídeo, isoschaftosídeo, diosmina-8-C-glicosídeo, orientina e 4\',5\'-di-O-metil-luteolina-8-C-glicosídeo na garapa. Estes resultados justificam estudos nutricionais e/ou farmacológicos mais aprofundados a fim de classificar (ou não) a cana-de-açúcar como alimento funcional. / In the present study, analytical methods (TLC and HPLC/UV) were used to evaluate the antioxidant activity of the flavonoids of sugar cane (Saccharum officinarum L.) regarding to its possible use as a functional food. HPLC/UV and HPLC microfractionation techniques allowed fractionation and isolation of the flavonoids, which were evaluated by TLC using specific reagents to detect the antioxidant compounds. The results allowed to identify the flavonoids of sugar cane with antioxidant activity, based on comparison of UV-DAD spectra and retention time: orientin-O-rhamnoside in the leaves and schaftoside, isoschaftoside, diosmetin-8-C-glycoside, orientin and 4\'-5\'-dimethyl-luteolin-8-C-glycoside in the juice. Theses results justify more detailed nutricional and pharmacologic studies to classify (or not) sugar cane as a functional food.

Ngai Lei-Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 113-125). / Abstracts in English and Chinese. / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.1.1 --- The six major classes of flavonoids --- p.1 / Chapter 1.1.1.1 --- Flavonones --- p.1 / Chapter 1.1.1.2 --- Flavones --- p.2 / Chapter 1.1.1.3 --- Flavonols --- p.2 / Chapter 1.1.1.4 --- Isoflavonoids --- p.2 / Chapter 1.1.1.5 --- Anthocyanidins --- p.3 / Chapter 1.1.1.6 --- Flavans --- p.3 / Chapter 1.1.2 --- Structural variation of flavonoids --- p.3 / Chapter 1.1.3 --- The roles of flavonoids --- p.6 / Chapter 1.2 --- "Free radicals, oxidative stress and antioxidants" --- p.7 / Chapter 1.2.1 --- Oxidants and free radicals --- p.7 / Chapter 1.2.2 --- Lipid peroxidation (LPO) --- p.8 / Chapter 1.2.3 --- Oxidative stress and human diseases --- p.9 / Chapter 1.2.4 --- Role of food antioxidants --- p.10 / Chapter 1.2.5 --- Synthetic and natural food antioxidants --- p.11 / Chapter 1.3 --- Antioxidant activity of flavonoids --- p.12 / Chapter 1.4 --- Determination of antioxidant activity --- p.13 / Chapter 1.4.1 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.13 / Chapter 1.4.2 --- DPPH radical scavenging assay --- p.14 / Chapter 1.4.3 --- β-carotene bleaching assay --- p.15 / Chapter 1.5 --- Single cell gel electrophoresis assay (Comet assay) --- p.16 / Chapter 1.6 --- Determination of the genotoxicity --- p.18 / Chapter 1.7 --- Research objectives --- p.19 / Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Standards and reagents --- p.26 / Chapter 2.2 --- Sample Preparation --- p.26 / Chapter 2.3 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.27 / Chapter 2.4 --- DPPH´Ø radical scavenging assay --- p.28 / Chapter 2.5 --- β-carotene bleaching assay --- p.29 / Chapter 2.6 --- The comet assay --- p.30 / Chapter 2.6.1 --- Preparation of reagents --- p.31 / Chapter 2.6.2 --- Blood sample --- p.32 / Chapter 2.6.3 --- Optimal condition of comet assay --- p.32 / Chapter 2.6.3.1 --- Induction of DNA damage --- p.32 / Chapter 2.6.3.2 --- Antioxidant pre-treatment --- p.32 / Chapter 2.6.4 --- Hydrogen peroxide (H2O2) treatment --- p.32 / Chapter 2.6.4.1 --- Pre-incubation system --- p.32 / Chapter 2.6.4.2 --- Co-incubation system --- p.33 / Chapter 2.6.5 --- Slide preparation --- p.33 / Chapter 2.6.6 --- Cell lysis --- p.33 / Chapter 2.6.7 --- Alkaline treatment and electrophoresis --- p.34 / Chapter 2.6.8 --- Neutralization --- p.34 / Chapter 2.6.9 --- Quantification of DNA damage --- p.34 / Chapter 2.6.10 --- Cell viability analysis --- p.35 / Chapter 2.6.11 --- Statistical Analysis --- p.35 / Chapter 2.7 --- Mutatox® test --- p.36 / Chapter 2.8 --- Statistical analysis --- p.36 / Chapter 3 --- Result --- p.45 / Chapter 3.1 --- Determination of antioxidant activity using Trolox equivalent antioxidant capacity (TEAC) assay --- p.45 / Chapter 3.1.1 --- Trolox standard reference --- p.45 / Chapter 3.1.2 --- Antioxidant activity: ABTS´Ø+ scavenging capacity --- p.45 / Chapter 3.2 --- Evaluation of antioxidant activity using free radical scavenging assay --- p.46 / Chapter 3.2.1 --- Free radical scavenging abilities at 5 minutes --- p.46 / Chapter 3.2.2 --- Antiradical efficiency --- p.47 / Chapter 3.3 --- Evaluation of antioxidant activity using β-carotene bleaching assay --- p.47 / Chapter 3.3.1 --- Optimal incubation time --- p.47 / Chapter 3.2.2 --- Antioxidant activities on β-carotene bleaching assay --- p.47 / Chapter 3.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids --- p.48 / Chapter 3.4.1 --- Effect of hydroxylation on the antioxidant activities of flavonoids --- p.48 / Chapter 3.4.1.1 --- Hydroxylation positions --- p.48 / Chapter 3.4.1.2 --- Polyhydroxylation --- p.48 / Chapter 3.4.2 --- Importance of B ring structure --- p.49 / Chapter 3.4.2.1 --- Increase hydroxylation in B ring --- p.49 / Chapter 3.4.2.2 --- The othro-dihydroxyl arranagement in B ring --- p.49 / Chapter 3.4.3 --- Importance of C ring structure --- p.50 / Chapter 3.4.3.1 --- The presence of a hydroxyl group at C3 --- p.50 / Chapter 3.4.3.2 --- The blockage of hydroxylation at C3 --- p.50 / Chapter 3.4.3.3 --- The presence of a double bond between C2 to C3 --- p.50 / Chapter 3.4.3.4 --- The presence of a carbonyl group at C4 --- p.50 / Chapter 3.4.3.5 --- The conjugation of a carbonyl at with a double bond between C2 to C3 --- p.51 / Chapter 3.4.4 --- Effect of glycosylation --- p.51 / Chapter 3.5 --- Evaluation of protective effects on DNA damage using comet assay --- p.51 / Chapter 3.5.1 --- Optimization of conditions fro the determination of H2O2-induced DNA damage --- p.52 / Chapter 3.5.1.1 --- H2O2 concentration & treatment temperature --- p.52 / Chapter 3.5.1.2 --- H2O2 treatment time --- p.52 / Chapter 3.5.1.3 --- Sample volume --- p.52 / Chapter 3.5.2 --- Protective effect of vitamin C on DNA damage --- p.53 / Chapter 3.5.3 --- Protective effect of selected flavonoids on DNA damage --- p.53 / Chapter 3.5.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids in comet assay --- p.54 / Chapter 3.5.4.1 --- The importance of B ring structures --- p.54 / Chapter 3.5.4.2 --- The importance of C ring structures --- p.54 / Chapter 3.6 --- Evaluation of genotoxicity of flavonoids using Mutatox test --- p.55 / Chapter 4 --- Discussion --- p.96 / Chapter 4.1 --- Comparison of antioxidant activities between hydrophilic and lipophilic assays --- p.97 / Chapter 4.2 --- Structure and antioxidant activity relationship of flavonoids --- p.98 / Chapter 4.3 --- The comet assay --- p.103 / Chapter 4.4 --- Comparison of protective effect on DNA damage between pre-incubation and co-incubation systems --- p.105 / Chapter 4.5 --- Structure and protective effect of flavonoids in the comet assay --- p.106 / Chapter 4.6 --- Genotoxicity of flavonoids --- p.108 / Chapter 4.7 --- Significance and future works --- p.108 / Chapter 5 --- Conclusion --- p.111 / References --- p.113

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-16Bitstream added on 2014-06-13T19:19:53Z : No. of bitstreams: 1 salvaro_lms_dr_botib.pdf: 542129 bytes, checksum: 9f3ea451106084bdce151c44b55ad171 (MD5) / O objetivo deste trabalho foi verificar a eficiência da técnica de milcropropagação para a espécie Piper hispidinervium, utilizando reguladores vegetais e poliaminas e verificar a influência destes sobre o desenvolvimento dos explantes (número de raízes, número de brotações, formação de calo e altura), bem como verificar influência da época de avaliação das plântulas sobre os teores de flavonóides totais, atividade antioxidante, atividade da peroxidase e teores endógenos de poliaminas livres. Microestacas obtidas da germinação in vitro foram inoculadas nos devidos tratamentos, os quais foram divididos em dois experimentos, o primeiro, utilizando concentrações de benzilaminopurina (BAP) isoladas e combinadas com ácido indolilbutírico (IBA) e o segundo, utilizando poliaminas (espermina- Spm e espermidina-Spd) adicionadas ao meio de cultura, isoladas, combinadas entre si e com BAP. Desta forma, no primeiro experimento os tratamentos foram: BAP (0,25; 0,50; 0,75 e 1,0 mg L-1), isoladas ou combinadas com IBA (0,25 mg L-1 ), o qual também foi utilizado isolado, totalizando 10 tratamentos, 3 repetições com 6 microestacas por repetição. No segundo experimento os tratamentos foram: T1: MS (testemunha); T2: 1mg L-1 BAP; T3:1mM Spd; T4: 1mM pd + 1mg L-1 BAP; T5: 1mM Spm; T6: 1mM Spm + 1mg L-1 BAP e T7: 1mM Spd + 1mM Spm. Aos 30 e 60 dias após a inoculação (DAI) foram realizadas as análises biométricas (formação de calo, número de raízes, número de brotações e altura das plântulas). No momento da inoculação (tempo 0), aos 30 e 60 DAI foram coletadas amostras para a realização das análises bioquímicas do teor de flavonóides totais, atividade antioxidante, atividade da peroxidase e teor endógeno de poliaminas livres (experimento 2). Nas condições realizadas no primeiro experimento, o IBA propiciou o maior crescimento das plântulas e induziu maior... / The purpose of this paper was to evaluate the efficiency of the technique of micropropagation for the Piper hispidinervium species, using plant growth regulators and polyamines and also evaluate their influence on formation of callus, number of roots, number of shootings and height of explants and the influence of cutting time on the tenors of contents of totals flavonoids, antioxidant activity, activity of the peroxidase and endogenous contents of free polyamines. Microcuttings obtained of the germination in vitro were inoculated in the proper treatments, they were divided into two experiments, the first experiment, using benzilaminopurine (BAP) isolated and combined with indolilbutiric acid (IBA), and the second one, using polyamines (Espermine- Spm and Espermidine- Spd) added to the culture medium, isolated, combined among them and with BAP. Thus, in the first experiment, the treatments were: BAP (0.25; 0.50; 0.75 and 1.0 mg L-1), isolated or combined with IBA (0.25 mg L-1), and it was also used isolated, totalizing 10 treatments, 3 repetitions with 6 microcuttings for repetition. In the second experiment the treatments were: T1: MS (control); T2: 1 mg L-1 BAP; T3:1 mM Spd; T4: 1 m M Spd + 1 mg L-1 BAP; T5: 1 mM Spm; T6: 1 mM Spm + 1mg L-1 BAP and T7: 1 mM Spd + 1 mM Spm. After 30 and 60 days of the inoculation (DAI) analysis were carried out (formation of callus, number of roots, number of shootings and height of explants). At the moment of the inoculation (time 0), at 30 and 60 DAI, samples were collected for the realization of biochemical analyses of contents totals of flavonoids, antioxidant activity, activity of the peroxidase and endogenous contents of free polyamines (only in the experiment 2). In the realized conditions, in the first experiment, the IBA favored the highest growth of the seedlings and induced a higher number of roots, as well as a higher content... (Complete abstract click electronic access below)