Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Full text also available online. Scroll down for electronic link.

Thesis (M.S.)--Universidad de Puerto Rico, Recinto Universitario de Mayagüez, 2005. / Tables. Printout. Includes bibliographical references (leaves 68-73)

Concern that youth do not have enough fruit and vegetable intake lead to two strategies implemented to influence intake in the school environment: the Healthy Hunger-Free Kids Act (HHFKA) and Fighting with Food: Battling Chemical Toxicity with Good Nutrition program (FF), which could influence phytochemical content and knowledge regarding phytochemicals, respectively. Individual food logs (n=468) were assessed upon completion of FF curriculum to determine whether students were able to correctly apply their nutrition knowledge regarding FF. Menus from one district were analyzed pre-HHFKA and post-HHFKA in elementary (n=156), middle (n=171), and high schools (n=171), for change in the frequency of fruit and vegetables, and for changes in select phytochemical and vitamin content. In food logs, students correctly identified fighting foods 71% of the time. School menus showed an increase in dark green, red/orange vegetables, with significant increases in carotenoid and flavonol content. Results suggest students are applying their nutrition knowledge. Also, more variety of fruit and vegetables are being offered, despite lack of a robust increase in all phytochemicals, which can help to lower inflammation and oxidative stress. Both strategies have the potential to work together as a multi-level intervention that can encourage more fruit and vegetable consumption among youth.

Doctor of Philosophy / Food Science Institute / Channa B. Rajashekar / Weiqun Wang / As health-promoting agents, phytochemicals are biosynthesized in the plants that typically respond to environmental stresses. This study focused on the analysis of phytochemical contents in vegetables in response to environmental changes of high tunnel and light spectra. A potential anticancer activity was further studied by developing a novel 3D cell culture model. Three specific studies were conducted as follows. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 μM. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application. Study 1: High tunnel production has been applied in mid-west for many years due to the advantages of extending growing season and increasing crop yield. Previous studies, however, showed high tunnel resulted in reduction of phenolic contents in vegetables. Therefore, the first study was to confirm the effect of high tunnel on phenolic contents in two varieties of lettuce (‘Two Star’ and ‘Red Fire’) and carotenoid contents in two varieties of tomatoes (‘Mountain Fresh’ and ‘Celebrity’). Phenolics in lettuce and carotenoids in tomato were isolated and quantitated, respectively, by HPLC. High tunnel resulted in a significant reduction of phenolic contents in ‘Two Star’ but not in ‘Red Fire’ lettuce when compared with open field. A significant decrease of carotenoid contents in ‘Celebrity’ but not in ‘Mountain Fresh’ tomato was also observed. Therefore, this study confirmed that high tunnel application reduced phenolic or carotenoid contents in one of the two lettuce or tomato varieties, suggesting the effect of high tunnel production is variable and genotype specific. Study 2: Light is an important environmental factor influenced not only photosynthesis but also phenolic biosynthesis in vegetables. The objective of this study was to investigate the effect of supplemental light spectra including red, far-red, and blue light on phenolic contents in two varieties of lettuce (green-leaf variety ‘Two Star’ and red-leaf variety ‘Red Fire’) and two varieties of spinach (‘Avon’ and ‘Bloomsdale’). The phenolics were extracted and quantitated by HPLC. Far-red and blue light but not red light resulted in an increase of phenolic contents in ‘Two Star’ lettuce. In ‘Red Fire’ lettuce, a significant increase in phenolic contents were observed when exposed to red light, while far-red and blue light reduced phenolic contents. Supplemental lighting did not alter flavonoid contents in two varieties of spinach. Taking together, the results showed that supplemental lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach, and the impact varied depending upon the variety or species. Study 3: Traditionally, cancer research is primarily relied on in vitro 2D monolayer cell culture and in vivo animal model studies. Given a flat 2D cell culture that usually lacks 3D microenvironmental cell-cell interaction and considering an animal model that is typically expensive and time-consumed, an alternative 3D cell culture has been promising. This pilot study was to develop a novel 3D hydrogel cell culture model of human hepatocarcinoma HepG2 cells or colorectal adenocarcinoma SW480 cells by treating with chlorogenic acid (CGA) at 0-40 M. The results showed both HepG2 and SW480 cells grew much better in 3D hydrogel culture system than 2D by extended exponential phase and high proliferation. CGA treatment resulted in a dose- and time-response inhibition of HepG2 and SW480 growth in exponential phase, while HepG2 cells were more susceptible than SW480 cells. Establishment of this novel 3D hydrogel culture model for future phytochemical function may bridge the gap between 2D cell culture and in vivo animal model studies. Taken together, this dissertation of three studies focused on phytochemicals from quantitation analysis in vegetables in response to environmental factors of high tunnel and light spectra to a novel 3D hydrogel cell culture development for potential phytochemical anti-cancer function. The conclusions, i.e., (1). high tunnel application reduced phenolic or carotenoid contents in special genotype of lettuce or tomato varieties; (2). lighting and its spectral quality had significant effect on the phytochemical contents of lettuce but not spinach; (3). establishment of a novel 3D hydrogel culture model for phytochemical treatment may bridge the gap between 2D cell culture and in vivo animal model studies, could be of particular significance in health-promoting phytochemical research and functional food application.

O estudo envolveu dois experimentos com o objetivo de avaliar o efeito de estratégias de alimentação sobre o estresse oxidativo, consumo, metabólitos sanguineos, a evolução da produção e composição do leite de vacas de diferentes composições raciais durante o período de transição. Estudo 1:Foi utilizado o delineamento inteiramente casualizado, com medidas repetidas no tempo e 24 vacas da raça Jersey foram distribuídas nos tratamentos controle - sem a adição de extratos vegetais (CON), adição de 10 gramas/dia de extrato de orégano (EO) e adição de 5,0 g/dia de extrato de chá verde (ECV).O fornecimento de extrato de orégano melhorou a eficiência alimentare a produção de leite em relação ao extrato de chá verde. A adição de extratos vegetais na dieta reduziu o radical livre(ROS) DCFE, contribuindo para redução do estresse oxidativo. Estudo 2:Foi utilizado o delineamento experimental inteiramente casualizado, em arranjo fatorial 2x2 (duas composições raciais e dois planos nutricionais no pré parto) com seis repetiçõese medidas repetidas no tempo. Durante 21 dias pré parto, novilhas Girolando F1 (½ Holandês ½ Gir) e Holandês receberam a mesma dieta total misturada (TMR), variando somente a quantidade ofertada (plano nutricional): 1,69 ou1,89% do peso corporal(PC) de matéria seca No pós parto, os animais receberam dietas semelhantes formuladas conforme as recomendações do NRC (prevista para raça Holandês). O plano nutricional de 1,89% do PC aumentou a concentração de ácidos graxos não esterificados (AGNE) no pré parto,a concentração de glicose no pós parto e o estresse oxidativo durante todo o período experimental em relação ao plano nutricional 1,69% do PC. Os animais da raça Girolando F1apresentaram os maior ECC durante todo o período experimental, menor ingestão de matéria seca no pré e pós parto e menor consumo de energia metabólica no pós parto;aumento da concentração de creatinina e AGNE no pré parto e maior concentração de glicose no pós parto; menor produção de leite, menor concentração de lactose em relação a animais Holandês.Diferenças metabólicas como as concentrações de AGNE, creatinina e glicose entre as composições raciais podem ter influenciado as variáveis indicativas de estresse oxidativo. O plano nutricional 1,89% do PC durante o período seconão deve ser oferecido, independentemente da composição racial. Animais Girolando F1 devem ser manejados de forma distinta de animais Holandês durante o período de transição, pois apresentam diferenças de consumo e metabolismo. / The study was performed based on two trialsand aimed of evaluating the effect of feeding strategies on oxidative stress, consumption, blood metabolites, milk production and milk composition of cows of different racial compositions during the transition period. Trial 1: we used a completely randomized design, with repeated measures along the time, where 24 Jersey cows were distributed in two diets: control - without the addition of plant extracts (CON), addition of 10 grams per day of oregano extract (OE) and addition of 5.0 grams per day of green tea extract (GTE). The supply of OE improvedfeed conversion during the postpartum period and milk production compared to GTE. The addition of plant extracts in the diet reduces the free radical (ROS) DCFE, contributing to the reduction of oxidative stress. Trial 2: A completely randomized experiment design was used, in a 2x2 factorial arrangement (two racial compositions and two nutritional plans in prepartum), with time-repeated measures (during days), with four treatments and six repetitions (heifers) in each treatment. For 21 day before of prepartum, nuluparous females Girolando F1 (½ Holstein ½ Gir) and Holstein received the same total mixed diet (TMR), varying only the amount offered (nutritional plan): 1.69 and 1.89% of (BW) of dry matter. In the postpartum the animals received the same basal diet formulated according to the recommendations of the NRC (predicted for Holstein) The nutritional plane of 1.89% showed a higher concentration of calcium and non-esterified fatty acids (NEFA) in the prepartum, higher concentration of glucose in the postpartum period and greater oxidative stress throughout the experimental period in relation to the nutritional plane 1,69% of the PC. The animals Girolando F1 had the highest ECC during the experimental period, lower intake of dry matter in the pre and postpartum, and lower metabolic energy consumption in the postpartum period; increased creatinine concentration and NEFA in prepartum and higher postpartum glucose concentration; lower milk yield, lower lactose concentration in relation to Hostein animals. Metabolic differences such as the concentrations of NEFA, creatinine and glucose among racial compositions may have influenced the variables indicative of oxidative stress. The nutritional plan 1.89%CP supply during the dry period should not be indicated. Girolando F1 animals should be handled differently from Holstein animals during the transition period, as they exhibit differences in intake and metabolism.

Made available in DSpace on 2015-03-26T13:42:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 244413 bytes, checksum: f5f18043e48f747c94912ce637ef46aa (MD5) Previous issue date: 2009-02-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is an important pest of Lycopersicon esculentum. The injuries caused by adults and nymphs are sucking of sap, injection of toxins and transmission of fitoviroses. The use of resistants varieties is a alternative to managmant of this pest. In order to have these varieties is important the selection of sources of resistance, study of mechanisms and causes this resistance. Thus, this work aimed to select sources of resistance to B. tabaci of the 103 tomato plants accessions of the Horticultural Germplasm Bank of Federal University of Viçosa (HGB-UFV). The experiment was conducted in greenhouse of the DBA-UFV. The characteristics evaluated were number of adults/plant, eggs/plant and nymphs/plant. Were calculates the number of eggs/adult, nymphs/egg and the index resistance of the accessions compared with commercial cultivar Santa Clara. Were evaluated the number of trichome/0.04 cm2 of the leaf, the identification and the quantification of the chemical compounds from in the hexanic extraction in each tomato accessions. Differences were observed in the densities adults/plant, eggs/plant, nymphs/plant, eggs/adult and e nymphs/egg. 20 peaks were identified in the gas chromatogram from hexanic extracts of the leaves of 103 tomato plants accessions (HGB-UFV). Observed significant positive correlation (p<0.05) between hydrocarbons undecane, tritecane, octadecane, hexacosane and nonacosane with adults and nymphs B. tabaci. Significative differences were observed in the trichomes density/0.04 cm2 and significant positive correlation betweem the trichomes density and the number of eggs/plant. The accections HGB-225, HGB-327, HGB-630, HGB- 813, HGB-985, HGB-2029, HGB-2030, HGB-2055, HGB-2057, HGB- 2060, HGB-2062 e HGB-2068 were the more resistants to B. tabaci in all characteristics evaluated. The resistance mechanisms associated to these HGBs were antixenosis and antibiosis. The causes were chemical and morphological. / A mosca-branca Bemisia tabaci (Hemiptera: Aleyrodidae) é uma importante praga de Lycopersicon esculentum. Os danos causados pelos adultos e ninfas consistem na sucção de seiva, injeção de toxinas e transmissão de fitoviroses. O uso de variedades resistentes é uma alternativa no manejo dessa praga. No processo de obtenção dessas variedades é fundamental a seleção de fontes de resistência, o estudo dos mecanismos e causas da resistência. Assim, o objetivo deste trabalho foi selecionar fontes de resistência a B. tabaci dentre 103 subamostras de tomateiro do Banco de Germoplasma de Hortaliças da Universidade Federal de Viçosa (BGH-UFV). O experimento foi conduzido em casa de vegetação do DBA- UFV. As características avaliadas foram os números de adultos/planta, ovos/planta e ninfas/planta. Calcularam-se os números de ovos/adulto, ninfas/ovo e o índice de resistência das subamostras em relação ao padrão de suscetibilidade (cultivar Santa Clara). Realizou-se contagem do número de tricomas/0,04 cm2 do limbo foliar, a identificação e a quantificação dos compostos químicos encontrados na extração hexânica em cada subamostra de tomateiro. Verificou-se diferença nas densidades de adultos/planta, ovos/planta, ninfas/planta, ovos/adulto e ninfas/ovo. Foram identificados 20 picos nos cromatogramas dos extratos hexânicos das folhas de 104 subamostras de tomateiro do BGH-UFV. Observou-se correlação positiva e significativa (p<0,05) dos hidrocarbonetos undecano, tritecano, octadecano, hexacosano e nonacosano com adultos e ninfas de B. tabaci. Verificaram-se diferenças significativas na densidade de tricomas/0,04 cm2 e correlação positiva e significativa entre a densidade de tricomas e o número de ovos/planta. As subamostras BGH-225, BGH-327, BGH-630, BGH-813, BGH-985, BGH-2029, BGH-2030, BGH-2055, BGH- 2057, BGH-2060, BGH-2062 e BGH-2068 são fontes de resistência ao biótipo B de B. tabaci considerando todas as características avaliadas. Os mecanismos de resistência associados a estes BGHs foram antixenose e antibiose e as causas da resistência foram químicas e morfológicas.

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2017. / The continued use and popularity of plant-based traditional medicine demands scientific validation of the therapeutic potential of the medicinal plants used in disease management and treatment. These medicinal plants are to be evaluated for phytochemical constituents and pharmacologically screened for their bioactivity and include the isolation and identification of their bioactive compounds. The diabetes tea and its eight individual plants constituents were collected from Sing Fefur Herbs in McGregor, Western Cape. The plant material was ground to a fine powder form using a milling machine. The powdered plant material was sequentially extracted with hexane, 1:1 DCM, DCM:MeOH, MeOH and water. The antioxidant activity of the tea and its plants was evaluated with comparison to the antioxidant activity of brewed rooibos tea in literature. The concentration of antioxidants in the plants and the tea were found to be significantly high. The ORAC assay results of the water extracts were significantly higher than that of rooibos tea in all plants. Salvia africana-caerulea water extract ORAC results were 14147.10±1.02 μmol TE/g and this is 10 times better than the brewed rooibos tea results of 1402±44.1 μmol TE/g. The alpha-amylase enzyme inhibition assay showed no significant results while the alpha-glucosidase enzyme inhibition assays showed significant results in some of the extracts. The highest inhibitory activity towards alpha-glucosidase was found in the Urtica urens hexane extract and the Thymus vulgaris hexane extract (69.66% and 68.43%, respectively). This observation suggests that alpha-glucosidase enzyme is inhibited mostly by the less polar or medium polarity chemical components of the plant extracts. The crude plant extracts that showed significant activity in the antidiabetic bioassays were further subjected to cytotoxicity assay to ascertain the safety of extracts. The T. vulgaris DCM extract, Salvia officinalis DCM extract and Salvia officinalis hexane extract showed a cell growth inhibition of 54.91%, 62.14% and 63.87% at 100 μg/ml, respectively. The Salvia africana-caerulea DCM extract showed a cell growth inhibition of 59.10% at 50 μg/ml and 62.14% at 100 μg/ml. In the cytotoxicity analysis Salvia africana-caerulea DCM extract is the only extract that showed cell viability below 50% for both concentrations. Phytochemical screening of selected methanolic and aqueous extracts of the diabetes tea and the Salvia africana-caerulea showed the presence of alkaloids, sugars, flavonoids, glycosides, proteins & amino acids, phenolics & tannins and saponins. Furthermore isolation, purification and analysis of two Salvia africana-caerulea crude extracts (DCM and DCM:MeOH) were done in order to try and obtain pure compounds. The compound characterization was done through the use of chromatographic techniques. Thin layer chromatography (TLC), flash chromatography and column chromatography resulted in the generation of 29 fractions. Spectroscopic techniques utilized for chemical structural elucidation for compounds of interest included Liquid chromatography mass spectrometry and Nuclear Magnetic Resonance Spectroscopy. Of all the fractions generated, DM 23 was the purest and its structural elucidation was attempted.

CAPES / A família Fabaceae (Leguminosae) é constituída por 750 gêneros e mais de 18000 espécies, distribuídas em todo o mundo. No Brasil esta é representada por aproximadamente 175 gêneros e 1500 espécies. As espécies da família Fabaceae são particularmente ricas em flavonoides e compostos biossinteticamente relacionados, como rotenóides e isoflavonoides. Alcalóides, terpenóides e esteróides são exemplos de outras classes de substâncias que ocorrem em muitos exemplares da família. Dentre as espécies representantes desta família na região de Porto Rico-PR, área de proteção ambiental, está Aeschynomene fluminensis e Machaerium hirtum. Uma revisão bibliográfica destas espécies indicou a ausência de estudos fitoquímicos e biológicos, o que motivou a escolha das mesmas para investigação. Ainda foi verificado que M. hirtum é utilizada na medicina popular contra tosse, diarréia e câncer. O estudo fitoquímico para as duas espécies foi iniciado a partir da obtenção dos extratos brutos metanólicos das folhas e dos galhos, por maceração, e as obtenções de frações destes extratos a partir de partições em solventes de polaridades diferentes. As frações obtidas foram submetidas a purificações cromatográficas em colunas de gel de sílica e/ou Sephadex LH-20. Os tratamentos cromatográficos realizados para as frações de A. fluminensis levaram ao isolamento dos flavonóides canferitrina, α-raminoisorobina, quercitrina, 3-O-apiofuranosil-7-O-raminopiranosil-canferol e avicularina, da procianidina A2; da flavanona iso-hemifolina; da isoflavona daidzeína e do açúcar 3- O-metil-quiro-inositol. O estudo das frações obtidas de M. hirtum, resultou no isolamento das flavonas isovitexina e swertisina; do alcalóide 4-hidróxi-N-metilprolina; dos triterpenos friedelan-3-ona e lupen-20(29)-3β-ol e da mistura dos esteróides estigmasterol e sitosterol, a qual também foi encontrada em A. fluminensis. A elucidação das estruturas foi realizada mediante análise de dados espectroscópicos de RMN de 1H e 13C e de técnicas bidimensionais gCOSY, ii gHSQC e gHMBC) e por comparação com os dados relatados na literatura. Os extratos brutos e frações de A. fluminensis foram submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha e se mostraram todos ativos, sendo mais significativos os extratos e frações hexânicas das folhas e dos galhos. Nos ensaios de atividade da enzima de mieloperoxidase (MPO) apresentaram resultados significativos todas as frações de A. fluminensis com maiores atividades as frações hexânicas e CHCl3 das folhas e dos galhos. Os extratos brutos e todas as frações de M. hirtum apresentaram bons resultados quando submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha, para a substância 4-hidróxi-N-metil-prolina este ensaio resultou em redução moderada. Na atividade da enzima de mieloperoxidase apresentaram resultados significativos os extratos e todas as frações de M. hirtum, sendo mais ativas as frações hexânicas e AcOEt das folhas e dos galhos e CHCl3 dos galhos. A mistura swertisina e isovitexina e a substância 4-hidróxi-N-metil-prolina apresentaram redução pronunciada da enzima. O teste de pleurisia foi significativo apenas para o extrato bruto das folhas de M. hirtum. No teste da atividade antitumoral de A. fluminensis sobre células Caco-2 foi observado que a fração CHCl3 dos galhos e a canferitrina apresentaram os resultados mais promissores. / The Fabaceae family (Leguminosae) comprises 750 genera and over 18000 species, distributed worldwide. In Brazil it is represented by approximately 175 genera and 1500 species. The Fabaceae species are particularly rich in flavonoids and biosynthetically related compounds, such as isoflavonoids and rotenoids. Alkaloids, terpenoids and steroids are examples of other classes of substances that occur in many samples of the family. Aeschynomene fluminensis and Machaerium hirtum are among the representative species of this family in the region of Porto Rico-Paraná state, an environmental protection area. A literature review of these species indicated the absence of phytochemical and biological studies, which motivated their choice for investigation. It was also found that M. hirtum is used in folk medicine against cough, diarrhea and cancer. The phytochemical study for both species was initiated after obtaining the methanol crude extracts of leaves and twigs, by percolation, and the varieties of extract fractions by means of partitions in different polarities solvents. The obtained fractions were subjected to purification on chromatographic columns of silica gel and/or Sephadex LH-20. The chromatographic treatments performed for A. fluminensis fractions led to the isolation of the following flavonoids: kaempferitrin, α-rhamnoisorobin, quercitrin, 3-Oapiofuranosyl- 7-O-rhamnopyranosyl-kaempferol and avicularin, A2 procyanidin; the flavanone iso-hemipholin; the isoflavone daidzein and the 3-O-methyl-chiro-inositol sugar. The study of the obtained fractions of M. hirtum resulted in the isolation of the isovitexin and swertisin flavones; of the 4-hydroxy-N-methyl-proline alkaloid, the friedelan-3-one and lupen-20(29)-3β-ol triterpenes and the stigmasterol and sitosterol steroids mixture, which was also found in A. fluminensis. The elucidation of the structures was performed by analysis of spectroscopic data of 1H and 13C NMR and bidimensional techniques (gCOSY, gHSQC and gHMBC) and by comparison with the data reported in the literature. The crude extracts and the A. fluminensis iv fractions were tested for anti-inflammatory activity in the ear edema model and showed all active; the most significant were the extracts and hexane fractions from leaves and twigs. The tests of the myeloperoxidase (MPO) enzyme activity showed significant results for all A. fluminensis fractions with more activities for the CHCl3 and hexane fractions of leaves and twigs. The crude extracts and all M. hirtum fractions showed good results when tested for anti-inflammatory activity in the ear edema model; for the substance 4-hydroxy-N-methyl-proline this test resulted in moderate reduction. For the activity of the myeloperoxidase enzyme, the M. hirtum extracts and all fractions showed significant results, being more active the hexane and EtOAc fractions of leaves and twigs and CHCl3 of twigs. The swertisin and isovitexin mixture and the substance 4-hydroxy-N-methyl-proline showed pronounced enzyme reduction. The pleurisy test was significant only for the crude extract of M. hirtum leaves. When testing the A. fluminensis antitumor activity on Caco-2 cells, it was observed that the CHCl3 fraction of the twigs and the kaempferitrin showed the most promising results.

CAPES; Fundação Araucária / A espécie Moringa oleifera (Moringaceae) é uma planta que possui ampla aplicação industrial, alto valor nutricional e que, além disso, também exibe diversas atividades biológicas. Utilizadas na medicina popular, as folhas de M. oleifera já demonstraram possuir grande variedade de moléculas bioativas, inclusive compostos fenólicos, os quais são, possivelmente, os responsáveis pelo potencial antioxidante desta parte da planta. Apesar do crescente interesse sobre a espécie e, especificamente, sobre o seu potencial fitoquímico, são escassos os trabalhos que relatam o isolamento e a identificação dos compostos bioativos presentes nas suas folhas, principalmente em exemplares cultivados no Brasil. Sendo assim, os objetivos deste trabalho foram comparar dois métodos de extração de compostos bioativos e, na sequência, isolar bioguiadamente compostos com atividade antioxidante das folhas de M. oleifera coletadas no município de Itajaí (Santa Catarina, Brasil). O monitoramento bioguiado foi realizado com ensaios in vitro de determinação da atividade antioxidante: capacidade de redução do reagente Folin-Ciocalteau, FRAP, sequestro dos radicais DPPH e ABTS, além do ORAC. A técnica de CLAE-DAD foi utilizada para a caracterização química e acompanhamento das etapas do isolamento. A principal diferença prática entre os métodos de extração avaliados foi o preparo de um extrato hidroalcoólico inicial, no processo de extração 1. A partir dos resultados de determinação da atividade antioxidante, interpretados com o auxílio de ferramentas estatísticas (teste de Tukey e teste t pareado), foi possível perceber que o potencial das folhas de M. oleifera sofreu variações em função da forma de extração e dos solventes utilizados. Em geral, as frações produzidas a partir do processo de extração 1 apresentaram maior atividade antioxidante e perfis cromatográficos com sinais mais intensos. Com base nestes resultados, a fração obtida com acetato de etila, no processo de extração 1, foi selecionada para dar sequência ao isolamento bioguiado. A purificação desta fração em coluna aberta preenchida com sílica gel gerou 61 subfrações, as quais, após análise de CCD, foram agrupadas em 18. A avaliação da atividade antioxidante das subfrações agrupadas mostrou que cinco apresentavam grande potencial. Contudo, em função do rendimento, apenas três puderam dar sequência ao isolamento. Nesta etapa, uma análise adicional foi realizada: a determinação da atividade antioxidante por CLAE on-line com o ABTS•+, que permitiu definir quais dos compostos presentes nas três subfrações possuíam maior potencial e, por isso, seriam isolados. Desta forma, cinco compostos foram isolados pela técnica de CLAE semipreparativa, sendo que dois foram testados frente ao ensaio de sequestro do DPPH•. Os valores de EC50 obtidos, 30,34 e 38,72 μg/mL, estão próximos aos encontrados na literatura para substâncias isoladas de outras matrizes naturais. A técnica de RMN permitiu identificar um flavonol glicosilado. Os resultados deste trabalho mostraram que as folhas de M. oleifera coletadas em Itajaí são fonte de compostos fenólicos com potencial antioxidante e, por isso, promissoras para aplicação nas indústrias de cosméticos, alimentos e farmacêutica. / Moringa oleifera (Moringaceae) is a plant that has wide industrial application, high nutritional value and, also, exhibits several biological activities. Used in folk medicine, M. oleifera leaves have already been shown to possess a wide variety of bioactive molecules, including phenolic componds, which are possibly responsible for the antioxidant potential of this part of the plant. Despite the growing interest in this species and, specifically, in its phytochemical potential, there are few studies about the isolation and identification of bioactive compounds present in M. oleifera leaves, especially in specimens grown in Brazil. Therefore, the aims of this work were to compare two methods for extracting bioactive componds and, than, to isolate compounds with antioxidant activity of M. oleifera leaves collected in Itajaí (Santa Catarina, Brazil) by a bioguided study. The bioguided monitoring was carried out with in vitro assays to determine the antioxidant activity: Folin-Ciocalteau reagent reduction capacity, FRAP assay, DPPH and ABTS radical scavenging methods and, also, the ORAC assay. HPLC-DAD technique was used for chemical characterization and monitoring of the isolation stages. The main practical difference between the evaluated extraction methods was the preparation of an initial hydroalcoholic extract, in the extraction process 1. From the results of the antioxidant activity determination, interpreted with the aid of statistical tools (Tukey’s test and paired t-test), it was possible to see that the potential of M. oleifera leaves varied depending on the extraction form and on the solvents used. In general, the fractions prepared from the extraction process 1 showed higher antioxidant activity and chromatographic profiles with more intense signals. Based on these results, the fraction obtained with ethyl acetate, in the extraction process 1, was selected for the bioguided isolation. The purification of this fraction on an open column of silica gel generated 61 subfractions, which, after TLC analysis, were grouped in 18. The evaluation of the antioxidant activity of grouped subfractions showed that five of them presented great potential. However, depending on the yield, only three could follow the isolation. In this step, an additional analysis was performed: the determination of the antioxidant activity by an on-line HPLC method with the ABTS•+. This technique allowed defining which of the compounds presented in each subfraction had higher potential and, therefore, would be isolated. In this way, five compounds were isolated by semipreparative HPLC, two of them were tested by the DPPH• scavenging assay. The obtained EC50 values, 30.34 and 38.72 μg/mL, are close to those found in literature for substances isolated from other natural matrices. The NMR technique allowed identifying a flavonol glucoside. The results of this work showed that M. oleifera leaves collected in Itajaí are source of phenolic compounds with antioxidant potential and, therefore, are promising for the application in cosmetics, food and pharmaceutical industries.

CAPES / A família Fabaceae (Leguminosae) é constituída por 750 gêneros e mais de 18000 espécies, distribuídas em todo o mundo. No Brasil esta é representada por aproximadamente 175 gêneros e 1500 espécies. As espécies da família Fabaceae são particularmente ricas em flavonoides e compostos biossinteticamente relacionados, como rotenóides e isoflavonoides. Alcalóides, terpenóides e esteróides são exemplos de outras classes de substâncias que ocorrem em muitos exemplares da família. Dentre as espécies representantes desta família na região de Porto Rico-PR, área de proteção ambiental, está Aeschynomene fluminensis e Machaerium hirtum. Uma revisão bibliográfica destas espécies indicou a ausência de estudos fitoquímicos e biológicos, o que motivou a escolha das mesmas para investigação. Ainda foi verificado que M. hirtum é utilizada na medicina popular contra tosse, diarréia e câncer. O estudo fitoquímico para as duas espécies foi iniciado a partir da obtenção dos extratos brutos metanólicos das folhas e dos galhos, por maceração, e as obtenções de frações destes extratos a partir de partições em solventes de polaridades diferentes. As frações obtidas foram submetidas a purificações cromatográficas em colunas de gel de sílica e/ou Sephadex LH-20. Os tratamentos cromatográficos realizados para as frações de A. fluminensis levaram ao isolamento dos flavonóides canferitrina, α-raminoisorobina, quercitrina, 3-O-apiofuranosil-7-O-raminopiranosil-canferol e avicularina, da procianidina A2; da flavanona iso-hemifolina; da isoflavona daidzeína e do açúcar 3- O-metil-quiro-inositol. O estudo das frações obtidas de M. hirtum, resultou no isolamento das flavonas isovitexina e swertisina; do alcalóide 4-hidróxi-N-metilprolina; dos triterpenos friedelan-3-ona e lupen-20(29)-3β-ol e da mistura dos esteróides estigmasterol e sitosterol, a qual também foi encontrada em A. fluminensis. A elucidação das estruturas foi realizada mediante análise de dados espectroscópicos de RMN de 1H e 13C e de técnicas bidimensionais gCOSY, ii gHSQC e gHMBC) e por comparação com os dados relatados na literatura. Os extratos brutos e frações de A. fluminensis foram submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha e se mostraram todos ativos, sendo mais significativos os extratos e frações hexânicas das folhas e dos galhos. Nos ensaios de atividade da enzima de mieloperoxidase (MPO) apresentaram resultados significativos todas as frações de A. fluminensis com maiores atividades as frações hexânicas e CHCl3 das folhas e dos galhos. Os extratos brutos e todas as frações de M. hirtum apresentaram bons resultados quando submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha, para a substância 4-hidróxi-N-metil-prolina este ensaio resultou em redução moderada. Na atividade da enzima de mieloperoxidase apresentaram resultados significativos os extratos e todas as frações de M. hirtum, sendo mais ativas as frações hexânicas e AcOEt das folhas e dos galhos e CHCl3 dos galhos. A mistura swertisina e isovitexina e a substância 4-hidróxi-N-metil-prolina apresentaram redução pronunciada da enzima. O teste de pleurisia foi significativo apenas para o extrato bruto das folhas de M. hirtum. No teste da atividade antitumoral de A. fluminensis sobre células Caco-2 foi observado que a fração CHCl3 dos galhos e a canferitrina apresentaram os resultados mais promissores. / The Fabaceae family (Leguminosae) comprises 750 genera and over 18000 species, distributed worldwide. In Brazil it is represented by approximately 175 genera and 1500 species. The Fabaceae species are particularly rich in flavonoids and biosynthetically related compounds, such as isoflavonoids and rotenoids. Alkaloids, terpenoids and steroids are examples of other classes of substances that occur in many samples of the family. Aeschynomene fluminensis and Machaerium hirtum are among the representative species of this family in the region of Porto Rico-Paraná state, an environmental protection area. A literature review of these species indicated the absence of phytochemical and biological studies, which motivated their choice for investigation. It was also found that M. hirtum is used in folk medicine against cough, diarrhea and cancer. The phytochemical study for both species was initiated after obtaining the methanol crude extracts of leaves and twigs, by percolation, and the varieties of extract fractions by means of partitions in different polarities solvents. The obtained fractions were subjected to purification on chromatographic columns of silica gel and/or Sephadex LH-20. The chromatographic treatments performed for A. fluminensis fractions led to the isolation of the following flavonoids: kaempferitrin, α-rhamnoisorobin, quercitrin, 3-Oapiofuranosyl- 7-O-rhamnopyranosyl-kaempferol and avicularin, A2 procyanidin; the flavanone iso-hemipholin; the isoflavone daidzein and the 3-O-methyl-chiro-inositol sugar. The study of the obtained fractions of M. hirtum resulted in the isolation of the isovitexin and swertisin flavones; of the 4-hydroxy-N-methyl-proline alkaloid, the friedelan-3-one and lupen-20(29)-3β-ol triterpenes and the stigmasterol and sitosterol steroids mixture, which was also found in A. fluminensis. The elucidation of the structures was performed by analysis of spectroscopic data of 1H and 13C NMR and bidimensional techniques (gCOSY, gHSQC and gHMBC) and by comparison with the data reported in the literature. The crude extracts and the A. fluminensis iv fractions were tested for anti-inflammatory activity in the ear edema model and showed all active; the most significant were the extracts and hexane fractions from leaves and twigs. The tests of the myeloperoxidase (MPO) enzyme activity showed significant results for all A. fluminensis fractions with more activities for the CHCl3 and hexane fractions of leaves and twigs. The crude extracts and all M. hirtum fractions showed good results when tested for anti-inflammatory activity in the ear edema model; for the substance 4-hydroxy-N-methyl-proline this test resulted in moderate reduction. For the activity of the myeloperoxidase enzyme, the M. hirtum extracts and all fractions showed significant results, being more active the hexane and EtOAc fractions of leaves and twigs and CHCl3 of twigs. The swertisin and isovitexin mixture and the substance 4-hydroxy-N-methyl-proline showed pronounced enzyme reduction. The pleurisy test was significant only for the crude extract of M. hirtum leaves. When testing the A. fluminensis antitumor activity on Caco-2 cells, it was observed that the CHCl3 fraction of the twigs and the kaempferitrin showed the most promising results.