A ansiedade é uma enfermidade psiquiátrica que acomete milhões de pessoas no mundo todo. Dentre as substâncias que podem ser ligadas ao efeito ansiolítico de ervas medicinais, os flavonóides se destacam porque freqüentemente são detectados nestas ervas. Neste estudo procurou-se avaliar se compostos bioativos da classe dos flavonóides, presentes no quiabo (Abelmoschus esculentum), poderiam exercer atividade ansiolítica, empregando-se modelos comportamentais para análise de ansiedade em camundongos. Avaliou-se a quantidade e a qualidade dos flavonóides do quiabo, evidenciando-se que o quiabo liofilizado possui 16mg do flavonóide rutina/ 100g. Ratos mostraram-se capazes de absorver rutina, quando esta foi perfundida in situ no duodeno destes animais. Os comportamentos relacionados à ansiedade foram a avaliados no Campo Aberto, no Labirinto em Cruz Elevado e na Placa Perfurada após a ingestão de rações com diferentes concentrações de quiabo (4,5; 7 e 10%) por 7, 14 e 21 dias. Camundongos foram tratados com pentilenotetrazol, droga ansiogênica, e diazepam, droga ansiolítica, para comparar os resultados com aqueles de animais que receberam ração com quiabo. Os resultados obtidos no Campo Aberto mostraram que a suplementação dietética com quiabo aumentou o tempo de permanência na zona interna e também o número de quadros percorridos nesta região, de maneira semelhante ao diazepam, o que evidencia um efeito com perfil ansiolítico. No Labirinto, tanto o tempo de permanência, como o número de entradas nos braços abertos aumentaram em camundongos que consumiram rações suplementadas com quiabo e naqueles tratados com diazepam, corroborando os resultados obtidos no Campo Aberto. Na Placa Perfurada, o tempo de latência para o primeiro mergulho em um dos orifícios diminuiu em camundongos que se alimentaram com ração suplementada com quiabo, de modo similar ou superior ao efeito obtido pelo tratamento com diazepam e significativamente menor que o efeito produzido pelo tratamento com pentilenotetrazol. Não foram detectados flavonóides em tecidos biológicos de ratos e camundongos tratados com quiabo ou com rutina. Assim, concluiu-se que a suplementação dietética com o flavonóide rutina, proveniente do quiabo, resultou em efeito ansiolítico em camundongos, mostrando que o enriquecimento da dieta com alimentos ricos em rutina pode contribuir para a prevenção e tratamento do comportamento relacionado à ansiedade em camundongos. / Anxiety is a psychiatric disorder that affects millions of people around the world. Among the substances that may be linked to the anxiolytic effect of medicinal herbs, the flavonoids stand out because they are often found in these herbs. In the present study we sought to evaluate whether the c1ass of bioactive compounds flavonoids, present in okra (Abelmoschus esculentum), could exert anxiolytic activity, using models for behavioral analysis of anxiolytic activity in mice. We evaluate the quantity and quality of flavonoids of okra, showing that the okra lyophilisate has 16mg of flavonoid rutin/100g. Rats were shown able of absorb rutin, when it was perfused in situ in the duodenum of these animals. Behaviors related to anxiety were evaluated in the Open Field, Elevated Plus Maze and Hole Board after the intake of diets with different concentrations of okra (4.5, 7 and 10%) for 7, 14 and 21 days. Mice were treated with pentilenotetrazol, an ansiogenic drug, and diazepam, an anxiolytic drug, to compare the results with those of animals that received diet with okra. The results obtained in the Open Field showed that dietary supplementation with okra increased the permanence in the inner zone and also the number of tables covered in this region, so similar to diazepam, shows a anxiolytic profile. In the Elevated Plus Maze, the time, as the number of entries in open arms increased in mice that consumed diets supplemented with okra and those treated with diazepam, corroborating the results obtained in the Open Field. In the Hole Board, the latency time to first diving in one of the holes decreased in mice that are treated with diet supplemented with okra, so similar to or greater than the effect obtained by treatment with diazepam and significantly smaller than the effect produced by treatment with pentilenotetrazol. Flavonoids were not detected in biological tissues of rats and mice treated with okra or with rutin. Thus, it was concluded that dietary treatment with the flavonoid rutin, from okra, resulted in anxiolytic effect in mice, showing that the enrichment of the diet with foods rich in rutin can contribute to the prevention and treatment of anxiety-related behavior in mice.

Os chás destacam-se como a segunda bebida mais consumida no mundo. Além disso, seu consumo tem se modernizado, adequando-se aos anseios dos consumidores. São uma boa fonte de compostos com atividade antioxidante, os quais podem trazer benefícios à saúde humana pelo retardamento do processo de envelhecimento, assim como pela prevenção de doenças crônicas, tais como o câncer e doenças cardiovasculares. Infusões de ervas processadas e frescas foram estudadas quanto a teor de compostos fenólicos totais, perfil de flavonóides e atividade antioxidante. Os teores de compostos fenólicos totais, determinados pelo método de Folin–Ciocalteu, variaram de zero a 46,46 mg/g. Os flavonóides miricetina, quercetina e campferol foram identificados e quantificados por cromatografia líquida de alta eficiência (CLAE) nos chás preto, verde e de camomila. A atividade antioxidante foi avaliada pelo método do seqüestro do radical 2,2'-difenil-1-picrilhidrazil (DPPH) e pelo sistema modelo β-caroteno/ácido linoléico (BCAL), usando hidroxitolueno butilado (BHT) e α- tocoferol como padrões. As maiores atividades encontradas pelo sistema modelo BCAL foram para as infusões de chá preto, mate, capim-limão, camomila e funcho, enquanto as infusões de ervas frescas apresentaram as menores atividades. Entretanto, quando a atividade antioxidante foi avaliada pelo método do DPPH, as infusões de ervas frescas de menta e funcho foram as que apresentaram as maiores atividades. Os chás processados que apresentaram os menores valores de IC50 foram verde e preto (147,63 µg/mL e 288,60 µg/mL, respectivamente). Os resultados obtidos demonstraram que as infusões analisadas são boas fontes de compostos com atividade antioxidante. / Teas are the second most consumed beverage in the world. Besides this, their consumption has been modernized to suit the consumers? desires. They are a good source of compounds presenting antioxidant activity, which can bring benefits to human health by retarding the process of aging, as well as by preventing chronic diseases, such as cancer and cardiovascular diseases. Processed teas and herbal infusions were studied for their total polyphenol content, flavonoid profile, and antioxidant activity. Total phenolics, determined by Folin?Ciocalteu procedure, ranged from zero to 46.46 mg/g. Flavonoids myricetin, quercetin, and kaempferol were identified and quantified by High Efficiency Liquid Chromatography (HPLC) in black, green, and chamomile tea. Antioxidant activity was evaluated using 2,2 Diphenyl-1-picryl-hydrazyl method (DPPH) and β-carotene bleaching test (BCB), with butylated hydroxytoluene (BHT) and α-tocopherol as reference substances. The highest activities using BCB were found for infusions of black tea, maté, lemongrass, chamomile, and fennel, whereas fresh herbal infusions presented the lowest activities. However, when the antioxidant activity was evaluated by DPPH, fresh herbal infusions of mint and fennel presented the highest activities. Processed teas with the lowest IC50 values were green and black (147.63 µg/mL and 288.60g/mL and 288.60 µg/mL, respectively). The results obtained demonstrate that the infusions analyzed are good sources of compounds presenting antioxidant activity.

Methods. The intestinal absorption and metabolism of W and OA as well as the potential interactions among B, Wand OA were investigated at in vitro, in situ and in vivo levels. Various models were employed including Caco-2 cell monolayer model, in vitro enzymatic kinetics study, rat in situ single-pass intestinal perfusion model and in vivo pharmacokinetic study in rats. / Purpose. Scutellariae baicalensis Georgi is a medicinal plant widely distributed in Asia. Its dried root, Radix Scutellariae (RS), has been extensively used in Chinese and Japanese medicine. Six flavones including baicalein (B), wogonin (W), oroxylin A (OA) and their corresponding glucuronic acid conjugates (BG, WG, OAG) are the major bioactive components in RS. Our previous studies on B revealed an extensive first-pass metabolism during its absorption. Hence, it is expected that W and OA which have the similar structures as B, may share similar absorption and metabolic pathways as B. The present project aims to (1) establish an assay method for better quality control of RS; (2) provide further biopharmaceutic characterizations ofW and OA in RS; (3) investigate the potential pharmacokinetic interactions among B, Wand OA. / Results. Similar to B, Wand OA showed favorable permeability in both the Caco-2 cell and the rat in situ single-pass perfusion models. However, they experienced extensive first-pass metabolism, mainly in the form of glucuronidation. Intracellularly formed WG and OAG could be effluxed to both the apical side (lumen side) and basolateral side (mesenteric blood side) mainly by MRPs, which was confirmed by inhibition transport studies in Caco-2 cells and transfected MDCK cells. The glucuronidation rate of OA was higher than that of W, which was observed by enzymatic kinetics studies by sub-cellular fractions with intrinsic clearances (Vmax/K m, mul/min/mg) of 456 to 4170 for W and 509∼5038 for OA. UGT 1A9 was the most potent metabolic enzyme for hepatic glucuronidation, while UGTs 1A8 and 1AlO were responsible for the intestinal glucuronidation of W and OA. The in vivo rat pharmacokinetics studies showed that W and OA may be readily absorbed and extensively metabolized with no parent compound detectable in blood after oral administration of W and OA. A new metabolite of W was identified to be the glucuronic acid conjugate at 5-0H of W. After co-administration of B, W and OA, decreased formation of BG, WG and OAG was observed in in vitro enzymatic kinetics study. Further studies in absorption models of Caco-2 cell monolayer and rat in situ single-pass intestinal perfusion demonstrated the enhancement in absorption of B, W and OA and decrease of BG, WG and OAG after the co-administration of B, W and OA. The ultimate pharmacokinetics interaction study revealed that glucuronides were the predominant form in systemic circulation and the AUC of OAG significantly increased after co-administration of B, Wand OA. Conclusion: Similar to B, Wand OA may be well absorbed followed by extensive first-pass metabolism, which was mediated by various UGT isozymes. During absorption, the intracellularly formed WG and OAG were mainly effluxed by MRPs to both the lumen and mesenteric blood side of the intestine. Both in vitro and in situ models indicated that interactions among B, W and OA would lead to decreased glucuronidation and increased absorption of parent flavones. Due to extensive metabolism in vivo, only glucuronides appeared in systemic circulation after co-administration of B, W and OA in rats. The resulted increased systemic exposure of OAG indicated that the co-administration might lead to the enhancement of bioavailability for the studied flavones in the form of glucuronides. / Li, Chenrui. / Adviser: Zuo Zhong. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 201-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-02-05T16:22:24Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Carlos Dávila Sánchez.pdf: 22396898 bytes, checksum: 5122ed06da0d2dc71b9cbf4253c97cda (MD5) / Made available in DSpace on 2018-02-05T16:22:24Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Carlos Dávila Sánchez.pdf: 22396898 bytes, checksum: 5122ed06da0d2dc71b9cbf4253c97cda (MD5) Previous issue date: 2017-12-07 / O objetivo deste estudo foi avaliar o efeito da aplicação de primers experimentais à base de flavonoides na resistência de união, nanodureza, módulo de elasticidade e micromorfologia da interface adesiva criada em dentina hígida e dentina afetada por cárie utilizando um adesivo universal aplicado em modo convencional. Superfícies planas de dentina foram expostas em 182 molares permanentes. Noventa e um molares foram armazenados em água, enquanto os outros 91 foram imersos em uma solução de BHI/Sacarose (pH 4.0) e cepas de Streptococcus mutans por 14 dias para induzir lesões artificiais de cárie em dentina. Quarenta e nove dentes foram aleatoriamente alocados em 7 grupos (n=7) de acordo com o primer experimental usado em cada tipo de substrato (hígido ou afetado por cárie): Controle, sem aplicação de primer (CON), Placebo solução hidroalcoólica com tensoativo (PLA), Proantocinidina (PRO), Quercetina (QER), Naringina (NAR), Rutina (RUT), e Hesperidina (HES). Após o condicionamento, lavagem e secagem das superfícies de dentina, os primers foram ativamente aplicados por 1 min para reumedecer a dentina. Após serem restaurados, os espécimes foram seccionados no seu longo eixo até obter palitos retangulares (0,9 mm2) que foram testados em tração com velocidade de 1 mm/min, tanto após 24 h quanto após 25.000 ciclos em uma máquina de ciclagem térmica (5 ºC / 55 ºC). Para os testes de nanodureza e módulo de elasticidade (n=3), os espécimes foram cortados longitudinalmente até obter duas fatias planas (1,2 mm) e foram analisados em um nanoindentador com uma ponta Berkovich tanto após 24 h quanto após 25.000 ciclos térmicos. Para a análise qualitativa de microinfiltração, 21 dentes (n=3) assistida por corantes em Microscópio confocal (CSLM), o adesivo e a resina composta foram misturados com 0,2% de Rhodamina B (massa) antes dos procedimentos restauradores e duas fatias planas foram obtidas para análise após 24 h e 25.000 ciclos térmicos. As fatias foram imersas em uma solução de Fluoresceína (0,1%) por 4 horas. Quatro imagens da interface adesiva de cada dente foram obtidas em cada momento experimental (24 h ou 25.000 ciclos) a 5 e 10 μm de profundidade. Os resultados foram analisados usando uma análise de variância de medidas repetidas e pós-teste de Bonferroni (= 0,05). Resultados: Os grupos PLA e RUT mostraram os valores mais altos de μTBS em dentina hígida em ambos intervalos (p > 0,05). Na dentina afetada por cárie os grupos RUT e NAR apresentaram os valores mais altos em ambos intervalos. As imagens de CSLM mostraram sinais de infiltração em todos os grupos. PLA, RUT, NAR e PRO mostraram áreas sem infiltração em ambos intervalos e tipo de substrato. As nanopropriedades foram influenciadas pelos grupos experimentais em profundidade dependendo do flavonoide usado. Conclusão: O uso de flavonoides poderia ser uma alternativa terapêutica em dentina hígida e afetada por cárie para melhorar as propriedades e longevidade da interface adesiva. / The aim of this study was to evaluate the effect of experimental flavonoid primers on bond strength, nanohardness, elastic modulus, and micromorphology of bonding interfaces created on sound and caries-affected dentin using one commercially available universal bonding agent applied in the etch-and-rinse mode. Flat midcoronal dentin surfaces were exposed in 182 permanent molars. Ninety-one molars were stored in water while the other 91 molars were kept in a BHI/Sucrose solution (pH 4.0) with Streptococcus mutans strain for 14 days to induce artificial caries-affected dentin. Forty-nine teeth were randomly assigned to 7 groups (n=7) according to the experimental primer applied to each dentin surface condition (sound or caries-affected dentin): Control, in which no primer was applied (CON), Hydroalcoholic solution with surfactant Placebo (PLA), Proanthocianidin (PRO), Quercetin (QER), Naringin (NAR), Rutin (RUT) and Hesperidin (HES). After etching, rinse and drying the dentin surfaces, the primers were actively applied for 1 min to rewet the dentin surface. After bonding and restorative procedures, specimens were sectioned in the long axis to obtain rectangular sticks (0.9 mm2) that were subjected to microtensile tests (μTBS) at a crosshead speed of 1 mm/min, either 24 h or after 25.000 cycles in a thermocycling machine (5ºC / 55ºC). For nanohardness and elastic modulus (n=3), the specimens were longitudinally sectioned to obtain 2 flat silces (1,2 mm) and were analyzed in a nanohardness device with a Berkovich indenter either after 24 h or after 25.000 (5 ºC / 55 ºC) thermal cycles. For the dye assisted microinfiltration qualitative analysis 21 teeth (n=3) was performed with a Confocal Scanning Laser Microscope (CSLM), both adhesive system and resin composite were mixed with 0.2% (weight) Rhodamine B prior to the restorative procedure and two flat slices were obtained for analysis in 24 h and after 25.000 thermal cycles. The bonding interfaces were immersed in a 0,1%. Fluorescein solution for 4 hours. Four images of bonding interface per specimen where obtained in each experimental time (24 h and 25.000 cycles) at 5 and 10 m depth. The data were analyzed using two-way repeated measures ANOVA and Bonferroni post-hoc test ( = 0.05). Results. PLA and RUT showed the highest TBS values in sound teeth at both intervals (24h and ageing) (p>0.05), while RUT and NAR showed the highest TBS values in carious teeth in both stages. CLSM analysis showed signs of microinfiltration for all groups. PLA, RUT, NAR and PRO showed areas of no infiltration in both experiments and intervals. Nanomechanical properties were influenced by experimental groups in deep, dependending on the flavonoid used. Conclusion: The use of flavonoids may be an alternative for therapeutic substances in sound or carious dentin to improve their properties and longevity in the adhesive interphase.

New drugs are needed for better cancer management. Clinical trials are currently underway to assess the use of flavonoids (natural polyphenols) as anticancer agents. Among them, myricetin has been shown to induce cell cycle arrest and apoptosis in pre-clinical cancer models. We hypothesised that myricetin-derived novel flavonoids designed to enhance this natural potential and improve on the drug-likeness limitations of myricetin might have increased potential for their application in the management of breast and ovarian cancer. The effect of a library of novel flavonoids was screened on 3 panels of breast and ovarian cancer cell lines, representing different molecular subtypes and phenotypes, to assess their potency. The second-generation bi-methoxylated analogue AO-1530-OMe (Oncamex) was identified as the most effective candidate in the library, with sub-micromolar concentrations exerting a strong antiproliferative effect across almost all models studied. Results suggested that changes in the hydroxylation profile, the addition of methoxylations and a decyl alkyl chain were some of the structure-activity relationships contributing to this improved efficacy. Plate assays showed 8 h treatment with Oncamex reduced cell viability and induced cytotoxicity and apoptosis, concomitant with caspase activation and PARP cleavage. Pre-incubation with an antioxidant partially blocked these effects, suggesting the possible involvement of ROS modulation in the mechanism of action of Oncamex. Fluorescence microscopy reported the quick and stable delivery of Oncamex to the mitochondria. Fluorescent probes showed that Oncamex can induce mitochondrial superoxide production at concentrations associated with its antiproliferative effects. Study of the electrochemical properties of Oncamex by cyclic voltammetry supported this. Differential gene expression analysis following a microarray experiment showed Oncamex induces changes in the expression of genes controlling cell cycle and apoptosis. Together with previous results, the findings from this analysis led to the postulation of a model for the mechanism of action of Oncamex: due to its enhanced reactivity and mitochondrial targeting, Oncamex can generate mitochondrial superoxide, leading to mitochondrial dysfunction, membrane permeabilisation and the activation of the JNK pathway and the transcription factor FOXO3, which together contribute to the induction of intrinsic apoptosis and the inhibition of proliferation. Further proliferation assays on cell culture models also reported enhanced effect of Oncamex when administered in combination with paclitaxel and TRAIL. These improved responses were observed in breast and ovarian cancer models, including cells lines characterised by their treatment-resistant phenotype. Cotreatment with Oncamex also improved the effect of tamoxifen on anti-oestrogen resistant LCC9 breast cancer cells. Results from preliminary in vivo studies in mice implanted with the MDA-MB-231 breast cancer xenograft were consistent with an antiproliferative effect of Oncamex (25mg/kg/day) in vivo, as treatment inhibited tumour growth and reduced the expression of the marker of proliferation Ki-67 without signs of systemic toxicity. Tissues from this experiment also allowed for preliminary in vivo validation of the proposed mechanism of action of Oncamex by immunohistochemistry. The in vivo cytostatic effect of Oncamex was confirmed in a second in vivo experiment, which also investigated the effect of Oncamex at higher doses or in combination with paclitaxel. In conclusion, the novel flavonoid Oncamex has shown a promising antiproliferative effect in pre-clinical models of breast and ovarian cancer, including models of treatment-resistant cancers. Preliminary in vivo studies have demonstrated a partial recapitulation of the effect of Oncamex. A mechanistic model has been proposed by which Oncamex induces intrinsic apoptosis through its redox reactivity and mitochondrial targeting. These results support the potential of this prototypic candidate, although possible work in the structure and formulation of this candidate and further study and validation of its mechanism of action is needed for its continued development as an anticancer agent.

Submitted by Verena Bastos (verena@uefs.br) on 2015-07-24T13:45:42Z No. of bitstreams: 1 ?UDER REIS DIAS -DISSERTA??O MESTRADO 2013 -RGV.pdf: 1805178 bytes, checksum: fa9b0a71b8366540b825499da5adb0f8 (MD5) / Made available in DSpace on 2015-07-24T13:45:42Z (GMT). No. of bitstreams: 1 ?UDER REIS DIAS -DISSERTA??O MESTRADO 2013 -RGV.pdf: 1805178 bytes, checksum: fa9b0a71b8366540b825499da5adb0f8 (MD5) Previous issue date: 2013-10-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The Melastomataceae family comprises 4,570 species in approximately 180 genera present in all tropical and subtropical regions of the planet. This family, in Brazil, is the sixth largest family of Angiosperms, with over 1500 species. The genus Tibouchina presents, approximately, 350 species, however, are poorly studied. The Tibouchina pereirae Aubl., a shrub, is popularly used for the treatment of kidney diseases. In this study, we show the antioxidant and antinociceptive effects of T. pereirae. The hexane (EHTP), ethanol (EETP) and aqueous (EATP) extracts were obtained by maceration from the aerial parts of T. pereirae. The flavonoid rich fraction (FFTP) was obtained from fractionation of EHTP. The analysis of FFTP by HPLC-DAD and HPLC-MS/MS led to characterization of these flavonoids. The antioxidant activity of extracts was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay system and the in vivo experiments were conducted on Swiss mice using the acetic acid-induced writhing test and the formalin-induced pain test. Results showed that extracts (EHTP, EETP and EATP) and FFTP present antioxidant activity, when compared to standard butylated hydroxytoluene (BHT) and ascorbic acid (AA). The EHTP and FFTP (i.p., 100mg/Kg) reduced the nociception produced by acetic acid by 90,36% and 94,74%, respectively. The FFTP reduced the formalin effects in both phases by 54, 35% and 92,05%, while the EHTP only protected the second phase by 83,39%. / A fam?lia Melastomataceae apresenta cerca de 4.570 esp?cies, distribu?das em, aproximadamente, 180 g?neros presentes em todas as regi?es tropicais e subtropicais do planeta, sendo que no Brasil ? a sexta maior fam?lia de angiospermas, com mais de 1500 esp?cies. O g?nero Tibouchina possui aproximadamente 350 esp?cies, por?m s?o muito pouco estudadas, sendo que a esp?cie Tibouchina pereirae Aubl. ? popularmente utilizada para o tratamento de problemas renais. Nesse estudo, foram avaliadas as atividades antioxidante e antinociceptiva de T. pereirae. Foram obtidos os extratos hex?nico (EHTP), etan?lico (EETP) e aquoso (EATP) a partir da macera??o das partes a?reas de T. pereirae. A fra??o rica em flavonoides (FFTP) foi obtida a partir do fracionamento do EHTP. A an?lise por CLAE-DAD e CLAE-EM/EM desta fra??o possibilitou caracterizar estes flavonoides. A atividade antioxidante dos extratos foi avaliada utilizando o sistema de ensaio 2,2-difenil-1-picrilhidrazil (DPPH) e os experimentos in vivo foram realizados em camundongos Swiss utilizando os testes de contor??o abdominal induzida por ?cido ac?tico e da formalina. Os resultados obtidos demonstraram que o EHTP, EETP e EATP, assim como a fra??o FFTP apresentaram atividade seq?estradora radicalar, quando comparados aos padr?es butilhidroxitolueno (BHT) e ?cido asc?rbico (AA). O EHTP e a FFTP reduziram a nocicep??o induzida pelo ?cido ac?tico em 90,6% e 94,74%, respectivamente. A FFTP reduziu os efeitos da formalina em ambas fases em 54,35% e 92,05%, respectivamente, enquanto que o EHTP foi ativo somente na segunda fase do teste, com uma redu??o no tempo de lambida de 83,39%.

Camarea St.-Hil. (Malpighiaceae) é um gênero endêmico da América do Sul, constituído por nove espécies. O objetivo do trabalho foi a reconstrução da filogenia do gênero, por meio de evidências químicas e moleculares. Foram avaliados nove terminais, sete dos quais são espécies correntemente reconhecidas, um é uma espécie que entrou em sinonímia (C. triphylla = C. axillaris) e outro é um suposto híbrido. Como grupos externos, foram utilizadas as espécies Peixotoa reticulata e Janusia guaranitica. Foram analisados os n-alcanos das ceras epicuticulares e os flavonóides de todas as espécies. Os n-alcanos principais foram C29, C31 e C33, todos da série normal. Como flavonóides característicos de Camarea, foram identificados glicosídeos de apigenina, luteolina, crisoeriol, campferol e quercetina. A análise de agrupamentos baseada na distribuição de alcanos, usando UPGMA e distâncias euclideanas, resultou em dois grupos principais, um com C29 como homólogo principal, constituído por C. hirsuta, C. affinis x C. hirsuta, C. affinis e C. ericoides. O outro grupo caracteriza-se por homólogos principais C31 ou C33, e é formado por C. elongata, C. humifusa, C. sericea, C. axillaris e C. triphylla (= C. axillaris). Esses dois principais agrupamentos contêm grupos internos menores. Uma análise de UPGMA usando coeficiente de DICE e baseada na distribuição de agliconas de flavonóides forneceu um dendrograma com alguns agrupamentos coerentes com as afinidades reveladas pela distribuição de alcanos, como as associações: 1) C. hirsuta, C. affinis e C. affinis x hirsuta; 2) C. elongata e C. axillaris; 3) C. sericea e C. humifusa. Uma inferência filogenética molecular foi obtida com seqüências de duas regiões do cloroplasto (trnL-F e rps16) e uma nuclear (ITS). Dentre as análises com um só marcador, resultados mais consistentes foram conseguidos com ITS, que forneceu 49 caracteres filogeneticamente informativos, enquanto trnL-F e rps16 forneceram 10 e 18 caracteres informativos, respectivamente. A análise de consenso estrito de quatro árvores mais parcimoniosas de uma análise combinando-se as três seqüências resultou em um cladograma em que Camarea é um grupo monofilético, com \"bootstrap\" (BS) 100, várias politomias e clados com baixa consistência. Foi realizada análise por AFLP utilizando quatro combinações de iniciadores seletivos, obtendo-se 217 fragmentos polimórficos. Uma análise combinando as evidências moleculares resultantes de seqüências de DNA e marcadores AFLP forneceu uma única árvore mais parcimoniosa com uma politomia agrupando C. affinis, C. ericoides, C. hirsuta e C. affinis x C. hirsuta, mas boa resolução e elevada sustentação em outros clados. A combinação de todas as evidências moleculares e químicas (estas compreendendo três caracteres derivados da análise de alcanos e cinco de flavonóides) resultou numa única árvore mais parcimoniosa completamente resolvida. Os resultados apóiam a fusão de C. triphylla em sinonímia com C. axillaris e indicam forte associação entre: 1) C. humifusa e C. sericea (BS 94); 2) C. affinis, C. affinis x hirsuta, C. hirsuta e C. ericoides (BS 83); 3) C. axillaris e C. elongata (BS 100). C. affinis, C. hirsuta e C. affinis x C. hirsuta compartilham a presença crisoeriol. C. affinis e C. affinis x C. hirsuta, compartilham também luteolina e formam um clado com BS 70. O presente trabalho demonstra a utilidade de caracteres químicos para melhorar a resolução de filogenias e elevar a sustentação de clados. / Camarea St.-Hil. (Malpighiaceae) is a genus with eight species endemic in South America. The purpose of the present work was the attainment of a phylogenetic inference of the genus by means of chemical and molecular evidences. Nine accessions were analyzed: seven correspond to currently recognized species; one is a species sunk into synonymy (C. triphylla = C. axillaris); and a last one is a hypothesized hybrid. Peixotoa reticulata and Janusia guaranitica were used as out-groups. n-Alkanes from epicuticular waxes and flavonoids were analyzed from all species. The main alkanes of all distributions were either C29 or C31 or C33, all from the normal series. The characteristic flavonoids of Camarea were shown to be apigenin, luteolin, chrysoeriol, kaempferol and quercetin. A cluster analysis based on the alkane distribution using UPGMA and Euclidean distances provided two main clusters. One cluster is characterized by C29 as main homologue and is formed by C. hirsuta, C. affinis, C. affinis x hirsuta and C. ericoides. The other cluster has species with either C31 or C33 as main homologue and is formed by C. elongata, C. humifusa, C. sericea, C. axillaris and C. triphylla (= C. axillaris). These two main clusters contain smaller inner clusters. An analysis using UPGMA and DICE coefficients and based on the distribution of flavonoid aglycones provided a dendrogram with clusters congruent with affinities revealed by the alkane evidence, such as the groupings: 1) C. hirsuta, C. affinis and C. affinis x hirsuta; 2) C. elongata and C. axillaris; 3) C. sericea and C. humifusa. A phylogenetic molecular inference was obtained with sequences from two chloroplast (trnL-F and rps16) and one nuclear (ITS) DNA regions. Among the analyses based on a single marker, more consistent results were obtained with ITS, which provided 49 informative phylogenetic characters, while trnL-F and rps16 provided 10 and 18 informative characters, respectively. A strict consensus analysis based on four more parsimonious trees from an analysis combining sequences of the three DNA regions gave a cladogram showing Camarea as a monophyletic group with bootstrap support (BS) 100. The cladogram contains several polytomies and clades with low support. An AFLP analysis, using four combinations of selective primers, provided 217 polymorphic fragments. Data from sequencing and AFLP were combined in a phylogenetic analysis. A sole more parsimonious tree was obtained, with most clades completely resolved with and high support. A polytomy remained, grouping C. affinis, C. ericoides, C. hirsuta and C. affinis x hirsuta. The combination of all molecular and chemical evidences (the latter comprising three alkane and five flavonoid characters) was used for a phylogenetic analysis. A sole more parsimonious tree was obtained, completely resolved and with clades highly supported. The results support sinking C. triphylla into synonymy of C. axillaris and indicate strong kinship: 1) between C. humifusa and C. sericea (BS 94); 2) among C. affinis, C. affinis x C. hirsuta, C. hirsuta and C. ericoides (BS 83); 3) between C. axillaris and C. elongata (BS 100). C. affinis, C. hirsuta and C. affinis x C. hirsuta share the possession of chrysoeriol. C. affinis and C. affinis x C. hirsuta share the possession also of luteolin and form a clade with BS 70. The present work reveals the utility of chemical characters to improve resolution of phylogenies and increment clade support.

Foram estudados os efeitos da deficiência hídrica e da aplicação de ácido abscísico (ABA) sobre alguns aspectos fisiológicos e a produção de flavonóides em plantas de calêndula. Testaramse quatro intervalos de suspensão da irrigação (controle - irrigação diária; 3; 6 e 9 dias sem irrigar) acompanhados por três doses de ABA (0, 10 e 100 µM), resultando em 12 tratamentos. O experimento foi instalado em condições de casa de vegetação com plantas envasadas. Os tratamentos foram aplicados no início do florescimento e seus efeitos foram avaliados pelo conteúdo relativo de água na folha (CRA) e trocas gasosas (A= fotossíntese líquida, gs= condutância estomática, E= transpiração, Ci= concentração intercelular de CO2 e TL= temperatura da folha); por meio de analisador portátil por infra-vermelho. A eficiência do uso da água (EUA) foi calculada como A/E. Aos 3 dias de suspensão da irrigação as plantas de calêndula não apresentaram alterações significativas nas variáveis de trocas gasosas avaliadas. Aos 6 dias de deficiência hídrica as variáveis Ci, CRA e EUA não apresentaram diferença em relação aos tratamentos com suprimento constante de água, acrescidos ou não de ABA. Porém, a imposição de estresse hídrico somada à aplicação de ABA nas plantas resultou em diminuição nos valores de E, gs, A e TL em relação aos tratamentos controle. Aos 9 dias sem irrigar ocorreram mudanças drásticas nas plantas, sendo que todos os parâmetros de trocas gasosas avaliados apresentaram reduções significativas em relação aos tratamentos controle (irrigação diária com ou sem ABA). Concluiu-se que a aplicação exógena de ABA mimetizou as respostas fotossintéticas da planta ao estresse hídrico, sendo que o efeito principal deste biorregulador foi o de causar diminuição na gs. Porém, esta redução na gs só foi acompanhada de uma redução em A quando as plantas estavam submetidas à deficiência hídrica. O efeito residual do ABA nas plantas foi de apenas 7-8 dias e portanto, as variações observadas aos 6 dias de deficiência hídrica foram provavelmente induzidas pelo ABA ; enquanto que as diferenças observadas aos 9 dias foram exclusivamente causadas pela deficiência hídrica. Não houve diferença significativa entre as diferentes intensidades de deficiência hídrica testadas com relação ao acúmulo de flavonóides nas inflorescências de calêndula. Entretanto, a aplicação de ABA provocou uma diminuição de até 50% no teor de flavonóides, tanto nas plantas controle (bem hidratadas) como nas plantas submetidas ao estresse hídrico, para todas as intensidades testadas. Concluiu-se que o ABA restringe a rota biossintética de flavonóides. / It was evaluated the effects of water deficit and abscisic acid (ABA) application on some physiological parameters and flavonoid production in marigold plants. It was tested four intervals of withholding water (control - diary irrigation, 3, 6 and 9 days without irrigation) accomplished of 3 concentrations of ABA (0, 10 e 100 µM), resulting in 12 treatments. The experiment was performed under nursery conditions with plotted plants. The treatments were applied in the beginning of blooming and their effects were evaluated by the relative water content (RWC) and leaf gas exchange (A= net photosynthesis, gs= stomatal conductance, E= evaporation, Ci= CO2 intercellular concentration and TL=leaf temperature); using a portable infrared gas analyzer. The water use efficiency (EUA) was calculated as A?E. At 3 days of water suppression the marigold plants did not showed significant alterations in leaf gas exchange evaluated parameters. At 6 days of water deficit the parameters Ci, RWC and EUA did not showed difference in relation to the treatments with daily irrigation, added or not of ABA. However, the water stress plus ABA application resulted in smaller values of E, gs and A in relation to control plants. At 9 days of water deficit there were drastic changes in the plants, with significant reductions in all leaf gas exchange parameters evaluated in relation to control treatments (daily irrigation with or without ABA). It was concluded that exogenous ABA application mimics the plant photosynthetic responses to water stress, and the main ABA effect was to cause a reduction on gs. However, this gs reduction only was accomplished of a reduction in A when the plants were submitted to the water deficit. The residual effect of ABA in plants was only 7-8 days and so, the differences observed at the 6 days of water deficit were probably inducted by the ABA, yet differences observed at the 9 days were exclusively caused by the water deficit. There was no significant difference among the different levels of water deficit used in relation to total flavonoids content in inflorescences of marigold. However, ABA application resulted in a reduction of almost 50% in flavonoids content, in well watered plant as well as stressed plants, for all levels of water deficit tested. It was concluded that ABA restricts the biosynthetic rote of flavonoids.

Lipossomas (LUV) são estruturas compostas por uma bicamada lipídica que se organizam de forma semelhante a vesículas, contendo um compartimento aquoso em seu interior. Têm sido avaliados como potenciais carreadores de fármacos. No entanto, após sua administração, in vivo, opsoninas do soro adsorvem-se em sua superfície contribuindo para que o sistema fagocitário mononuclear (SFM) reconheça essas partículas, favorecendo sua remoção da circulação. O sistema complemento (SC) parece ter papel importante neste processo, principalmente por gerar fragmentos ativos do componente C3 (C3b/iC3b) que se depositam nas vesículas lipossomais e são reconhecidos por receptores do complemento presentes, por exemplo, nos polimorfonucleares. Antioxidantes, como a quercetina, têm demonstrado importantes e benéficos efeitos sobre a saúde humana, porém sua baixa solubilidade em água e biodisponibilidade limitam seu uso. Assim, o desenvolvimento apropriado de carreadores de flavonoides seria de grande importância para sua aplicabilidade in vivo. O objetivo do presente trabalho é avaliar a ativação das proteínas do SC por lipossomas compostos de fosfatidilcolina de soja e colesterol (PC:CHOL) ou colesteril-etil-éter (PC:CHOL-OET), contendo ou não quercetina. O consumo das vias clássica (VC) e alternativa (VA) provocado pelas diferentes vesículas foi analisado por ensaio hemolítico e a quantificação de iC3b e anticorpos naturais (IgG e IgM) na superfície dessas partículas foi realizada através de kits de ELISA. A ativação de C3 por vesículas contendo ou não quercetina foi avaliada por imunoeletroforese bidimensional (IEF). Os resultados mostram que lipossomas vazios, compostos por grande quantidade de colesterol, consomem mais os componentes do complemento para ambas as vias, VC e VA. Ainda, a substituição de colesterol por colesteril-etil éter reduziu o consumo das duas vias, mas a ativação do SC ainda é dependente da quantidade deste composto. A incorporação de quercetina não alterou o consumo de ambas as vias. O depósito de iC3b, IgG ou IgM nas vesículas compostas de PC:CHOL-OET na proporção de massa 3:1 foi o menor comparado aos demais. A IEF mostrou que vesículas PC:CHOL vazias induzem maior clivagem de C3 em relação às vesículas PC:CHOL-OET. Ainda, a incorporação de quercetina reduz a conversão de C3 em seus fragmentos. Essas observações sugerem que a preparação lipossomal PC:CHOL-OET em proporção de massa 3:1 parece ser a mais adequada para dar continuidade aos estudos que deverão culminar na tentativa de utilizá-la como carreadora de quercetina para administração in vivo / Liposomes (LUV) are structures composed by lipid bilayer that are organized similarly to vesicles, containing an aqueous compartment inside. They have been evaluated as potential drug carriers, however, after in vivo administration, serum opsonins are adsorb on the surface, contributing to their clearance from the circulation by mononuclear phagocytes system (MPS). The complement system (CS) seems to play an important role in this process, mainly to generate active fragments of the C3 component (C3b/iC3b) that are deposited in the liposomal vesicles and are recognized by complement receptors present, for example, in polymorphonuclear cells. Antioxidants such as quercetin have demonstrated significant and beneficial effects on human health, but its low water solubility and bioavailability limit their use. Thus, the proper development of flavonoids carriers would be of great importance to its applicability in vivo. The objective of this study is to evaluate the activation of SC proteins by liposomes composed of soy phosphatidylcholine and cholesterol (PC: CHOL) or cholesteryl ethyl ether (PC: CHOL-OET), with or without quercetin. The consumption of the classical (CP) and alternative pathway (AP) caused by the different vesicles was analyzed by hemolytic assay and quantification of iC3b and natural antibodies (IgG and IgM) on the surface of these particles was performed using ELISA kits. The activation of C3 by vesicles with or without quercetin was assessed by two-dimensional immunoelectrophoresis (IEF). The results show that empty liposomes, composed of large amounts of cholesterol, consume more CS components in both pathways, CP and AP. Moreover the replacement of cholesterol by cholesteryl ethyl ether reduced the consumption of both pathways, but the activation of the SC is still dependent on the amount of the compound. The incorporation of quercetin did not alter the consumption of both pathways. The deposition of iC3b, IgG or IgM in vesicles composed of PC: CHOL-OET at mass ratio of 3:1 was the lowest compared to the others. The IEF showed that empty vesicles of PC:CHOL induce less cleavage of C3 in relation to vesicles of PC: CHOL-OET. In addition, the incorporation of quercetin reduces the conversion of C3 into its fragments. These observation suggest that the liposomes PC:CHOL at mass ratio 3:1 seems to be the most appropriate to continue the studies that could culminate in an attempt to use it as a carrier to administrate quercetin in vivo

Propolis is an approved food additive containing flavonoids as a major active constituent. Variability has been found in the composition of propolis in distinctive regions and it was noticed that there are limitations in the analysis of propolis. In this study, the identification of ten flavonoids and residual antibiotics in propolis was investigated by using several liquid chromatography techniques, including reversed-phase high-performance liquid chromatography (RP-HPLC), microemulsion LC (MELC) and ultra-performance LC (UPLC). The ten flavonoids that were selected for this research include rutin, myricetin, quercetin, apigenin, kaempferol, pinocembrin, CAPE, chrysin, galangin and acacetin while chlortetracycline, oxytetracycline and doxycycline were selected to examine the residual antibiotics in propolis. For the analysis of the selected flavonoids, routine RP-HPLC method was found to be the best method, while MELC technique was found more efficient for the analysis of the selected antibiotics. Solid phase extraction with HLB sorbent was utilised in the analysis of antibiotics for clean-up of propolis. In method development studies for flavonoids and antibiotics, one-factor-at-a-time (OFAT) approach was followed. The final optimised method for the analysis of flavonoids as well as the method. for the analysis of antibiotics was validated using the ICH guidelines, and various aspects, such as the linearity, selectivity, accuracy, recovery, robustness and stability parameters, were examined. Development of efficient conventional method for the extraction of flavonoids from propolis was studied extensively in the present research work using different extraction techniques such as maceration, hot extraction, ultrasound assisted extraction. Among all extraction experiments, ethanolic extraction using ultrasound extraction method was the best efficient approach. This thesis shows that, in general, the performance of O/W MELC is superior to that of conventional HPLC for the determination of residual antibiotics in propolis. UPLC was not suitable for the analysis of flavonoids and antibiotics. The conventional LC was the only technique to separate the ten flavonoids but MELC was able to separate nine of the flavonoids with faster analysis time. This work also showed that MELC uses cheaper solvents. This considerable saving in both cost and time will potentially improve efficiency within quality control.