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Colon cancer is the third most common cancer in the United States. However, evidence indicates that a proper diet abundant in fruits and vegetables may be protective against colon cancer development. Bioactive compounds in fruits and vegetables, such as flavonoids and limonoids, have been shown to possess anti-proliferative and antitumorigenic effects in various in vitro and in vivo models of cancer. Since there are few animal studies involving flavonoids and limonoids and colon cancer, this experiment investigated the potentially protective effects of four citrus flavonoids and one limonoid mixture against the promotion stage of chemically-induced colon cancer in rats. Male SD rats (n =60; 10 rats/group) were assigned to receive diets containing 0.1% apigenin, 0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin glucoside/obacunone glucoside mixture, or a control diet (0% flavonoid/limonoid). Rats received the diets for 10 wk and were injected with azoxymethane (15 mg/kg) at wk 3 and 4. The excised colons were evaluated for aberrant crypt foci (ACF) formation, cell proliferation (PCNA assay), apoptosis (TUNEL assay), and iNOS and COX-2 expression. When compared to the control diet, apigenin lowered the number of high multiplicity ACF (> 4 AC/focus) by 57% (P<0.05) and tended to lower the proliferative index (28%; P=0.07), while naringenin lowered both the number of high multiplicity ACF by 51% (P<0.05) and the proliferative index by 32% (P<0.05). Both apigenin and naringenin increased apoptosis of surface colon cells (78% and 97%, respectively; P<0.05) when compared to control diet. Hesperidin, nobiletin, and the limoninglucoside/obacunone glucoside mixture did not have any effects on the above variables measured in this model of colon carcinogenesis. The colonic mucosal protein levels of iNOS or COX-2 were not different among the six diet groups. Evidence suggests that high multiplicity ACF are indicative of future tumor development in both humans and rats. Furthermore, dysregulated proliferation and apoptosis may also lead to tumorigenesis. Therefore, the ability of dietary apigenin and naringenin to reduce high multiplicity ACF, lower proliferation, and increase apoptosis may contribute toward colon cancer prevention. However, their protection is not due to their influence on iNOS and COX-2 protein levels.

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Orientador: Rubens Monti / Resumo: ---- / Mestre

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-20T12:34:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CarlaFontouraFerrreira.pdf: 19008440 bytes, checksum: b5b118a8e2abb810388a00dc8e27ac80 (MD5) / Made available in DSpace on 2018-08-20T12:34:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CarlaFontouraFerrreira.pdf: 19008440 bytes, checksum: b5b118a8e2abb810388a00dc8e27ac80 (MD5) Previous issue date: 2017-03-30 / Reactive species (ERs) and oxidative stress contribute to the pathophysiology of several diseases including dementia. An innovative alternative for protecting organs such as the brain is the use of antioxidant-like flavonoids such as naringin and naringenin. However, these two compounds have low bioavailability when administered orally. An alternative to overcome this limitation is the use of nanocarriers, such as nanocapsules (NC). Therefore, the present study aimed to evaluate the neuroprotective effect of naringin and naringenin in a model of streptozotocin (STZ) -induced dementia in mice. The profile of drug release from in vitro suspensions was evaluated as well as the effects of nanoencapsulated drugs on cell viability with Vero cell line. In addition, the involvement of oxidative stress in the brain tissue of mice was verified. Naringin and naringenin NC had acid pH, particle diameter less than 95 nm, polydispersity index (IPD) of less than 0.2. The zeta potential was negative, between -12.46 and -17.6 mV, and the encapsulation efficiency was 93% for naringin and 95% for naringenin. For the study of the release profile, the dialysis technique was used, with 46.5% of naringin and 6.9% of naringenin being released in the medium, in a period of 9h. The results of the cell viability assay showed that the NC suspensions caused a reduction in cell viability of Vero cells at concentrations of 5, 50 and 500 μg / ml when compared to the control at the 24 and 72h incubation periods. For in vivo evaluation of neuroprotection the mice were pretreated for 15 days by oral (vo) with the suspension of white nanocapsules (NB), NC containing the mixture naringin and naringenin (N-NANG), suspension of naringin and naringenin in the form Free (NANG) and vehicle (Sham) at dose 10 mg / kg body weight. Subsequently, stereotactic surgery was performed for intracerebroventricular (i.c.v) infusion of STZ or FAC (cerebral artificial fluid). Behavioral tasks were then initiated. In the open field task the locomotor activity differed in the test section in the NB + STZ group evidenced by a hyperlocomotion between the mice of this experimental group. For exploratory activity there was no significant difference between groups. In the evaluation of the short-term memory in the task of object recognition the animals treated with N-NANG did not recognize the new object. For the long-term memory there was learning only for the mice treated with NB. The mice treated with NB + STZ had memory impairment assessed in the task of inhibitory avoidance, and pre-treatment with N-NANG prevented this effect. In the evaluation of the anti-depressive and anxiolytic-type effects, it was observed that the NNANG + STZ induced greater body activity in the twist parameter in the tail suspension task and in the high "zero" labyrinth there was an increase in the number of spies Relative to the free drug. Regarding the levels of brain antioxidants, there were no changes in the levels of reduced glutathione (GSH) and catalase activity (CAT). The NB + STZ group significantly increased levels of brain lipid peroxidation of thiobarbituric acid reactive species (TBA). N-NANG administration reduced the peroxidation induced by STZ. Thus, it can be concluded that the CNs present characteristics suitable for oral administration. In vivo studies demonstrate that pre-treatment with naringin and naringenin NC was preventative and able to reverse memory deficits, type-depressive and anxiolytic behavior caused by STZ in mice. However, this preventive effect was not observed with the flavonoids in the free form. / As espécies reativas (ERs) e o estresse oxidativo contribuem para a fisiopatologia de diversas doenças entre elas a demência. Uma alternativa inovadora de proteção aos órgãos como o cérebro é o uso de flavonoides com ação antioxidante como a naringina e naringenina. No entanto, esses dois compostos apresentam baixa biodisponibilidade quando administrados pela via oral. Uma alternativa para contornar esta limitação é o uso de nanocarreadores, como as nanocápsulas (NC). Diante disso, o presente estudo teve por objetivo avaliar o efeito neuroprotetor de NC contendo naringina e naringenina em modelo de demência induzida por estreptozotocina (STZ) em camundongos. Foram avaliados o perfil de liberação dos fármacos a partir das suspensões in vitro assim como determinados os efeitos dos fármacos nanoencapsulados na viabilidade celular com linhagem de células Vero. Além disso, foi verificado o envolvimento do estresse oxidativo no tecido cerebral dos camundongos. As NC de naringina e naringenina apresentaram pH ácido, diâmetro de partícula inferior a 95 nm, índice de polidispersão (IPD) menor que 0,2. O potencial zeta foi negativo, entre -12,46 e -17,6 mV e a eficiência de encapsulação foi de 93% para naringina e 95% para a naringenina. Para o estudo do perfil de liberação, foi empregada a técnica de diálise, sendo que 46,5% da naringina e 6,9% da naringenina foram liberadas no meio, em um período de 9h. Os resultados do ensaio de viabilidade celular mostraram que as suspensões de NC causaram redução da viabilidade celular das células Vero nas concentrações de 5, 50 e 500 μg/mL quando comparadas ao controle nos períodos de 24 e 72h de incubação. Para avaliação in vivo da neuroproteção os camundongos foram pré-tratados durante 15 dias via oral (v.o) com a suspensão de nanocápsulas brancas (NB), NC contendo a mistura naringina e naringenina (N-NANG), suspensão de naringina e naringenina na forma livre (NANG) e veículo (Sham) na dose 10 mg/kg corporal. Subsequentemente foi realizada a cirurgia estereotáxica para a infusão via intracerebroventricular (i.c.v) de STZ ou FAC (fluido artificial cerebral). Posteriormente foram iniciadas as tarefas comportamentais. Na tarefa do campo aberto a atividade locomotora diferiu na seção teste no grupo NB+STZ evidenciada por uma hiperlocomoção entre os camundongos deste grupo experimental. Para atividade exploratória não houve diferença significativa entre os grupos. Na avaliação da memória de curto prazo na tarefa de reconhecimento de objetos os animais tratados com N-NANG não reconheceram o objeto novo. Para a memória de longo prazo houve aprendizagem somente para os camundongos tratados com NB. Os camundongos tratados com NB+STZ tiveram prejuízo de memória avaliado na tarefa da esquiva inibitória, e o pré-tratamento com N-NANG preveniu esse efeito. Na avaliação do efeito tipo anti-depressivo e tipo-ansiolítico observa-se que as N-NANG+STZ induziram maior atividade corporal no parâmetro torções na tarefa da suspensão da cauda e no labirinto em “zero” elevado houve um aumento do número de espiadas em relação ao fármaco livre. Com relação aos níveis de antioxidantes cerebrais não houve alterações nos níveis de Glutationa reduzida (GSH) e na atividade da catalase (CAT). O grupo NB+STZ aumentou significativamente os níveis de peroxidação lipídica cerebral de espécies reativas ao ácido tiobarbitúrico (TBA). Já administração de N-NANG reduziu a peroxidação induzida pela STZ. Com isso pode-se concluir que as NC apresentam características adequadas para administração via oral. Os estudos in vivo demonstram que o pré- tratamento com as NC de naringina e naringenina foi preventivo e capaz de reverter os déficits de memória, comportamento tipo-depressivo e ansiolítico causado pela STZ nos camundongos. Contudo, esse efeito preventivo não foi observado com os flavonoides na forma livre.

Philosophiae Doctor - PhD / Typha capensis (T. capensis), commonly known as bulrush, is a medicinal plant found growing in the wetland areas of South Africa. In traditional medicine, rhizome decoctions of T. capensis are used to treat a wide variety of ailments, including venereal disease, dysentery, diarrhoea and low libido in men. Previously, T. capensis rhizomes were shown to be a rich source of antioxidants, such as catechin and epicatechin, inhibiting both reactive oxygen species and reactive nitrogen species. The antioxidant capacity of such plant species serves as a reservoir of electrons to transport them into gold salt for the production of gold nanoparticles through green nanotechnology. Therefore, this study aimed to investigate the application of T. capensis in green nanotechnology and nano-medicine.

A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/μL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/ or in small tissue samples and requires only 100 mg of tissue.

Flavonoids play crucial roles in plant life cycles and in human welfare. Grapefruit plants produce several glycosylated flavonoids and the glycosylation reactions are catalyzed by UDP-glucose: glucosyltransferases (GTs). Our objective was to use the SMART RACE RT-PCR strategy to obtain cDNA sequences of putative grapefruit flavonoid GTs. Gene specific primers were designed from the plant secondary product glucosyltransferase (PSPG) box and used to amplify 5’GT clones. Clone-specific primers were designed from 5’clones to amplify 3’ GT clones. We obtained 5 5’ clones and 1 3’ clone as candidate GTs. We used 3 of these clones to predict 2 compiled GT sequences. Structural comparison of the putative GTs with functionally known GTs from other plants showed an overall low pair-wise sequence identity (11-31%), but high identity (52.2-75%) within the PSPG box. We conclude that assignment of function from sequence information is not appropriate; assignment should depend on biochemical characterization of expressed GT enzymes.