Flavonoids are plant secondary metabolites that are integral to our lives. Grapefruits are well-known for production of unique glucosylated products and the enzymes responsible are UDP-glucose:glucosyltransferases (GTs). The objective of this research was to obtain full-length clones of putative grapefruit GTs, express them, and characterize them. Previously, gene specific primers (from conserved PSPG box) and clone specific primers (from partial 5' clones) were designed, and a compiled sequence attained using SMART RACE RT-PCR. A full-length clone was obtained using primers designed from the extreme ends of the compiled sequence. The full-length clone was inserted into expression vector (pET32a) and transformed into expression host BL21(DE3)RIL. Expressed protein was tested for GT activity using different flavonoid aglycones and UDP-14C-glucose as glucose donor. Results indicated that the expressed protein was probably not a flavonoid GT. A directionally cloned grapefruit leaf cDNA library is undergoing EST mining to identify additional GT candidates.

Atualmente, devido ao conhecimento dos danos causados pela exposição da pele à radiação UV, existe uma tendência em utilizar, além dos filtros solares convencionais, substâncias naturais com potencial antioxidante. Entretanto, o uso de associações fotoinstáveis, pode, além de comprometer a capacidade fotoprotetora, levar à formação de intermediários reativos que podem ocasionar dermatites de contato e reações fototóxicas na pele. Dessa forma, o objetivo deste trabalho foi avaliar a fotoestabilidade e fototoxicidade de formulações fotoprotetoras contendo diferentes associações de filtros solares acrescidas ou não dos polifenóis mangiferina e naringenina. Para o estudo de fotoestabilidade, amostras das formulações foram aplicadas em lâminas de vidro e expostas à radiação UVA e, a seguir, foram feitas análises por cromatografia líquida de alta eficiência (CLAE), para dosagem do teor de filtros solares e antioxidantes, e por espectrofotometria, para determinação da razão UVA/UVB. A fototoxicidade foi avaliada por meio do uso de cultura de fibroblastos 3T3, submetida ou não à radiação UVA, para determinação da viabilidade celular. Os resultados de fotoestabilidade por CLAE demonstraram que a naringenina, o metoxicinamato de etilexila e avobenzona foram considerados fotoinstáveis. Além disso, foi demonstrado que a adição de mangiferina à associação contendo avobenzona e naringenina, proporcionou um aumento da fotoestabilidade das mesmas. A presença do dietilamino hidroxibenzoil hexil benzoato (DHHB) na formulação contendo avobenzona e naringenina promoveu melhora da fotoestabilidade da naringenina, no entanto não houve melhora da fotoestabilidade da avobenzona. As análises espectrofotométricas demonstraram que as formulações contendo a associação de avobenzona e DHHB (associação 3) apresentaram uma proteção no UVA superior às demais associações, ou seja, que continham avobenzona e DHHB isoladamente. O estudo de fototoxicidade mostrou que a avobenzona e o DHHB apresentaram potencial fototóxico. Quando as associações de filtros solares e antioxidantes foram analisadas, foi observado que a associação de avobenzona e naringenina, combinada ou não ao DHHB, apresentou potencial fototóxico. Dessa forma, esta não é recomendada para o desenvolvimento de formulações fotoprotetoras. A associação DHHB / avobenzona, por apresentar elevada proteção UVA, pode ser vantajosa para o desenvolvimento de fotoprotetores, entretanto a sua associação com antioxidantes deve ser utilizada com cautela e analisada caso a caso. A naringenina não é indicada para fotoprotetores contendo avobenzona, uma vez que esta associação foi considerada fotoinstável e fototóxica. Já a utilização da mangiferina pode ser considerada mais segura, e, além disso, a sua elevada atividade antioxidante pode complementar a eficácia de fotoprotetores. / Nowadays, due to the knowledge of UV-induced skin damage, there is a tendency to use natural substances with antioxidant potential, beyond conventional UV-filters. However, the use of photounstable combinations can compromise the photoprotective capacity and lead to formation of reactive intermediates that can cause contact dermatitis and phototoxic skin reactions. Thus, the objective of this study was to evaluate the photostability and phototoxicity of sunscreen formulations containing different UV-filter combinations supplemented or not with the polyphenols mangiferin and naringenin. For photostability studies, samples of the formulations were spread onto glass plates, exposed to UVA radiation and then analyzed by HPLC to determine the UV-filters and antioxidants concentrations, and also by spectrophotometry to determine the UVA/UVB ratio. Phototoxicity was evaluated by using the 3T3 fibroblast cultures, which was exposed to UVA radiation for the determination of cell viability. The results of photostability by HPLC analysis showed that naringenin, ethylhexyl methoxycinnamate and avobenzone were considered photounstable. Furthermore, it was demonstrated that the addition of mangiferin combined with avobenzone and naringenin, provided an increase in their photostability. The presence of DHHB in the formulation containing avobenzone and naringenin promoted an improvement in naringenin photostability, what was not observed for avobenzone. The spectrophotometric analysis showed that the formulations containing the combination of avobenzone and DHHB (combination 3) presented higher protection against UVA than the other combinations, i.e. the combinations containing avobenzone and DHHB, separately. The phototoxicity study showed that avobenzone and DHHB presented phototoxic potential. When UV-filters and antioxidants combinations were analyzed, it was observed that the combination of avobenzone and naringenin, combined or not with DHHB, presented phototoxic potential. Therefore, this is not recommended for the development of sunscreens. The combination DHHB / avobenzone, due to its high UVA protection, may be suitable on the development of sunscreens, however its combination with antioxidants should be used with caution and analyzed case-by-case. Naringenin is not indicated for sunscreens containing avobenzone, since this combination was considered photounstable and phototoxic. Moreover, the use of mangiferin can be considered safer than naringenin and, besides that, its high antioxidant activity can improve the photoprotective effects of sunscreens.

P-glycoprotein (Pgp), which leads to multidrug resistance in tumour cells, is an ATP-dependent secretory drug efflux pump. In the intestine, as well as at specific other epithelial and endothelial sites, P-glycoprotein expression is localised to the apical membrane, consistent with secretory detoxifying and absorption limitation functions. The primary function of Pgp is to clear the membrane lipid bilayer of lipophilic drugs. Results from in vitro studies with human Caco-2 cells provide direct evidence for Pgp limiting drug absorption. Limitation has non-linear dependence of absorption on substrate (eg. vinblastine) concentration, increased absorption upon saturation of secretion and increased absorption upon inhibition of Pgp function, with modulators such as verapamil. The aim of this study was to investigate the effect of a known Pgp inhibitor (verapamil) and grapefruit juice components (naringenin, quercetin and bergamottin) on the transport of Rhodamine 123 across rat jejunum and to compare these results with those obtained in similar studies done in Caco-2 cells and in rat intestine (monodirectional). Verapamil, naringenin (442 µM, 662 µM and 884 µM), quercetin (73 µM, 183 µM and 292 µM) and bergamottin (12 µM, 30 µM and 48 µM) were evaluated as modulators of rhodamine 123 transport across rat jejunum using Sweetana-Grass diffusion cells. This study was done bidirectionally, with three cells measuring transport in the apical to basolateral direction (AP / BL) and three cells measuring transport in the basolateral to apical direction (BL / AP). The rate of transport was expressed as the apparent permeability coefficient (Papp) and the extent of active transport was expressed by calculating the ratio of BL/AP to AP/BL. The BL-AP/AP-BL ratio calculated for Rhodamine 123 with no modulators added was 2.31. The known modulator verapamil decreased the BL-AP/AP-BL ratio to 1.52. This was statistically significant and inhibition of active transport was clearly demonstrated. All modulators inhibited active transport. Only naringenin 884 µM, quercetin 183 µM and bergamottin 30 µM did not show a statistically significant decrease in the BL-AP/AP-BL ratio. All three components of grapefruit juice showed inhibition of active transport and should have an effect on the bioavailability of the substrates of Pgp and other active transporters. The results obtained in this study are similar to the results found in Caco-2 cells, which suggests that Sweetana-Grass diffusion method can be used for diffusion studies. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

P-glycoprotein (Pgp), which leads to multidrug resistance in tumour cells, is an ATP-dependent secretory drug efflux pump. In the intestine, as well as at specific other epithelial and endothelial sites, P-glycoprotein expression is localised to the apical membrane, consistent with secretory detoxifying and absorption limitation functions. The primary function of Pgp is to clear the membrane lipid bilayer of lipophilic drugs. Results from in vitro studies with human Caco-2 cells provide direct evidence for Pgp limiting drug absorption. Limitation has non-linear dependence of absorption on substrate (eg. vinblastine) concentration, increased absorption upon saturation of secretion and increased absorption upon inhibition of Pgp function, with modulators such as verapamil. The aim of this study was to investigate the effect of a known Pgp inhibitor (verapamil) and grapefruit juice components (naringenin, quercetin and bergamottin) on the transport of Rhodamine 123 across rat jejunum and to compare these results with those obtained in similar studies done in Caco-2 cells and in rat intestine (monodirectional). Verapamil, naringenin (442 µM, 662 µM and 884 µM), quercetin (73 µM, 183 µM and 292 µM) and bergamottin (12 µM, 30 µM and 48 µM) were evaluated as modulators of rhodamine 123 transport across rat jejunum using Sweetana-Grass diffusion cells. This study was done bidirectionally, with three cells measuring transport in the apical to basolateral direction (AP / BL) and three cells measuring transport in the basolateral to apical direction (BL / AP). The rate of transport was expressed as the apparent permeability coefficient (Papp) and the extent of active transport was expressed by calculating the ratio of BL/AP to AP/BL. The BL-AP/AP-BL ratio calculated for Rhodamine 123 with no modulators added was 2.31. The known modulator verapamil decreased the BL-AP/AP-BL ratio to 1.52. This was statistically significant and inhibition of active transport was clearly demonstrated. All modulators inhibited active transport. Only naringenin 884 µM, quercetin 183 µM and bergamottin 30 µM did not show a statistically significant decrease in the BL-AP/AP-BL ratio. All three components of grapefruit juice showed inhibition of active transport and should have an effect on the bioavailability of the substrates of Pgp and other active transporters. The results obtained in this study are similar to the results found in Caco-2 cells, which suggests that Sweetana-Grass diffusion method can be used for diffusion studies. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Orientador: Ana Angélica Henrique Fernandes / Resumo: Introdução: A dieta ocidental rica em carboidrato e lipídio promove o desenvolvimento da síndrome metabólica. Alterações do metabolismo energético provocadas por um desbalanço redox em virtude de dieta hipercalórica, gera danos oxidativos e deficiência no sistema enzimático antioxidante, intensificando fatores de risco e eventos que levam à doença cardiovascular. Os flavonoides são compostos de origem vegetal com propriedades antioxidante e antiaterogênico. O objetivo do estudo foi avaliar as alterações metabólicas séricas e cardíacas em ratos submetidos a dieta hipercalórica e tratados com naringenina. Material e Métodos: Foram utilizados 32 ratos Wistar machos, distribuídos em 4 grupos: (C) Controle, (N) naringenina, (H) hipercalórico, (HN) hipercalórico tratado com naringenina. Os grupos (C e N, n=16) receberam dieta padrão enquanto que os grupos (H e HN, n=16) receberam dieta hipercalórica durante 30 dias. Após este período iniciou-se o tratamento com naringenina 50mg/kg (grupos N e HN) durante 43 dias à intervalos de 7 dias via intra gástrica. Ao final do experimento, os animais foram anestesiados para eutanásia. Foram coletadas duas porções de tecido cardíaco e amostra sérica para análise de perfil lipídico, glicemia, metabolismo energético, estresse oxidativo e glicogênio cardíaco. Resultados: O grupo (HN) tiveram uma diminuição na ingestão hídrica e ingestão alimentar, peso final, ganho de peso e glicemia. Não houve diferença significativa no peso inicial entre os gru... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Western diet rich in carbohydrates and lipids is an important role in metabolic syndrome development. Energy metabolism alterations caused by redox unbalance due to high fat diet, generated oxidative damage and deficiency in the antioxidant enzyme system, increasing risk factors and events that lead to cardiovascular disease. Flavonoids are natural compounds with antioxidant and antiatherogenic properties. This study assessed serum and cardiac metabolic alterations in rats fed with high fat diet and treated with naringenin. Material and Methods: We used 32 male Wistar rats, divided into 4 groups: (C) control, (N) naringenin, (H) high fat diet, (HN) high fat diet treated with naringenin. Groups (C and N, n=16) received standard diet whereas the groups (H and HN, n=16) received high fat diet during 30 days. After this period, the rats were treated with naringenin 50mg/kg (groups N and HN) during 43 days and at 7 days interval by gavage. Animals were anesthetized and led to euthanasia. Two cardiac tissue portions were collected as well as blood serum to evaluate lipid profile, glycemia, energy metabolism, oxidative stress and cardiac glycogen. Results: Group (HN) showed a decreased in water and food intake, final body weight, body weight gain and glycemia. There wasn’t significant difference in initial body weight between the groups. There was an improvement in group (HN) on lipid profile, energy metabolism and oxidative stress. There wasn’t significant difference ... (Complete abstract click electronic access below) / Mestre

Atualmente, devido ao conhecimento dos danos causados pela exposição da pele à radiação UV, existe uma tendência em utilizar, além dos filtros solares convencionais, substâncias naturais com potencial antioxidante. Entretanto, o uso de associações fotoinstáveis, pode, além de comprometer a capacidade fotoprotetora, levar à formação de intermediários reativos que podem ocasionar dermatites de contato e reações fototóxicas na pele. Dessa forma, o objetivo deste trabalho foi avaliar a fotoestabilidade e fototoxicidade de formulações fotoprotetoras contendo diferentes associações de filtros solares acrescidas ou não dos polifenóis mangiferina e naringenina. Para o estudo de fotoestabilidade, amostras das formulações foram aplicadas em lâminas de vidro e expostas à radiação UVA e, a seguir, foram feitas análises por cromatografia líquida de alta eficiência (CLAE), para dosagem do teor de filtros solares e antioxidantes, e por espectrofotometria, para determinação da razão UVA/UVB. A fototoxicidade foi avaliada por meio do uso de cultura de fibroblastos 3T3, submetida ou não à radiação UVA, para determinação da viabilidade celular. Os resultados de fotoestabilidade por CLAE demonstraram que a naringenina, o metoxicinamato de etilexila e avobenzona foram considerados fotoinstáveis. Além disso, foi demonstrado que a adição de mangiferina à associação contendo avobenzona e naringenina, proporcionou um aumento da fotoestabilidade das mesmas. A presença do dietilamino hidroxibenzoil hexil benzoato (DHHB) na formulação contendo avobenzona e naringenina promoveu melhora da fotoestabilidade da naringenina, no entanto não houve melhora da fotoestabilidade da avobenzona. As análises espectrofotométricas demonstraram que as formulações contendo a associação de avobenzona e DHHB (associação 3) apresentaram uma proteção no UVA superior às demais associações, ou seja, que continham avobenzona e DHHB isoladamente. O estudo de fototoxicidade mostrou que a avobenzona e o DHHB apresentaram potencial fototóxico. Quando as associações de filtros solares e antioxidantes foram analisadas, foi observado que a associação de avobenzona e naringenina, combinada ou não ao DHHB, apresentou potencial fototóxico. Dessa forma, esta não é recomendada para o desenvolvimento de formulações fotoprotetoras. A associação DHHB / avobenzona, por apresentar elevada proteção UVA, pode ser vantajosa para o desenvolvimento de fotoprotetores, entretanto a sua associação com antioxidantes deve ser utilizada com cautela e analisada caso a caso. A naringenina não é indicada para fotoprotetores contendo avobenzona, uma vez que esta associação foi considerada fotoinstável e fototóxica. Já a utilização da mangiferina pode ser considerada mais segura, e, além disso, a sua elevada atividade antioxidante pode complementar a eficácia de fotoprotetores. / Nowadays, due to the knowledge of UV-induced skin damage, there is a tendency to use natural substances with antioxidant potential, beyond conventional UV-filters. However, the use of photounstable combinations can compromise the photoprotective capacity and lead to formation of reactive intermediates that can cause contact dermatitis and phototoxic skin reactions. Thus, the objective of this study was to evaluate the photostability and phototoxicity of sunscreen formulations containing different UV-filter combinations supplemented or not with the polyphenols mangiferin and naringenin. For photostability studies, samples of the formulations were spread onto glass plates, exposed to UVA radiation and then analyzed by HPLC to determine the UV-filters and antioxidants concentrations, and also by spectrophotometry to determine the UVA/UVB ratio. Phototoxicity was evaluated by using the 3T3 fibroblast cultures, which was exposed to UVA radiation for the determination of cell viability. The results of photostability by HPLC analysis showed that naringenin, ethylhexyl methoxycinnamate and avobenzone were considered photounstable. Furthermore, it was demonstrated that the addition of mangiferin combined with avobenzone and naringenin, provided an increase in their photostability. The presence of DHHB in the formulation containing avobenzone and naringenin promoted an improvement in naringenin photostability, what was not observed for avobenzone. The spectrophotometric analysis showed that the formulations containing the combination of avobenzone and DHHB (combination 3) presented higher protection against UVA than the other combinations, i.e. the combinations containing avobenzone and DHHB, separately. The phototoxicity study showed that avobenzone and DHHB presented phototoxic potential. When UV-filters and antioxidants combinations were analyzed, it was observed that the combination of avobenzone and naringenin, combined or not with DHHB, presented phototoxic potential. Therefore, this is not recommended for the development of sunscreens. The combination DHHB / avobenzone, due to its high UVA protection, may be suitable on the development of sunscreens, however its combination with antioxidants should be used with caution and analyzed case-by-case. Naringenin is not indicated for sunscreens containing avobenzone, since this combination was considered photounstable and phototoxic. Moreover, the use of mangiferin can be considered safer than naringenin and, besides that, its high antioxidant activity can improve the photoprotective effects of sunscreens.

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 μM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavonoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.

Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization.

No description available.

No description available.