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In dentistry, repair and restoration of tooth structure to regain its mechanical properties is the ultimate aim of caries management. Currently, preventive therapies are preferred to operative interference. New preventive approaches are necessary to treat dental caries. Dentine is composed of an organic matrix, consisting of type 1. Degradation of collagen matrix is the main reason for breakdown of resin-dentine bonds and development of secondary caries over time. Hence, preservation of collagen fibrils is essential for preventive and reparative procedures in minimum intervention dentistry. Proanthocyanidin (PA) is a natural collagen cross-linker, which can be obtained from grape seeds, pine bark or elm tree. It has a high affinity to proline-rich proteins, like collagen. Cross-linking of the collagen fibrils increases their mechanical properties and their resistance to breakdown by proteolytic enzymes. Thus, in the first part of the study, with the aim of discovering other possible natural dentine collagen cross-linkers, the effect of two smaller molecular size flavonoids, naringin and quercetin, on the mechanical properties of demineralized dentine was compared to PA. Demineralized dentine treated with PA showed the greatest increase in mechanical properties, followed by quercetin and naringin. The protease inhibitory effect of PA was evaluated by examining its actions on soluble and collagen-bound matrix metalloproteinases and cysteine cathepsins. Proanthocyanidin, even at low concentration of 1%, exhibited excellent inhibitory effects on soluble and matrix-bound proteases. Secondly, PA was incorporated in a dental adhesive to facilitate its application in clinical situations. The effect of PA incorporation on durability of resin-dentine bond was evaluated. Up to 2% of PA could be added to dental adhesive with no adverse effects on immediate resin-dentine bond strength. However, the bond strengths of PA-incorporated adhesives dropped significantly following ageing. This could be attributed to the free radical scavenging effect of PA, which might also have interfered with polymerization of dental adhesive. The mechanical properties of PA-incorporated adhesive were evaluated and it was shown that up to 1% PA could be incorporated into an adhesive resin. With the addition of higher concentration of PA, the mechanical properties of the adhesive resin were reduced with increased solubility. Proanthocyanidin release from the cured resin showed an initial burst for 48 hours and was stabilized after five days. Finally, the remineralization potential of PA on artificial root caries was also compared with quercetin and naringin. All three flavonoids showed remineralization potential; however, their effects were inferior to fluoride. Proanthocyanidin formed a precipitate band on the superficial layer of carious lesion, preventing further mineral deposition. Subsequently, PA was incorporated in a CPP-ACFP (casein phosphoproteins amorphous calcium fluorophosphates) containing-paste to increase the mineral uptake in subsurface layer of caries lesion. Simultaneous application of PA and CPP-ACFP was shown to have a favourable outcome on mineral deposition in root caries lesion. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

A new leuco-anthocyanidin, (+)-7,4'dihydroxyflavan- 3:4-diol has been isolated from the heartwood of Guibourtia coleosperma. The heartwood extractives were fractionated by enrichment procedures involving fractional precipitation and Craig partitioning, to give a high RF (subscript) low molecular weight fraction containing the above leuco -anthocyanidin. This was further fractionated by "preparative" paper chromatography . The leuco-anthocyanidin was amorphous and present in low proportion (0.004%) in the wood. On boiling with alcoholic hydrochloric acid the compound gave an anthocyanidin which was identified as 3,7,4'-trihydroxyflavylium chloride. The degradation products formed by alkali fusion, on a micro-scale, were resorcinol and ♭-resorcylic acid, and also p (underscore)-hydroxybenzoic acid. This indicated resorcinol and phenol A and B nuclei respectively. The formation of amorphous dimethyl ether and tetra-acetoxy derivatives indicated two phenolic and two alcoholic hydroxyl groups. The above indicates that the compound was 7,4'-dihydroxyflavan- 3:4- diol and a molecular weight estimation showed it to be monomeric. Comparison of the infrared absorption spectra of the natural dimethyl ether with synthetic (±) dimethoxyflavan- 3:4-diol, to which a tentative 2:3- trans- 3:4-cis conformation had been assigned (Phatak and Kulkarni 94), showed close identity. Crystalline (±)-7, 4 '-dihydroxyflavan- 3:4 - diol was synthesised by catalytic hydrogenation over platinum oxide of the corresponoing (±) -7,4'-dihydroxyflavanonol. The flavanonol was synthesised by sodium hyposulphite reduction of the 7,4 ' -dihydroxyflavonol. The infrared absorption spectra of the natural and synthetic diols were similar but not identical. Chromatographic evidence showed the apparent identity of the synthetic and natural flavan- 3:4- diols, and two possible configurations were assigned for the natural flavan-3:4-diol. The new leuco-anthocyanidin was observed to form an O(underscore)-ethyl ether derivative on manipulation in ethanol. A monomeric leuco- fisetinidin from the heartwood was identified, by two dimensional paper chromatography, as (+) - mollisacacidin. The condensed tannins found also in the heartwood of G.(underscore)- coleosperma appear to be polymeric forms of leuco - fisetinidin and leuco - guibourtinidin. These polymeric tannins form a large proportion of the heartwood extractives and furnish high yields of fisetinidin and guibourtinidin chlorides on boiling with alcoholic hydrochloric acid. Examination of some other members of the Guibourtia spp . by paper chromatography showed that only G (underscore). coleosperma contained the new leuco - anthocyanidin as well as a leuco - fisetinidin. G. (underscore) tessmannii and G. (underscore) demeusii heartwoods contained only leuco-fisetinidin and the related polymers and a close chemical relationship to G.(underscore) coleosperma is thus apparent. G.(underscore) arnoldiana on the other hand is not chemically interrelated with the above members since it appeared to contain only leuco - cyanidin. (±) - 7,4 ' -Dihydroxyflavan- 4-ol was synthesised by hydrogenation of the corresponding flavanone over platinum oxide. Observations were made regarding its reddenig and ease of condensation.

The anthocyanins show efficient antioxidant properties and free radical scavenging properties which result in various health-promoting benefits. This research investigated the ability of anthocyanidins to distribute into mitochondria and protect mitochondria from oxidative stress. In an in vitro study, the uptake of pure cyanidin and quercetin, and their 3-glucosylated forms into isolated rat liver mitochondria was tested, along with their effects on mitochondrial oxidative stress parameters. The absorption of cyanidin was significantly higher (67% uptake of 125 µM) than the other three flavonoids. Measurements indicated that the cyanidin was taken up into or tightly bound by mitochondria. Also, results suggested that cyanidin uptake was partially dependent on membrane potential. When incubated together (internally and externally) with mitochondria all tested flavonoids decreased reactive oxygen species (ROS) generation during mitochondrial respiration, and inhibited lipid peroxidation to different extents. Importantly, pre-loaded CY showed much stronger effects against oxidative stress in two analyses than other flavonoids. Due to its greater uptake by mitochondria, cyanidin may provide greater protection in vivo. In an in vivo study, cyanidin, quercetin and their 3-glucosides were administered into rat tail vein to give a dose of 7.6 µmol/Kg body weight. Cyanidin and its glucoside had greater affinity to liver and kidney than did quercetin and its glucoside; particularly, all test tissues contained a significantly higher amount of cyanidin than other test flavonoids. Also, cyanidin accumulated more in liver mitochondria than other flavonoids, and consistent with in vitro results was present in mitochondria to a much greater extent than cyanidin glucoside. However, delivery of the flavonoids at this dose did not significantly affect the liver mitochondria susceptibility to lipid peroxidation or the level of endogenous tissue oxidative damage. Altogether the results show that cyanidin can rapidly and efficiently accumulate in mitochondria, wherein it exhibits strong bio-antioxidant activity against oxidative stress and may help protect mitochondrial function and integrity. Also, the anthocyanidin and its 3-glucoside have greater ability than flavonols to accumulate in organs; especially cyanidin presented in liver mitochondria to a much greater extent. Cyanidin could be a potent natural antioxidant compound that is effective in mitochondria-protective therapies.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 55-60). Also available via the World Wide Web.

Call number: LD2668 .T4 1961 A43

The principle calcium regulator in the muscle cell is the calcium ion release channel (RyR). Improper calcium homeostasis in the muscle cell is the foundation of many pathological states and has been targeted as a contributing factor to ventricular tachycardia, which is known to precede sudden cardiac arrest. Numerous endogenous and exogenous compounds can affect the way RyR regulates calcium. In this study the anthocyanidin Pelargonidin (Pg), an important natural colorant and dietary antioxidant, is evaluated for its effect on regulating the transport of calcium through the RyR1 of skeletal muscle sarcoplasmic reticulum. Pelargonidin undergoes time dependent structural changes in aqueous solutions at physiological pH and a mixture of up to seven forms of Pelargonidin are present in solution simultaneously. Pelargonidin is a unique RyR1 modulator. It can both stimulate and inhibit the RyR1 depending on the experimental conditions. In addition, when Pelargonidin is irradiated with white light, its inhibition properties on the RyR1 are essentially nullified. Proposed mechanisms include excited state charge shift within RyR1-Pg complexes.

Diversas características interferem na aceitabilidade de variedades de feijoeiro (Phaseolus vulgaris L.) no mercado consumidor, como o brilho do tegumento. Geralmente, o feijão com brilho é rejeitado para consumo, pois apresenta difícil cozimento. No entanto, o brilho pode conferir resistência ao ataque de insetos e patógenos à semente, podendo constituir uma vantagem para o melhoramento do feijão. A presença de brilho é devida à expressão do gene Asp (Asper) na forma alélica dominante. Genótipos aspasp, portanto, apresentam tegumento opaco. Outro gene, J (Joker), interfere na expressão da característica, de modo que o alelo recessivo j diminui a intensidade do brilho e altera a coloração do tegumento. J é considerado o precursor de polifenois denominados pró-antocianidinas. Na presença j estes compostos não são sintetizados. No presente trabalho foram genotipadas variedades de feijoeiro crioulas [Serro Azul Brilhante (SAB - tegumento brilhante) e Fosco (SAF - tegumento opaco) e Puebla-152 (P-152 - brilhante)] e a cultivar Diamante Negro (DN - opaco), contrastantes para o brilho do tegumento. Análises fenotípicas, morfológicas e bioquímicas permitiram inferir os genótipos das variedades e avaliar a segregação do brilho em populações derivadas de cruzamentos entre contrastantes: SAF x SAB e P-152 x DN. Os resultados mostraram que as variedades não variam para J, apenas para Asp, sendo que SAB e P-152 carregam Asp e SAF e DN o alelo asp. Em todos os cruzamentos a segregação foi de três brilhantes (Asp) para um opaco (asp) na geração F2 e de cinco brilhantes para três opacos em F3. A partir dos resultados, as variedades foram contrastadas por meio de marcadores de AFLP, analisando-se a similaridade genética e identificando-se bandas diferenciadoras entre as mesmas. A partir disso, foi empregada a metodologia de Bulk Segregant Analysis na população F2 de P-152 x DN, para encontrar marcadores AFLP candidatos ao mapeamento do brilho. Foram encontrados marcadores associados que, no entanto, revelaram serem falsos positivos. Análises utilizando marcadores microssatélites ou análises bioinformáticas de sintenia com a soja poderiam ser realizadas, visando detectar marcadores precisamente ligados à característica / Several characteristics interfere in acceptability of common bean varieties (Phaseolus vulgaris L.) by consumers, like seed coat shininess. Generally, varieties with shiny seed coat are rejected by consumers due to their difficult cooking. However, shininess could confer resistance to insects attak and also pathogens to the seed, giving an advantage for this trait in common bean breeding. The presence of shininess is due the expression of the Asp (Asper) gene in the dominant allelic form. Other gene, J (Joker), interferes on the expression of this characteristic, with j conditioning a less shiny seed coat and changing seed coat color. J is considered the precursor of a class of polyfenols called pro-anthocyanidins. In the presence of j these compounds are not synthesized. In the present work we genotyped common bean landraces [Shiny Serro Azul (SAB shiny seed coat) and Dull Serro Azul (SAF dull seed coat) and Puebla-152 (P-152 shiny)] and the cultivar Diamante Negro (DN dull), contrasting for seed coat shininess. Phenotypic, morphological and biochemical analyses allowed to genotype all varieties and evaluate the segregation of shinines in populations derived from crosses between contrasting genotypes: SAF x SAB and P-152 x DN. Results showed that varieties do not vary for J, only for Asp. SAB and P-152 carry Asp and SAF and DN carry asp. In all crosses segregation was of three shiny (Asp) for one opaque (asp) in F2 and of five shiny for three dull in F3. Based on the results, all varities were contrasted by AFLP markers, analyzing genetic similarity and identifying differencial bands among all of them. Further it was used the Bulk Segregant Analysis method in population F2 of the cross P-152 x DN, aiming to find candidate AFLP markers for shininess mapping. We found markers associated but they revealed to be false positives. Further analyses using microsatellite markers or sinteny comparisons with soybean genome could be done, aiming to find specific markers linked to this trait

Diversas características interferem na aceitabilidade de variedades de feijoeiro (Phaseolus vulgaris L.) no mercado consumidor, como o brilho do tegumento. Geralmente, o feijão com brilho é rejeitado para consumo, pois apresenta difícil cozimento. No entanto, o brilho pode conferir resistência ao ataque de insetos e patógenos à semente, podendo constituir uma vantagem para o melhoramento do feijão. A presença de brilho é devida à expressão do gene Asp (Asper) na forma alélica dominante. Genótipos aspasp, portanto, apresentam tegumento opaco. Outro gene, J (Joker), interfere na expressão da característica, de modo que o alelo recessivo j diminui a intensidade do brilho e altera a coloração do tegumento. J é considerado o precursor de polifenois denominados pró-antocianidinas. Na presença j estes compostos não são sintetizados. No presente trabalho foram genotipadas variedades de feijoeiro crioulas [Serro Azul Brilhante (SAB - tegumento brilhante) e Fosco (SAF - tegumento opaco) e Puebla-152 (P-152 - brilhante)] e a cultivar Diamante Negro (DN - opaco), contrastantes para o brilho do tegumento. Análises fenotípicas, morfológicas e bioquímicas permitiram inferir os genótipos das variedades e avaliar a segregação do brilho em populações derivadas de cruzamentos entre contrastantes: SAF x SAB e P-152 x DN. Os resultados mostraram que as variedades não variam para J, apenas para Asp, sendo que SAB e P-152 carregam Asp e SAF e DN o alelo asp. Em todos os cruzamentos a segregação foi de três brilhantes (Asp) para um opaco (asp) na geração F2 e de cinco brilhantes para três opacos em F3. A partir dos resultados, as variedades foram contrastadas por meio de marcadores de AFLP, analisando-se a similaridade genética e identificando-se bandas diferenciadoras entre as mesmas. A partir disso, foi empregada a metodologia de Bulk Segregant Analysis na população F2 de P-152 x DN, para encontrar marcadores AFLP candidatos ao mapeamento do brilho. Foram encontrados marcadores associados que, no entanto, revelaram serem falsos positivos. Análises utilizando marcadores microssatélites ou análises bioinformáticas de sintenia com a soja poderiam ser realizadas, visando detectar marcadores precisamente ligados à característica / Several characteristics interfere in acceptability of common bean varieties (Phaseolus vulgaris L.) by consumers, like seed coat shininess. Generally, varieties with shiny seed coat are rejected by consumers due to their difficult cooking. However, shininess could confer resistance to insects attak and also pathogens to the seed, giving an advantage for this trait in common bean breeding. The presence of shininess is due the expression of the Asp (Asper) gene in the dominant allelic form. Other gene, J (Joker), interferes on the expression of this characteristic, with j conditioning a less shiny seed coat and changing seed coat color. J is considered the precursor of a class of polyfenols called pro-anthocyanidins. In the presence of j these compounds are not synthesized. In the present work we genotyped common bean landraces [Shiny Serro Azul (SAB shiny seed coat) and Dull Serro Azul (SAF dull seed coat) and Puebla-152 (P-152 shiny)] and the cultivar Diamante Negro (DN dull), contrasting for seed coat shininess. Phenotypic, morphological and biochemical analyses allowed to genotype all varieties and evaluate the segregation of shinines in populations derived from crosses between contrasting genotypes: SAF x SAB and P-152 x DN. Results showed that varieties do not vary for J, only for Asp. SAB and P-152 carry Asp and SAF and DN carry asp. In all crosses segregation was of three shiny (Asp) for one opaque (asp) in F2 and of five shiny for three dull in F3. Based on the results, all varities were contrasted by AFLP markers, analyzing genetic similarity and identifying differencial bands among all of them. Further it was used the Bulk Segregant Analysis method in population F2 of the cross P-152 x DN, aiming to find candidate AFLP markers for shininess mapping. We found markers associated but they revealed to be false positives. Further analyses using microsatellite markers or sinteny comparisons with soybean genome could be done, aiming to find specific markers linked to this trait

Cardiovascular disease (CVD) claims more lives than any other disease in the world. Although numerous biological pathways share the blame, ventricular tachyarrhythmia (VT) is estimated to account for ~25% of all CVD deaths. A complete understanding of the molecular mechanisms underlying VT is unknown but recent studies have linked VT to improper calcium handling in the heart (canine). The principle calcium regulator in the muscle cell is the calcium ion release channel (aka RyR). Numerous endogenous and exogenous compounds can affect the way the RyR regulates calcium. In particular, abnormal levels of oxidants (reactive oxygen species) can oxidize critical thiol groups on the RyR and modulate its activity. Interestingly, high levels of oxidants are also associated with numerous bodily disease states including cancers, muscle fatigue/failure, and CVD. In this thesis, two important dietary antioxidant compounds, the anthocyanidins pelargonidin and delphinidin, are evaluated for their effects on regulating the transport of calcium through the calcium release channel (RyR1) of the sarcoplasmic reticulum of skeletal muscle. Pelargonidin and delphinidin are structurally similar with delphinidin only differing from pelargonidin by the addition of two hydroxyl groups. Both compounds undergo time dependent structural changes in aqueous solutions at physiological pH and a mixture of more than four structures of each compound can be present in solution simultaneously. Pelargonidin and delphinidin show distinct differences in their calcium flux regulating effect on the RyR1. Delphinidin stimulates calcium flux and RyR1 activity where as pelargonidin can cause both inhibition and stimulation of the RyR1. The strength of stimulation and inhibition of calcium transport through the RyR by delphinidin and pelargonidin may be attributed to the structural and chemical changes in those compounds that occur in solutions near physiological pH and the subsequent chemical characteristics of the diverse set of structures that are simultaneously present in solution.