Cranberry Proanthocyanidins may be the constituents responsible for the association between consumption of cranberry juice and decreased risks of urinary tract infections because these compounds prevent the adhesion of uropathogenic p-fimbriated E. coli to uroepithelial cells in vitro. Proanthocyanidins are oligomeric flavonoids present in many botanicals and nutritional supplements. However, cranberry proanthocyanidins exhibit structural heterogeneity in the degree of polymerization, nature of interflavan bonds (A and B type), pattern of hydroxylation of flavan units and substitution with anthocyanins. This structural heterogeneity creates difficulty in structure to bioactivity. Biomedical research on the health benefits and risks of increased consumption of cranberry proanthocyanidins is severely limited by lack of information on structure/bioactivity relationships that can be used in standardization of cranberry products. Our core hypothesis is that the bioactivity of cranberry proanthocyanidin oligomers is a function of specific structure. Therefore, given the large diversity of individual oligomers, we would expect large variation in bioactivity. The goals of this proposal are: 1. Characterize structural heterogeneity (i.e. intra-molecular bonds, nature of the substitutions and degree of polymerization) of proanthocyanidin structures in the standardized NIH-NCCAM cranberry products using online and offline liquid chromatographic separations coupled with mass spectrometry, and 2. Relate the structural characteristics of the proanthocyanidin fractions to bioactivity in cell culture and in vitro models of inflammation, bacterial adherence and oxidation. We will carry out 3 types of experiments to characterize and prepare cranberry proanthocyanidins for assays of bioactivity; 1, experiments to optimize online and offline chromatography, 2, chemical degradation studies coupled with mass spectrometry of products to rapidly characterize structure, and 3, fragmentation studies using tandem mass spectrometry. We will then assay proanthocyanidin fractions for bioactivity using 3 methods; 1. Attenuation of LPS induced expression of cycloxygenase 2 in macrophages, 2. In vitro model of adherence of p-fimbriated E. coli to uroepithelial cells, and 3. specific association of proanthocyanidins to low density lipoproteins and inhibition of copper induced oxidation. ? ? ?
