Chemical and Biological Methods to Characterize Cranberry Proanthocyanidins: Relevance to Urinary Tract Health

Urinary tract infections (UTI) are one of the most frequent extra-intestinal infections caused by Escherichia coli (ExPEC). Cranberry juice has been used for decades to alleviate symptoms and prevent recurrent UTI. The putative active compounds in cranberries are proanthocyanidins (PAC), specifically PAC with "A-type" interflavan bonds. The development of a standard isolated from cranberry was pursued to more accurately estimate PAC content in cranberry products using the 4-(dimethylammino) cinnamaldeyde (DMAC), rather than using commercially available standards. Despite being fast, inexpensive and convenient, the DMAC method conveys the same PAC content to products that have different ratios of "A-type" to "B-type" interflavan bonds. Matrix assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the most powerful technique to study PAC. MALDI-TOF MS was applied to deconvolute PAC isotope patterns with "A-type" and "B-type" interflavan bonds in order to quantify the percentages of each type of bond per degree of polymerization (DP). The method was validated using standards and applied to cranberry PAC. Since PAC are not absorbed, their health benefits in UTI may occur through interactions at the mucosal surface in the gut. Two novel in vitro methods were developed: 1) agglutination of ExPEC and 2) inhibition of ExPEC invasion on Caco-2 cells. As a proof of concept the effect of PAC from cranberries ("A-type") with PAC from apples ("B-type") was compared and "A-type" were significantly more bioactive then "B-type" PAC. A "green chemistry" extraction method (supercritical fluid extraction, SFE), with potential to be scaled-up, was developed. The ratios of "A-type" to "B-type" interflavan bonds and PAC DP in fractions prepared with and without SFE were compared. In addition, these fractions were tested for biological activity by ExPEC agglutination and invasion in a Caco-2 cell model and greater bioactivity was associated with higher DP and higher number of "A-type" bonds for the SFE sample. Fluorescent derivatization was used to label cranberry PAC. Reaction of PAC with ([5-(4,6-dichlorotriazin-2-yl-amino)-fluorescein] in alkaline media generated fluorescent PAC which were detected and structurally characterized by MALDI-TOF MS. Fluorescent labeled PAC were mixed with ExPEC and agglutination with fluorescence emission was detected by microscopy.