Cranberry fruit (Vaccinium macrocarpon Ait.) was collected at the Marucci Center for Blueberry and Cranberry Research, Chatsworth, NJ. Purified C-PAC extract was isolated from cranberries of the ‘Early Black’ cultivar utilizing solid-phase chromatography according to well established methodology [9 (link)–12 (link)]. In brief, the fruit was homogenized in 70% aqueous acetone, filtered and the pulp discarded. Collected cranberry-derived proanthocyanidins were concentrated under reduced pressure and purified extract isolated using bioassay-directed fractionation. The absence of absorption at 360 nm and 450 nm confirm all but proanthocyanidins are removed. Additional methods including 13C NMR, electrospray mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and acid catalyzed degradation with phloroglucinol were utilized to verify the presence of A-type linkages as well as to determine the concentration of proanthocyanidins in the purified extract. C-PAC is comprised of five main proanthocyanidins as previously characterized by Dr. Howell and colleagues [11 (link)]. The proanthocyanidin molecules largely consist of epicatechin units with degrees of polymerization of 4 or 5, as well as epigallocatechin and catechin. C-PAC contains three types of linkages, two common B-type linkages (C4→C6 and C4→C8) and at least one unique A-type ether linkage (C2→O→C7) found only in cranberry, chokeberry, plums and avocado [14 (link), 15 (link)]. Purified C-PAC was freeze-dried and stored at −80°C. C-PAC concentrations chosen for study were informed by our earlier research which determined the LD50 to be in the 50 to 100 μg/ml range in various cancer cell lines [16 (link)–18 (link)]. Consideration was also given to earlier evaluations by Howell and colleagues showing 50 μg/ml of C-PAC inhibits adhesion of p-fimbriated uropathogenic E. coli bacteria in vitro and that 36 mg/day of C-PAC delivered in 10 ounces of juice inhibits bacterial adhesion in the urinary tract wall of humans [9 (link)–13 (link)]. Importantly, the concentrations of C-PAC under evaluation in this series of preclinical investigations are readily achievable in humans and are already under evaluation for oral and urinary tract health benefits.
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