Abstract
Cranberries (Vaccinium macrocarpon) are a low bush fruit native to North America that have been studied extensively due to their many purported health benefits. Health benefits associated with cranberry consumption include antibacterial activity, prevention of tumor cell proliferation, preventing oxidative stress and reducing inflammation. Additionally, consumption of fruits and berries, including cranberries, has been shown to shift the overall composition of the gut microbiome towards a composition that is considered healthier, resulting in the prevention or reduction of diseases such as obesity, Crohn’s disease and cardiovascular complications. Cranberry can be consumed in many forms including whole berry, juices, jams or in supplement form. However, the supplement industry is generally unregulated in terms of manufacturing an authentic product. Therefore, many who consume cranberry in supplement form may not reap the benefits the whole fruit provides. The aims of this study were to first apply chemometric tools to the phytochemical analysis of commercially available cranberry supplements in comparison to a whole cranberry reference powder, and secondly to examine the effects these supplements of varying composition may have on commonly occurring bacteria found in the gut. Quantitative ¹H NMR was applied to the analysis of common organic acids and triterpenoids produced by cranberries. Whole cranberry powder was found to contain organic acids malic acid, citric acid, quinic acid and triterpenoid isomers ursolic acid and oleanolic acid. Of the nine commercially available supplements analyzed, only two were found to contain all these secondary metabolites. Three supplements contained only one of the aforementioned secondary metabolites, with the remaining four supplements containing some but not all of the compounds of interest. PCA was then applied to ¹H NMR data to examine similarities and differences between samples and groups of samples. Scores plots indicated significant deviation from whole cranberry powder for most supplements tested. Corresponding loadings plots were used to identify specific compounds contributing the variation between samples. Common outliers were oleanolic and ursolic acid, ascorbic acid, citric acid, and in some cases quercetin, proanthocyanidins and hyperoside. Furthermore, SIMCA was applied to ¹H NMR data to generate a representative class model of authentic cranberry powder. Supplements screened against the SIMCA class model were determined to be outliers at 95% confidence, indicating that their metabolic profiles according to ¹H qNMR did not match that of an authentic cranberry powder. HPLC-DAD was used to assess flavonol glycosides and anthocyanin content. Three supplements did not contain quantifiable amounts of anthocyanins while one supplement was found to contain no flavonols at all. Two samples, S6 and JE were found to have higher concentrations of both families of compounds than whole cranberry reference powder. The BL-DMAC assay was used to assess total proanthocyanidin (PAC) content for all samples. Four supplements were found to contain no PACs, while S6 and JE were found to contain approximately ten times the amount of PACs as whole cranberry powder. FP, S6 and JE were subjected to MALDI-TOF-MS to determine PAC degrees of polymerization and linkage types. The DPPH radical scavenging colorimetric assay was used to determine the efficacy of each vsupplement at neutralizing free radicals. S4 demonstrated no radical scavenging activity, while S6 and S9 demonstrated nearly twice the effectiveness of whole cranberry reference powder. Lastly, the Folin-Ciocalteau assay was performed to determine comparative total phenolic content present within the samples. Most samples had a lower total content of phenolic compounds compared to whole cranberry powder except for S6 and S9, which were both estimated to contain significantly higher quantities of phenolic compounds than whole cranberry powder based on the Folin-Ciocalteu method. Results of all analyses indicate drastic variation between samples, with many samples possessing secondary metabolite profiles vastly different from that of authentic cranberry powder. Study results emphasize the need for stricter regulations and guidelines regarding the manufacturing and production of supplements. The second aim of the study was to assess the effects the previously characterized supplements may have on common pathogenic and probiotic bacterial species. The bacterial species studied were Lactobacillus plantarum, Lactobacillus rhamnosus, Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium. A disk diffusion assay was used to efficiently screen various supplements and extracts against each species. Extracts that demonstrated activity during the disk diffusion assay were selected for further bioassays. The crystal violet biofilm formation assay was used to assess the efficacy of cranberry extracts at inhibiting pathogenic biofilm formation. No trends were observed for the inhibition of biofilms by Escherichia coli or Klebisella oxytoca, however, significant effects were observed when treating Salmonella typhimurium and Klebsiella pneumoniae with particular extracts. Salmonella typhimurium biofilm formation was inhibited by three of the cranberry products, FP, JE, and S6, and two commercial standards, PAC A2 and hyperoside. Additionally, Klebsiella pneumoniae biofilm formation was inhibited by S6 alone. Growth analysis of probiotic species was performed as they do not readily form biofilms. Results indicate slight but statistically significant reductions in growth of both Lactobacillus species by FP, JE and S6, however there was no bactericidal activity observed. Results suggest that cranberry extracts may aid overall gut health by inhibiting certain pathogenic species, while allowing for probiotic species to thrive.