Abstract
Cranberries (Vaccinium macrocarpon) are a commonly grown crop in Massachusetts (MA), Oregon (OR) and other parts of North America. Cranberries have gained attention in natural products research, as they contain phytochemical constituents relevant to human health, including flavonoids, phenolics and terpenoids. Reported cranberry bioactivities include inhibition of antioxidant activity, anti-inflammatory response, anticancer and anti-proliferative properties. Cranberry breeding programs were to develop multiple varieties of cranberry fruit. As with all plants, the content of phytoconstituents in cranberry fruit may vary based on cultivars and growing regions. Therefore, the aim of this study is to investigate the effects of these genetic and environmental factors on cranberry fruit’s phytochemical composition and the associated bioactivities. Cranberry fruit of nine cultivars was collected from MA and OR bogs during the 2016 and 2017 growing seasons. HPLC-DAD, qNMR and UPLC-MS methods were developed and applied to study the variation in concentrations of constituents in a broad range of fruit samples. HPLC-DAD was used for quantitative analysis of anthocyanins and flavonols. Quantitative ¹H NMR (qNMR) methods combined with AssureNMR software (Bruker Biospin) were developed to quantify triterpenoids and organic acids which were not easily detected by absorbance-based methods. These include anti-inflammatory triterpenoid compounds ursolic and oleanolic acid, as well as organic acids. Factors influencing the content of triterpenoids in North American cranberries were largely unexplored. 1H NMR combined with Principle Component Analysis provided non-targeted analysis of variation in fruit composition among samples, revealing similarities and differences between cultivars and regions. Regional trends were found in metabolites contents for cranberry fruit from east and west coast MA and OR: OR fruit was higher in anthocyanins and flavonols, and lower in triterpenoids. The content of ursolic acid in all samples ranged from 2.72-16.8 g kgˉ¹ dry weight. While qNMR is effective for the two major triterpenoids, it lacks sensitivity compared to LCMS. A UPLC-MS method was therefore developed for simultaneous determination of low concentration triterpenoids and their esters including maslinic acid, corosolic acid and 3-O-p-hydroxycinnamoyl ursolic acid. We hypothesized that variations in these compounds based on various growing regions and cultivars will impact the antioxidant and anti-inflammatory activities of the fruit. To evaluate antioxidant activity arising from key compounds in cranberries, a microplate DPPH assay was applied to compare the free-radical scavenging antioxidant activities of crude cranberry fruit extracts. The results showed that OR fruits had significantly higher in radical scavenging activity than MA fruits, correlating positively with the content of flavonoids. To evaluate anti-inflammatory activity, concentrated de-sugared fruit extracts were prepared and investigated for their inhibitory effects on production of pro-inflammatory cytokines in lipopolysaccharide (LPS)-treated THP-1 monocytic cell line by ELISA assay. All concentrated cranberry extracts exhibited anti-inflammation based on decreased interleukin-6 (IL-6) secretion. A subgroup of cranberry extracts exhibited dose-dependent inhibition of IL-6 at 10 and 100 μg/mL. The observed inhibition of pro-inflammatory cytokine production suggested that anthocyanins, proanthocyanidins and triterpenoids in cranberries may all contribute to their anti-inflammatory activities. The results of this study are expected to improve our understanding of how cranberry quality and potential health benefits are impacted by cultivar and growing conditions.