Metabolite profiling of polyphenols in Vaccinium berries and determination of their chemopreventive properties

Berries and their derived products have demonstrated to possess several positive effects on human health, due to their high content of polyphenols and their chemopreventive activity against many degenerative diseases, involving oxidative stress, inflammation and carcinogenesis. Amongst berry fruits, Vaccinium myrtillus L. (bilberry), which is a species native to Europe, has received special attention, due to its traditional use in folk medicine. Several studies have demonstrated the benefits of bilberry in the inhibition of cancer cell growth and management of visual disorders. Vaccinium floribundum Kunth, also called Morti\uf1o, is a species from South America and belongs to the Ericaceae family. V. froribundum berries are widely consumed in Ecuador as the fresh fruit or as processed products. Local communities have also used this plant to treat various medical conditions, including diabetes and inflammation. The chemical composition of V. floribundum has been reported in the literature, but the polyphenol characterization and the biological properties have not been deeply investigated. In this study, the complete metabolite profiling of polyphenols in V. floribundum berries from Ecuador was carried out my means of HPLC-UV/DAD, HPLC-ESI-MS and MS2, using an ion trap mass analyzer. As a comparison, V. myrtillus berries from Italian Northern Apennine were analyzed in parallel. A sequential selective extraction procedure was developed to maximize the yields of the secondary metabolites from the berries, using ethyl acetate for flavonoids and phenolic acids, followed by acidified methanol for anthocyanins. The HPLC analysis of flavonoids and phenolic acids was carried out on an Ascentis C18 column (250 7 4.6 mm, 5 \ub5m), with a gradient mobile phase consisting of H2O with 0.1 M formic acid and acetonitrile. Anthocyanins were analyzed using a Zorbax SB-C18 column (150 7 4.6 mm, 5 \ub5m), with a gradient mobile phase composed by H2O-formic acid (9:1, v/v) and methanol-H2O-formic acid (5:4:1, v/v/v). The results of the HPLC analyses indicated several representative differences in both the quali- and quantitative composition of flavonoids and anthocyanins in the berry extracts obtained from V. floribundum and V. myrtillus, which showed typical fingerprints of their phenolic secondary metabolites. Thanks to its unique polyphenol composition, V. floribundum can be easily differentiated from common bilberries using the proposed techniques. The chemopreventive activity of these berries and their fractions was evaluated by the determination of their antioxidant activity using a panel of in vitro tests (DPPH, ABTS) and their antimutagenic properties against dietary and environmental, direct and indirect, mutagens. The results of these biological assays were finally correlated with the chemical composition of the two investigated Vaccinium species, to establish the potential application of V. floribundum as a source of phytochemicals in the nutraceutical and functional food ambit