Diet derived anthocyanins (ACN) such as cyanidin-3-glucoside (C3G) are believed to reduce the risk of cardiovascular disease (CVD)⁽ Reference Cassidy, O'Reilly and Kay ^{1 ,} Reference Hooper, Kroon and Rimm ^{2 )} despite their apparent low bioavailability⁽ Reference Kay ^{3 )}. However, given that ACN are likely to degrade following ingestion, their degradation products and metabolites are likely to contribute to their bioactivity⁽ Reference Kay, Kroon and Cassidy ^{4 )}.

In our recently completed human feeding study in which stable isotope labelled C3G was fed to healthy male participants (500 mg bolus of ¹³C₅–C3G; n=8), a total of 22 metabolites were identified. This included protocatachuic acid (PCA) which had a C_Max of 0.23±0.12 μM and its methylated derivative vanillic acid (VA) which had a C_Max of 0.66±0.41 μM. We explored the bioactivity of these metabolites on vascular health by measuring the effects on mediators of nitric oxide bioavailability including endothelial nitric oxide synthase (eNOS), NADPH oxidase (NOX4) and superoxide. Protein expression of eNOS was measured by ELISA and NOX4 by western blotting, while superoxide was measured indirectly via ferrocytochrome C oxidation (A₅₅₀) in cultured human umbilical cord vascular endothelial cells treated with either 0.1, 1 or 10 μM of C3G, PCA or VA.

Values are given as percentage relative to control and are the mean of 3 independent experiments *P<0.05 (ANOVA with Tukey Kramer test, n=3).

C3G significantly increased eNOS at a concentration of 10 μM and significantly decreased superoxide at 0.1 and 1 μM but had no effect on NOX4 protein expression. PCA signficantly decreased eNOS protein at all concentrations tested, reduced NOX4 protein at 1 μM and decreased superoxide at 0.1 and 10 μM. VA significantly increased eNOS at 0.1 and 10 μM, but did not affect NOX4 expression or superoxide production. These studies provide early evidence that metabolites of anthocyanins may exhibit greater bioactivity than the parent molecule. In addition, these activities are apparent at relatively low and dietary achievable concentrations.