Role and mechanism of action of the anthocyanin, delphinidin, in protecting endothelial cells against oxidative stress

Abstract

Diet-derived polyphenols are believed to have health benefits on account of their antioxidant properties. Cardiovascular health is considered to be a suitable target for antioxidant therapy because oxidative stress is implicit in atherogenesis – the disease process that underpins heart attacks, ischaemic strokes and peripheral vascular disease. There is a wealth of in vitro, in vivo and clinical data indicating that dietary polyphenols are protective in cardiovascular disease, but there is a substantial mis-match between the concentrations of these agents used in vitro to determine mechanism of action (generally > 10 M), and the bioavailability in vivo (often < 1 M). This discrepancy is largely overlooked, but could be important because the antioxidant mechanism identified through in vitro studies might not be a true representation of the mechanism involved in vivo. This thesis describes a wide range of studies to establish the polyphenol constituents present in widely consumed berries, to characterize the activity and stability of a key polyphenol, delphinidin, and ultimately to test the hypothesis that delphinidin, at concentrations that are relevant to bioavailability, protects cultured human umbilical vein endothelial cells (HUVECs) against oxidative damage via a mechanism that is independent of direct antioxidant activity. Further experiments were then conducted to test the hypothesis that the beneficial effects were mediated by up-regulation of intracellular antioxidant systems. The results found that a range of indigenous Scottish berries (raspberries, blackberries, blackcurrants) were rich in polyphenolic compounds that conferred substantial reducing capacity, which is often associated with antioxidant capacity. Tannins and anthocyanins were particularly prominent; the anthocyanin, delphinidin, was selected for further exploration on account of its higher bioavailability. Delphinidin aglycone was found to be unstable in solution, particularly tissue culture medium, in which it decomposed rapidly to simple phenolic compounds, including gallic acid. Electron paramagnetic resonance spectroscopy indicated that, if anything, both delphinidin and gallic acid were pro-oxidant rather than antioxidant. High concentrations of both delphinidin and gallic acid induced rapid morphological changes in HUVECs, most notably in the formation of vacuoles or vesicles: delphinidin and some other metabolites and intermediates were found inside the cells, whereas gallic acid was only found in the culture medium. Treatment of HUVECs with a range of concentrations of delphinidin and gallic acid that spanned the concentrations that might be found in vivo showed that high (100 M) concentrations of both were cytotoxic. However, both agents were found to have a protective effect in cells exposed to oxidative stress which was most evident at concentrations of ~ 1 M – too low to be due to direct antioxidant activity. Deeper examination of cells treated with delphinidin and gallic acid indicated that the protective effect was perhaps partially mediated by changes in expression of the intracellular antioxidant, glutathione; catalase did not seem to be affected. Taken together, the results in this thesis suggest that metabolic products of delphinidin might be important in conferring the protective effects via a mechanism that is independent of any direct antioxidant activity. This finding has profound implications for the importance of polyphenols in health benefits, suggesting that simple phenolic agents instead of the parent compounds, might be responsible for the antioxidant effects seen on account of initiating cellular defence responses.

Date of Award	31 Jul 2016
Original language	English
Awarding Institution	University of Edinburgh
Sponsors	Scottish Crop Research Institute
Supervisor	Ian Megson (Supervisor), Derek Stewart (Supervisor) & Garry Duthie (Supervisor)