The protective effect of blackberry anthocyanins against free radical-induced oxidation and cytotoxicity in multiple cell lines

Anthocyanins are the blue and red pigments found in berries, with known antioxidant properties that may be associated with several health benefits, such as a reduction in the risk of heart disease and cancer. Blackberry in particular, is a rich source of anthocyanins and has notable antioxidant activity. Although the antioxidant capacity of anthocyanins has been well established in cell free in vitro system, there is very little evidence that links this antioxidant activity with protection against free radical associated oxidative damage in biological systems. The ultimate goal of this thesis, therefore, was to evaluate the protective effect of blackberry anthocyanins against free radical-induced oxidative stress and the resulting cytotoxicity using multiple cultured cell lines. Anthocyanins of both crude blackberry extracts as well as an anthocyanin enriched fraction were identified and quantified using HPLC. Different cytotoxicity assays (MTT, CellTiterGlo, BrdU) were validated against cell counting method to determine the most appropriate cytotoxicity assay(s) for the evaluation of blackberry anthocyanins in cultured cells. The effect of blackberry anthocyanins were individually evaluated in five distinct cell lines: two breast cancer lines (MDA-MB-453 and MCF-7), two intestinal cell lines (Caco-2 and INT-407), and one prostate cancer cell line (LNCaP), using M T T and CellTiter-Glo assay. In other tests, AAPH (2, 2' -azobis (2-amidinopropane) dihydrochloride), a free radical generator, was used to initiate intracellular oxidation and induce cytotoxicity. The effect of the blackberry extracts against AAPH initiated intracellular oxidation was monitored with a dichlorofluorescin diacetate (DCFH-DA) probe. The protective effect of blackberry anthocyanins against AAPH-induced cytotoxicity was measured with MTT and CellTiterGlo assays. Cell cycle analysis was also performed to determine possible protective mechanisms of blackberry anthocyanins against free radical associated damages. Cyanidin-3-glucoside was found to be the major anthocyanin (85 %) in blackberry. Blackberry anthocyanins were demonstrated to have no cytotoxic properties at physiological concentration. In addition, blackberry anthocyanins were found to suppress AAPH-initiated intracellular oxidation. This effect corresponded a protection effect against free radical-induced cytotoxicity. Cell cycle analysis with propidium iodide staining demonstrated that blackberry anthocyanins prevented cytotoxicity by scavenging the generated peroxyl radicals, thus alleviating AAPH-induced apoptosis.Land and Food Systems, Faculty ofGraduat