Effects of Commercial Anthocyanin-Rich Extracts on Colonic Cancer and Nontumorigenic Colonic Cell Growth CUIWEI ZHAO, M. MONICA GIUSTI,MINNIE MALIK,MARY P. MOYER, AND BERNADENE A. MAGNUSON* Department of Nutrition and Food Science, 0112 Skinner Building, University of Maryland, College Park, Maryland 20742-7521 Commercially prepared grape (Vitis vinifera), bilberry (Vaccinium myrtillus L.), and chokeberry (Aronia meloncarpa E.) anthocyanin-rich extracts (AREs) were investigated for their potential chemopreventive activity against colon cancer. The growth of colon-cancer-derived HT-29 and nontumorigenic colonic NCM460 cells exposed to semipurified AREs (10-75 μg of monomeric anthocyanin/mL) was monitored for up to 72 h using a sulforhodamine B assay. All extracts inhibited the growth of HT-29 cells, with chokeberry ARE being the most potent inhibitor. HT-29 cell growth was inhibited ∼50% after 48 h of exposure to 25 μg/mL chokeberry ARE. Most importantly, the growth of NCM460 cells was not inhibited at lower concentrations of all three AREs, illustrating greater growth inhibition of colon cancer, as compared to nontumorigenic colon cells. Extracts were semipurified and characterized by high- pressure liquid chromatography, spectrophotometry, and colorimetry. Grape anthocyanins were the glucosylated derivatives of five different anthocyanidin molecules, with or without p-coumaric acid acylation. Bilberry contained five different anthocyanidins glycosylated with galactose, glucose, and arabinose. Chokeberry anthocyanins were cyanidin derivatives, monoglycosylated mostly with galactose and arabinose. The varying compositions and degrees of growth inhibition suggest that the anthocyanin chemical structure may play an important role in the growth inhibitory activity of commercially available AREs. KEYWORDS: Anthocyanins; colon cancer; HT-29 colon cancer cells; NCM460 colon cells; cell growth; chokeberry; bilberry; grape INTRODUCTION Anthocyanins, natural pigments present in fruits and veg- etables, have shown considerable potential in the food industry as safe and effective food colorants (1, 2). Recently, interest in anthocyanin-rich foods and extracts has intensified because of their possible health benefits. Anthocyanins are potent antioxi- dants (3-6). Anthocyanin fractions extracted from different sources, including flower petals (7), grape rinds and red rice (8), red soybeans and red beans (9), Vaccinium species (10), purple corn (11), and different cherry and berry extracts (12-14), have demonstrated anticancer activity. In vitro, anthocyanin fractions more effectively inhibited growth of human intestinal carcinoma HCT-15 cells than did flavonoids (7, 9, 15). HCT116 colon cancer cells were inhibited by anthocyanin-containing berry extracts including cowberry, strawberry, blueberry, and bilberry extracts (13). Similarly, tart cherry anthocyanins and their aglycon cyanidin were shown to inhibit the growth of human colon cancer cell lines HT-29 and HCT116 (14). In vivo, the tart cherry extract inhibited the intestinal tumor development in Apc(min) mice (14), suggesting that anthocyanins as well as the aglycons may reduce the risk of intestinal cancer. Freeze-dried black raspberries (12) and purple corn (11) have been shown to inhibit azoxymethane- induced colon tumors in rats. However, the relationship of anthocyanin structure to anti- carcinogenic activity has not been well established. Koide and co-workers (8, 9) reported that different sources of anthocyanins and the presence of glycosylations might affect the inhibition of HCT-15 cancer cell growth. Bioabsorption studies have demonstrated that the structure of anthocyanins greatly affects uptake (16, 17). We (unpublished data), and others (18), have observed that the method of preparation of anthocyanin extracts greatly affects the biological activity. Therefore, we investigated the chemopreventive activity of anthocyanin extracts com- mercially prepared for the food industry as natural colorants. The first objective of the present study was to compare the effects of commercially available anthocyanin-rich extracts (AREs) with different anthocyanin profiles on the growth of colon cancer cells. The AREs from grape, bilberry, and chokeberry were selected on the basis of their anthocyanin high- pressure liquid chromatography (HPLC) profiles provided by the commercial companies. Grapes contain acylated glucoside derivatives of a variety of anthocyanidins, whereas bilberry * To whom correspondence should be addressed. E-mail: bmagnuso@ umd.edu. Phone: (301) 405-4523. Fax: (301) 314-3313. 6122 J. Agric. Food Chem. 2004, 52, 6122-6128 10.1021/jf049517a CCC: $27.50 © 2004 American Chemical Society Published on Web 09/14/2004