H: Health, Nutrition, & Food JFS H: Health, Nutrition, and Food Bog Bilberry ( Vaccinium uliginosum L.) Extract Reduces Cultured Hep-G2, Caco-2, and 3T3-L1 Cell Viability, Affects Cell Cycle Progression, and Has Variable Effects on Membrane Permeability JIA LIU,WEI ZHANG,HAO JING, AND DAVID G. POPOVICH ABSTRACT: Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 ± 2.6 μg/mg of dry weight), followed by malvidin-3-glucoside (10.3 ± 0.3 μg/mg) and malvidin-3-galactoside (8.1 ± 0.4 μg/mg). Hep- G2 LC50 was calculated to be 0.563 ± 0.04 mg/mL, Caco-2 LC50 was 0.390 ± 0.30 mg/mL and 0.214 ± 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P ≤ 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition. Keywords: anthocyanins, bog bilberry, LDH, Vaccinium uliginosum L., viability Introduction B og bilberry (Vaccinium uliginosum L.) is an edible blue- colored berry classified in the same genus as the common blueberry (Vaccinium corymbosum) and bilberry (Vaccinium myr- tillus L.). Bog bilberries are typically found in northern hemisphere countries such as Russia and Norway (Andersen 1987) and north- ern China. Bilberries and blueberries along with other pigmented fruits such as blackberry (Rubus fruticosus), cranberry (Rubus occi- dentallis), raspberry (Rubus idaeus), and many other anthocyanin containing fruits and vegetables have been reported to possess a wide variety of biological activities (Seeram 2008). Strong free rad- ical scavenging antioxidant properties of berry extracts have been the focus of many reports along with an ability to inhibit the growth of variety of cultured cancer cell lines (Kong and others 2003; Elisia and Kitts 2008; Wang and Stoner 2008). Anthocyanins are respon- sible for the color of bog bilberries with over 15 different antho- cyanins identified from bog bilberries grown in the west coast of Norway (Andersen 1987). Reports on bog bilberry composition and bioactive proper- ties are limited and a greater amount of literature is currently available on related species of bilberry (Vaccinium myrtillus L.). Bilberries contain cyanidin, delphinidin, peronidin, and malvidin MS 20091065 Submitted 10/26/2009, Accepted 1/4/2010. Authors Liu and Jing are with College of Food Science & Nutritional Engineering, China Agri- cultural Univ., Nr 17 Qinghua East Rd., Beijing, China, 100083. Authors Zhang and Popovich are with Dept. of Chemistry, Natl. Univ. of Singapore, 3 Science Drive 3, Singapore, 117543. Direct inquiries to author Popovich (E-mail: chmpdg@nus.edu.sg). glycosides making up the majority of reported anthocyanin con- tent (Upton 2001). Anthocyanins are typically found as glycosides in berries shown by the chemical structure in Figure 1. The antho- cyanin content of bog bilberry shares similar composition to other blue-pigmented fruit of the Vaccinium species (Andersen 1987). With regard to cancer cell proliferation, anthocyanin containing extracts from a variety of different berries have been reported to inhibit the growth of human colon (HT-29), prostate (LNCaP), breast (MCF-7), and oral (CAL27) cancer cell lines and stimulate apoptosis in cultured human colon cells (HT-29) (Seeram and oth- ers 2006). Specific anthocyanins such as cyanidin-3-glucoside and peonindin-3-glucoside have been shown to inhibit growth and in- duce apoptosis in human breast carcinoma cells (HS578T) (Chen and others 2005). There is a growing interest in the potential health promoting aspects of berries for their chemopreventative proper- ties but the literature specifically focused on bog bilberries is scarce. Therefore, the objective of this study was to assess a bog bilberry extract propensity to selectively inhibit the growth, influence mem- brane permeability, and affect the cell cycle of 3 distinct cell lines. The cells included 2 human cancer cell line models, representing the colonocytes (Caco-2), hepatocytes (Hep-G2), and a nonmalig- nant murine fibroblast cell line (3T3-L1). Materials and Methods Plant material and HPLC analysis Anthocyanin standards, cyanidin-3-glucoside, malvidin-3- galactoside, and malvidin-3-glucoside were purchased from Sigma C  2010 Institute of Food Technologists R  Vol. 75, Nr. 3, 2010—JOURNAL OF FOOD SCIENCE H103 doi: 10.1111/j.1750-3841.2010.01546.x Further reproduction without permission is prohibited