pubs.acs.org/JAFC Published on Web 05/27/2010 © 2010 American Chemical Society 7458 J. Agric. Food Chem. 2010, 58, 7458–7464 DOI:10.1021/jf101485r Characterization of Anthocyanins and Proanthocyanidins in Wild and Domesticated Mexican Blackberries (Rubus spp.) EDITH O. CUEVAS-RODRI ´ GUEZ, † GAD G. YOUSEF, ‡ PEDRO A. GARCI ´ A-SAUCEDO, § JOSE ´ LO ´ PEZ-MEDINA, § OCTAVIO PAREDES-LO ´ PEZ, ) AND MARY ANN LILA* ,‡,^ † Programa de Posgrado en Alimentos del Centro de la Republica (PROPAC), Universidad Auto´ noma de Quere´taro, Santiago de Quere´taro, Quere´taro, Mexico, ‡ Plants for Human Health Institute, North Carolina State University, Kannapolis, North Carolina 28081, § Universidad Michoacana de San Nicola´s de Hidalgo, Uruapan, Michoaca´ n, Mexico, ) Centro de Investigacio´ n y de Estudios Avanzados del Instituto Polite´cnico Nacional, Irapuato, Guanajuato, Mexico, and ^ Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, Illinois 61802 This study was designed to characterize and compare wild, commercial, and noncommercial cultivated blackberry genotypes grown in Michoaca ´ n, Mexico. Six genotypes, including WB-3, WB-7, WB-10, and WB-11 (all wild blackberry types), Tupy (a commercial cultivar), and UM-601 (a cultivated breeding line), were selected and profiled for anthocyanins and proanthocyanidins by separating extracts over Amberlite XAD-7 resin and Sephadex LH-20 columns. Subsequent high- performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization- mass spectrometry (LC-ESI-MS) analyses revealed that the major anthocyanin for all genotypes was cyanidin 3-O-glucoside. The proanthocyanidins (condensed tannins) were present in mono- to hexamer forms. Also, hydrolyzable tannins, ellagitannins, were characterized in the blackberry fruits. The average anthocyanin concentration in Sephadex LH-20 fractions was 49.2 mg/g in the commercial cultivar Tupy, while in the wild genotypes and the breeding line, the range was 361.3-494.9 mg/g (cyanidin 3-O-glucoside equivalent). The proanthocyanidin concentration varied widely among wild genotypes (417.5-1343.6 mg/g, catechin equivalent). This study demonstrated that the use of Amberlite XAD-7 followed by Sephadex LH-20 chromatography, with subsequent HPLC and LC-ESI-MS analyses, was able to effectively separate and characterize the diverse polyphenolics in blackberry genotypes. These results suggest that recommendations for dietary intake of blackberries for human health benefits need to take into account the source, because of the wide inherent variation in bioactive polyphenolic content in different blackberry genotypes. KEYWORDS: Anthocyanin; proanthocyanidin; ellagitannins; Rubus; HPLC-ESI-MS INTRODUCTION The Rubus genus (Rosaceae family), which includes the black- berry, is cultivated worldwide but primarily concentrated in the northern hemisphere. The blackberry was among the earliest fruits used for medicinal purposes; as early as the 16th century, black- berry juice was used in Europe to treat infections of the mouth and eyes ( 1 ). Recent studies have demonstrated very high antioxidant capacity in blackberries and their enhanced potential to reduce risks of cancer and cardiovascular diseases ( 2 -5 ) compared to other fruits and vegetables ( 6 ). The genotype, species, environment, and cultivation conditions have all been shown to exert a profound influence on the content of bioactive compounds in berries ( 7 -9 ). Wild berries may provide a preferred resource for investigating phytochemicals that can improve human health, because they contain natural inherent levels of compounds that have not been influenced by commercial breeding and selection ( 10 ). In addition to their fiber content, wild berries are rich in vitamins, minerals, various organic acids, and phenolic com- pounds ( 11 ). Because blackberries can contain a unique and intense complement of polyphenolic compounds, research on their com- position has grown. The major polyphenolic compounds in berries are anthocyanins, hydrolizable tannins (gallo- and ellagitannins), flavonols, and flavan-3-ols, including proanthocyanidins ( 1 , 3 ). Anthocyanins are the predominant group of flavonoids present in berries ( 11 , 12 ). They are water-soluble glycosides and acylglyco- sides, which are polyhydroxy and polymethoxy derivatives of the 2-phenylbenzopyrylium (flavylium) cation ( 13 ). Flavan-3-ols, bet- ter known as proanthocyanidins or condensed tannins, are poly- mers of flavan-3-ols and/or flavan-3,4-diol mixtures ( 14 ). These compounds give a characteristic astringent or bitter taste to many berries ( 11 ). They are ubiquitous in nature and are the second most abundant group of natural phenolics after lignin ( 15 ). Anthocya- nins and proanthocyanidins are found in a wide range of common foods, including cereals, legumes, fruits, vegetables, and wines, and affect texture, color, and taste. These polyphenolics are also of *To whom correspondence should be addressed. Telephone: 704- 250-5407. E-mail: mlila@ncsu.edu.