pubs.acs.org/JAFC Published on Web 06/09/2010 © 2010 American Chemical Society 7572 J. Agric. Food Chem. 2010, 58, 7572–7579 DOI:10.1021/jf100700p Release of Bound Procyanidins from Cranberry Pomace by Alkaline Hydrolysis BRITTANY L. WHITE, § LUKE R. HOWARD,* ,§ AND RONALD L. PRIOR # § Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, Arkansas 72703, and # USDA - ARS Arkansas Children’s Nutrition Center, 1120 Marshall Street, Little Rock, Arkansas 72202 Procyanidins in plant products are present as extractable or unextractable/bound forms. We optimized alkaline hydrolysis conditions to liberate procyanidins and depolymerize polymers from dried cranberry pomace. Alkaline extracts were neutralized (pH 6-7) and then procyanidins were extracted with ethyl acetate and analyzed by normal phase high performance liquid chromatogra- phy. Alkaline hydrolysis resulted in an increase in low molecular weight procyanidins, and the increase was greater at higher temperature, short time combinations. The most procyanidins (DP1-DP3) were extracted at 60 °C for 15 min with each concentration of NaOH. When compared to conventional extraction using homogenization with acetone/water/acetic acid (70:29.5:0.5 v/v/v), treatment with NaOH increased procyanidin oligomer extraction by 3.8-14.9-fold, with the greatest increase being DP1 (14.9Â) and A-type DP2 (8.4Â) procyanidins. Alkaline treatment of the residue remaining after conventional extraction resulted in further procyanidin extraction, indicating that procyanidins are not fully extracted by conventional extraction methods. KEYWORDS: Alkaline; cranberry; hydrolysis; pomace; procyanidins; unextractable INTRODUCTION Cranberries (Vaccinium macrocarpon) are growing in popular- ity due to the increasing information regarding their health benefits. Cranberry juice has long been recognized for its ability to prevent urinary tract infections; however, there are several other health benefits associated with cranberries, which include antioxidant, antitumor, antiulcer, anti-inflammatory, and anti- atherosclerotic activities ( 1 -4 ). Cranberry pomace is composed primarily of seeds, skins, and stems, which are leftover from the juicing and canning processes of the cranberry processing indus- try ( 5 ). The seeds and skins of berries are a rich source of polyphenolic compounds, which have shown to be responsible for the numerous health benefits associated with the berries. Procyanidins are a class of polyphenolic compounds that impart astringency and bitterness to many plant products. In plants, they are believed to serve as a defense mechanism against potential predators because their bitterness and astringency is undesirable to animals, insects, and microbes ( 6 ). Procyanidins are formed via the condensation of the flavan-3-ols catechin and epicatechin and consist of two to several monomeric units ( 6 ). Structurally, the monomeric units may be linked in one of three ways. The “B”-type linkage is the most common and consists of 4β f 8 linkage between units. Units connected by both a 2β f O-7 and a 4β f 8 linkage are more rigid than “B”-type linkages and are denoted as “A”-type. The final type of linkage is the “C”- type linkage, which consists of a C-4 f C-6 linkage ( 6 ). Recently, the ability of cranberries to prevent urinary tract infections has been attributed to the presence of procyanidins containing “A”- type linkages ( 7 ). The bioavailability of procyanidins is dependent upon the size of the molecule with monomers and dimers being absorbed and present in blood at relatively low levels, but those larger than trimers are not absorbed ( 8 , 9 ). Whether absorption is required for procyanidins to impart their health benefits is still unknown. Polyphenolic compounds, including procyanidins, are com- monly perceived to be found mainly in the vacuoles of plants where they are separated from other cellular components. How- ever, many may also be associated with cellular components, such as the cell wall, especially after cell injury when vacuoles may rupture. This results in the release of phenolic compounds which may then associate with cell wall polysaccharides through hydro- gen bonding and hydrophobic interactions ( 10 ). Procyanidins in particular have a strong affinity for cell wall material ( 11 ), with higher molecular weight compounds having a greater affinity for binding than smaller compounds. The idea of “unextractable” procyanidins has been of great interest recently because it is believed that the procyanidin contents in plant materials has been underestimated due to the presence of procyanidins bound so tightly to cell wall material that they are not released by normal extraction methods ( 12 -16 ). Alkaline treatments are commonly used to extract bound phenolic acids and other phenolic compounds from grains such as rice, wheat, and corn. It is known that phenolic compounds, namely, ferulic acid, are insoluble and bound to cell wall materials. Treatment with different concentrations of sodium hydroxide for varying lengths of time has proven to be effective in releasing these bound phenolic compounds ( 17 , 18 ). There is *Author to whom correspondence should be addressed [telephone (479) 575-2978; fax (479) 575-6936; e-mail lukeh@uark.edu].