Food Chemistry Food Chemistry 100 (2007) 956–963 www.elsevier.com/locate/foodchem Phenolic profiles of Andean purple corn (Zea mays L.) Romina Pedreschi 1, Luis Cisneros-Zevallos * Department of Horticultural Sciences, Texas A&M University, College Station, HFSC Building, Room 202, MS 2133, TX 77843, USA Received 14 July 2005; received in revised form 7 October 2005; accepted 9 November 2005 Abstract Qualitative and quantitative high performance liquid chromatography with diode array detection (HPLC-DAD) was performed to characterize the presence of phenolic compounds in Andean purple corn. Phenolic compounds were analyzed by separating them in two main fractions: a water fraction (WF) and an ethyl acetate fraction (EAF). The WF rich in anthocyanins revealed the presence of cyanidin-3-glucoside, pelargonidin-3-glucoside and peonidin-3-glucoside. The respective acylated anthocyanin glucoside forms of these compounds were also detected following alkaline hydrolysis. The EAF was composed of phenolic acids such as p-coumaric, vanillic acid, protocatechuic acid, flavonoids such quercetin derivatives and a hesperitin derivative. Alkaline and acid hydrolysis of the EAF revealed the presence of p-coumaric and ferulic acid as main components in four bound hydroxycinnamic acid forms present in the ethyl acetate fraction. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Andean purple corn; Anthocyanins; Phenolic profiles 1. Introduction & Heinonen, 2003; Kamei et al., 1995; Katsube, Iwashita, Tsushida, Yamaki, & Kobori, 2003; Remeuf, Dorange, & Purple corn has been cultivated for centuries in the Dupuy, 1983; Yoshimoto, Okuno, Kumagai, Yoshinaga, Andean Region. In Peru, people consume a typical drink & Yamakawa, 1999; Yoshimoto, Okuno, Yamaguchi, & made from purple corn called ‘‘Chicha Morada’’ which is Yamakawa, 2001). In addition, anthocyanins have been uti- believed by folklore use to improve health (Brack-Egg, lized in the treatment of various blood circulation disorders 1999). Purple corn is an important source of anthocyanins, resulting from capillary fragility (Wang, Cao, & Prior, 1997), natural pigments widely distributed in the plant kingdom in vaso-protective and anti-inflammatory studies (Lietti, with peculiar features regarding coloring of foods. Antho- Cristoni, & Picci, 1976), in the inhibition of platelet aggrega- cyanins already characterized in purple corn cobs and seeds tion (Morazzoni & Magistretti, 1986), and in the mainte- include cyanidin-3-glucoside, pelargonidin-3-glucoside, nance of normal vascular permeability (Wang et al., 1997). peonidin-3-glucoside and their respective malonated coun- Beneficial health-related effects of other non-anthocya- terparts (Aoki, Kuze, & Kato, 2001; Pascual-Teresa, San- nin phenolic compounds have also been extensively tos-Buelga, & Rivas-Gonzalo, 2002). reported including antioxidant (Adom & Liu, 2002; Cakir Anthocyanins are attributed with many biological activi- et al., 2003; Dewanto, Wu, & Liu, 2002; Friedman, 1997; ties. Antioxidant, antimutagenic and anticarcinogenic activ- Ghiselli, Nardini, Baldi, & Scaccini, 1998; Lien, Ren, Bui, ities of anthocyanins have been extensively reported & Wang, 1999; Rice-Evans, Miller, & Paganga, 1996; Stint- (Bomser, Madhavi, Singletary, & Smith, 1996; Ka¨hko¨nen zing, Stintzing, Carle, Frei, & Wrolstad, 2002; Wang & Lin, 2000); antimutagenic (Cardador-Martinez, Castan˜o- Tostado, & Loarca-Pin˜a, 2002; Edenharper, Keller, Platt, * Corresponding author. Tel./fax: +1 979 845 3244. E-mail addresses: [email protected], [email protected] (L. & Unger, 2001; Edenharper, von Petersdoff, & Rauscher, Cisneros-Zevallos). 1993; Miyazawa & Hisama, 2003; Wang & Lee, 1996), anti- 1 Present address: Katholieke Universiteit Leuven, Belgium. carcinogenic activity (Ferguson, 2001; Hertog, Van Poppel, 0308-8146/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2005.11.004 R. Pedreschi, L. Cisneros-Zevallos / Food Chemistry 100 (2007) 956–963 957 & Verhoeven, 1997; Iwashita, Kobori, Yamaki, & Tsush- myricetin, kaempferol), flavones (apigenin, luteolin), flava- ida, 2000; Katsube et al., 2003; Loo, 2003; Surh, 1999; none (hesperitin) were purchased from Sigma Chemical Yang, Landau, Huang, & Newmark, 2001) and other bio- Co. (St. Louis, MO, USA). For tannin determination, cat- logical properties. echin and vanillin were obtained from Sigma Chemical Co. In general, corn is considered an important crop world- (St. Louis, MO, USA). Sugar standards D-glucose, rhanm- wide. There have been some studies in the identification nose, galactose and organic acids: malonic acid, fumaric and quantification of phenolic compounds from yellow acid, quinic acid were purchased from Sigma Chemical corn including p-hydroxybenzoic acid, vanillic, proto- Co. (St. Louis, MO, USA). catechuic, syringic, p-coumaric, ferulic, caffeic and sinapic HPLC grade solvents: acetonitrile, methanol, hydro- acids (Adom & Liu, 2002; Dewanto et al., 2002; Gueldner, chloric acid were purchased from EM Science (a division Snook, Widstrom, & Wiseman, 1992; Hedin & Callahan, of EM Industries Inc., Gibbstown, NJ, USA). Trifluoro- 1990; Snook et al., 1995; Sosulski, Krygier, & Hogge, acetic acid was purchased from Fisher Scientific (Fair 1982). However, the characterization of non-anthocyanin Lawn, NJ, USA). Other chemicals such as sodium hydrox- phenolic compounds present in purple corn has not been ide, potassium hydroxide were purchased from Sigma reported to our knowledge. Chemical Co, (St. Louis, MO, USA). Silica gel 60 F254 Most of the properties attributed to purple corn plates from Fisher Scientific (Fair Lawn, NJ, USA) were extracts, including coloring attributes (Duhard, Garner, used in the separation and identification of sugars by thin & Megard, 1997; Cevallos-Casals & Cisneros-Zevallos, layer chromatography. Diphenylamine and phosphoric 2004), antioxidant (Cevallos-Casals & Cisneros-Zevallos, acid were purchased from Riedel de Ha¨en (Seelze, Ger- 2003a), antimicrobial (Cevallos-Casals & Cisneros-Zevallos, many) and aniline was purchased from Sigma Chemical 2003b), anti-obesity activity and amelioration of hypergly- Co. (St. Louis, MO, USA). cemia (Tsuda, Horio, Uchida, Aoki, & Osawa, 2003), and anticarcinogenic properties (Hagiwara et al., 2001) were 2.2. Sample preparation related to anthocyanins. Thus, there is need in characteriz- ing other phenolic compounds present in purple corn in A 0.5 g of purple corn extract (PCE) was partitioned in a addition to anthocyanins. We believe, these phenolic com- decantation flask with 100 mL water and 400 mL of ethyl pounds might be involved in health-related properties and acetate. The mixture was shaken vigorously and let to could have been missed in previous studies in which bioac- stand until two phases were observed. The water fraction tive properties were attributed to anthocyanins only. (WF) was freeze-dried in a freeze dryer FTSÒ Systems, The objectives of this study were: (1) The identification Inc (Stone Ridge, NY, USA) at À50 °C and at 200 lm and quantification of the main anthocyanins present in Hg of pressure. The ethyl acetate fraction (EAF) was con- Andean purple corn extracts and (2) the identification and centrated at 240 mbar pressure in a roto-evaporator quantification of other phenolic compounds present in (Bu¨chi, Switzerland) at 40 °C until dryness and then re-sus- Andean purple corn extracts using HPLC-DAD as a tool. pended in methanol. The dried WF and EAF represented 92% and 8% of the PCE by weight, respectively. 2. Materials and methods 2.3. Tannin determination 2.1. Sample material, standards and reagents For tannin determination the vanillic assay was carried Purple corn extract (PCE) was kindly given by Fitof- out as recommended by Price, Van Scoyoc, and Butler (1978) arma (Lima, Peru). The powder extract was prepared by extraction of ground cobs (mesh 60) in a 60% aqueous eth- 2.4. HPLC-DAD analysis of anthocyanins and other anol solution at room temperature for 48 h (1 kg cobs/7L phenolic compounds solvent). The obtained extract was filtered and spray dried (180 °C inlet and 85 °C outlet temperatures) using malto- Phenolic compounds were separated using a binary dextrins as carrier (0.5 kg maltodextrin/100 L extract). Waters 515 HPLC pump system, a Waters 717 plus auto- One kilogram of dried PCE contained 40% maltodextrin. sampler automated gradient controller, a SP8792 tempera- Anthocyanin standards composed of a mixture of ture controller and a Waters 996 Photodiode array detector. pelargonidin-3-glucoside, cyanidin-3-glucoside, peonidin- For peak integration a Millenium32 software from Waters 3-glucoside, delphinidin-3-glucoside, petunidin-3-glucoside (Milford, MA, USA) was used. An Atlantisä C18 5 lm, and malvidin-3-glucoside were purchased from Polyphe- 4.6 mm · 150 mm column and a 4.6 mm · 20 mm guard nols Laboratories AS (Sandnes, Norway). The anthocya- column was used for the separation of phenolic compounds. nin aglycons from bilberry: cyanidin, pelargonidin, The mobile phase was composed of solvent A (nanopure peonidin, petunidin, delphinidin and malvidin were pur- water adjusted at pH 2.3 with 2 N HCl) and solvent B (ace- chased from ChromaDexä (Santa Clara, CA, USA). Phe- tonitrile HPLC grade). The elution was as follows: isocratic nolic acids (vanillin, p-coumaric, protocatechuic, ferulic conditions from 0 to 5 min with 85% A and 15% B. Gradi- acid, gallic acid, benzoic acid), flavonols (quercetin, rutin, ent conditions from minute 5 to 30 starting with 85% A and 958 R. Pedreschi, L.