Antioxidant Activity of Pomegranate Juice and Its Relationship with Phenolic Composition and Processing Marı ´a I. Gil, † Francisco A. Toma ´ s-Barbera ´n, † Betty Hess-Pierce, ‡ Deirdre M. Holcroft, § and Adel A. Kader* ,‡ Department of Pomology, University of California, Davis, California 95616, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 4195, Murcia 30080, Spain, and Department of Horticultural Science, Private Bag X1, Matieland 7602, South Africa The antioxidant activity of pomegranate juices was evaluated by four different methods (ABTS, DPPH, DMPD, and FRAP) and compared to those of red wine and a green tea infusion. Commercial pomegranate juices showed an antioxidant activity (18-20 TEAC) three times higher than those of red wine and green tea (6-8 TEAC). The activity was higher in commercial juices extracted from whole pomegranates than in experimental juices obtained from the arils only (12-14 TEAC). HPLC- DAD and HPLC-MS analyses of the juices revealed that commercial juices contained the pomegranate tannin punicalagin (1500-1900 mg/L) while only traces of this compound were detected in the experimental juice obtained from arils in the laboratory. This shows that pomegranate industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. This could account for the higher antioxidant activity of commercial juices compared to the experimental ones. In addition, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were detected and quantified in the pomegranate juices. Keywords: Pomegranate; Punica granatum; Punicaceae; juice; phenolics; anthocyanins; ellagic acid; punicalagin; tannins; antioxidant activity; ABTS; DPPH; DMPD; FRAP INTRODUCTION Epidemiological studies show that consumption of fruits and vegetables with high phenolic content cor- relate with reduced cardio- and cerebrovascular diseases and cancer mortality (Hertog et al., 1997a,b). Phenolic compounds may produce their beneficial effects by scavenging free radicals. In the past few years there has been an increasing interest in determining relevant dietary sources of antioxidant phenolics. Thus, red fruit juices such as grape and different berry juices have received attention due to their antioxidant activity. Pomegranate juice has become more popular because of the attribution of important biological actions (Lansky et al., 1998). Thus, the antioxidant and antitumoral activity of pomegranate bark tannins (punicacortein) (Kashiwada et al., 1992; Su et al., 1988) and the antioxidant activity of the fermented pomegranate juice (Schubert et al., 1999) have been reported. However, detailed investigations of the phenolic compounds and the antioxidant activity of the juice have not yet been carried out. Pomegranate juice is an important source of antho- cyanins, and the 3-glucosides and 3,5-diglucosides of delphinidin, cyanidin, and pelargonidin have been reported (Du et al., 1975). It also contains 1 g/L citric acid and only 7 mg/L ascorbic acid (El-Nemr et al., 1990). In addition, pomegranate bark (Tanaka et al., 1986b), leaf (Tanaka et al., 1985; Nawwar et al., 1994b), and the fruit husk (Mayer et al., 1977) are very rich in ellagitannins and gallotannins. Several apigenin and luteolin glycosides from pomegranate leaves (Nawwar et al., 1994a) and the hydrolyzable tannins punicalagin and punicalin from pomegranate husk have previously been identified (Mayer et al., 1977; Tanaka et al., 1986a). We report here on the evaluation of the antioxidant activity of pomegranate juice extracted by different procedures and the identification of the compounds responsible for this activity. MATERIALS AND METHODS Materials. Four types of pomegranate juices were produced from “Wonderful” pomegranates harvested in California during October 1998. Juice 1 was obtained in the laboratory from pomegranate arils by a hand press reaching a soluble solids (SS) value of 15.5%. Juice 2 was obtained as for juice 1, but in this case, the arils were frozen and stored for 9 months at -20 °C prior to juice preparation with a SS content of 16.6%. Juice 3 was a single-strength commercial juice produced by Green- Valley Packers (Arvin, CA) with a 16.6% SS, and juice 4 was a commercial concentrate juice produced by the same company. The juice 4 was reconstituted in the laboratory by adding water to decrease SS from 65.0 to 16.3% as in the original juice. Both commercial and experimental juices were stored frozen (-20 °C) until analyzed. HPLC-DAD Analyses. Three replicates from each juice were centrifuged in an eppendorf tube (2 min at 1400 rpm) and filtered through a 0.45 µm filter. Samples of 20 µL of juice were analyzed using an HPLC system (Hewlett-Packard 1050 pump) coupled with a photodiode array detector (DAD) (series 1040M, series II) and an autosampler (series 1050), operated by HP ChemStation software. A reversed-phase C 18 Nucleosil column (150 × 4.6 mm; particle size 5 µm) with a guard column * To whom correspondence should be addressed. Phone: (530) 752-0909. Fax: (530) 752-8502. E-mail: aakader@ ucdavis.edu. † Department of Food Science and Technology. ‡ Department of Pomology. § Department of Horticultural Science. 4581 J. Agric. Food Chem. 2000, 48, 4581-4589 10.1021/jf000404a CCC: $19.00 © 2000 American Chemical Society Published on Web 09/15/2000