Cent. Eur. J. Biol. • 4(4) • 2009 • 499–506 DOI: 10.2478/s11535-009-0041-1 Central European Journal of Biology Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn Research Article Wende Li1, Arnold W. Hydamaka 1, Lynda Lowry2, Trust Beta1,3* 1Department of Food Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 2Food Development Centre, Portage la Prairie, Manitoba R1N 3J9, Canada 3Richardson Centre for Functional Foods & Nutraceuticals, Smartpark, University of Manitoba, Winnipeg, Manitoba R3T 6C5, Canada Received 9 June 2009; Accepted 21 July 2009 Abstract: Antioxidant capacity and phenolic compounds (phenolic acids and anthocyanins) of four berry fruits (strawberry, Saskatoon berry, raspberry and wild blueberry), chokecherry and seabuckthorn were compared in the present study. Total phenolic content and total anthocyanin content ranged from 22.83 to 131.88 g/kg and 3.51 to 13.13 g/kg, respectively. 2,2-Diphenyl-1-picryhydrazyl free radical scavenging activity ranged from 29.97 to 78.86%. Chokecherry had the highest antioxidant capacity when compared with berry fruits and seabuckthorn. The highest caffeic acid, gallic acid and trans-cinnamic acid levels were found in chokecherry (6455 mg/kg), raspberry (1129 mg/kg) and strawberry (566 mg/kg), respectively. Caffeic acid was also the major phenolic acid in Saskatoon berry (2088 mg/kg) and wild blueberry (1473 mg/kg). The findings that chokecherry has very high antioxidant capacity and caffeic acid levels, are useful for developing novel value-added antioxidant products and also provide evidence essential for breeding novel cultivars of fruit plants with strong natural antioxidants. Keywords: Berry fruits • Chokecherry • Antioxidant capacity • Radical scavenging • Phenolic compounds • Anthocyanin • Phenolic acids © Versita Warsaw and Springer-Verlag Berlin Heidelberg. 1. Introduction in black currant berries [11] and raspberry [12] have been reported. The benefit of these phytochemicals in promoting health is believed to be achieved through Many epidemiological studies show that consumption of possible mechanisms of directly reacting with and fruits and vegetables is able to reduce the risk for some quenching free radicals, chelating transition metals, major human chronic diseases such as age-related reducing peroxides, and stimulating the antioxidative degenerative diseases, cancer and cardiovascular defense enzyme system [13]. Anticancer effects of berry diseases [1-3]. Beneficial effects of promoting health fruit bioactives have been confirmed, such as strawberry and preventing diseases are due to an enrichment of extracts inhibiting the growth of human oral, colon and phytochemicals present in various fruits and vegetables prostate cancer cells [10], cranberry proanthocyanidin [4,5]. The presence of phytochemicals including vitamin extract inhibiting the viability and proliferation of human C and tocopherols in seabuckthorn berries [6], phenolic esophageal adenocarcinoma cells [14]. Anticancer compounds in strawberry fruits [7-10], and anthocyanins impact of berry bioactives is believed to be achieved * E-mail: [email protected] 499 W. Li et al. through their ability to counteract, reduce, and repair ambient temperature in a rotary shaker (Fermentation damage resulting from oxidative stress and inflammation, Design Inc., Allentown, PA) set at 300 rpm. The mixture and also through regulating carcinogen and xenobiotic was then centrifuged at 7,800 x g (10,000 rpm, SS-34 metabolizing enzymes, various transcription and growth Rotors, RC5C Sorvall Instruments) at 5°C for 15 min. factors, inflammatory cytokines, and subcellular signaling The supernatant fluid was kept at -20°C in the dark until pathways of cancer cell proliferation, apoptosis, and further analysis for DPPH• scavenging activity, and TPC. tumor angiogenesis [15]. Various fruits and vegetables Extraction of each sample was carried out in duplicate. are an important part of our daily diets. Hence, fruits Fruit samples were subjected to alkaline hydrolysis and vegetables with a high level of antioxidant capacity for determination of their phenolic acid composition by should have potential for disease prevention and health HPLC according to our previous method with some promotion. modification [16]. The hydrolysis procedure was as Four berry fruits, chokecherry and seabuckthorn follows. Ground sample (2 g) was hydrolyzed using were selected for comparison of their antioxidant 2 M NaOH (60 mL) containing ascorbic acid (1% w/v) capacity and phenolic compounds in this study. The main and ethylenediaminetetraacetic acid (10 mM) for 2 h objective was to obtain an in-depth understanding of at 25oC in a rotary shaker (MaxQ 5000, BI Barnstead/ their antioxidant activity and phenolic acid composition. Lab-Line) set at 280 rpm. The hydrolyzed mixture was Antioxidant-enriched fruits have potential in value-added adjusted to pH 1.5-2.5 using ice-cold 6 M HCl and then applications in addition to their health benefits. centrifuged at 7,800 x g (10,000 rpm, RC5C, Sorvall Instruments, DuPont, Wilmington, DE) at 5oC for 20 min. The supernatant was extracted with hexane to remove 2. Experimental Procedures lipids. The final solution was extracted three times with ethyl acetate. The combined ethyl acetate fraction was 2.1 Chemicals evaporated to dryness at 35oC by using a rotary vacuum Folin-Ciocalteau reagent, 2,2-diphenyl-1- evaporator (RotaVapor R-205, BÜCHI Labortechnik AG, picryhydrazyl free radical (DPPH•), 2,2΄-azobis(2- Switzerland). The residue was dissolved in 5 mL of 50% methylpropionamidine) dihydrochloride (AAPH), and methanol, filtered through a 0.45 µm nylon filter, and 16 phenolic acid standards such as gallic, gentisic, analyzed by HPLC to obtain phenolic acid composition. p-coumaric, m-coumaric, caffeic, sinapic, ferulic, syringic, Hydrolysis of each sample was carried out in duplicate. o-coumaric, vanillic, protocatechuic, chlorogenic, isoferulic, ellagic, trans-cinnamic, and p-hydroxybenzoic 2.3 TPC determination acids were from Sigma-Aldrich (St. Louis, MO). All other TPCs were determined by using modified procedures chemicals and solvents were of the highest commercial of the Folin-Ciocalteau method [17]. The extract grade and used without further purification. (200 µL) was added to 1.9 mL of freshly diluted 10-fold Folin-Ciocalteau reagent. Sodium carbonate solution 2.2 Sample preparation (1.9 mL) (60 g/L) was then added to the mixture. After Four berry fruits (strawberry, Saskatoon berry, raspberry, 120 min of reaction at ambient temperature, the wild blueberry), chokecherry and seabuckthorn were absorbance of the mixture was measured at 725 nm selected for comparison of their antioxidant capacity and against a blank of distilled water. Ferulic acid was used phenolic compounds. Each of the fruit samples came as a standard and results expressed as ferulic acid from one variety. Each variety was randomly picked at equivalents. All analyses were performed in duplicate. the peak of their ripeness from several trees in orchards surrounding Winnipeg (Manitoba, Canada) and then 2.4 TAC determination combined. All fruits were first freeze-dried using Genesis TACs were determined according to the pH-differential 25 Freeze Dryer (SP Industries, Gardiner, NY) and then method [18]. Briefly, the extract (1 mL) was placed into a ground to powder prior to analyses. The stone seed 25 mL volumetric flask, made up to final volume with pH (stone) present in fresh chokecherry and seabuckthorn 1.0 buffer (1.49 g of KCl/100 mL water and 0.2 N HCl, was removed prior to freeze-drying. Their antioxidant with a ratio of 25:67), and mixed. Another 1 mL of extract components were extracted using ethanol (95%)/1N was also placed into a 25 ml volumetric flask, made up HCl (85:15, v/v) for analysis of total phenolic content to final volume with pH 4.5 buffer (1.64 g of sodium (TPC), total anthocyanin content (TAC), and DPPH• acetate/100 mL of water), and mixed. Absorbance was scavenging activity. The extraction procedure involved measured at 510 nm and at 700 nm. Absorbance was adding 15 mL of solvent to 1.0 g of ground sample in calculated as: D 50-mL brown bottles and shaking the sample for 6 h at A = (A510nm – A700nm)pH1.0 – (A510nm – A700nm)pH4.5. 500 Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn Results were calculated using the following equation 2.7 Statistical analysis and expressed as equivalents of cyanidin 3-glucoside: Data were subjected to one-way analysis of variance Total anthocyanin content (mg/kg) = (DA/εL) × MW for comparison of means, and significant differences × D × (V/G) × 1,000 were calculated according to Duncan’s multiple range where DA is absorbance, ε is cyanidin 3-glucoside molar test at the 5% level. Data were reported as means absorbance (26900), L is cell path length (1 cm), MW ± standard deviation. Differences at P<0.05 were is the molecular weight of cyanidin 3-glucoside (449.2), considered statistically significant. Quantitative results D is a dilution factor, V is the final volume (mL), G is were expressed on a dry weight basis (dwb). the sample weight (g), 1,000 is a conversion factor from gram to kilogram. All determinations were carried out at least in duplicate. 3. Results 2.5 DPPH• scavenging activity assay 3.1 TPC DPPH• scavenging activity was measured according to a The TPC, expressed as ferulic acid equivalents, of fruit method previously reported [19] with some modification. extracts are shown in Table 1. Significant differences in Briefly, a 60 µm DPPH• solution was freshly made in TPC were found between chokecherry and seabuckthorn, 95% ethanol solution. The extracts (200 µL) were between chokecherry and four berry fruits (strawberry, reacted with 3.8 mL of the DPPH• solution for 60 min. Saskatoon berry, raspberry and wild blueberry) and The absorbance (A) at 515 nm was measured against a between seabuckthorn and four berry fruits (strawberry, blank of pure 95% ethanol at t = 0, 5, 10, 20, 30, 40, 50, Saskatoon berry, raspberry and wild blueberry).