Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn

Transcription

1 Cent. Eur. J. Biol. 4(4) DOI: /s Central European Journal of Biology Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn Research Article Wende Li 1, Arnold W. Hydamaka 1, Lynda Lowry 2, Trust Beta 1,3* 1 Department of Food Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada 2 Food Development Centre, Portage la Prairie, Manitoba R1N 3J9, Canada 3 Richardson 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 to g/kg and 3.51 to g/kg, respectively. 2,2-Diphenyl-1-picryhydrazyl free radical scavenging activity ranged from 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 Many epidemiological studies show that consumption of fruits and vegetables is able to reduce the risk for some major human chronic diseases such as age-related degenerative diseases, cancer and cardiovascular diseases [1-3]. Beneficial effects of promoting health and preventing diseases are due to an enrichment of phytochemicals present in various fruits and vegetables [4,5]. The presence of phytochemicals including vitamin C and tocopherols in seabuckthorn berries [6], phenolic compounds in strawberry fruits [7-10], and anthocyanins 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 possible mechanisms of directly reacting with and quenching free radicals, chelating transition metals, reducing peroxides, and stimulating the antioxidative defense enzyme system [13]. Anticancer effects of berry fruit bioactives have been confirmed, such as strawberry extracts inhibiting the growth of human oral, colon and prostate cancer cells [10], cranberry proanthocyanidin extract inhibiting the viability and proliferation of human esophageal adenocarcinoma cells [14]. Anticancer impact of berry bioactives is believed to be achieved 499 * Trust_Beta@umanitoba.ca

2 W. Li et al. through their ability to counteract, reduce, and repair damage resulting from oxidative stress and inflammation, and also through regulating carcinogen and xenobiotic metabolizing enzymes, various transcription and growth factors, inflammatory cytokines, and subcellular signaling pathways of cancer cell proliferation, apoptosis, and tumor angiogenesis [15]. Various fruits and vegetables are an important part of our daily diets. Hence, fruits and vegetables with a high level of antioxidant capacity should have potential for disease prevention and health promotion. Four berry fruits, chokecherry and seabuckthorn were selected for comparison of their antioxidant capacity and phenolic compounds in this study. The main objective was to obtain an in-depth understanding of their antioxidant activity and phenolic acid composition. Antioxidant-enriched fruits have potential in value-added applications in addition to their health benefits. 2. Experimental Procedures 2.1 Chemicals Folin-Ciocalteau reagent, 2,2-diphenyl-1- picryhydrazyl free radical (DPPH ), 2,2 -azobis(2- methylpropionamidine) dihydrochloride (AAPH), and 16 phenolic acid standards such as gallic, gentisic, p-coumaric, m-coumaric, caffeic, sinapic, ferulic, syringic, o-coumaric, vanillic, protocatechuic, chlorogenic, isoferulic, ellagic, trans-cinnamic, and p-hydroxybenzoic acids were from Sigma-Aldrich (St. Louis, MO). All other chemicals and solvents were of the highest commercial grade and used without further purification. 2.2 Sample preparation Four berry fruits (strawberry, Saskatoon berry, raspberry, wild blueberry), chokecherry and seabuckthorn were selected for comparison of their antioxidant capacity and phenolic compounds. Each of the fruit samples came from one variety. Each variety was randomly picked at the peak of their ripeness from several trees in orchards surrounding Winnipeg (Manitoba, Canada) and then combined. All fruits were first freeze-dried using Genesis 25 Freeze Dryer (SP Industries, Gardiner, NY) and then ground to powder prior to analyses. The stone seed (stone) present in fresh chokecherry and seabuckthorn was removed prior to freeze-drying. Their antioxidant components were extracted using ethanol (95%)/1N HCl (85:15, v/v) for analysis of total phenolic content (TPC), total anthocyanin content (TAC), and DPPH scavenging activity. The extraction procedure involved adding 15 ml of solvent to 1.0 g of ground sample in 50-mL brown bottles and shaking the sample for 6 h at ambient temperature in a rotary shaker (Fermentation Design Inc., Allentown, PA) set at 300 rpm. The mixture was then centrifuged at 7,800 x g (10,000 rpm, SS-34 Rotors, RC5C Sorvall Instruments) at 5 C for 15 min. The supernatant fluid was kept at -20 C in the dark until further analysis for DPPH scavenging activity, and TPC. Extraction of each sample was carried out in duplicate. Fruit samples were subjected to alkaline hydrolysis for determination of their phenolic acid composition by HPLC according to our previous method with some modification [16]. The hydrolysis procedure was as follows. Ground sample (2 g) was hydrolyzed using 2 M NaOH (60 ml) containing ascorbic acid (1% w/v) and ethylenediaminetetraacetic acid (10 mm) for 2 h at 25 o C in a rotary shaker (Max Q 5000, BI Barnstead/ Lab-Line) set at 280 rpm. The hydrolyzed mixture was adjusted to ph using ice-cold 6 M HCl and then centrifuged at 7,800 x g (10,000 rpm, RC5C, Sorvall Instruments, DuPont, Wilmington, DE) at 5 o C for 20 min. The supernatant was extracted with hexane to remove lipids. The final solution was extracted three times with ethyl acetate. The combined ethyl acetate fraction was evaporated to dryness at 35 o C by using a rotary vacuum evaporator (RotaVapor R-205, BÜCHI Labortechnik AG, Switzerland). The residue was dissolved in 5 ml of 50% methanol, filtered through a 0.45 µm nylon filter, and analyzed by HPLC to obtain phenolic acid composition. Hydrolysis of each sample was carried out in duplicate. 2.3 TPC determination TPCs were determined by using modified procedures of the Folin-Ciocalteau method [17]. The extract (200 µl) was added to 1.9 ml of freshly diluted 10-fold Folin-Ciocalteau reagent. Sodium carbonate solution (1.9 ml) (60 g/l) was then added to the mixture. After 120 min of reaction at ambient temperature, the absorbance of the mixture was measured at 725 nm against a blank of distilled water. Ferulic acid was used as a standard and results expressed as ferulic acid equivalents. All analyses were performed in duplicate. 2.4 TAC determination TACs were determined according to the ph-differential method [18]. Briefly, the extract (1 ml) was placed into a 25 ml volumetric flask, made up to final volume with ph 1.0 buffer (1.49 g of KCl/100 ml water and 0.2 N HCl, with a ratio of 25:67), and mixed. Another 1 ml of extract was also placed into a 25 ml volumetric flask, made up to final volume with ph 4.5 buffer (1.64 g of sodium acetate/100 ml of water), and mixed. Absorbance was measured at 510 nm and at 700 nm. Absorbance was calculated as: DA = (A 510nm A 700nm )ph 1.0 (A 510nm A 700nm )ph

3 Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn Results were calculated using the following equation and expressed as equivalents of cyanidin 3-glucoside: Total anthocyanin content (mg/kg) = (DA/εL) MW D (V/G) 1,000 where DA is absorbance, ε is cyanidin 3-glucoside molar absorbance (26900), L is cell path length (1 cm), MW is the molecular weight of cyanidin 3-glucoside (449.2), D is a dilution factor, V is the final volume (ml), G is the sample weight (g), 1,000 is a conversion factor from gram to kilogram. All determinations were carried out at least in duplicate. 2.5 DPPH scavenging activity assay DPPH scavenging activity was measured according to a method previously reported [19] with some modification. Briefly, a 60 µm DPPH solution was freshly made in 95% ethanol solution. The extracts (200 µl) were reacted with 3.8 ml of the DPPH solution for 60 min. The absorbance (A) at 515 nm was measured against a blank of pure 95% ethanol at t = 0, 5, 10, 20, 30, 40, 50, and 60 min. The chemical kinetics of antioxidant activity of fruit extracts was also recorded. Antioxidant activity was calculated as follows: % DPPH scavenging activity = (1 - [A sample t /A control t=0 ]) 100. DPPH tests were all carried out in duplicate. 2.6 Determination of phenolic acid composition HPLC analysis for phenolic acid compositions of fruit hydrolysate was performed on a Waters model 2695 chromatograph instrument (Waters, Mississauga, ON, Canada) equipped with a Waters 2996 photodiode array detector. Phenolic acids were separated on a reversephase Phenomenex C18 column (150 mm 4.6 mm) with a gradient of solvent A (water containing 1% (v/v) formic acid) and solvent B (methanol containing 0.1% (v/v) formic acid) for 72 min at a flow rate of 0.7 ml/min. The column temperature was set at 30 o C. The solvent gradient was programmed as follows: at 0 min 15% B, 7 min 20% B, at 8 to 20 min 15% B, 21 to 33 min 24% B, 34 to 36 min 13% B, 37 to 45 min 20% B, 46 to 62 min 42% B, 63 to 68 min 100% B, and 69 to 72 min 15% B. Phenolic acids in the eluants were monitored at 270 and 325 nm synchronously. Identification of the phenolic acids was accomplished by comparing the retention times of peaks in samples to those of 16 phenolic acid standards. The HPLC profile of 16 phenolic acid standards is shown in Figure 2A. Isoferulic acid was used as the internal standard. The HPLC analyses were carried out in duplicate. 2.7 Statistical analysis Data were subjected to one-way analysis of variance for comparison of means, and significant differences were calculated according to Duncan s multiple range test at the 5% level. Data were reported as means ± standard deviation. Differences at P<0.05 were considered statistically significant. Quantitative results were expressed on a dry weight basis (dwb). 3. Results 3.1 TPC The TPC, expressed as ferulic acid equivalents, of fruit extracts are shown in Table 1. Significant differences in TPC were found between chokecherry and seabuckthorn, between chokecherry and four berry fruits (strawberry, Saskatoon berry, raspberry and wild blueberry) and between seabuckthorn and four berry fruits (strawberry, Saskatoon berry, raspberry and wild blueberry). Chokecherry had the highest TPC ( g/kg) among the six fruits. The TPC decreased in the following order: chokecherry without stone (CCWS) > Saskatoon berry (SKB, g/kg) > wild blueberry (WBB, g/kg) > raspberry (RB, g/kg) > strawberry (SB, g/kg) > seabuckthorn without stone (SWS, g/kg). However, significant differences were not found among the berry fruits (SB, SKB, RB and WBB). Name Strawberry Saskatoon berry Raspberry Wild blueberry Chokecherry without stone Seabuckthorn without stone Equivalent of ferulic acid (g/kg) ± 1.86 b ± 0.61 b ± 0.95 b ± 0.93 b ± 8.09 a ± 1.40 c Table 1. Total phenolic content of extracts from Manitoba fruits a. a Values are mean ± standard deviation. Values with the same letter are not statistically different at the 5% level (Duncan s multiple range test). 3.2 TAC The TAC, expressed as cyanidin 3-glucoside equivalents, of fruit extracts are shown in Table 2. The colour of extracts from berry fruits (SB, SKB, RB and WBB) and CCWS was red in acidic media. The red colour indicates the presence of anthocyanin compounds in fruits. However, anthocyanins in SWS were not detectable. CCWS had a higher TAC (13.13 g/kg) than other berry fruits (SB, SKB, RB and WBB). The TAC decreased in 501

4 W. Li et al. the order CCWS > SKB (10.79 g/kg) > WBB (9.97 g/kg) > RB (6.62 g/kg) > SB (3.51 g/kg). The TAC levels in CCWS were 20 to 270% higher in comparison to the berry fruits (SB, SKB, RB and WBB) studied. Significant differences in TAC were found among CCWS and berry fruits (SKB, WBB, RB and SB). The results indicated that anthocyanins present in fruits were significantly affected by the varieties or genotypes of fruits. A significant correlation (r=0.6739) was found between TAC and TPC at P<0.05. Name Strawberry Equivalent of cyanidian-3-glucoside (g/kg) 3.51 ± 0.01 e Name DPPH scavenging (%) Strawberry Saskatoon berry Raspberry Wild blueberry Chokecherry without stone Seabuckthorn without stone ± 0.96 c ± 0.67 d ± 0.38 b ± 1.20 e ± 0.54 a ± 0.51 f Table 3. DPPH scavenging activity after 60 min reaction time a. a Values are mean ± standard deviation. Values with the same letter are not statistically different at the 5% level (Duncan s multiple range test). DPPH radical scavenging (%) was determined at 60-fold dilution for extracts of 1 g (dwb) powder. Saskatoon berry Raspberry Wild blueberry Chokecherry without stone ± 0.18 b 6.62 ± 0.25 d 9.97 ± 0.45 c ± 0.03 a Seabuckthorn without stone Table 2. Total anthocyanin content of extracts from Manitoba fruits a. nd a Values are mean ± standard deviation. Values with the same letter are not statistically different at the 5% level (Duncan s multiple range test). nd, not detectable. 3.3 DPPH scavenging activity DPPH scavenging activity of fruit extracts is shown in Table 3. Significant differences in scavenging activity were found among six types of fruits. DPPH scavenging activity (78.86%) of CCWS was the highest among the fruit extracts. DPPH scavenging activity decreased in the order of CCWS > RB (51.23%) > SB (40.33%) > SKB (36.59%) > WBB (34.13%) > SWS (29.97%). CCWS had 0.54 to 1.63 times greater scavenging activity than berry fruits (SB, SKB, RB and WBB) and seabuckthorn. The reaction kinetics of fruit extracts with DPPH is shown in Figure 1. The kinetic curves clearly indicated that CCWS extract had high scavenging activity during the reaction period. CCWS extract still kept a high reaction rate after 10 min, however the reaction rate of berry fruit and seabuckthorn extracts became progressively slow and stable (Figure 1). A highly significant (P<0.05) correlation (r=0.9362) was found between DPPH scavenging activity and TPC. However, the correlation (r=0.5909) between DPPH scavenging activity and TAC was relatively low but significant (P<0.05). 3.4 Phenolic acid composition The phenolic acid composition of berry fruits (SB, SKB, RB and WBB), chokecherry and seabuckthorn is shown in Table 4. The HPLC profile of a representative Figure 1. Kinetics of antioxidant activity of fruit extracts using the DPPH free radical. Notes: SB, strawberry; SKB, Saskatoon berry; RB, Raspberry; WBB, Wild blueberry; CCWS, Chokecherry without stone; SWS, Seabuckthorn without stone. DPPH radical scavenging (%) was determined at 60-fold dilution for extracts of 1 g (dwb) powder. chromatogram of WBB is listed in Figure 2B. Nine types of phenolic acids were detected in SB and WBB, eight types in RB, seven types in SKB, five types in SWS and four types in CCWS. The major phenolic acid compounds (>100 mg/kg) were trans-cinnamic (566 mg/kg), p-coumaric (213 mg/kg), gallic (212 mg/kg) and p-hydroxybenzoic (124 mg/kg) acids in SB, caffeic (2088 mg/kg) and protocatechuic (132 mg/kg) acids in SKB, gallic (1129 mg/kg) and protocatechuic (102 mg/kg) acids in RB, caffeic (1473 mg/kg), syringic (286 mg/kg) and gallic (190 mg/kg) acids in WBB, and caffeic (6455 mg/kg), p-coumaric (953 mg/kg) and protocatechuic (214 mg/kg) acids in CCWS. Chokecherry showed very high levels of caffeic acid. Chokecherry had 2.1, 3.4, 188.9, and times higher caffeic acid levels than the berry fruits (SKB, WBB, RB, SB) and seabuckthorn, respectively. Chokecherry also contained a high p-coumaric acid content with levels 3.5, 10.6, 12.2, 502

5 Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn Phenolic acids SB SKB RB WBB CCWS SWS GA 212 ± 18 b 22 ± 4 c 1129 ± 62 a 190 ± 3 b nd 42 ± 10 c PA 34 ± 2 d 132 ± 3 b 102 ± 26 c 76 ± 8 c 214 ± 6 a 42 ± 2 d p-ha 124 ± 5 a 7 ± 4 c 54 ± 9 b nd nd nd VA nd nd nd 56 ± 3 nd nd CA 24 ± 2 d 2088 ± 31 b 34 ± 4 d 1473 ± 6 c 6456 ± 503 a 10 ± 2 d SYA nd nd nd 286 ± 3 nd nd p-ca 213 ± 2 b 82 ± 4 c 68 ± 7 c 39 ± 2 c 953 ± 43 a 72 ± 3 c FA 14 ± 1 c 50 ± 3 a 35 ± 6 b 41 ± 6 ba 43 ± 5 ba 15 ± 2 c SIA nd nd 18 ± 2 nd nd nd o-ca 16 ± 2 nd nd nd nd nd EA 20 ± 9 b 20 ± 6 b 52 ± 8 a 24 ± 5 b nd nd trans-ca 566 ± 9 a nd nd 9 ± 3 b nd nd Table 4. Phenolic acid composition (mg/kg) of fruit hydrolysates a. a Values are mean ± standard deviation. Values with the same letter in the rows are not statistically different at the 5% level (Duncan s multiple range test). GA, gallic acid; PA, protocatechuic acid; p-ha, p-hydroxybenzoic acid; VA, vanillic acid; CA, caffeic acid; SYA, syringic acid; p-ca, p-coumaric acid; FA, ferulic acid; SIA, sinapinic acid; o-ca, o-coumaric acid; EA, ellagic acid; trans-ca, trans-cinnamic acid. SB, strawberry; SKB, Saskatoon berry; RB, Raspberry; WBB, Wild blueberry; CCWS, Chokecherry without stone; SWS, Seabuckthorn without stone. nd, not detectable and 23.4 times higher in comparison with those found in SB, SKB, SWS, RB and WBB, respectively. Protocatechuic acid level contained in chokecherry was also the highest with 1.6, 2.1, 2.8, 5.1 and 6.3 times higher in comparison with those detected in SKB, RB, WBB, SWS and SB, respectively. The mapping of phenolic acid composition was achieved for berry fruits, chokecherry and seabuckthorn; that is, SB contained higher trans-cinnamic acid content and RB was richer in gallic acid in comparison to other fruits. Higher contents of caffeic acid were also present in WBB and SKB when compared to the levels in SB, RB and SWS. 4. Discussion Figure 2. (A) The HPLC profile of 16 standard phenolic acids. (B) The HPLC profile of wild blueberry. Numbers in (A) indicate the following standard chemicals: 1, gallic acid; 2, protocatechuic acid; 3, p-hydroxybenzoic acids; 4, gentisic acid; 5, chlorogenic acid; 6, vanillic acid; 7, caffeic acid; 8, syringic acid; 9, p-coumaric acid; 10, ferulic acid; 11, m-coumaric acid; 12, sinapic acid; 13, isoferulic acid; 14, o-coumaric acid; 15, ellagic acid; 16, trans-cinnamic acid. Numbers in (B) indicate the same chemicals as in (A) and isoferulic acid was used as internal standard in (B). Solid and dotted lines in (A) and (B) indicate 270 nm and 325 nm, respectively. TPC was affected by fruit type [7,9]. High TPC is generally regarded as an indication of high total antioxidant capacity [20]. The very high TPC of chokecherry (Table 1), up three to six times greater than the other five fruits, showed its potent potential as a source of natural antioxidants. The benefits of phenolic-rich juice from grapes, some cherries and berries are to prevent cell death and DNA single-strand breakage against induced oxidative stress through an iron-chelating mechanism [21]. Phenolic compound fractions from muscadine grapes and blueberries demonstrate inhibition effects on the growth of HepG2 liver cancer cell [22]. Phenolic compounds in fruits also contribute to the in vitro inhibition of tumour-cell proliferation [4]. 503

6 W. Li et al. Anthocyanins belong to a class of important antioxidants and are enriched in colorful fruits. They are quantitatively the most important type of fruit polyphenol compounds. Anthocyanin compounds, such as pelargonidin-glucoside, pelargonidin-malosideglucoside, pelargonidin-rutinoside and cyanidinglucoside, exist in different strawberry genotypes [9]. The benefits of anthocyanins in improving health and preventing disease have received great attention in recent years. Some berry and cherry fruits are rich sources of natural anthocyanins. Anthocyanins in grape juice had the ability to reduce in vitro oxidation of human low-density lipoprotein [23]. Anticancer and antitumour activities of anthocyanins from various sources have been demonstrated, such as inhibition of tumor development and reduction in the proliferation of colon cancer cells [24], prevention of carcinogen-induced colorectal cancer [25], and blocking of breast cell DNA damage [26]. It was reported that anthocyanins from some fruit plants had inhibitory effects on the growth of colonic cancer cell [27] and HepG2 liver cancer cell [22]. Free radicals may attack and damage important biological components in biological systems and lead to illnesses, such as cancers, heart diseases, and other aging-associated health problems [28]. A diet rich in antioxidants will be an effective pathway for reducing the risk of these illnesses because antioxidants may terminate the attack of free radicals. Consumers prefer natural antioxidants as they are concerned about the safety of the synthetic antioxidants. In addition, antioxidants are important food additives to enhance the quality, stability, and safety of food products [28]. Fruits with high free radical scavenging activity, especially chokecherry (Table 3) found in this study, have potential as a source of novel natural antioxidants for disease prevention and health promotion, and also as natural food additives to improve the quality, stability, and safety of food products. It is very useful to evaluate phenolic acid composition in plants for application purposes. Glucose esters of p-coumaric and ellagic acids were found in strawberry extracts [10]. The antioxidant capacity of some phenolic acids decreased in the order protocatechuic acid > chlorogenic acid > caffeic acid > p-hydroxybenzoic acid > gentistic acid > ferulic acid > vanillic acid > syringic acid > p-coumaric acid [29]. Oxygen radical absorbance capacity method also confirmed that protocatechuic acid (18.61 µmol Trolox equ.mg -1 ), caffeic acid (15.28 µmol Trolox equ.mg -1 ) and gentistic acid (13.85 µmol Trolox equ.mg -1 ) had high antioxidant capacity when compared with p-coumaric acid (10.16 µmol Trolox equ.mg -1 ), ferulic acid (8.48 µmol Trolox equ.mg -1 ), vanillic acid (7.22 µmol Trolox equ.mg -1 ) and gallic acid (6.97 µmol Trolox equ.mg -1 ) [30]. High antioxidant activity of protocatechuic and caffeic acids is possible to have a positive relation with their two ortho hydroxyl groups present in benzenic ring (2, 5 in Figure 2) according to established structure-activity relation from Villaño et al. [30]. Antiradical efficiencies of caffeic acid, gallic acid, tannic acid, and ferulic acid were 2.75, 2.62, 0.57, and 0.12, respectively [31]. In comparison with berry fruits (SKB, WBB, RB, SB) and seabuckthorn, rich caffeic (up to 6456 mg/kg) and high protocatechuic (214 mg/kg) acid levels found in chokecherry (Table 4) indicated the potential of chokecherry as novel source of natural antioxidants because of protocatechuic and caffeic acids with strong antioxidant capacity. The impact of some phenolic acids on health has been studied. Caffeic acid has novel and therapeutic effects on hepatocarcinoma cells [32] and protects WI-38 human lung fibroblast cells against H 2 O 2 damage [33]. It was reported that caffeic, ellagic, chlorogenic and ferulic acids had inhibition effect on 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis [34]. Ferulic acid may offer beneficial effects against cancer, cardiovascular disease, diabetes and Alzheimer s disease [35]. p-coumaric acid protects the heart against doxorubicin-induced oxidative stress [36] and demonstrates good antiplatelet activity for the prevention of vascular disease [37]. Protocatechuic acid has been shown to induce hepatocellular carcinoma cell death [38]. It was reported that gallic acid had anticancer effects by inhibiting cancer cell proliferation through inducing apoptosis in cancer cells, such as against esophageal cancer cells [39], against stomach cancer and colon adenocarcinoma cells [40], and against lung cancer cells [41]. When trans-cinnamic acid was used in combination with anti-tuberculosis drugs, synergistic activities against Mycobacterium tuberculosis were enhanced [42]. Because of the important benefist of phenolic acids in promoting health, it is valuable to fully understand phenolic acid composition of fruits as a novel source of natural antioxidants for developing functional food. 5. Conclusion The study reported the antioxidant capacity of four berry fruits, chokecherry and seabuckthorn. Differences in antioxidant capacity were found among some of them. Antioxidant capacity was closely related to fruit type. Chokecherry showed very high antioxidant capacity including its TPC, TAC, DPPH scavenging activity and caffeic acid level in comparison with the four berry fruits and seabuckthorn studied. The findings on phenolic 504

7 Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn acid composition indicated that trans-cinnamic acid and gallic acid were present at high levels in strawberry and raspberry, respectively. Although the highest caffeic acid levels were observed in chokecherry, Saskatoon berry and wild blueberry also contained high caffeic acid levels. The results provide evidence essential for breeding novel cultivars of fruit plants with strong natural antioxidants and developing new functional food products. Further research is required to identify anthocyanin composition from the tested fruits and to investigate effect of phenolic acid composition on fruit quality and to demonstrate the inhibition effects of chokecherry antioxidants on cancer cells. Acknowledgements We are grateful for the financial support provided by the Canada Foundation for Innovation (CFI New Opportunities Fund), and Canada Research Chairs Program and the Manitoba Agri-Food Research and Development Initiative (ARDI). We thank ARDI for facilitating the provision of samples used for this study. We are thankful for the technical assistance from Wan Yuin (Alison) Ser of the Department of Food Science at University of Manitoba. References [1] Ames B.M., Shigens M.K., Hagen T.M., Oxidants, antioxidants and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A., 1993, 90, [2] Halliwell B., Free radical, antioxidants and human disease: curiosity, cause or consequence, Lancet, 1994, 344, [3] Ribolin E., Norat T., Epidemiological evidence of the protective effect of fruit and vegetables on cancer risk, Am. J. Clin. Nutr., 2003, 78, 559S-569S [4] Eberhardt M.V., Lee C.Y., Liu R.H., Antioxidant activity of fresh apples, Nature, 2000, 405, [5] Arts I.C., Hollman P.C., Polyphenols and disease risk in epidemiologic studies, Am. J. Clin. Nutr., 2005, 81, 243S-255S [6] Kallio H., Yang B., Peippo P., Effects of different origins and harvesting time on Vitamin C, tocopherols and tocotrienols in seabuckthorn (Hippophane rhamnoides) berries, J. Agric. Food Chem., 2002, 50, [7] Scalzo J., Politi A., Pellegrini N., Mezzetti B., Battino M., Plant genotype affects total antioxidant capacity and phenolic contents in fruit, Nutrition, 2005, 21, [8] Aaby K., Ekeberg D., Skrede G., Characterization of phenolic compounds in strawberry (Fragaria ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity, J. Agric. Food Chem., 2007, 55, [9] Tulipani S., Mezzetti B., Capocasa F., Bompadre S., Beekwilder J., Vos C.H.R.D., et al., Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes, J. Agric. Food Chem., 2008, 56, [10] Zhang Y., Seeram N.P., Lee R., Feng L., Heber D., Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties, J. Agric. Food Chem., 2008, 56, [11] Slimestad R., Solheim H., Anthocyanins from black currants (Ribes nigrum L.), J. Agric. Food Chem., 2002, 50, [12] McDougall G.J., Dobson P., Smith P., Blake A., Stewart D., Assessing potential bioavailability of raspberry anthocyanins using an in vitro digestion system, J. Agric. Food Chem., 2005, 53, [13] Zhou K., Yu L., Effects of extraction solvent on wheat bran antioxidant activity estimation, LWT- Food Sci. Technol., 2004, 37, [14] Kresty L.A., Howell A.B., Baird M., Cranberry proanthocyanidins induce apoptosis and inhibit acid-induced proliferation of human esophageal adenocarcinoma cells, J. Agric. Food Chem., 2008, 56, [15] Seeram N.P., Berry fruits for cancer prevention: current status and future prospects, J. Agric. Food Chem., 2008, 56, [16] Li W., Wei C., White P.J., Beta T., High-amylose corn exhibits better antioxidant activity than typical and waxy genotypes, J. Agric. Food Chem., 2007, 55, [17] Li W., Pickard M.D., Beta T., Evaluation of antioxidant activity and electronic taste and aroma properties of antho-beers from purple wheat grain, J. Agric. Food Chem., 2007, 55, [18] Guisti M.M., Wrolstad R.E., Characterization and measurement of anthocyanins by UV-visible spectroscopy, In: Wrolstad R.E., (Ed.), Current Protocols in Food Analytical Chemistry, John Wiley & Sons Inc, New York, 2000 [19] Brand-Williams W., Cuvelier M.E., Berset C., Use of a free radical method to evaluate antioxidant activity, LWT-Food Sci. Technol., 1995, 28, [20] Li W., Pickard M.D., Beta T., Effect of thermal processing on antioxidant properties of purple wheat bran, Food Chem., 2007, 104,

8 W. Li et al. [21] Garcia-Alonso F.J., Guidarelli A., Periago M.J., Phenolic-rich juice prevents DNA single-strand breakage and cytotoxicity caused by tertbutylhydroperoxide in U937 cells: the role of iron chelation, J. Nutr. Biochem., 2007, 18, [22] Yi W., Akoh C.C., Fischer J., Krewer G., Effects of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis, Food Res. Int., 2006, 39, [23] Kay C.D., Holub B.J., The effect of wild blueberry (Vaccinium angustifolium) consumption on postprandial serum antioxidant status in human subjects, Br. J. Nutr., 2002, 88, [24] Kang S.Y., Seeram N.P., Nair M.G., Bourquin L.D., Tart cherry anthocyanins inhibit tumor development in Apc Min mice and reduce proliferation of human colon cancer cells, Cancer Lett., 2003, 194, [25] Cooke D., Schwarz M., Boocock D., Winterhalter P., Steward W.P., Gescher A.J., et al., Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the Apc Min mouse model of intestinal carcinogenesis - Relationship with tissue anthocyanin levels, Int. J. Cancer, 2006, 119, [26] Singletary K.W., Jung K.J., Giusti M., Anthocyaninrich grape extract blocks breast cell DNA damage, J. Med. Food, 2007, 10, [27] Zhao C., Monica G., Malik M., Moyer M.P., Magnuson B.A., Effects of commercial anthocyaninrich extracts on colonic cancer and nontumorigenic colonic cell growth, J. Agric. Food Chem., 2004, 52, [28] Zhou K., Yu L., Antioxidant properties of bran extracts from Trego wheat grown at different locations, J. Agric. Food Chem., 2004, 52, [29] Onyeneho S.N., Hettiarachchy N.S., Antioxidant activity of durum wheat bran, J. Agric. Food Chem., 1992, 40, [30] Villaño D., Fernández-Pachón M.S., Troncoso A.M., García-Parrilla M.C., Comparison of antioxidant activity of wine phenolic compounds and metabolites in vitro, Anal. Chim. Acta, 2005, 538, [31] Sánchez-Moreno C., Larrauri J.A., Saura-Calixto F., A procedure of measure the antiradical efficiency of polyphenols, J. Sci. Food Agric., 1998, 76, [32] Chung T.-W., Moon S.-K, Chang Y.-C, Ko J.-H., Lee Y.-C., Cho G., et al., Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism, FASEB J., 2004, 18, [33] Kang K.A, Lee K.H., Zhang R., Piao M., Chae S., Kim K.N., et al., Caffeic acid protects hydrogen peroxide induced cell damage in WI-38 human lung fibroblast cells, Biol. Pharm. Bull., 2006, 29, [34] Tanaka T., Kojima T., Kawamori T., Wang A., Suzui M., Okamoto K., et al., Inhibition of 4-nitroquinoline- 1-oxide-induced rat tongue carcinogenesis by the naturally occurring plant phenolic caffeic, ellagic, chlorogenic and ferulic acids, Carcinogenesis, 1993, 14, [35] Zhao Z., Moghadasian M.H., Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: A review, Food Chem., 2008, 109, [36] Abdel-Wahab M.H., Ei-Mahdy M.A., Abd-Ellah M.F., Helal G.K., Khalifa F., Hamada F.M.A., Influence of p-coumaric acid on doxorubicin-induced oxidative stress in rat s heart, Pharmacol Res., 2003, 48, [37] Luceri C., Giannini L., Lodovici M., Antonucci E., Abbate R., Masini E., et al., p-coumaric acid, a common dietary phenol, inhibits platelet activity in vitro and in vivo, Br. J. Nutr., 2007, 97, [38] Yip E.C.H., Chan A.S.L., Pang H., Tam Y.K., Wong Y.H., Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-jun N-terminal kinase-dependent mechanism, Cell Biol. Toxicol., 2006, 22, [39] Faried A., Kurnia D., Faried L.S., Usman N., Miyazaki T., Kato H., et al, Anticancer effects of gallic acid isolated from Indonesian herbal medicine, Phaleria macrocarpa (Scheff.) Boerl, on human cancer cell lines, Int. J. Oncol., 2007, 30, [40] Yoshioka K., Kataoka T., Hayashi T., Hasegawa M., Ishi Y., Hibasami H., Induction of apoptosis by gallic acid in human stomach cancer KATO III and colon adenocarcinoma COLO 205 cell lines, Oncol. Rep., 2000, 7, [41] Ohno Y., Fukuda K., Takemura G., Toyota M., Watanabe M., Yasuda N., et al., Induction of apoptosis by gallic acid in lung cancer cells, Anti- Cancer Drugs, 1999, 10, [42] Rastogi N., Goh K.S., Horgen L., Barrow W.W., Synergistic activities of antituberculous drugs with cerulenin and trans-cinnamic acid against mycobacterium tuberculosis, FEMS Immunol. Med. Microbiol., 1998, 21,