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* Department of Food Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
1
2 Food Development Centre, Portage la Prairie, Manitoba R1N 3J9, Canada
Richardson Centre for Functional Foods & Nutraceuticals, Smartpark, University of Manitoba, Winnipeg, Manitoba R3T 6C5, Canada
3
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 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
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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
* E-mail:
[email protected]
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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-1picryhydrazyl free radical (DPPH•), 2,2΄-azobis(2methylpropionamidine) 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 25oC in a rotary shaker (MaxQ 5000, BI Barnstead/ Lab-Line) set at 280 rpm. The hydrolyzed mixture was adjusted to pH 1.5-2.5 using ice-cold 6 M HCl and then 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 lipids. The final solution was extracted three times with ethyl acetate. The combined ethyl acetate fraction was evaporated to dryness at 35oC 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 = (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 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.
2.7 Statistical analysis
2.5 DPPH• scavenging activity assay
3.1 TPC
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 - [Asample t/Acontrol t=0]) × 100. DPPH tests were all carried out in duplicate.
2.6
Determination composition
of
phenolic
acid
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 30oC. 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.
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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 Saskatoon berry (SKB, 37.91 g/kg) > wild blueberry (WBB, 37.76 g/kg) > raspberry (RB, 36.81 g/kg) > strawberry (SB, 33.74 g/kg) > seabuckthorn without stone (SWS, 22.83 g/kg). However, significant differences were not found among the berry fruits (SB, SKB, RB and WBB). Name
Equivalent of ferulic acid (g/kg)
Strawberry
33.74 ± 1.86b
Saskatoon berry
37.91 ± 0.61b
Raspberry
36.81 ± 0.95b
Wild blueberry
37.76 ± 0.93b
Chokecherry without stone
131.88 ± 8.09a
Seabuckthorn without stone
22.83 ± 1.40c
Table 1. Total phenolic content of extracts from Manitoba fruitsa. 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
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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 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 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 H2O2 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
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.
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