Antioxidant Activities of Selected Berries and Their Free, Esterified, and Insoluble-Bound Phenolic Acid Contents.

To explore the potential of berries as natural sources of bioactive compounds, the quantities of free, esterified, and insoluble-bound phenolic acids in a number of berries were determined. In addition, the antioxidant activities of the berries were determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric reducing antioxidant power, and Trolox equivalent antioxidant capacity assays, in addition to determination of their metal ion chelating activities. Furthermore, several phenolic compounds were detected using high-performance liquid chromatography. Of the 6 tested berries, black chokeberry and blackberry exhibited the strongest antioxidant activities, and the various berry samples were found to contain catechin, caffeic acid, p-coumaric acid, epicatechin, vanillic acid, quercitrin, resveratrol, morin, naringenin, and apigenin. Moreover, the antioxidant activities and total phenolic contents of the fractions containing insoluble-bound phenolic acids were higher than those containing the free and esterified phenolic acids. The results imply that the insoluble-bound fractions of these berries are important natural sources of antioxidants for the preparation of functional food ingredients and preventing diseases associated with oxidative stress.

INTRODUCTION

Berries, which are particularly important in the Finnish diet, contain many essential functional components, including flavonoids and phenolic acids, which constitute two large and heterogeneous groups of biologically active non-nutrients. Although the contents of flavonoids and phenolic acids vary widely within berries, the contents of phenolic compounds within a single species also vary due to differences in the berry varieties (1) and growth conditions (2).

In a structural context, phenolic compounds are composed of aromatic rings that bear one or more hydroxyl groups, and are generally categorized as phenolic acids, flavonoids, anthocyanins, or tannins (3). Indeed, such phenolic compounds constitute one of the most numerous and ubiquitously distributed groups of plant secondary metabolites, and have been demonstrated to exhibit various beneficial effects in the treatment of a multitude of diseases (4). In the case of the phenolic acids, these compounds exist in three main forms, namely soluble free acids, esterified acids, which are esterified with sugars and low-molecular mass components, and insoluble-bound acids, which are covalently bound to the structural components of cell walls (5,6). In terms of their biological activities, phenolic compounds are considered to contribute to the antioxidant, anti-carcinogenic, anti-inflammatory, and anti-angiogenic properties of berries (7–9), while also improving the nutritional value of processed foods by preventing the oxidation of lipids and proteins in such products (10). Although a number of studies have examined the nutritional and chemical components of berries in addition to their bioactivities (11,12), few reports on the free, esterified, and insoluble-bound phenolic acid contents of berries and their resulting antioxidant capacities are available. Thus, we herein aim to investigate the contents and antioxidant capacities of these three classes of phenolics following their isolation from selected berries. In addition, the polyphenols present in the free, esterified, and insoluble-bound components were identified and quantified by high-performance liquid chromatography (HPLC).

MATERIALS AND METHODS

Chemicals and reagents

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), gallic acid, p-hydroxybenzoic acid, chlorogenic acid, catechin, caffeic acid, epicatechin, epigallocatechin gallate, p-coumaric acid, ferulic acid, m-coumaric acid, o-coumaric acid, quercitrin, myricetin, resveratrol, morin, quercetin, naringenin, apigenin, vanillic acid, kaempferol, and formic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aluminum chloride, anhydrous sodium sulfate, Folin-Ciocalteu reagent, methanol, n-hexane, sodium carbonate, and sodium nitrite were obtained from Merck (Darmstadt, Germany). All other chemicals used in the experiments were of analytical grade, and deionized water was used throughout.

Sample preparation

Six berries were studied, namely, raspberry (Rubus idaeus L.), blackcurrant (Ribes nigrum), blackberry (Rubus croceacanthus), cranberry (Vaccinium macrocarpon), black chokeberry (Aronia melanocarpa), and blueberry (Vaccinium spp). The berries were collected from local markets in Gimhae, Korea. After washing with deionized water and immediately freeze-drying, the lyophilized berries were ground into powders using a grinder and stored at −80°C prior to analysis.

Extraction and separation of the phenolic fractions

Free phenolic acid fractions

Extraction of the free phenolic acid fraction was carried out following the method described by Neo et al. (13) with slight modifications. More specifically, a sample of the desired berry powder (1 g) was homogenized using a mixture of aqueous methanol (MeOH) and acetone (both 70%, 1:1, v/v) at room temperature. Each homogenized mixture was then subjected to centrifugation and the supernatants were combined prior to reducing their volumes by evaporation under reduced pressure. The pH of the resulting aqueous suspension was adjusted to pH 2, then extracted with n-hexane to remove lipid contaminants. Subsequently, the free phenolics present in the aqueous phase were extracted using a mixture of diethyl ether/ethyl acetate (EtOAc) (1:1, v/v), and the combined organic extracts were dried over anhydrous Na2SO4, filtered (Whatman No.1 filter paper), and evaporated to dryness under reduced pressure at 45°C, prior to re-dissolving the residue in MeOH (5 mL). The recovered aqueous extract was then combined with the precipitate obtained following the initial centrifugation step for extraction of the esterified phenolic acid components.

Esterified phenolic acid fractions

The esterified phenolic acid fraction was extracted according to the method described by Neo et al. (13) using the aqueous phase obtained following extraction of the free phenolic acid-containing fraction. Initially, this aqueous phase was directly hydrolyzed using 4 N NaOH at room temperature, after which the pH of the hydrolyzed solution was adjusted to pH 2, and n-hexane was added to remove any residual oils. The pH-adjusted extract was then washed using the above described diethyl ether/EtOAc mixture, and the resulting extracts were combined and evaporated to dryness under vacuum at 45°C to give the esterified phenolic compounds, which were re-dissolved in MeOH (5 mL).

Insoluble-bound phenolic acid fractions

The insoluble-bound phenolic acids were extracted from the above residue according to the method described by Neo et al. (13). In this case, the berry samples obtained following extraction with methanol/acetone were hydrolyzed using 4 N NaOH at room temperature. The pH of the solution was then adjusted to pH 2, and the resulting solution subjected to centrifugation. The obtained supernatant was extracted using n-hexane, followed by diethyl ether/EtOAc, prior to evaporation to dryness and re-dissolving in MeOH (5 mL).

Qualitative and quantitative analysis of the phenolic acid fractions

Determination of the total phenolic content (TPC)

The TPC of each extract was colorimetrically estimated using the Folin-Ciocalteu method (14). More specifically, a portion of the Folin-Ciocalteu reagent (0.5 mL) was added to each extract (0.1 mL), after which distilled water was added (7 mL). The resulting mixtures were allowed to stand at room temperature for 5 min prior to the addition of an aqueous 7.5% Na2CO3 solution (1.5 mL). The obtained solutions were then allowed to stand at room temperature for a further 2 h, after which time their absorbances at 765 nm were measured using gallic acid and methanol as the reference standard and blank solutions, respectively. All values were expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample dry matter.

HPLC analysis of the phenolic fractions

HPLC was used to separate and identify the individual polyphenolic compounds present in the berry samples according to a previously reported method (15) with slight modifications. Each extract was filtered through a 0.45 μm filter prior to injection into the HPLC system (Agilent 1260 Infinity Quaternary liquid chromatograph, Hewlett Packard, Wilmington, NC, USA), which was equipped with a multiple wavelength detector operating at 280 nm. Chromatographic separations were achieved using an Agilent Zorbax RRHD SB-C18 column (2.1 mm i.d.×100 mm, 1.8 μm particle size; Agilent Technologies, Santa Clara, CA, USA). The column temperature and flow rate were set at 30°C and 0.3 mL/min, respectively. Two solvents (solutions A and B) were used to achieve a gradient elution. Solution A was composed of water containing 0.1% formic acid, while solution B was composed of acetonitrile containing 0.1% formic acid, and the following gradient was employed: 0% B (0 min), 5% B (0~3.5 min), 15% B (3.5~7.1 min), 40% B (7.1~25 min), 40% B (25~26 min), 100% B (26~27 min), 100% B (27~29 min), and 0% B (29~35 min). The standards employed for analysis were caffeic acid, chlorogenic acid, ferulic acid, gallic acid, m-coumaric acid, o-coumaric acid, p-coumaric acid, p-hydroxybenzoic acid, vanillic acid, catechin, epicatechin, epigallocatechin gallate, quercitrin, myricetin, resveratrol, morin, quercetin, naringenin, apigenin, and kaempferol.

Evaluation of the antioxidant activities of the phenolic acid fractions

DPPH radical scavenging activity

The radical scavenging activities of the extracts were estimated according to the procedure described by Delgado-Andrade et al. (16). An aliquot of the desired extract (200 μL) was added to a solution of the DPPH radical in MeOH (1 mL, 74 mg/L). It should be noted that a freshly prepared solution of the DPPH radical gave a final absorption of 1.8 AU at 520 nm. The above mixture (i.e., containing the DPPH radical and the sample extract) was then shaken for 30 min, after which time its absorption at 520 nm was measured. Trolox solutions of various concentrations were used for calibration (0.15~1.15 mM), and the results were expressed as mM equivalents of Trolox (TE)/g of sample.

Ferric reducing antioxidant power (FRAP) assay

The ferric reducing antioxidant power of each extract was evaluated according to the Benzie and Strain method (17). A portion of the freshly prepared FRAP reagent warmed to 37°C (900 μL) was mixed with distilled water (90 μL), and either the desired extract or water (as a blank) was added (30 μL). The final dilution of each test sample was 1:34. The FRAP reagent employed herein contains a 10 mM 2,4,6-tripyridyl-S-triazine solution (2.5 mL) prepared in 40 mM aqueous HCl, in addition to 20 mM FeCl3·6H2O (2.5 mL), and 0.3 M acetate buffer (25 mL) at pH 3.6. The absorption of each solution was recorded at 595 nm every 15 s using a Synergy HTX spectrophotometer (Biotech Instruments, Winooski, VT, USA), and the reaction was monitored for 30 min at 37°C. Trolox solutions of various concentrations were used for calibration, and the results were expressed as mM TE/g of sample.

Determination of the total antioxidant capacity using the Trolox equivalent antioxidant capacity (TEAC) assay

The antioxidant capacity of each extract was estimated according to the procedure reported by Re et al. (18). Thus, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS+·) was prepared by reacting a 7 mM ABTS stock solution with a 2.45 mM aqueous solution of K2S2O8. The resulting ABTS+· solution was diluted using 5 mM phosphate-buffered saline buffer (pH 7.4) to obtain an absorbance of 0.70±0.02 at 730 nm. Following addition of the desired extract (10 μL), the Trolox standard was added to a portion of the diluted ABTS+· solution (4 mL), and the absorbance was recorded over 20 min (Synergy HTX spectrophotometer, Biotech Instruments). Trolox solutions of various concentrations were used for calibration, and the results were expressed as mM TE/g of sample.

Metal ion chelating activity

The metal ion chelating abilities of the extracts were investigated according to the procedures reported by Wang and Xiong (19) and Dinis et al. (20). To determine the Cu2+ chelating ability, a 2 mM solution of CuSO4 (1 mL) was mixed with pyridine (1 mL) at pH 7.0, and a 0.1% solution of pyrocatechol violet (20 μL) was added. Following the addition of the desired extract (1 mL), the blue color of the CuSO4 solution disappeared, due to dissociation of the Cu2+ ions. After allowing the reaction to proceed for 5 min, the absorbance was recorded at 632 nm (Synergy HTX spectrophotometer). To determine the Fe2+ chelating ability, a portion of each extract (100 μL) was added to a mixture of distilled water (600 μL) and 0.2 mM FeCl2·4H2O (100 μL), and the resulting mixture allowed to stand at room temperature for 30 s. After this time the reaction mixture was added to a 1 mM solution of ferrozine (200 μL), and the absorbance was recorded at 562 nm (Synergy HTX spectrophotometer, Biotech Instruments) after allowing to stand for 10 min at room temperature. The Cu2+ and Fe2+ chelating activities were calculated using the following equation:

where A0 and As are the absorbance values of the control and the extract samples, respectively.

Statistical analysis

The experimental data were analyzed by analysis of variance (ANOVA), and the significant differences between the mean values determined from measurements carried out in quintuples (i.e., P<0.05) were obtained by Duncan’s multiple range test using statistical analysis software (SPSS 17.0, IBM Inc., Armonk, NY, USA).

RESULTS AND DISCUSSION

Total phenolic contents of the berry samples

The TPCs of the berry samples and their isolated fractions are presented in Table 1. As indicated, the TPCs of the free, esterified, and insoluble-bound phenolic acids from the various berry samples were within the range of 86.67~372.33, 130.00~735.00, and 272.67~801.67 mg GAE/100 g of dry weight (DW) samples, respectively. More specifically, the free phenolic acid fraction of black chokeberry had the highest TPC, followed by cranberry, blackberry, blueberry, and blackcurrant, while raspberry had the lowest TPC. For the esterified phenolic acid fraction, the highest TPC was observed for black chokeberry, followed by blackberry, cranberry, blueberry, blackcurrant, and raspberry. In the insoluble-bound phenolic acid fraction, black chokeberry again exhibited the highest TPC, followed by blackberry, cranberry, blueberry, blackcurrant, and raspberry. Upon combination of the obtained values for the three fractions of the different berry samples, the total TPCs ranged from 489.67 to 1,909.33 mg GAE/100 g DW, with black chokeberry, blackberry, and cranberry giving the highest values. In addition, we found that for all samples, the TPCs in the insoluble-bound phenolic acid fractions were significantly (P<0.05) higher than those of the free and esterified phenolic acid fractions, with a contribution of 39.78~55.68%.

Table 1

The total phenolic contents of the berry samples and their isolated fractions (mg gallic acid equivalents/100 g of sample)

	Free	Esterified	Insoluble-bound	Total
Raspberry	86.67±1.53fC	130.00±1.00fB	272.67±0.58fA	489.67±2.52f
Blackcurrant	189.67±0.58eC	227.67±1.53eB	292.33±1.53eA	710.33±2.52e
Blackberry	272.00±1.73cC	476.67±2.08bB	559.00±5.20bA	1,307.33±9.24b
Cranberry	285.00±1.73bC	355.67±2.08cB	492.33±2.31cA	1,133.00±3.00c
Black chokeberry	372.33±2.08aC	735.00±2.65aB	801.67±1.53aA	1,909.33±5.51a
Blueberry	214.67±3.06dC	349.33±2.52dB	372.67±2.52dA	936.67±2.08d

Data represent the mean value for each sample±standard deviation (n=5).

Different letters within the same row (A–C) and column (a–f) indicate significant differences at P<0.05.

More specifically, the TPCs of the insoluble-bound fractions obtained from the raspberry, blackcurrant, blackberry, cranberry, black chokeberry, and blueberry samples were 55.68, 41.15, 42.76, 43.45, 41.99, and 39.79%, respectively. Interestingly, Acosta-Estrada et al. (5) reported that insoluble-bound phenolic acid constituents were covalently bound to structural components of the cell walls, and that they play important roles in providing chemical and physical barriers, in determining the antioxidant properties of the berries, and in protecting against pathogen invasion by animals, insects, and microorganisms (21). In addition, release of the phenolic compounds was previously reported to be more efficient by alkaline hydrolysis than by acid hydrolysis (22), as alkaline hydrolysis resulted in cleavage of the ester bonds linking the phenolic acids to the polysaccharides present in the cell walls (5).

Identification and quantification of the phenolic acid compounds

The phenolic acid compounds present in the various berry samples were then identified and quantified as outlined in Table 2, with catechin, caffeic acid, p-coumaric acid, epicatechin, vanillic acid, quercitrin, resveratrol, morin, naringenin, and apigenin being present in all berry samples. In the free phenolic acid fraction, the TPC ranged from 2,960.71 to 18,936.78 μg/100 g DW, with the blackberry sample containing the highest content, followed by the blueberry, black chokeberry, cranberry, blackcurrant, and raspberry fractions. In addition, the esterified fraction contained a TPC ranging from 6,910.83 to 32,644.08 μg/100 g DW. In this case, black chokeberry contained the highest TPC, followed by blueberry, raspberry, blackberry, cranberry, and blackcurrant. Furthermore, the TPC of the insoluble-bound phenolic acid fraction ranged from 16,152.67 to 35,497.91 μg/100 g DW, with the raspberry extract containing the highest TPC, followed by the black chokeberry, blueberry, blackberry, blackcurrant, and cranberry extracts. Although the TPC of the esterified fraction obtained from the blueberries was significantly (P<0.05) higher than those of the free and insoluble-bound fractions, a different trend was observed for the other berries, with higher TPCs being detected in their insoluble-bound fractions. More specifically, high catechin contents were found in the free phenolic acid fractions of blueberry (12,072.87 μg/100 g DW), black chokeberry (10,985.48 μg/100 g DW), blackberry (4,150.90 μg/100 g DW), cranberry (1,982.41 μg/100 g DW), and blackcurrant (626.05 μg/100 g DW), while high contents of p-coumaric acid were detected in the free phenolic acid fractions of blackberry (10,491.54 μg/100 g DW), blueberry (2,170.81 μg/100 g DW), and black chokeberry (1,576.64 μg/100 g DW). In addition, high contents of morin were found in the cranberry (1,047.81 μg/100 g DW) and raspberry (407.38 μg/100 g DW) extracts, while in the case of the blackcurrant and cranberry samples, high contents of vanillic acid were detected in the free phenolic fractions (i.e., 1,102.24 and 584.78 μg/100 g DW, respectively). In the case of the esterified fraction, high catechin contents were found in the blueberry (10,615.92 μg/100 g DW), black chokeberry (5,288.70 μg/100 g DW), and blackcurrant (1,565.89 μg/100 g DW) samples, while high caffeic acid contents were detected for the blackberry, blueberry, blackcurrant, and raspberry extracts (i.e., 2,526.80, 1,804.70, 1,546.96, and 766.47 μg/100 g DW). For the insoluble-bound fractions, high catechin contents were found in the blueberry (6,970.62 μg/100 g DW), black chokeberry (4,391.78 μg/100 g DW), blackberry (1,708.61 μg/100 g DW), and blackcurrant (1,216.04 μg/100 g DW) samples, whereas high p-coumaric acid contents were detected in the insoluble-bound fractions of raspberry, blueberry, and blackberry (i.e., 10,597.95, 3,311.30, and 3,254.54 μg/100 g DW, respectively). To date, a number of studies have reported that the polyphenol components present in plants correlated with their respective antioxidant activities (23, 24). Indeed, Intra and Kuo (25) reported that the antioxidant activity of catechin was related to its free radical scavenging and metal chelating activities, which rendered catechin a more potent lipid antioxidant than vitamins C and E. In addition, it should be noted that catechin does not exhibit pro-oxidant activity in physiological achievable concentrations. In terms of its structure, catechin contains two aromatic rings and a dihydropyran heterocyclic moiety bearing a hydroxyl group at the C-3 position (26), which can chelate to metal ions (27). As such, catechin could be considered a powerful antioxidant for the neutralization of free radicals (28). In addition, vanillic acid is a phenolic derivative present in numerous edible plants and fruits, and is also an intermediate in the production of vanillin from ferulic acid (29). Furthermore, morin is a type of flavonoid belonging to the flavonol group, while p-coumaric acid is a hydroxyl derivative of cinnamic acid. Interestingly, this compound exhibits a range of medicinal properties, including antifilarial, anticancer, and antimicrobial activities (30), which can be attributed to its free-radical scavenging ability (31).

Table 2

The main phenolic compounds and their contents of selected berries (unit: μg/100 g dry weight)

Phenolics	Free	Esterified	Insoluble-bound
Raspberry
p-Hydroxybenzoic acid	ND	ND	ND
Gallic acid	ND	10.64±0.58B	51.95±3.43A
Chlorogenic acid	ND	18.90±0.25B	701.07±37.55A
Catechin	107.86±4.32C	657.72±4.68A	189.66±7.09B
Caffeic acid	139.95±1.05C	766.47±10.05A	636.23±24.01B
p-Coumaric acid	66.11±1.51C	5,518.41±168.49B	10,597.95±101.55A
Epicatechin	168.97±2.63C	10,838.60±145.40A	9,561.10±123.49B
Epigallocatechin gallate	ND	250.09±3.64A	186.61±9.27B
Ferulic acid	467.26±7.87A	5.18±0.43C	66.15±4.29B
m-Coumaric acid	339.71±3.90C	6,281.16±31.04B	9,369.64±180.92A
o-Coumaric acid	ND	ND	48.02±6.01
Vanillic acid	18.66±0.83C	703.40±31.31A	376.55±26.26B
Quercitrin	939.21±8.28A	30.93±0.22C	146.99±5.03B
Myricetin	11.89±0.33B	ND	39.15±1.32A
Resveratrol	237.19±1.63A	53.89±0.06C	72.25±1.34B
Morin	407.38±2.26B	1,006.23±3.74A	173.46±2.28C
Quercetin	1.86±0.08C	7.79±0.26B	17.43±1.08A
Naringenin	14.06±0.30B	9.34±0.28C	38.24±2.46A
Apigenin	40.59±0.39B	12.65±0.17C	103.88±1.95A
Kaempferol	ND	ND	3,121.59±31.77
Total	2,960.71±21.97C	26,171.39±80.97B	35,497.91±227.42A
Blackcurrant
p-Hydroxybenzoic acid	575.37±2.17A	495.47±6.69B	ND
Gallic acid	51.13±1.83B	27.74±4.36C	66.98±2.39A
Chlorogenic acid	382.59±40.76A	37.13±4.04C	173.18±1.04B
Catechin	626.05±15.14C	1,565.89±10.51A	1,216.04±4.90B
Caffeic acid	167.01±14.08C	1,546.96±48.97A	774.27±12.39B
p-Coumaric acid	474.76±21.44A	299.85±14.93B	168.97±15.28C
Epicatechin	194.24±2.66B	152.97±2.43C	236.56±6.52A
Epigallocatechin gallate	16.17±1.03C	2,216.32±43.98A	1,269.71±29.88B
Ferulic acid	ND	222.47±2.54B	283.20±13.66A
m-Coumaric acid	ND	ND	ND
o-Coumaric acid	29.88±1.48	ND	ND
Vanillic acid	1,102.24±21.19A	14.95±1.50C	696.85±9.63B
Quercitrin	155.97±2.00A	164.02±8.93A	91.07±7.34B
Myricetin	20.44±0.95A	3.68±0.48B	ND
Resveratrol	55.59±2.15C	88.22±3.78B	151.87±6.04A
Morin	11.50±0.95C	45.00±2.76A	24.74±1.47B
Quercetin	ND	2.28±0.21B	3.91±0.16A
Naringenin	27.66±0.84A	9.31±0.36C	22.98±0.66B
Apigenin	28.88±1.75B	18.59±1.04C	80.89±2.57A
Kaempferol	ND	ND	11,466.97±149.81
Total	3,919.47±38.51C	6,910.83±31.69B	16,728.16±140.56A
Blackberry
p-Hydroxybenzoic acid	685.01±2.16	ND	ND
Gallic acid	163.39±3.27A	21.11±2.80B	ND
Chlorogenic acid	ND	ND	63.24±17.81
Catechin	4,150.90±183.57A	2,364.52±28.53B	1,708.61±43.24C
Caffeic acid	1,296.79±54.11C	2,526.80±10.2A	1,587.30±15.06B
p-Coumaric acid	10,491.54±152.65A	1,560.84±101.78C	3,254.54±102.45B
Epicatechin	176.51±6.80C	2,418.42±33.17B	5,023.41±55.78A
Epigallocatechin gallate	226.39±13.44C	2,114.32±12.47A	1,155.83±18.02B
Ferulic acid	179.18±5.16A	92.94±7.98C	127.75±4.09B
m-Coumaric acid	363.10±32.54C	7,046.05±334.46A	5,686.20±213.23B
o-Coumaric acid	191.08±2.22	ND	ND
Vanillic acid	432.90±19.20C	519.66±47.92B	693.35±21.43A
Quercitrin	129.25±13.30C	868.90±15.50A	307.57±41.50B
Myricetin	6.62±0.59B	4.33±0.44B	25.07±5.84A
Resveratrol	228.89±8.16A	104.14±0.41C	181.59±2.68B
Morin	98.40±0.33B	257.49±2.61A	86.27±7.29C
Quercetin	8.12±0.94B	3.81±0.26C	24.42±0.50A
Naringenin	20.63±0.68A	13.05±0.38B	13.59±0.60B
Apigenin	88.09±1.54A	9.72±0.88C	49.94±5.21B
Kaempferol	ND	ND	249.48±14.83
Total	18,936.78±101.81C	19,926.11±273.06B	20,238.14±272.13A
Cranberry
p-Hydroxybenzoic acid	435.41±0.06A	415.26±0.21B	413.64±5.47B
Gallic acid	ND	27.03±1.10	ND
Chlorogenic acid	ND	41.95±1.12B	1,134.36±25.22A
Catechin	1,982.41±5.05A	294.40±14.87C	676.34±20.34B
Caffeic acid	651.92±9.54B	37.71±20.65B	1,523.64±56.92A
p-Coumaric acid	94.01±2.99C	228.38±6.42B	1,051.06±54.95A
Epicatechin	1,343.81±9.28A	26.32±1.90C	1,140.79±9.24B
Epigallocatechin gallate	373.38±11.62C	4,001.21±54.26B	4,870.57±82.50A
Ferulic acid	129.23±5.43C	350.46±9.39B	444.71±30.91A
m-Coumaric acid	110.14±8.30A	24.70±0.96B	ND
o-Coumaric acid	39.66±1.01C	57.96±1.04B	368.49±14.95A
Vanillic acid	584.78±13.69B	119.20±1.93C	1,626.01±55.84A
Quercitrin	97.87±0.91B	75.58±1.41C	366.36±11.65A
Myricetin	113.93±5.39A	7.27±0.84B	ND
Resveratrol	233.78±1.58A	79.29±1.96C	87.10±3.22B
Morin	1,047.81±15.70A	912.02±9.24B	434.03±10.79C
Quercetin	5.06±0.17C	24.57±0.15A	22.88±0.50B
Naringenin	17.25±0.28B	23.01±3.39A	10.11±0.38C
Apigenin	29.60±0.38A	7.18±0.08C	19.73±1.22B
Kaempferol	ND	ND	1,962.86±93.33
Total	7,290.03±25.27B	7,353.50±112.99B	16,152.67±133.48A
Black chokeberry
p-Hydroxybenzoic acid	424.40±7.33	ND	ND
Gallic acid	289.27±17.13A	211.19±16.94B	ND
Chlorogenic acid	1,707.39±21.75C	4,896.15±131.98B	13,253.03±579.46A
Catechin	10,985.48±80.44A	5,288.70±497.19B	4,391.78±339.28C
Caffeic acid	235.20±4.32C	2,603.96±401.22B	11,818.82±177.50A
p-Coumaric acid	1,576.64±85.08B	1,718.38±19.09A	796.57±94.40C
Epicatechin	30.28±6.07C	1,760.75±77.11A	518.93±10.51B
Epigallocatechin gallate	ND	2,348.95±23.43A	1,426.52±23.86B
Ferulic acid	ND	1,212.88±23.81A	539.83±12.67B
m-Coumaric acid	73.17±4.83	ND	ND
o-Coumaric acid	12.99±0.72B	29.56±4.65A	ND
Vanillic acid	325.76±33.36B	1,152.81±91.05A	21.62±3.09C
Quercitrin	603.76±15.18B	9,754.80±176.79A	215.68±12.90C
Myricetin	38.79±0.15B	151.35±3.72A	28.36±1.33C
Resveratrol	246.32±1.88A	92.23±1.22B	95.13±5.38B
Morin	177.90±9.91C	1,386.48±5.57A	530.75±27.82B
Quercetin	5.80±0.55B	1.77±0.10C	14.94±1.69A
Naringenin	18.28±1.23A	18.82±0.88A	19.51±1.67A
Apigenin	13.82±0.35B	15.30±0.59B	912.63±17.98A
Kaempferol	ND	ND	501.35±29.62
Total	16,765.25±197.45C	32,644.08±716.22B	34,952.25±477.52A
Blueberry
p-Hydroxybenzoic acid	454.35±5.38A	ND	421.76±0.79B
Gallic acid	ND	ND	ND
Chlorogenic acid	ND	29.56±1.97B	156.06±1.92A
Catechin	12,072.87±189.74A	10,615.92±436.88B	6,970.62±109.02C
Caffeic acid	286.52±19.74C	1,804.70±90.11B	6,680.09±115.81A
p-Coumaric acid	2,170.81±320.36C	10,598.66±145.29A	3,311.30±78.62B
Epicatechin	334.77±28.30B	71.19±5.62C	366.69±19.32A
Epigallocatechin gallate	ND	709.56±21.56B	759.16±22.42A
Ferulic acid	4.42±0.52C	302.70±3.74A	248.16±11.52B
m-Coumaric acid	ND	102.42±12.89	ND
o-Coumaric acid	458.18±18.26A	122.62±8.67B	ND
Vanillic acid	571.09±31.64C	3,261.67±9.81A	975.37±9.15B
Quercitrin	344.30±11.18A	111.68±4.28B	112.45±2.28B
Myricetin	69.87±2.43A	34.62±0.80A	54.48±52.93A
Resveratrol	147.73±4.35A	98.74±2.99B	148.60±17.82A
Morin	45.26±1.13C	403.05±40.44B	841.43±2.61A
Quercetin	50.03±0.31A	28.29±1.95C	31.71±0.83B
Naringenin	24.62±0.70A	17.02±1.52B	15.93±0.75B
Apigenin	8.45±0.15C	10.02±0.25B	14.64±1.42A
Kaempferol	ND	ND	2,995.57±263.63
Total	17,043.27±565.13C	28,322.43±489.79A	24,104.01±372.49B

Data represent the mean value for each sample±standard deviation (n=5).

Different letters (A–C) within a row indicate significant differences at P<0.05 level.

ND, not detected.

DPPH radical scavenging activity

The scavenging activities of the phenolic acids extracted from the various berry samples were then investigated using the DPPH radical scavenging assay. This assay was selected due to the greater stability of the DPPH radical compared to the superoxide and hydroxyl radicals (32). Thus, Table 3 shows the DPPH radical scavenging capacities of the free, esterified, and insoluble-bound phenolic acid fractions obtained from the berry samples, which were in the range of 86.67~156.95, 78.11~183.11, and 97.90~272.75 μmol TE/g DW, respectively. Upon combination of the results obtained for these three fractions, the total radical scavenging activities were calculated to range from 265.58 to 568.80 μmol TE/g DW. Interestingly, for all samples the DPPH radical scavenging activities of the insoluble-bound fractions were significantly (P<0.05) higher than those of the free and the esterified fractions. In addition, black chokeberry exhibited the highest DPPH scavenging activity (i.e., 568.80 μmol TE/g DW), followed by the raspberry, blackberry, cranberry, blueberry, and blackcurrant extracts. Interestingly, Madhujith and Shahidi (33) reported that the DPPH radical scavenging activities of insoluble-bound phenolic acids were higher than of free and esterified phenolic acids. Other studies have also indicated that insoluble-bound phenolics acids exhibit higher antioxidant activities than free phenolics (34,35). It therefore appears that the antioxidant activities of plant extracts are related to the presence of certain individual phenolic compounds and their corresponding structures (36), where the positions and quantities of the hydroxyl groups are likely of particular importance (37). The results obtained herein therefore indicate that the insoluble-bound phenolic acids present in the various berry samples could effectively react with DPPH radicals to convert them into stable products and terminate the radical chain reaction.

Table 3

The DPPH radical scavenging capacities of the free, esterified, and insoluble-bound phenolic acid fractions obtained from the berry samples (unit: μmol Trolox equivalent/g dry weight)

	Free	Esterified	Insoluble-bound	Total
Raspberry	86.67±6.92cC	130.37±1.60bB	272.75±5.22aA	489.79±13.75b
Blackcurrant	67.63±7.28dB	100.04±6.02cA	97.90±4.04dA	265.58±17.34e
Blackberry	156.95±6.68aB	98.33±4.01cC	199.58±4.47bA	454.87±15.16c
Cranberry	93.30±6.13cB	99.67±4.42cB	123.58±5.22cA	316.55±15.77d
Black chokeberry	114.59±6.92bC	183.11±2.02aB	271.10±3.62aA	568.80±12.56a
Blueberry	109.99±6.57bA	78.11±4.84dB	116.14±5.26cA	304.25±16.67d

Data represent the mean value for each sample±standard deviation (n=5).

Different letters within the same row (A–C) and column (a–e) indicate significant differences at P<0.05.

FRAP assay

Two main mechanisms exist for the scavenging of free radicals by antioxidants, namely hydrogen atom transfer and single electron transfer (38,39). The FRAP assay is a typical electron transfer-based method that measures the reduction of ferric ions (Fe3+) to intensely blue colored ferrous ions (Fe2+) by antioxidants in acidic media. Thus, we determined the FRAP values of the free, esterified, and insoluble-bound phenolic acid fractions from the various berry samples, and the results are outlined in Table 4. In general, the samples exhibiting higher ferric-reducing antioxidant powers also contained the highest TPCs. As shown in Table 4, for the fraction containing the free phenolic acids, the FRAP values varied from 17.27 to 111.63 μmol TE/g DW, with blackberry giving the highest value, followed by the black chokeberry, raspberry, cranberry, blackcurrant, and blueberry samples. In the fraction containing the esterified phenolic compounds, the FRAP values ranged 18.27 to 177.90 μmol TE/g DW. In this case, black chokeberry gave the highest FRAP value, followed by raspberry, blackberry, blackcurrant, cranberry, and blueberry. Furthermore, for the insoluble-bound phenolic acid fraction, the FRAP values ranged from 23.55 to 238.80 μmol TE/g DW, with black chokeberry again exhibiting the highest FRAP value, in this case followed by raspberry, blackberry, blueberry, cranberry, and blackcurrant. With the exception of the blackcurrant sample, the FRAP values of the insoluble-bound phenolic acid fractions were significantly (P<0.05) higher than those of the free and esterified phenolics. Upon combination of the results obtained for the three fractions, the total FRAP values ranged from 65.84 to 449.86 μmol TE/g DW. Similarly, Liyana-Pathirana and Shahidi (40) reported that the contribution of insoluble-bound phenolic acids to the TPC in wheat was significantly higher than those of the free and esterified fractions, with the insoluble-bound phenolics demonstrating a significantly higher antioxidant capacity. The berry samples examined herein that contain significant quantities of insoluble-bound phenolic acids should therefore be considered a good source of phenolics that exhibit numerous potential health benefits.

Table 4

The ferric reducing antioxidant power of free, esterified, and insoluble-bound phenolic fractions obtained from the berry samples (unit: μmol Trolox equivalent/g dry weight)

	Free	Esterified	Insoluble-bound	Total
Raspberry	28.11±5.91bcC	133.97±2.47bB	218.36±2.90bA	380.44±11.29b
Blackcurrant	17.81±1.46dC	27.46±1.50dA	23.55±1.33dB	68.83±4.29d
Blackberry	111.63±7.26aB	63.51±8.13cC	135.35±6.70cA	310.49±22.09c
Cranberry	22.41±3.26cdB	22.87±0.77deB	29.38±1.15dA	74.65±5.17d
Black chokeberry	33.16±4.75bC	177.90±4.67aB	238.80±6.90aA	449.86±16.31a
Blueberry	17.27±2.33dB	18.27±0.97eB	30.30±2.18dA	65.84±5.48d

Data represent the mean value for each sample±standard deviation (n=5).

Different letters within the same row (A–C) and column (a–e) indicate significant differences at P<0.05.

Determination of the total antioxidant capacity using the TEAC assay

The total antioxidant activities of the free, esterified, and insoluble-bound phenolic acid components isolated from the various berry samples were determined using the TEAC assay, and the results are given in Table 5, where values ranging from 47.10~178.35, 58.15~231.69, and 80.44~243.35 μmol TE/g DW are indicated, respectively. In this case, the black chokeberry samples in the insoluble-bound fraction exhibited the highest TEAC value (243.35 μmol TE/g DW), followed by blackberry, cranberry, raspberry, blueberry, and blackcurrant. In addition, the TEAC values of the raspberry, blackberry, cranberry, and black chokeberry extracts containing the insoluble-bound phenolic acids were significantly (P<0.05) higher than those containing the free and esterified phenolics. However, a different trend was observed for the blackcurrant and blueberry samples, with the fractions containing the esterified and free phenolic acids giving higher TEAC values than that containing the insoluble-bound phenolics. Combination of the results for the three fractions gave total TEAC values ranging from 230.27 to 552.15 μmol TE/g DW, where the black chokeberry sample exhibited the highest total TEAC value, followed by the blackberry, cranberry, raspberry, blueberry, and blackcurrant samples.

Table 5

The ABTS radical cation scavenging activity of the free, esterified, and insoluble-bound phenolic acid components isolated from the various berry samples (unit: μmol Trolox equivalent/g dry weight)

	Free	Esterified	Insoluble-bound	Total
Raspberry	51.48±1.57eC	111.06±1.88cB	158.56±3.13cA	321.10±6.58d
Blackcurrant	47.10±0.96fC	102.73±2.53dA	80.44±1.88eB	230.27±5.36f
Blackberry	178.35±2.20aB	99.19±1.86dC	186.90±2.53bA	464.44±6.60b
Cranberry	88.77±1.91cC	125.23±2.82bB	162.31±1.66cA	376.31±6.38c
Black chokeberry	77.10±1.58dC	231.69±3.75aB	243.35±1.58aA	552.15±6.90a
Blueberry	102.31±2.26bA	58.15±2.20eB	100.44±1.88dA	260.90±6.33e

Data represent the mean value for each sample±standard deviation (n=5).

Different letters within the same row (A–C) and column (a–f) indicate significant differences at P<0.05.

However, the total TEAC values of the tested samples did not appear to show any clear relationship with the TPCs. In this case, the TEAC value of a compound represents the concentration of Trolox that exhibits the same antioxidant capacity as the compound or compounds of interest (41). Thus, the TEAC value can be considered to be a stoichiometric number related to a Trolox value of 1. Indeed, Alshikh et al. (39) suggested that the TEAC value was dependent not only on the phenolic concentration but also on the identity of the phenolic compounds and the reaction mechanisms taking place. This suggests that the TPCs may not sufficiently explain the observed antioxidant activities of fruit and plant phenolic extracts, which are mixtures of different compounds exhibiting variable activities.

Metal ion chelating activity

In their higher valence states, metals such as iron, copper, manganese, nickel, and cobalt are known to participate in the direct initiation of lipid oxidation via electron transfer and lipid alkyl radical formation, while lower valence metals can directly initiate lipid oxidation via the formation of reactive oxygen species (ROS) (42). In addition, the chelation of iron can prevent the formation of free radicals in addition to preventing the impairment of vital organ functions in vivo. More specifically, the formation of a complex between the antioxidant and the metal renders the metal ions inactive and so they cannot act as initiators of lipid oxidation (43). Thus, the metal ion chelating activities of the free, esterified, and insoluble-bound phenolic acid fractions from the various berry samples are given in Table 6. In the case of the cupric ion (Cu2+) chelating ability, values of 33.58~54.34, 41.60~54.95, and 42.61~59.23% were obtained for the berry extracts containing the free, esterified, and insoluble-bound phenolics, respectively. Combination of the results for the three different fractions gave total Cu2+ chelating abilities ranging from 135.35 to 160.18%, where the black chokeberry sample exhibited the highest Cu2+ chelating ability. In addition, in the case of ferrous ion (Fe2+) chelating ability, the values obtained for the berry extracts containing free phenolic acids and those released from their esterified and insoluble-bound forms were in the range of 33.25~47.60, 44.21~53.56, and 44.21~55.53%, respectively, with combination of these results giving total Fe2+ chelating abilities of 132.19~145.51%. However, the Cu2+ and Fe2+ chelating abilities of the tested samples showed no clear trend between the free, esterified, and insoluble-bound phenolic acid fractions. Although the phenolic compounds present in these berry samples are the main components responsible for chelation to metal ions, it is also possible that nonphenolic constituents present in the extracts may also participate in this process (44).

Table 6

The metal ion chelating activity of the free, esterified, and insoluble-bound phenolic acid fractions from the various berry samples (unit: %)

	Cu2+ chelating ability				Fe2+ chelating ability
	Free	Esterified	Insoluble-bound	Total	Free	Esterified	Insoluble-bound	Total
Raspberry	54.34±2.19aA	43.40±0.11eB	42.61±0.19fB	140.36±2.48b	37.27±0.41dC	53.56±0.30aA	47.60±0.39bB	138.43±1.10b
Blackcurrant	44.32±1.67cB	46.32±0.10dA	46.64±0.09dA	137.28±1.86c	45.57±0.39bA	44.21±0.40dB	44.89±0.40dAB	134.66±1.18c
Blackberry	49.51±1.21bA	47.68±0.82cB	44.64±0.10eC	141.83±2.13b	45.57±0.39bB	47.35±0.08cA	44.21±0.40dC	137.12±0.86b
Cranberry	33.58±0.50dC	53.36±0.87bA	48.50±0.09bB	135.44±1.45c	47.60±0.39aA	45.11±0.40dB	45.79±0.40cB	138.50±1.18b
Black chokeberry	45.99±0.25cC	54.95±0.14aB	59.23±0.10aA	160.18±0.48a	42.84±0.40cC	47.15±1.06cB	55.53±0.38aA	145.51±1.80a
Blueberry	46.37±0.25cB	41.60±0.11fC	47.38±0.16cA	135.35±0.52c	33.25±0.42eC	52.47±0.75bA	46.47±0.39cB	132.19±1.56d

Data represent the mean value for each sample±standard deviation (n=5).

Different letters within the same row (A–C) and column (a–f) indicate significant differences at P<0.05.

Thus, in conclusion, we herein investigated the antioxidant capacities and total phenolic acid contents (free, esterified, and insoluble-bound fractions) of 6 selected berries, in addition to identifying and quantifying a number of phenolic compounds present in the berry samples using HPLC. Interestingly, we found that the contents and antioxidant capacities of the free, esterified, and insoluble-bound phenolic acid components varied considerably. More specifically, the black chokeberry and blackberry samples exhibited superior antioxidant activities to the other berry samples, as determined by a combination of the results obtained from DPPH, FRAP, and TEAC assays, in addition to determination of their metal ion chelating activities.

Furthermore, catechin, caffeic acid, p-coumaric acid, epicatechin, vanillic acid, quercitrin, resveratrol, morin, naringenin, and apigenin were found to be widely abundant in the selected berry samples. Moreover, the antioxidant activities and TPCs of the fractions containing the insoluble-bound phenolics were higher than those of the fractions containing the free and esterified phenolic acids. Our results therefore suggest that the insoluble-bound fractions of the various berries examined herein could be regarded as good sources of natural antioxidants, and so may be suitable for application in the preparation of functional food ingredients and preventing diseases associated with oxidative stress.