Comparative Polyphenolic Content and Antioxidant Activities of Some Wild and Cultivated Blueberries from Romania

Two wild and three cultivated blueberry varieties (‘Elliot’, ‘Bluecrop’ and ‘Duke’) from Romania were analyzed comparatively in order to determine the total polyphenols, total anthocyanins, total flavonoids content and measuring the antioxidant activity using three different single electron transfer-based assays, Trolox equivalent antioxidant capacity (ABTS), ferric reducing ability (FRAP), 2,2-diphenylpicrylhydrazil radical scavenging capacity (DPPH) and one hydrogen atom transfer-assay, oxygen radical absorbance capacity (ORAC). Total polyphenols content ranged from 424.84 - 819.12 mg GAE/100 g FW, total flavonoids ranged from 84.33-112.5 mg QE /100 g FW and total anthocyanins ranged from 100.58-300.02 C3GE/100g FW. The anthocyanins were separated and quantified using RP-HPLC-DAD. In Vaccinium myrtillus , petunidin-3-glucoside and delphinidin-3-glucoside have the highest contribution to the anthocyanin content while in Vaccinium corymbosum , peonidin-3-galactoside represent the major anthocyanin. Except for ORAC assay (r=0.765), all antioxidant activity values obtained were highly correlated with total polyphenol content (0.923≤ r ≤0.986). Wild blueberries had higher total polyphenols content and also antioxidant activity compared with cultivated ones.


Introduction
The interest for natural antioxidants, especially from fruits and vegetables, has increased in recent years. Epidemiological studies indicated that a higher level of natural antioxidants (ascorbic acids, vitamin E, carotenoids and phenolics) in human everyday diet can protect against cardiovascular diseases, cataract, cancer and aging-related disorders (Steffen et al., 2003).
Berries contain high concentration in bioactive compounds such as polyphenols, including anthocyanins, phenolic acids, tannins, carotenoids, vitamin A, C, E, folic acid and minerals such as calcium, selenium and zinc (Kresty et al., 2001;Pineli et al., 2011). Among them, blueberries became well known and often consumed due to their uses for treating biliary disorders, coughs, tuberculosis, diabetes (Martineau et al., 2006;Valentová et al., 2007) and visual disorders (Canter and Ernst, 2004). Blueberries contain high level of anthocyanin and phenolic compounds with high in vitro antioxidant capacities compared with other fruits (Wang and Jiao, 2000). The blueberries phenolic content are affected by genetic differences, the cultivar type, growing location and the degree of maturity at harvest (Zadernowski et al., 2005). The total amount and the proportion between different classes of phenolic compounds in berries cultivar may vary (Beekwilder et al., 2005).
The methods used to determine the total antioxidant capacity can be divided in two major groups: methods based on single electron transfer (SET) and hydrogen atom transfer reaction (HAT). The SET methods include ABTS/TEAC assay (Trolox equivalent antioxidant capacity), FRAP (ferric reducing ability), CUPRAC assay (copper reduction) and DPPH assay (2,2-diphenylpicrylhydrazil radical scavenging capacity). Hydrogen atom transfer reaction (HAT) assay include ORAC (oxygen radical absorbance capacity) and TRAP (total radicaltrapping antioxidant parameter) assay. These assays are frequently used to measure the total antioxidant capacity of food extracts. Because their characteristics and because of the differences in the mechanisms of the reaction, a single assay will not reflect all the antioxidants present in the system (Li et al., 2009). Hence, only by combining different assays, information about the response of the compounds present in samples in different experimental conditions can be achieved (Sariburun et al., 2010). was carried out under stirring. The filtrates were combined in a total extract, which was dried by vacuum rotary evaporator at 40°C. Prior to each analysis, the dry residues were redisolved in 10 ml of methanol, the samples were centrifuged at 5000 rpm and filtered through 0.45 μm nylon filter (Millipore).

Total phenolics
The amount of total polyphenol in the blueberry extracts was determined using modified Folin-Ciocalteu colorimetric method (Singleton et al., 1999). Stock solution of sample extracts (25 μl each) were dissolved in methanol and further dilution were performed to obtain readings within the standard curve made with gallic acid (R=0.997). The extracts were oxidized by the Folin-Ciocalteu reagent (120 μl) and the neutralization was made with Na 2 CO 3 (340 μl), after 5 minutes. The absorbance was measured at 750 nm after 90 minute in the dark, at room temperature. The results were expressed as milligram of gallic acid per 100 grams.

Total flavonoids
The total flavonoids content was determined using a colorimetric method (Kim et al., 2003). The alcoholic extract was diluted to a final volume of 5 ml with distilled water. After adding 300 μl 5% NaNO 2 the mixture was allowed to stay 5 min. Then 300 μl AlCl 3 10% was added and, after 6 minutes, 2 ml NaOH 1N. The solution was mixed well and the absorbance was measured against prepared water blank at 510 nm. Total flavonoid content was expressed as mg quercetin equivalents/100 g fresh weight basis.

Quantification of the anthocyanin content
The monomeric anthocyanin content of the blueberry extract was measured using the pH-differential method (Giusti and Wrolstad, 2001). The blueberry extracts dissolved in methanol were diluted with 0.025 mol/l potassium chloride (adjusted with HCl to pH 1.0) and 0.4 mol/l sodium acetate (pH 4.5). Each sample and the standard (cyanidin-3-glucoside) were diluted with the buffer solution pH 1 and the absorbance was measured at 520 nm and 700 nm using a UV spectrophotometer ( Jasco V-630, International Co. Ltd, Japan). A second aliquot of each sample was diluted to the same value with the buffer solution pH 4.5 and measured at 520 and 700 nm. The absorbance values were calculated as follows: A = (A λ520 -A λ700 ) pH1.0 -(A λ520 -A λ700 ) pH4.5 The total anthocyanin content was calculated according to the formula: The results were expressed as mg cyanidin-3-glucoside chloride per 100 g fruit using the molar absorbtivity (ε) The literature data about blueberries antioxidant activity and total polyphenol content are diverse, more on cultivated than wild varieties (Castrejón et al., 2008;Dragović-Uzelac et al., 2010;Garzón et al., 2010;Giovanelli and Buratti, 2009;Howard et al., 2003;Koca and Karadeniz, 2009;Prior et al., 1998;You et al., 2011).
The objectives of this study were (1) to investigate total polyphenols, total flavonoids, total anthocyanins content of blueberry extracts, (2) to separate and characterize the anthocyanin content using RP-HPLC-DAD, (3) to measure the antioxidant activity using three different SET-based assays (ABTS, FRAP, DPPH) and one HATbased method (ORAC), (4) to correlate the antioxidant methods applied with total polyphenol, total flavonoid and total anthocyanin content.

Samples extraction
Three varieties of cultivated highbush blueberries (Vaccinium corymbosum) 'Elliot' , 'Bluecrop' and 'Duke' were purchased directly from the producers, a farm situated in North-West of Romania. The two types of wild blueberries (Vaccinium myrtillus, Wild 1 and Wild 2) were harvested from two different mountainous geographical zones: 45°24´44˝N and 46°44´37˝E of Romania. All berries were picked at the commercially ripe stage. Samples were stored in a freezer at -20°C until analyzed.
For sample extraction, 5 g of blueberries, in three replicated each, was extracted by grinding the sample 1 min at 20,000 rpm in a blender (Ultra-Turrax Miccra D-9 KT Digitronic, Germany) with 10 ml of acidified methanol (85:15 v/v, MeOH:HCl) (El-Sayed and Hucl, 2003). The homogenate was centrifuged at 3500 rpm for 10 min. The extract was separated and the residual tissue was reextracted until the extraction solvents became colorless (the total solvent volume was between 100-250 ml). After adding 10 ml of the same solvent mixture, the extraction in methanol/HCl of 34300 (M -1 cm -1 ), molecular weight (MW) of 484.8 and dilution factor (DF).

Oxygen radical absorbance activity (ORAC)
The oxygen radical absorbance capacity (ORAC) measure the peroxyl radical scavenging activity using as standard 6-hydroxy-2,5,7,8-tetrametylchroman-2-carboxylic acid (Trolox) (Huang et al., 2002). A fluorescein stock solution (4x10 -3 μM) was made in phosphate buffer (75 mM, pH 7.4) and kept in the dark at 4°C. Before utilization, the fluorescein stock solution was diluted with the phosphate buffer. The fluorescein solution was added to each Trolox standard and blueberry sample (25 μl) made in phosphate buffer and incubated for 30 min, at 37°C. The reaction was initiated by adding 25 μl 2,2 ' -azobis-2amidinopropane (AAPH) and the fluorescence was measured kinetically at excitation wavelength 485 nm and emission wavelength 535 nm, every minute using a fluorescence microplate reader BioTek (Synergy HT, BioTek Instruments, Winooski, VT). The ORAC values for each blueberry extract were calculated using the net area under the decay curves and were expressed as micromoles Trolox equivalents per gram sample (μmol TE/g).

ABTS radical cation decolorization assay (ABTS)
The ABTS assay is based on the capacity of a sample to scavenge the ABTS radical cation (ABTS .+ ) compared to a standard antioxidant (Trolox), adapted to 96 wells microplate, described by Arnao et al. (2001). The ABTS .+ solution was produced by reacting 7mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) for 12-16 h, in the dark, at room temperature. Prior to use, the ABTS .+ working solution was prepared by diluting the stock solution with EtOH to an absorbance of 0.70 ±0.02 at 734 nm. The samples and Trolox standards (20 μl) were combined with the ABTS .+ working solution (170μl, absorbance 0.70 ±0.02) in 96-well microplate. After 6 min of incubation at 30°C, the absorbance at 734 nm was read with a microplate reader. If the absorbance of ABTS value was higher than the standard, the samples were diluted and re-evaluated. The results were expressed as micromoles Trolox equivalents per gram sample (TE μmol /g).

Reducing power (FRAP)
The reducing capacity of methanolic blueberry extracts was measured as ferric reducing antioxidant power (FRAP). Antioxidants are evaluated as reducers of Fe 3+ to Fe 2+ , which is chelated by TPTZ to form Fe 2+ -TPTZ complex, with a maximum absorbance at 593 nm (Benzie and Strain, 1996). The absorbance of the colored product was monitored by Biotek Synergy HT spectrophotometer. All solutions were used in the day of preparation. Briefly, 2.5 ml TPTZ (10 mM in 40 nM HCl), 25 ml acetate buffer (300 mM, pH=3.6) and 2.5 ml FeCl 3 (20 mM) were mixed and after adding 180 μl FRAP reagent the mixture was incubated for 3 min. Then, 20 μl of each sample added to each well and the absorbance was read immediately at 593 nm with a microplate reader. Samples dilution were perfomed when the values were over the linear range of the curve of 0 to 1 μM Fe 2+ /ml, using FeSO 4 x7H 2 O.

DPPH· scavenging activity assay
The DPPH scavenging activity assay was done according to a method reported by Brand-Williams et al. (1995). A DPPH· solution (80 μM) was freshly prepared in 95% methanol. A volume of 250 μl of this solution was allowed to react with 35 μl sample and the absorbance was measured at 515 nm, for 30 minutes. The chemical kinetics of blueberries extract was recorded. The antioxidant activity was calculated as follows: % DPPH· scavenging activity = (1-[A sample /A control t=o ]) 100

Statistical analysis
The data's are expressed as mean ± standard deviation (SD) from three parallel measurements. In order to determine the significant differences between values, analysis of variance (ANOVA) and Duncan's multiple range tests were performed. Significance of difference was defined at the 5% level (p<0.05). All statistical analysis was carried out using Graph Pad Version 4.0 (Graph Pad Software Inc; San Diego, CA, USA). Pearson's correlation coefficient was calculated using Microsoft Excel 2003.

Total polyphenols content
The comparative data about total polyphenols, flavonoids and anthocyanins content in wild and cultivated blueberries are presented in Tab. 1.
The total polyphenols content (TPC) was determined using the Folin-Ciocalteu method. Gallic acid was used as calibration standard and the results (expressed as gallic acid equivalents) were expressed as means ±standard deviation of triplicate analysis. The TPC values in the blueberry extracts analyzed was in the range of 424.84 -819.12 mg GAE/100 g fresh weight. Among all the varieties analyzed, the Wild 1 blueberry revealed the highest TPC at 819.12 gallic acid equivalents/100 g FW followed by Wild 2 blueberries (672.59 mg GAE/100g). Between the cultivated blueberries, 'Bluecrop' has the highest TPC at 652.27 mg GAE/100 g while the lowest value was found for 'Duke' variety (424.84 mg GAE/100g). Significant differences were found in total phenolic content when compared all the varieties analyzed (p<0.05).
The TPC data's obtained are comparable to previous findings which reported values between 251-310 mg GAE/100 g for some cultivated blueberries and between 577 and 614 mg GAE/100 g for wild Italian blueberries (Giovanelli and Buratti, 2009). Lee et al. (2004) obtained between 367-1286 mg GAE/100 g total polyphenols for V. membranaceum and for V. ovalifolium species 677-1054 mg GAE/100 g. Prior et al. (1998)

Total flavonoids content
The total flavonoids content of the extracts was determined using aluminium chloride colorimetric method and the results are shown in Tab. 1. The total flavonoids content ranged from 84.33 mg QE/100 g in 'Duke' variety to 112.5 mg QE/100 g in Wild 2 blueberries. For the other blueberry varieties the TFC were as follows: Wild 1 (110.36 mg QE/100 g), 'Bluecrop' (103.18 mg QE/100 g), 'Elliot' (92.82 mg QE/100 g) and 'Duke' (84.33 mg QE/100 g). There were no significant differences in total flavonoid content among 'Elliot' , 'Bluecrop' , Wild 1 and Wild 2 (p<0.05). 'Duke' variety has the lowest total flavonoid content, significantly different than other varieties analyzed. There are no data regarding the total content of flavonoids in blueberries. In blackberry varieties, the TFC content range between 29.07-82.21 mg QE/100 g (Sariburun et al., 2010).

Total anthocyanins content
The total anthocyanins content (TAC) of the blueberries extract were also determined (Tab. 1). The highest anthocyanins content was found in wild blueberries Wild 1 (300.02 mg/100g), followed by wild blueberries Wild 2 (252.23 mg/100g), 'Elliot' (163.4 mg/100g), 'Bluecrop' (160.76 mg/100g) and the lowest TAC was found in 'Duke' variety (69.58 mg/100g). There were significant differences (p<0.05) in anthocyanins content between Wild 1, Wild 2 and 'Duke' . However, significant differences in the total anthocyanin content were not observed between 'Elliot' and 'Bluecrop' (p<0.05). The levels of TAC in TPC are between 23% and 37% and they are in agreement with the data's reported by other authors (Giovanelli and Buratti, 2009). The present TAC values obtained are in agreement with other studies (Dragovič-Uzelac et al., 2010;Prior et al., 1998).

Total antioxidant activity
For measuring the total antioxidant activity of the blueberry extracts, four different assays were used. Tab. 3 includes the mean values for antioxidant activity measured.
ORAC assay is probably the most widely used HATbased assay and indicate the free-radical scavenging ability of antioxidant against peroxyl radical. The values obtained were not significantly different among samples, ranging from 34.85-38.49 μmol TE/g fresh weight. The present ORAC results are similar to those obtained by Prior et al. (1998) (17-37.1 μmol TE/g) and lower than the values reported for rabbiteye blueberries by You et al. (2011) (44-55 μmol TE/g). For 'Duke' variety, it has been obtained 34.85 μmol TE/g comparable with the values reported by Wang et al. (2009) (40.4 μmol TE/g) and Moyer et al. (2002) (32.6 μmol TE/g). Regarding the ORAC values for 'Bluecrop' variety it is a great discrepancy in the literature data. Prior et al. (1998), reported 17 mM TE/ kg, Moyer et al. (2002), 50 mM TE/kg and Howard et al. (2003), between 21-38 mM TE/kg, the last values being comparable with the present value. The last mention study indicates that genotypes influence more the TPC and ORAC values than growing season.
ABTS assay is based on the antioxidant ability of the extracts to react with ABTS ·+ radical cation generated in the system. In contrast, the ferric reducing antioxidant power assay (FRAP) measures the ability of the extracts to reduce ferric complex to the ferrous form. The averages values obtained for ABTS and FRAP assay are given in Tab. 2. Blueberries Wild 1 extract showed the highest antioxidant activity based on FRAP and ABTS assay (73.71 μM Fe 2+ /g and 56.65 μmol TE/g). The lowest level in both assays was obtained for 'Duke' variety. The antioxidant activity order in both assays, FRAP and ABTS, was: Wild 1>Wild 2>'Bluecrop'>'Elliot' >'Duke' . In FRAP assay, there were significant differences found between Wild 1, Wild 2, 'Bluecrop' and 'Duke' (p<0.05). In ABTS assay, significant differences were found between Wild 1, Wild 2, 'Elliot' and 'Duke' (p<0.05). There were no statistical differences between 'Elliot' and 'Bluecrop' varieties.

Identification and quantification of anthocyanins
Using HPLC-DAD, 13 anthocyanins were identified based on their retention time, UV-VIS spectra compared with standards and published data. HPLC chromatogram for anthocyanins separation in 'Bluecrop' variety is presented in Fig. 1.

Conclusions
There were analyzed, for the first time, the antioxidant activity in relation to the polyphenol content in wild and some cultivated blueberry fruits from Romania. Total polyphenol, total anthocyanin, total flavonoid content and antioxidant activity measured with six different assays were higher in wild blueberry varieties, as compared with cultivated ones. The FRAP, ABTS and DPPH assays showed higher correlation, statistically significant, with total polyphenol content compared with total anthocyanin content. To measure adequately the antioxidant potential ABTS and DPPH methods are considered to be most appropriate, in good agreement with the concentrations of phenolic derivatives (polyphenols, anthocyanins, flavonoids).
High level of antioxidant activity obtained for Wild 1 blueberries, by all the methods used, could be due to its high level of total polyphenol and total anthocyanin content.
The DPPH· scavenging activity of blueberries extract is presented in Tab. 4. It has been obtained significant differences between all the blueberry extracts analyzed, with the highest value for Wild 1 (59.79%) and the lowest for 'Duke' variety (29.96%). The present data are in agreement with previously reported data: 34.13% of inhibition for wild blueberries (Li et al., 2009) and 28% of inhibition (Ogawa et al., 2008).
The kinetic curves presented in Fig. 2 clearly indicated that Wild 1 and 2 blueberries had the highest scavenging activity during 30 minutes.
In Tab. 4 are presented the Pearson's coefficients, which indicate the possible correlation between polyphenol composition with different antioxidant activity and the correlation of different assays used with each other.
A correlation analysis was done among total polyphenols content, total flavonoid, total anthocyanin and the antioxidant activity values obtained (Tab. 4). The total polyphenols content (TPC), total flavonoids (TF) and total anthocyanins (TA) exhibit a significant correlation (p<0.05 and p<0.01) with different antioxidant methods with a decreasing order of TPC>TA>TF. This suggests that antioxidant activity is more correlated to TPC than TA. These results are in agreement with other studies (Giovanelli and Buratti, 2009;Koca and Karadeniz, 2009;Prior et al., 1998).The high correlation between the FRAP and TPC content can be attributed to the fact that both assays rely on the same reaction mechanism.
There were no statistically significant correlation between ORAC values and TPC, TF and TA. Tabart et al. (2009)