Biological activity and chemical composition of different berry juices

Food Chemistry 125 (2011) 1412–1417

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Analytical Methods

Biological activity and chemical composition of different berry juices Aleksandra Konic´-Ristic´ a, Katarina Šavikin b,⇑, Gordana Zdunic´ b, Teodora Jankovic´ b, Zorica Juranic c, Nebojša Menkovic´ b, Ivan Stankovic´ d a

Institute for Medical Research, Department of Nutrition and Metabolism, Tadeuša Košc´uška 1, 11000 Belgrade, Serbia Institute for Medicinal Plants Research, Tadeuša Košc´uška 1, 11000 Belgrade, Serbia c Institute for Oncology and Radiology, Dr. Subotic´a 10, 11000 Belgrade, Serbia d Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia b

a r t i c l e

i n f o

Article history: Received 21 January 2010 Received in revised form 29 September 2010 Accepted 3 October 2010

Keywords: Berry juices Anthocyanins Radical scavenging activity Antiproliferative activity

a b s t r a c t Total phenolics, total anthocyanins, mineral content, radical scavenging activity and antiproliferative activity against human cancer cell lines were evaluated in fresh pressed juices of five different berries. Total phenolic content ranged from 133.0 to 260.3 mg of gallic acid equivalents/100 g of fresh weight, for red currant and black currant, respectively. Bilberry juice contained the highest amount of total anthocyanins (0.18%). Significant correlation between total phenolics content and radical scavenging activity was observed (r = 0.980; p < 0.01). All examined juices showed antiproliferative activity in dose-dependent manner with IC50 ranging from 10.2 to 70.5 ll/ml. Black currant juice was the most effective inhibitor of proliferation in all cell lines tested (HeLa, Fem X, LS 174, MCF-7 and PC-3). Significant correlations of acidity and total anthocyanin content with antiproliferative activity of berry juices on HeLa cells, Fem X cells and MCF-7 cells were noticed. Berry juices are good sources of some minerals and contribute significantly to daily intake of these micronutrients. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Numerous epidemiological studies have shown that a diet rich in fruits and vegetables reduces the risk of many chronic diseases, such as cardiovascular diseases and cancer (Steinmetz & Potter, 1996). As a part of a diet, consumption of certain fruits may have noticeable long-term physiological effects. Among different fruit species, berries have attracted a great attention for their bioactivity and they are part of human diet all over the world (Amakura, Umino, Tsuji, & Tonogai, 2000). In addition to nutritive dietary components (vitamins, minerals, sugars, organic acids, dietary fibres, unsaturated fats) (Belitz, Grosch, & Schieberle, 2004), berries are also a good source of different classes of phytochemicals such as flavonoids (anthocyanins, flavonols, flavanols), tannins (proanthocyanidins, ellagitannins, gallotannins), stilbenoids (e.g., resveratrol), phenolic acids (hydroxybenzoic and hydroxycinnamic acid derivates) and lignans (Seeram et al., 2006). Many of the health benefits associated with berry fruits may be due to the high concentrations of anthocyanins. A growing number of scientific reports suggest that anthocyanin-rich berries or derived extracts exhibit a wide range of protective effects with potential benefits for human and animal health. Antioxidative, antiinflammatory, vasoprotective, neuroprotective, hepatoprotective, ⇑ Corresponding author. Tel.: +381 11 3031653; fax: +381 11 3031649. E-mail address: [email protected] (K. Šavikin). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.018

and inhibition of the growth of cancerous cells are biological effects that have been reported for anthocyanins (Kong, Chia, Goh, Chia, & Brouillard, 2003). As a result of many proposed beneficial effects, berry fruits and extracts are today often used as a compound of functional foods, dietary foods or dietary supplements. Plant species investigated in our study were red raspberry (Rubus idaeus L.), black raspberry (Rubus occidentalis L.), black currant (Ribes nigrum L.), red currant (Ribes rubrum L.) and bilberry (Vaccinium myrtillus L.) as very popular and readily consumed berries in European countries. We focused on berry juices, very often used as a major constituent of functional foods and beverages (Gruenwald, 2009). Furthermore, the consumption of fresh raw juices made in the households is becoming a part of a healthy lifestyle. The objectives of the current study were (1) chemical characterisation of berry juices, (2) investigation of radical scavenging activity and (3) investigation of in vitro antiproliferative activity on five human tumour cell lines. 2. Materials and methods 2.1. Plant material Raspberry (R. idaeus L.), black raspberry (R. occidentalis L.), red currant (R. rubrum L.) and black currant (R. nigrum L.) were harvested from commercial plantation, mountain Kopaonik, locality Lukovska Banja (1000 m), Serbia. Berries were hand-harvested in

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the third year of vegetation in June–July 2008, depending of the commercial ripening time for each species (85% coloured fruit). Wild bilberries (V. myrtillus L.) were harvested from nature, at three localities, near the commercial plantation in August 2008. Berry harvests were conducted once, and 150 g of each sample was collected. 2.2. Reagents and standards HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma (St. Louis, MO, USA). The anthocyanin standards, delphinidin chloride (purity 97.6%) and cyanidin chloride (purity 98.8%), were purchased from Chromadex (Irvine, CA, USA). Standards were kept at 20 °C. Gallic acid and Folin–Ciocalteu reagent were obtained from Fluka (Germany). Water was prepared by a Millipore Simplicity water purification system (Millipore, Bedford, MA, USA). 2.3. Sample preparation Fresh undamaged berries were selected, initially homogenised and manually pressed to separate juices. The yield of juices (expressed as ratio of juice weight after and weight of the fresh fruits before pressing) was 0.5 for both red raspberry juice (RJ) and black raspberry juice (BRJ), 0.31 for black currant juice (BCJ), 0.55 for red currant juice (RCJ) and 0.67 for bilberry juice (BJ). After pressing, juices were centrifuged at 14,000g for 20 min, divided into aliquots, and immediately frozen and stored at 20 °C. Gravimetric dry matter measurements of juices were performed before freezing, by heating the juices in an oven at 105 ± 5 °C until the constant mass. Dry matter contents, expressed as percentage of original juice samples, were 7.18%, 8.67%, 11.67%, 7.15% and 8.06%, for RJ, BRJ, BCJ, RCJ, and BJ, respectively. 2.4. Determination of titratable acidity, pH and refractometric dry substance Titratable acidity (TA) was determined by titration of 20 ml sample with standardised 0.1 N NaOH up to pH 8.1 by use of pH meter (AOAC official methods of analysis, 1984). TA was expressed as citric acid equivalents in percent (grams of citric acid per 100 grams of juice). The pH of the samples was measured using a pH meter (WTW, Munich, Germany) at room temperature. Refractometric dry substance (RSD) was measured using an Abbe refractometer (Carlo Erba, Milan, Italy) at a standardised temperature (21 °C) (AOAC official methods of analysis, 1995). All analyses were carried out in triplicate. 2.5. Determination of total phenolics The total concentration of phenols (TP) was estimated by Folin– Ciocalteu method (Waterman & Mole, 1994) with slight modifications. The juices samples (2 g) were diluted with distilled water up to 10 ml, centrifuged (2500g; 10 min) and supernatant was used for analyses. Two hundred microlitres of the samples were added to 1 ml of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 800 ll of sodium carbonate (75 g/l) were added. After 2 h of incubation at room temperature, the absorbance at 765 nm was measured. Gallic acid (0–100 mg/l) was used for calibration of a standard curve. The results were expressed as milligrams of gallic acid equivalents per 100 g of fresh weight (mg GAE/100 g FW). Triplicate measurements were taken and mean values were calculated.

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2.6. Determination of total anthocyanins Total anthocyanin (TAcy) content was investigated according to the procedure described in the European Pharmacopoeia 6.0. (2008), with slight modifications. To about 5.0 g of juice, 95 ml of methanol were added and mechanically stirred for 30 min and filtered into a 100 ml volumetric flask. An adequate dilution (1:10) of this solution in a 0.1% (v/v) solution of hydrochloric acid (36.2%) in methanol was prepared. The absorbance of the solution was measured at 528 nm against a 0.1% (v/v) solution of hydrochloric acid (36.2%) in methanol. The total content of anthocyanins (%) was expressed as cyanidin-3-glucoside chloride. Triplicate measurements were taken and mean values were calculated. 2.7. Quantitative analysis of anthocyanin aglycones Analysis was performed according to Nyman and Kumpulainen (2001), with slight modifications. Approximately 15 g of juice were extracted with 40 ml of a mixture, prepared with 50 ml MeOH, 33 ml H2O and 17 ml HCl (36.2%), on an ultrasonic bath during 20 min. Extract was filtered through a 0.45 lm filter (Agilent) into a PP vial and hydrolysed for 60 min at 100 °C. After quick cooling, samples were injected to HPLC. Separation and quantification was performed on an Agilent 1200 HPLC equipped with DAD detector, on a Lichrospher 100 RP-18 analytical column (250  4 mm i.d., 5 lm particle size) from Agilent. Mobile phase were A (H2O containing 10% HCOOH) and B (MeCN). Extracts were separated by gradient elution according to the following scheme: 1% B 0– 0.5 min; 1–7% B 0.5–1 min; 7% B 1–4 min; 7–10% B 4–7.5 min; 10–14% B 7.5–11.5 min; 14% B 11.5–15.5 min; 14–18% B 15.5– 18.5 min; 18% B 18.5–22 min. Flow was adjusted to 1 ml/min and detection was set at 290, 350 (for flavonoids) and 520 nm (for anthocyanins). The injection volume was 30 ll and the column temperature was maintained at 25 °C. The standard solutions of anthocyanins (1 mg/5 ml MeOH containing 0.01% HCl) at five concentrations (range 0.02–0.2 mg/ml) were injected in triplicate for each concentration to establish calibration curve, and results are expressed as mean value ± standard deviation. 2.8. Determination of radical scavenging activity The free radical scavenging activity (RSA) of juices on the stable 1 and 1-diphenyl-2-picrylhydrazyl (DPPH) radical was carried out according to the procedure described previously (Silva, Ferreres, Malva, & Dias, 2005), with slight modifications. The juices samples (2 g) were diluted with distilled water up to 10 ml, centrifuged (2500g; 10 min) and supernatant was used for analyses. The antiradical activity of each sample was evaluated using a dilution series, in order to obtain a large spectrum of sample concentrations. Extracts (100 ll) were mixed with 1400 ll of 80 lM methanolic solution of DPPH. Absorbance at 517 nm was measured after 20 min. The percentage of inhibition was calculated using the following equation:

Inhibition ¼ ½ðA0  Ai Þ=A0   100; where A0 is the absorbance of the control and Ai is the absorbance of the samples. Concentration of the sample which is required to scavenge 50% of the DPPH free radicals (IC50) was estimated using a nonlinear regression algorithm. All test analyses were run in triplicate. Trolox was used as a positive control. 2.9. Determination of mineral content For determination of mineral content all samples were homogenised, accurately weighed (10 g) and heated at 105 ± 5 °C for gravimetric dry matter measurements. Approximately 0.5 g of each

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dry sample was weighed and placed in a digestion vessel to which 5 ml of trace metal grade HNO3 acid (65%) and 1 ml of H2O2 (35%) was added. Vessels were tightly closed and transferred to the Ethos D Microwave Labstation (Milestone Microwave Lab Systems, Germany). Digestion followed a two step program recommended by the manufacturer for organic samples with fast exothermic reactions. In the first stage, the sample was brought to 180 °C in 10 min, the second stage allowed the digest to remain at 180 °C for an additional 10 min, and the program was finished with cooling for 30 min. All samples were filtered after digestion and diluted in deionised water. Reagent blanks and laboratory standards were routinely included in the analysis. Running standards were prepared daily, by dissolving standard solution of mineral salts (Merck) with ultra-pure water at five different concentrations in the range of 0.1–1 lg/ml for Mg, Cu, Zn, Mn and Fe, and 1–5 lg/ ml for K, Ca and Na. Calibration curves were obtained by plotting absorbencies of running standards, determined in the same experimental conditions. Mineral contents (Ca, Mg, Na, K, Cu, Fe, Mn, and Zn) of all samples were determined in triplicates by atomic absorption spectrometry (Avanta, GBC Scientific Equipment, Australia) in an air–acetylene flame using deuterium-background correction. For the determination of Na and K, Cs was added to standards and samples as an ionisation buffer (0.2% w/v), and for Ca and Mg measurements, La was used as a releasing agent (0.1% w/v).

in a 1 M potassium acetate in 70% ethanol. Absorbance (A) was measured 2 h later at 570 nm, and the percentage of cell survival was calculated using formula:

Sð%Þ ¼ ½As  Ab =Ac  Ab   100; where As is absorbance of sample with cells grown in the presence of various concentration of investigated compounds, Ac is absorbance of control, and Ab is absorbance of blank. Concentration IC50 was determined as a concentration of a compound or preparation that inhibited cell survival by 50%, compared with a vehicle-treated control. 2.11. Statistical analysis All results were expressed as mean values ± standard deviation for three replicates. Correlations among main components and biological activities of evaluated juices were computed following Pearson’s correlation coefficient method. Statistical analyses were performed using SPSS for Windows software, Rel. 11.0.1. (SPSS Inc., Chicago, USA). 3. Results and discussion 3.1. Titratable acidity, pH, refractometric dry substance

2.10. Determination of antiproliferative activity 2.10.1. Cell cultures and chemicals Five neoplastic human cell lines of different origin were used as target cells in the study (human cervix carcinoma cell line HeLa, human melanoma cell line Fem X, human colon cancer cell line LS-174, human breast carcinoma cell line MCF-7, and human prostate carcinoma cell line PC-3). Cell lines were cultured as monolayer in the nutrient medium (RPMI 1640 medium supplemented with L-glutamine (3 mM), streptomycin (100 lg/ml), penicillin (100 IU/ml) and 10% heat inactivated (56 °C) foetal calf serum (FCS), Hepes (25 mM) and adjusted to pH 7.2 by bicarbonate solution) and grown at 37 °C in 5% CO2 humidified air atmosphere. Frozen juices were thawed in room temperature, centrifuged at 550g, decanted, filtered through Millipore filter 0.22 lm and diluted in nutrient medium to the working concentrations. The highest concentration of the investigated juices was 80 ll/ml. 2.10.2. Treatment of cell lines HeLa, Fem X, MCF-7 and PC-3 cells were seeded at a density of 2000 cells/well in 100 lM of nutrient medium in 96-well plates. LS-174 cells were set up at a density of 7000 cells/well, and all cell lines were grown similarly. The following day, after the cell adherence, five different concentrations of the investigated juices (concentration range 5–80 ll/ml), were added to the wells. Wells with cells and nutrient medium were used as controls, while wells with different berry juices dilutions only were used as corresponding blanks. 2.10.3. Determination of cell survival Cell survival was assayed by measuring total cellular protein, using Kenacid Blue R (KBR) dye binding method (Clothier, 1995). Briefly, 72 h after the continuous action of investigated juices, medium was discarded and target cells were washed twice with warmed (37 °C) phosphate buffered saline (PBS). PBMC were centrifuged before discarding the medium and supernatants were removed by careful aspiration. After that, target cells were fixed with 150 ll of a methanol–acetic acid mixture (3:1) for 20 min, and then stained with 0.04% solution of Coomassie Brilliant Blue R250 (in 25% ethanol and 12% glacial acetic acid). After the staining (2–3 h), plates were washed with PBS and bound dye was dissolved

Acidity and sugar contents are characteristics usually used as an indicator of fruit maturity and ripeness. TA and RDS values, as a measure of acid and sugar content, are routinely determined during the quality control in the berry products industries (FAO Agricultural Services Bulletin No. 146, 2001). RDS value of investigated juices expressed as percentage of original juice samples was 5.9 ± 0.2%, 7.75 ± 0.3%, 12.4 ± 0.1%, 7.8 ± 0.2% and 7.8 ± 0.12%, for RJ, BRJ, BCJ, RCJ and BJ, respectively. Results obtained for TA and pH are given in Table 1. The highest TA value was detected for BCJ, followed by RJ and RCJ, while acidities of BRJ and BJ were the lowest. On the other hand, pH values did not follow the same order and values for all juices were similar, except for BRJ. Sugar/ acid ratio differs between different juices, and the calculated values for BRJ and BJ are above 10, while for other juices they are in the range between 3 and 4. A low value of sugar/acid ratio has been generally associated with an unpleasant character, although industrial processors of commercial juices have different requirements regarding acidity in the raw materials because this low ratio is a good indicator for prolonging fruit quality during storage (Kushman & Ballingen, 1969). 3.2. Total phenolics Total phenolics content of the juices is given in Table 1. Generally, total phenolics were higher in juices obtained from dark blue berries such as BRJ, BCJ and BJ. The highest amount was determined in BCJ while the lowest in RCJ. The amounts of total phenolics in the studied juice samples were lower compared to the values presented in literature for corresponding berries (Benvenuti, Pellati, Melegari, & Bertelli, 2004; Plessi, Bertelli, & Albasini, 2007). At the same time, results reported by these authors show that phenolic content of black currant and bilberry i.e. dark blue berries, is more than twofold higher than in red currant or raspberry, supporting the relation between phenolic content and the colour of investigated juices in our study. Smaller amounts of phenolic compounds in the studied juice samples, in comparison to the results published for related berries (Benvenuti et al., 2004; Plessi et al., 2007) could be explained by specific distribution of phenolics during juice preparation and their remain in the pressed residue (Sandell et al., 2009). Also, phenolic content of BCJ and BJ in our

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study was higher than phenolic content of commercial black currant and blueberry nectars evaluated by Piljac-Zegarac, Valek, Martinez, and Belscak (2009). Compared to the other commercially available fruit juices and fruit drinks (Mullen, Marks, & Crozier, 2007) investigated berry juices could be considered as a good source of phenolic compounds. 3.3. Total anthocyanins and anthocyanin aglycones Anthocyanins represent one of the most dominant classes of bioactive compounds in berry (Clifford, 2000). All investigated berries juices contained anthocyanins and the highest amounts were found in BJ and BRJ (Table 1). Correlation between TP and TAcy content could not be observed because BCJ which was the most abundant in TP, contained small amounts of TAcy. This is probably due to the fact that in black currant berries the majority of anthocyanins are localised in exocarp which remains as residue after the processing of black currant berries to juice. Sandell et al. (2009) showed that after pressing, with juice yield of 0.76, only 20% of total anthocyanins from black currant berry fruits were present in the juice. Anthocyanin aglycones contents in tested berry juices are shown in Table 2, and are consistent with anthocyanins profiles in related berries presented by other authors (Koponen, Happonen, Mattila, & Törrönen, 2007; Wu et al., 2006). Cyanidin was found to be the most ubiquitous anthocyanin aglycone and its amount was the highest in BRJ and RJ. Delphinidin was found in BCJ and BJ, while peonidin, petunidin and malvidin were characteristic for BJ only (Fig. 1). 3.4. Mineral content Macro and micro-elements contents in investigated fruit juices are given in Table 3. The most abundant macro-element in all juice

Table 1 Titrable acidity, pH, total phenolics, total anthocyanin content and radical scavenging activities of berry juices. Samples

TAa (%)

pH

TPb (mg GAE/ 100 g)

TAcyc (%)

RSAd (IC50 mg/ml)

RJ BRJ BCJ RCJ BJ

1.97 ± 0.02 0.74 ± 0.01 3.17 ± 0.03 1.83 ± 0.01 0.75 ± 0.01

2.87 3.81 2.96 2.62 3.04

164.4 ± 5.1 240.2 ± 5.3 260.3 ± 4.0 133.0 ± 2.1 236.3 ± 7.0

0.08 ± 0.01 0.15 ± 0.02 0.07 ± 0.02 0.03 ± 0.01 0.18 ± 0.03

2.40 ± 0.21 1.28 ± 0.09 0.91 ± 0.07 3.14 ± 0.23 1.68 ± 0.13

RJ – red raspberry juice; BRJ – black raspberry juice; BCJ – black currant juice; RCJ – red currant juice; and BJ – bilberry juice. Values represent means of three replicate determinations ± SD. a Titrable acidity expressed as g citric acid per 100 g of juice. b Total phenolic contents expressed as mg gallic acid equivalents per 100 g of juice. c Total anthocyanin contents. d Radical scavenging activity expressed as IC50 (mg/ml).

Table 2 Anthocyanin aglycones content (mg/ml of juice) in investigated berry juices. Samples

Delphinidin

Cyanidin

Peonidin

Malvidin

Petunidin

RJ BRJ BCJ RCJ BJ

– – 0.13 ± 0.02 – 0.16 ± 0.02

0.40 ± 0.03 0.42 ± 0.03 0.15 ± 0.02 0.11 ± 0.01 0.27 ± 0.01

– – – – 0.10 ± 0.01

– – – – 0.16 ± 0.02

– – – – 0.10 ± 0.01

RJ – red raspberry juice; BRJ – black raspberry juice; BCJ – black currant juice; RCJ – red currant juice; and BJ – bilberry juice. Values represent means of three replicate determinations ± SD.

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samples was K, its content ranged from 92.9 mg/100 g in BJ to 208.7 mg/100 g in BCJ. The content of Ca is twofold higher in BCJ (64.2 mg/100 g) than in RCJ and RJ (35.2 and 32.8 mg/100 g, respectively), while BJ and BRJ contain similar amounts (26.4 and 25.0 mg/100 g, respectively). The highest value of Mg was found in BRJ (12.7 mg/100 g), followed by BCJ (11.5 mg/100 g) and RJ (10.1 mg/100 g). Na content ranged between 1.2 mg/100 g (BJ) and 2.2 mg/100 g (BCJ). In general, the lowest contents of macroelements were found in BJ, while BCJ was the richest in K, Ca and Na. Evaluating micro-element content, BRJ had the highest values of Cu and Fe, with possible interactions beneficial in the prevention of anaemia (Sharp, 2004). On the other hand, BJ was the richest source of Zn, known for its positive role in immunity (Hirano et al., 2008). Mn content was similar in RJ and BJ, and it was more than 10 times higher than in BCJ and RCJ, providing almost 50% of adequate daily intake values for adults with 200 ml of each juice (Institute of Medicine, 2001). Current scientific literature lacks the data on mineral content in berry juices, as well as most of the open-access food composition databases (http://www.fineli.fi, http://www.foodcomp.dk, http:// www.nal.usda.gov/fnic/foodcomp/search/). Results on mineral content of berry juices, presented in our study, provide useful data and guidelines for characterisation and prioritisation of further use of berry juices, as part of the diet, in disease prevention and health promotion. Also, according to the results, berry juices are good sources of some minerals, and based on average intake of berries and related products contribute significantly to daily intake of these micronutrients. 3.5. Radical scavenging activity In vitro antioxidant activity of investigated juices expressed as radical scavenging activity determined by DPPH assay is shown in Table 1. As could be seen, BCJ has the highest radical scavenging activity (RSA), followed by BRJ, BJ and RJ. The lowest RSA is shown for RCJ. Bermúdez-Soto and Tomas-Barberán (2004) also showed that commercial juice of BCJ had the highest RSA, whereas RCJ exhibited higher activity than RJ. The relationship between total phenolic content and antiradical activity of berry juices was observed with statistically significant correlation (r = 0.980; p < 0.01). On the other hand, other constituents did not show significant correlation with radical scavenging activity, suggesting that not a single compound or only one group of compounds is responsible for such activity. This hypothesis is in accordance with the results of Nielsen, Haren, Magnussen, Dragsted, and Rasmussen (2003) who have found that vitamin C and anthocyanins accounted for about two-thirds of antioxidative potency of the black currants juices, indicating that other very potent antioxidants were present in phenolic fraction of this berry juice. 3.6. Antiproliferative activity Five berry juices were tested for their ability to inhibit the proliferation of human cancer cell lines such as cervix cell line (HeLa), melanoma cell line (Fem X), colon cell line (LS 174), breast cell line (MCF-7) and prostate cell line (PC-3). All examined juices showed antiproliferative activity in dose-dependent manner and IC50 values are presented in Table 4. Black currant juice (BCJ) showed the highest activity against all cell lines with IC50 values ranging between 10.2 and 26.8 ll/ml. Red currant (RCJ) and raspberry (RJ) juices exhibited good activity against LS 174, Fem X, HeLa and PC-3 cell lines (IC50 < 30 ll/ml), and bilberry juice (BJ) showed almost similar activity to that of RCJ for PC-3 cell lines and almost similar activity as RJ for Fem X and PC-3 cell lines. Black raspberry juice (BRJ) showed the least profound effects on all cell lines with IC50 values ranging between

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2

2 350

100

BRJ

300

BCJ 1

80

250 60 200 150

40

100 20 50 0

0 5

350

10

15

20

5

25

2

RJ

10

15

20

25

2

RCJ 80

300 250

60

200 40

150 100

20 50 0

0 5

10

15

20

25

5

10

15

20

25

2 140

BJ

120 100

1

80 60

3

40

4

20 0 5

10

15

20

25

30

Fig. 1. HPLC chromatogram at 520 nm of anthocyanin aglycones in juices. Peaks: (1) delphinidin; (2) cyanidin; (3) peonidin; and (4) malvidin.

Table 3 Mineral contents of berry juices expressed as mg/100 g of fresh weight. Samples

Ca

Mg

Na

K

Cu

Fe

Mn

Zn

RJ BRJ BCJ RCJ BJ

35.20 ± 5.55 25.09 ± 1.79 64.24 ± 1.76 32.77 ± 2.99 26.42 ± 4.04

10.13 ± 0.34 12.69 ± 6.45 11.46 ± 0.54 5.31 ± 0.29 5.54 ± 0.19

1.41 ± 0.65 1.58 ± 0.12 2.19 ± 0.16 1.70 ± 0.08 1.23 ± 0.26

145.95 ± 9.53 177.93 ± 8.21 208.73 ± 13.21 166.95 ± 12.42 92.96 ± 2.52

0.006 ± 0.002 0.022 ± 0.001 0.005 ± 0.004 0.007 ± 0.003 0.005 ± 0.001

0.07 ± 0.01 0.19 ± 0.02 0.15 ± 0.02 0.05 ± 0.01 0.03 ± 0.00

0.49 ± 0.01 0.18 ± 0.00 0.06 ± 0.01 0.06 ± 0.01 0.53 ± 0.01

0.13 ± 0.02 0.14 ± 0.01 0.17 ± 0.02 0.14 ± 0.01 0.31 ± 0.02

RJ – red raspberry juice; BRJ – black raspberry juice; BCJ – black currant juice; RCJ – red currant juice; and BJ – bilberry juice. Values represent means of three replicate determinations ± SD.

37.4 and 66.7 ll/ml. Among cell lines, MCF-7 cell line showed high resistance to all the tested juices. Cell lines of different origin possessed different sensitivity to growth inhibition toward different berry juices. Our results are consistent with previous studies investigating different berry extracts and fractions (Olsson, Gustavsson, Andersson, Nilsson, & Duan, 2004; Seeram, Adams, Hardy, & Heber, 2004). Significant correlation of titratable acidity and total anthocyanin content with antiproliferative activity of berry juices on HeLa cells, LS 174 cells and MCF-7 cells was noticed (Table 5). Seeram et al. (2004) showed that organic acids participated with the high percent

in the antiproliferative effects of total cranberries extracts against some cell lines. Moderate correlation was observed between cyanidin content and antiproliferative activity on all cell lines (r values between 0.41 and 0.63). On the other hand, correlation between total phenolics and antiproliferative activity was not observed. Sun, Chu, Wu, and Liu (2002) also showed that antiproliferative activities of fruit extracts did not correlate with total phenolic content as well, suggesting that a specific phenolic compound or class of phenolics in fruits was responsible for their antiproliferative activities. Results from correlation analysis among radical scavenging and antiproliferative activity on cancer cell lines used in the study

A. Konic´-Ristic´ et al. / Food Chemistry 125 (2011) 1412–1417 Table 4 Concentrations of juices (ll/ml) that induced 50% decrease in cell survival. Cell line

HeLa Fem X LS 174 MCF-7 PC-3

Antiproliferative activity, IC50 (ll/ml) RJ

BRJ

BCJ

RCJ

BJ

28.9 ± 1.9 20.7 ± 0.9 18.4 ± 1.8 41.0 ± 0.3 26.0 ± 2.2

43.4 ± 1.4 37.4 ± 0.9 66.7 ± 2.1 61.6 ± 1.1 60.8 ± 2.7

13.6 ± 3.3 13.6 ± 1.3 15.4 ± 2.3 26.8 ± 1.4 10.2 ± 2.2

22.1 ± 1.2 28.3 ± 1.9 21.1 ± 2.0 33.4 ± 1.5 29.3 ± 1.8

56.9 ± 1.9 20.3 ± 1.7 58.7 ± 3.1 70.5 ± 3.9 28.4 ± 3.5

RJ – red raspberry juice; BRJ – black raspberry juice; BCJ – black currant juice; RCJ – red currant juice; and BJ – bilberry juice.

Table 5 Correlation analysis of titratable acidity, total phenolics, total anthocyanins, radical scavenging activity and cyanidin content in berry juices with antiproliferative activity. Antiproliferative activity, IC50 (ll/ml)

Titratable acidity Total phenolics Total anthocyanins Radical scavenging activity Cyanidin content a

HeLa

Fem X

LS 174

MCF-7

PC-3

0.915a 0.261 0.910a 0.130

0.681 0.195 0.204 0.198

0.886a 0.453 0.889a 0.352

0.921a 0.329 0.935a 0.210

0.785 0.036 0.471 0.029

0.518

0.571

0.523

0.419

0.636

Indicates significant values at p < 0.05.

showed no significant relationship (Table 5). Similar results were obtained in the case of different berries where antiproliferative activity of berries was not a direct consequence of antioxidant activity measured by DPPH assay (Sun et al., 2002). 4. Conclusion This study demonstrated the antiradical and antiproliferative activities of different fresh pressed berry juices against five human cancer cell lines. Among investigated berry juices, black currant juice outstands with the highest total phenolic content which is in correlation with high radical scavenging activity. The highest total anthocyanin content and the most complex anthocyanin aglycone profile were determined in bilberry juice. Black currant juice showed the highest antiproliferative activity against all cell lines tested. Furthermore, regarding mineral content, consumption of these berry juices could be of importance for adequate daily intake of the analysed micronutrients. Data obtained in this study support the previous findings that berries and their products possess potent antioxidant and antiproliferative effects and make foundation for further research in order to enhance health benefits of berries. The obtained results could be used as guidelines and selection criteria for further use of berry juices as a part of conventional diet. Acknowledgement This work was supported by Ministry of Science and Technological Development of Republic of Serbia (Grant No. 046013). References Amakura, Y., Umino, Y., Tsuji, S., & Tonogai, Y. (2000). Influence of jam processing on the radical scavenging activity and phenolic content in berries. Journal of Agriculture and Food Chemistry, 48, 6292–6297. AOAC (1984). AOAC official methods of analysis (14th ed.). Washington, DC, USA: Association of Analytical Chemists. AOAC (1995). AOAC official methods of analysis (16th ed.). Arlington, WA, USA: Association of Analytical Communities.

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