Antioxidant and antiproliferative properties of strawberry ... - CiteSeerX

Journal of Berry Research 1 (2010) 3–12 DOI:10.3233/BR-2010-001 IOS Press

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Antioxidant and antiproliferative properties of strawberry tree tissues L. Tavaresa , S. Fortalezasa , C. Carrilhoa , G.J. McDougallb , D. Stewartb , R.B. Ferreiraa,c and C.N. Santosa,∗ a Disease

& Stress Biology Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal b Plant Products and Food Quality Programme, Scottish Crop Research Institute, Dundee, Scotland, UK c Departamento de Botânica e Engenharia Biológica – Instituto Superior de Agronomia, Lisboa, Portugal

Abstract. Strawberry tree (Arbutus unedo) belongs to the Ericaceae family and is endemic to the Mediterranean area. Its fruits are edible and its fruits and leaves are used in folk medicine for diverse purposes. Previous studies have shown that the fruits are rich in flavonoids, responsible for their antioxidant properties and compounds isolated from the entire plant were promising in cancer chemopreventive therapy. Strawberry tree fruits and leaves extracts enriched in polyphenols, but devoid of organic acids, carotenoids and sugars, were prepared by solid phase extraction (SPE) and tested for their antioxidant activities and their ability to inhibit metalloproteinases: attributes that could be related with initiation and proliferation of cancer cells. After fractionation by SPE, the apparent polyphenol yield was reduced for both leaf and fruit samples by the elimination of vitamins and organic acids, but the antioxidant and metalloproteinases inhibitory activities were potentiated. The antioxidant activity and the MMP-9 inhibitory activity of the polyphenol-enriched fractions of A. unedo tissues were similar or higher than those of blackberry and green tea, which have been recognized in the literature as highly effective. The phenolic profile of the fruit was dominated by gallic acid and quercetin derivatives with smaller amounts of proanthocyanidins and anthocyanins. The phenolic profile of the leaves was also dominated by gallic acid derivatives, flavonol derivatives and some tannins but lacked anthocyanins. The fractions obtained from both strawberry tree tissues seem to be quite promising as antioxidants and antiproliferative agents. Further cell-based assays are underway to study these possible outcomes. Keywords: Arbutus unedo, polyphenols, antioxidant activity, MMP-9, HPLC-MS

1. Introduction There is considerable epidemiological evidence that insufficient intake of fruits and vegetables may predispose the human body to a range of chronic health disorders, including cancer and cardiovascular disease [9]. Phytochemicals, such as polyphenols, present in many fruits and vegetables may participate in disease prevention and this has contributed to the growing interest in identifying components in edible plants responsible for anticancer effects [19]. Polyphenols demonstrate diverse biological activities attributed to their general free radical trapping capacity, or antioxidant activity per se, iron chelation, activation of survival genes, cell signalling pathways and regulation of mitochondrial function [9, 23]. In cancer, several studies point to the important role of the oxidative stress on the ∗ Corresponding author: C.N. Santos, Disease & Stress Biology Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Apartado 127, 2781-901 Oeiras, Portugal. Tel.: +351 214469651; Fax: +351 214433644; E-mail: [email protected].

1878-5093/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved

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L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

oncogenic stimulation. Various cancer cells have a cellular redox imbalance, i.e, a disruption between endogenous antioxidants and reactive oxygen and nitrogen species (ROS and RNS) balance when compared with normal cells, which could modify permanently the cell biology. This redox imbalance could lead to damage in cellular biomolecules that contribute to induction of mutagenesis and carcinogenesis [37–39]. Free radical damage could influence DNA mutations and altered gene expression, lipid peroxidation due to malondialdehyde (MDA) and 4-hydroxynonenal (HNE) formation [16, 25, 38], and protein oxidation [39]. Cancer cell invasion is a critical point for cancer metastasis. It is generally accepted that remodelling of the extracellular matrix (ECM) is required for cancer cell invasion [41]. The primary response for the degradation of ECM components was demonstrated to be the activation of zinc-dependent matrix metalloproteinases (MMPs) [27]. Although MMPs are expressed in normal tissue in remodelling conditions, such as during embryonic development, wound healing, uterine and mammary involution, cartilage-to-bone transition in ossification, and placenta development, the aberrant expression of various MMPs has been correlated with pathological conditions, such as rheumatoid arthritis, tumour cell invasion and metastasis [12]. Matrix metalloproteinase inhibitors (MMPIs) have been shown to inhibit angiogenesis in various models. However the synthetic inhibitors have demonstrated some disadvantages, such as the lack of selectivity and the secondary effects [24]. Strawberry tree (Arbutus unedo L.; Ericaceae family) is an evergreen shrub, a native Mediterranean species which is also cultivated in other regions of Eastern Europe [3]. Its fruits are spherical, about 2 cm in diameter, dark red, and tasty only when fully ripen in autumn. A. unedo berries are rarely eaten as fresh fruits but have some importance in local agricultural communities which use them for the production of alcoholic beverages, jams, jellies and marmalades [2, 28]. The fruits are also used in folk medicine as antiseptics, diuretics and laxatives, while the leaves have long been employed as an astringent, diuretic, urinary anti-septic agent and, more recently, in the therapy of hypertension and diabetes [4]. Recently, Carcache-Blanco and co-workers isolated ten compounds from A. unedo leaves exhibiting chemopreventive action through cyclooxygenase-2 inhibitory activity [6]. This work reinforces A. unedo as promising source of antiproliferative agents. Strawberry tree fruit contains several classes of naturally occurring antioxidants such as phenolic compounds (e.g. anthocyanins, gallic acid derivatives, tannins and flavonoids), vitamin C, vitamin E and carotenoids [2, 3, 22, 28, 29], and a high antioxidant capacity [28]. The aim of this work was to assess an in vitro potential of polyphenols from A. unedo fruits and leaves to provide an antiproliferative effect. The extracts prepared were enriched in polyphenols, but devoid of organic acids (vitamin C), carotenoids and sugars. Using this approach, we expect to maximize the activities of the compounds through positive synergisms between the different polyphenols or by eliminating antagonisms, in contrast to the Carcache-Blanco approach that tested pure compounds [6]. Synergistic positive effects are well described between polyphenols [1, 33, 35]. For example, Seeram et al. detected for cranberry extracts [33], after removing sugars and organic acids, a higher antiproliferative activity of polyphenols enriched fraction, when compared against the individual purified phytochemicals.

2. Material and methodology 2.1. Biological material Arbutus unedo L. fruits and leaves were collected in November 2007, in Arrábida Natural Park (southern region of Portugal) and frozen. For comparison purposes, the blackberry cv. Apache produced in Fataca experimental field (Odemira, Portugal) and commercial tea leaves (Lipton Green Tea Pure, Lipton) were used as control samples for fruits and leaves, respectively. The samples were freeze-dried, ground and stored at –80◦ C prior to extraction. 2.2. Extract preparation To each 1 g of lyophilized powder, 12 mL of hydroethanolic solvent (50% (v/v) ethanol/water) was added and the mixture was shaken for 30 min at room temperature in the dark. The mixture was then centrifuged at 12400 g for 10 min at room temperature. The supernatant was filtered through paper filter and then through 0.2 ␮m cellulose acetate membrane filters. The resulting extracts were stored frozen at –80◦ C.


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2.3. Fractionation by solid phase extraction Hydroethanolic extracts were fractionated by Solid Phase Extraction (SPE) using a Giga tubes 2 g/12 mL, C18-E units (Phenomenex® ). The columns were pre-washed in 0.5% (v/v) glacial acetic acid in acetonitrile and then preequilibrated in 0.5% (v/v) glacial acetic acid in water. The extracts were dried under vaccum and ressuspended in 0.5% (v/v) glacial acetic acid in water, then they were applied to the columns and unbound material, which contained the free sugars, organic acids or vitamin C, was discarded. The columns were washed with 0.5% (v/v) aqueous acetic acid and then polyphenol-enriched bound fractions were eluted with 0.5% (v/v) glacial acetic acid in acetonitrile [30, 32]. 2.4. Total phenolic measurement Determination of total phenolic compounds was performed by the Folin-Ciocalteau method [34]. Briefly, to each well of a microplate, 235 ␮L water, 5 ␮L sample (or solvent, in the blank), 15 ␮L Folin-Ciocalteau’s reagent (Fluka® ) and 45 ␮L saturated Na2 CO3 were added. The microplate was incubated for 30 min at 40◦ C and the absorbance at 765 nm measured. Gallic acid was used as the standard and the results were expressed as mg of gallic acid equivalents (mg GAE). 2.5. Peroxyl radical scavenging capacity assay Peroxyl radical scavenging capacity was determined by the ORAC (Oxygen Radical Absorbance Capacity) method [5, 40]. Briefly, the reaction mixture contained 150 ␮L of sodium fluorescein (0.2 nM) (Uranine, Fluorescein Sodium Salt® TCI Europe), 25 ␮L sample and 25 ␮L of 2,2 -azobis(2-amidopropane) dihydrochloride (41.4 g.L–1 ). All solutions are prepared in 75 mM phosphate buffer (pH 7.4). The blank contained 25 ␮L 75 mM phosphate buffer (pH 7.4) instead of sample, whereas the standards contained 25 ␮L of 10 to 50 ␮M 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox® ) in place of the sample. The fluorescent emission at 515 nm was then monitored kinetically during 30 min at 37◦ C, after excitation at 493 nm using a FLx800 Fluorescence Microplate Reader (Biotek). The final results were calculated using the area differences under the fluorescence decay curves between the blank and the sample, and were expressed as mM Trolox equivalents (mM TE). 2.6. MMP-9 inhibitory activity assay MMP-9 pro-enzyme was activated using 4-aminophenylmercuric acetate (APMA). The enzyme was incubated with 1 mM APMA in 100 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2 and 0.05% (v/v) Brij 35 and 2% (v/v) DMSO for 22 h at 37◦ C in the dark. Then the reaction was stopped with ice and the active MMP-9 was kept at 4◦ C. MMP-9 activity was measured by the direct hydrolysis of MCA-Pro-Leu-Gly-Leu-␤(Dnpa)-Ala-Ala-Arg-amida substrate [18], which has a fluorescent group 7-metoxicumarin-4-acetil (MCA) and a quenching group 2,4-dinitrofenilamine (Dnpa). Therefore, an increase in fluorescence intensity occurs as a result of substrate hydrolysis, namely the cleavage of peptide Gly-Leu. Before inhibition tests, enzymatic activity using different concentrations of MMP-9 was measured, which revealed a linear relationship between enzyme activity and enzyme concentration (data not shown). The highest concentration was chosen for inhibition assays. The assays were performed on 96 well plates measuring a residual enzymatic activity. The enzyme (3.2 nM) was pre-incubated with 100 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2 and 0.05% (v/v) Brij 35 and different concentrations (0–25 ␮g GAE. mL–1 ) of test fractions at 37◦ C for 30 min. The reaction was initiated by adding the substrate (12 ␮M in the same buffer as the enzyme) in a final volume of 100 ␮L. Fluorescence (␭emission = 393 nm, ␭excitation = 325 nm) was measured for 10 min at room temperature. Results were transformed using a non-linear regression with Origin Pro 6.1 software (OriginLab© , USA) and the IC50 values were determined. 2.7. HPLC-MS phenolic profile determination Phenolic extracts were dried by rotary evaporation, ressuspended in 5% (v/v) acetonitrile in water and were analyzed on a LCQ-DECA system controlled by the XCALIBUR software (2.0, ThermoFinnigan). The LCQ-Deca system

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comprised a Surveyor autosampler, pump and photo diode array detector (PDAD) and a Thermo Finnigan mass spectrometer iontrap. The PDA collected spectral data from 200–600 nm and scanned three discrete channels (at 280, 365 and 510 nm). The samples were applied to a C-18 column (Synergi Hydro C18 column with polar end capping, 4.6 mm × 150 mm, Phenomonex Ltd) and eluted over a gradient of 95:5 solvent A:B at time = 0 min to 60:40 A:B at time = 60 min at a flow rate of 400 ␮L/min. Solvent A was 0.1% (v/v) formic acid in ultra pure water and solvent B 0.1% (v/v) formic acid in acetonitrile. The LCQ-Deca LC-MS was fitted with an ESI (electrospray ionization) interface and analyzed the samples in positive and negative-ion mode. Two scan events, full scan analysis in mass range 80–2000 m/z followed by data dependent MS/MS of the most intense ions, were used for compounds detection and identification. The data-dependent MS/MS used collision energies (source voltage) of 45%. The capillary temperature was set at 275◦ C with sheath gas at 60 psi and auxiliary gas at 10 psi. Before the analysis, the system was tuned by using known concentrations of cyanidin-3-glucoside (positive mode) and quercetin-3-glucoside (negative mode) in ultrapure water. 2.8. Statistical analysis The results reported in this work are the averages of at least three independent experiments and are represented as the mean ± SD. Differences among treatments were detected by analysis of variance [31] with Tukey HSD (Honestly Significant Difference) multiple comparison test (α = 0.05) using SigmaStat 3.10 (Systat).

3. Results and discussion 3.1. Assessment of bioactivities recovery in polyphenols enriched fractions After the hydroethanolic extraction, crude extracts were fractionated by SPE column, to obtain polyphenol-enriched fractions. The crude extracts and enriched fractions were assessed for total phenolic content, antioxidant activity for peroxyl radical and the MMP-9 inhibitory activity (Fig. 1). After fractionation by the SPE column, strawberry tree fruit and leaf extracts showed a decrease in phenolic content (18% and 62% respectively). The decreases observed are probably due to molecules detected by FolinCiocalteu method, such as vitamins or organic acids that are eliminated by the SPE [13]. Indeed, components such 4 3.5 50 3 40

2.5

30

2 1.5

20

1 10

0.5

0

MMP-9 inhibitory activity (IC50)

Total phenol content/Anti oxidant activity

60

0

Extract

Enriched fraction Fruits

Extract

Enriched fraction Leaves

Fig. 1. Comparison of total phenol content, antioxidant capacity and metalloproteinases inhibitory activity between crude extracts and polyphenolenriched fractions obtained from A. unedo fruits and leaves. 䊏-total phenol content (mg GAE), - IC50 of MMP-9 inhibitory capacity (mg GAE. mL–1 ) and ⵧ antioxidant activity (×10 mmol TE) for peroxyl radical were determined for crude extracts and polyphenols enriched fractions obtained from A. unedo fruits and leaves. Vertical bars represent ± SD.


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as niacin, ascorbic acid, ␤-carotene, L-malic and quinic acids (detected and quantified in strawberry tree fruits by Alarcão-E-Silva et al. [2] or Ayaz et al. [3]) could account for the apparent loss in “total phenol content” after SPE. The greater decrease in the fruit fraction could be due to the higher contents in vitamins and organic acids in fruits. The strawberry tree fruit polyphenol-enriched fraction presented an increase of 100% of antioxidant activity, suggesting that the SPE fractionation eliminated some compounds with antagonistic effects. Strawberry tree leaves had an antioxidant capacity recovery of approximately 100%. Concerning the MMP-9 inhibitory capacity, the IC50 values for both fractions diminished 50%, which means that 50% of the amount of polyphenols is required to produce the same effect as the crude extract. Therefore, fractionation of strawberry tree tissues using SPE simultaneously eliminated molecules that could exert some antagonistic effects (e.g. in the antioxidant activity) and enriched the phenolic compounds (which diminished the IC50 value for MMP inhibition). However, further fractioning could not be so beneficial, since could eliminate positive synergistic effects, already detected by others [33]. In fact Carcache-Blanco have tested A. unedo isolated compounds and detected lower efficacies (high IC50 ) than their positive controls (13-cis-Retinoic acid and trans-Resveratrol) in chemopreventive assays [6]. 3.2. Antioxidant and MMP-9 inhibitory activities The strawberry tree fruit and leaf extracts gave significantly higher antioxidant capacities (211.66 ± 22.82 mM TE and 308.56 ± 26.74 mM TE, respectively) than blackberry and green tea, respectively (Table 1). The IC50 values of MMP-9 inhibitory activity for both strawberry tree tissues were not significantly different of the amount required for the respective control samples (1.68 ± 0.38 ␮g GAE. mL–1 for strawberry tree fruits and 1.31 ± 0.17 ␮g GAE. mL–1 for leaves) (Table 1). Control tissues used in this work, blackberry and green tea leaves, are recognized in the literature as highly antioxidants and anti-cancer agents [7, 8, 15, 36, 42–44], Blackberries are a rich source of polyphenols and present a recognized in vitro antioxidant activity (6221 ␮mol TE. 100 g–1 determined by ORAC method by Wolfe et al.) [42] as well as a good cellular antioxidant activity detected by a decrease in ROS in cells [42]. These fruits also have shown to inhibit MMP-2 and MMP-9 activities [36]. Moreover, they also could inhibit HT-29 colon tumor cell growth in a concentration-dependent manner [8]. Green tea leaves have been studied for many years and it has been shown their capacity to inhibit carcinogen-induced DNA damage in a number of cell line studies as reviewed by Yang et al. due to their direct radical scavenging and metal chelation [43, 44]. Tea and tea constituents have been shown to inhibit the development of several cancer in animal models like oral, esophageal, forestomach, stomach, intestinal, colon, skin, liver, bladder, prostate, and breast cancer [7, 15, 44]. In this work results for the A. unedo tissues, in comparison with the positive controls, revealed to be promising sources of effective antiproliferative extracts. To confirm these in vitro results, cell based assays should be performed as antiproliferative and intracellular antioxidant tests. Table 1 Comparison of antioxidant capacity and MMP-9 inhibitory capacity (IC50 ) values for polyphenol-enriched fractions from strawberry tree fruit and leaves with the respective positive controls (blackberry and green tea). Antioxidant capacity was determined by ORAC method and expressed as mM TE. IC50 values (␮g GAE. mL–1 ) were obtained by OriginPro 6.1 software. The values presented are the mean value of triplicates ± SD. The values of each tissue were statistically compared with the value of respective control. nd: not significantly different for p < 0.05; *: significantly different for p < 0.05; ***: significantly different for p < 0.001 Tissue

Sample

Fruits

Blackberry Strawberry tree Green tea Strawberry tree

Leaves

Antioxidant capacity (mM TE)

MMP-9 inhibitory capacity (IC50 ) (␮g GAE. mL–1 )

118.75 ± 17.15 211.66 ± 22.82*** 135.95 ± 2.99 308.56 ± 26.74*

1.52 ± 0.16 1.68 ± 0.38nd 1.94 ± 0.39 1.31 ± 0.17nd

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3.3. HPLC-MS phenolic profile determination To identify the main compounds present in fruits and leaves of strawberry tree, phenolic extracts were analysed by HPLC-ESI-PDA-MS. The compounds were tentatively identified in the bibliography by their PDA and MS fragmentation patterns [28, 29] (Fig. 2; Table 2). The fruits composition is relatively well-known compared to leaves [28, 29]. The fruits yielded mainly gallic acid derivatives (as glucosides, galloylquinic acid, galloylshikimic acid and gallotannins) but also some proanthocyanidins, quercetin derivatives and ellagic acid derivatives. The anthocyanins are not abundant in these fruits, but were identified in accordance with the bibliography as delphinidin-3-galactoside,

100

NL: 2.35E6 A

3

90

10

80 70 60

8

14

12 11 15

50

1

40

17 18 16 19 22 20 27

9 5 7

30

2 4

20

6

10 0

100

20

90

NL: 6.98E4 B

21 23 24

16

25

80 70

30

26 29

60 50 40

13

28

30 20 10

0 100

NL: 7.26E4 C

A2

90 80 70 60 50 40

A3

30

A1

20 10 0 0

10

20

30

40

50

60

Time (min) Fig. 2. HPLC phenolic profile assessed for A. unedo fruit extract. The chromatogram in (A) shows the absorbance of peaks at 280 nm, (B) peaks at 365 nm and (C) peaks at 560 nm. The peaks are numbered and assignments are given in Table 2 for phenols and Table 3 for anthocyanins.


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Table 2 Peaks assignments, retention times and mass spectral data of phenols present in A. unedo fruits extract Peak no.

RT

PDA

M/Z [M-H]

MS2

Putative identity

1 2 3 4

7.37 8.70 10.64 14.04

280 265 270 255–300

331.1 331.1 343.0 331.1

271.0, 211.1, 169.0 169.0, 125.0 191.2, 169.0 169.1

16.29 20.93 21.36 21.95 22.66 23.86 24.31 24.6 25.50 26.10 26.84 28.68 29.44

255–300 280 270–290 295 280 280 320 285 280, 525 275 270 360 275

169.0, 125.1 289.2 343.0, 191.0 343.0, 191.0 425.0, 407.2, 289.2 261.0, 175.0 577.1 453.1, 301.4, 169.2 325.0, 169.0 325.0, 169.0 463.0, 301.1 301.2 783.1, 492.8

18 19 20 21 22 23 24 25 26 27

31.09 33.08 33.36 34.67 35.21 36.30 36.69 36.96 37.40 38.09

280 275 360 260–355 260–355 260–355 255, 370 345 275–355 280

204.1, 186.1, 142.0 633.0, 463.2, 301.2 301.0 463.0, 301.1 463.0, 301.1 463.0, 301.1 301.2, 257.2 301.2 301.2 769.0, 617.2

Unknown Tannin Quercetin-3-xyloside Quercetin hexose galloyl derivative Quercetin hexose galloyl derivative Quercetin hexose galloyl derivative Ellagic acid Quercetin 3-galactoside Quercetin 3-glucoside Gallotannin

28 29 30

39.81 40.75 41.49

355 355 355

325.0 577.1 495.0, 465.0, 343.0, 191.2 495.0, 465.0, 343.0, 191.2 577.0, 423.2, 407.2, 289.2 289.1 865.1, 453.2, 325.1 541.1, 483.1, 467.3, 321.0, 301.2 477.0, 325.1 477.0, 325.1 633.1, 463.1, 301.2, 275.2 463.2, 301.3 1109.0, 972.9, 647.0, 635.1, 588.1, 441.0, 301.3 366.2, 186.0 953.0 433.1, 301.2 615.2, 463.2, 433.1, 301.1 615.2, 463.2, 433.1, 301.1 615.2, 463.2, 433.1, 301.1 301.2 463.1, 301.2 463.1, 301.2 939.1, 769.1, 729.0, 617.1, 544.2, 480.2, 469.2 599.0, 301.0 433.0, 301.1 447.0, 301.1

Gallic acid glucoside Galloyl glucoside 3-O- or 5-O-galloylquinic acid [29] Gallic acid 4-O-B-D-glucopyranoside or B-D-Glucogalline [29] Galloyl shikimic acid Proanthocyanidin dimer [29] Digalloylquinic acid Isomer of digalloylquinic acid Procyanidin dimer B2 [28] Catechin Procyanidin trimer Gallic acid derivative Digalloyl shikimic acid Digalloyl shikimic acid Strictinin ellagitannin Quercetin-3-glucoside Gallotannin derivative

5 6 7 8 9 10 11 12 13 14 15 16 17

463.1, 301.1 301.1 301.1

Ellagic acid-hexose derivative Ellagic acid arabinoside/xyloside Ellagic acid rhamnoside

The most abundant ions are shown in bold. Numbers in brackets are references.

cyanidin-3-galactoside, cyanidin-3-glucoside and cyanidin-3-arabinoside [28, 29]. The leaf extracts also yielded gallic acid derivatives common to fruits, (epi)catechin, tannins, myricetin derivatives and kaempferol derivatives. Some of these phytocompounds, such as tannins, quercetin derivatives and catechin gallate have already been described as responsible for anti-hypertensive and anti-aggregant effects using strawberry tree leaf extracts [20, 26].

4. Conclusion In this work the polyphenol-enriched fractions of Arbutus unedo fruits and leaves were characterized for antioxidant and MMP-9 inhibitory activity, and the results obtained suggest these tissues as promising sources of agents with pharmacological activities. Many naturally occurring agents have shown chemoprotective potential in a variety of

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L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues Table 3 Peaks assignments, retention times and mass spectral data of anthocyanins present in A. unedo fruits extract Peak no. A1 A2 A3

RT

PDA

20.60 22.94 25.28

280, 525 280, 515 280, 515

M/Z [M+H]

MS2

Pututative identity

579.1, 465.1, 303.2 449.0, 287.2 419.1, 287.2

303.2 287.2 287.2

Delphinidin-3-galactoside [28] Cyanidin 3-O-glucoside or cyanidin-3-galactoside [28] Cyanidin 3-O-arabinoside [28]

The most abundant ions are shown in bold. Numbers in brackets are references. Table 4 Peaks assignments, retention times and mass spectral data of phenols present in A. unedo leaves extract Peak No.

RT

PDA

M/Z [M-H]

MS2

Putative identity

1

9.57

280

10.98 12.80

280 260

839.5, 729.2, 676.1, 567.2, 270.9, 160.9, 109.2 271.0, 211.1, 169.1 271.1, 169.1, 125.1

Unknown

2 3

1111.2, 899.3, 839.3, 817.3, 567.3, 317.0 331.2, 271.1, 169.1 331.2, 271.2, 169.1

4

13.54

270

363.2, 169.2

169.1

5 6 7 8 9 10 11

14.80 20.57 26.48 26.94 27.92 30.82 33.26

275 275 275 275 280 270 275

12

36.91

355

13 14 15

38.57 41.26 43.04

280 360 280, 355

191.2, 169.1 169.1, 125.2 593.0, 575.1, 425.0, 407.2 423.2, 313.1, 169.1 245.2, 231.2, 205.2, 179.1 325.1 914.9, 896.9, 765.0, 613.1, 461.1, 445.1, 301.3 909.1, 801.0, 633.1, 463.2, 301.3 325.1 316.2 787.1, 769.1, 617.2, 601.2

16 17

46.51 50.25

255, 350 350

343.1, 191.2 325.2, 169.1, 125.2 745.1, 577.2, 495.1, 343.1, 289.1 423.3, 313.2 289.2 477.1, 325.2, 169.2 951.1, 933.1, 729.2, 477.1, 301.3, 273.2 1105.0, 1085.0, 953.2, 935.2, 785.1, 633.2, 479.2, 316.2, 301.3 629.0, 477.1, 325.2 463.2, 449.2, 316.2 939.2, 769.2, 617.2, 469.3, 301.3, 169.1 447.2, 300.2 583.2, 417.2, 300.2, 284.2

18 19 20

51.17 38.03 42.56

265, 340 355 350

476.8, 431.2, 285.2, 227.3 479.2, 317.2 463.0, 300.1

285.2 317.1 301.1

301.1 417.2, 327.1, 284.1, 255.3

Gallic acid glucose derivative B-D-Glucogalline or gallic acid 4-O-B-D-glucopyranoside B-D-Glucogalline or gallic acid 4-O-B-D-glucopyranoside derivative Galloylquinic acid derivative Galloylshikimic acid Proanthocyanidin derivative Galloyl derivative Catechin Digalloylshikimic acid derivative Tannin Tannin Galloylshikimic acid derivative Myricetin-3-rhamnose Gallotannin Quercetin 3-rhamnoside Kaempferol derivative (Arabinoside/Xyloside) Kaempferol derivative (Rhamnoside) Myricetin-Hexoside Quercetin-Hexoside

The most abundant ions are shown in bold.

bioassay systems and animal models. Phenolic compounds are reported to have antioxidant, antimutagenic and anticarcinogenic activity and are expected to reduce the risk of disease and to bring health benefits with daily intake [11]. Gallic acid derivatives dominate the profile of both fruits and leaves of Arbutus unedo. Gallic acid is an intermediate component of plant metabolism and, together with its analogs, has been associated with a wide variety of biological actions, including: antioxidant, antifungal, antimalarial, and antiherpetic action [10, 14, 17]. However, the main interest in gallic acid and its derivatives is related to its antitumoral activity, showing selective cytotoxicity toward a variety of tumor cells, more cytotoxic for tumor cells than for non-tumor cells [21]. Nevertheless, in studies using


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pure coumpounds, synergistic effects may be lost in determining anti-proliferative effects. In this study, the use of polyphenols-enriched fraction, devoid of ascorbic acid and carotenoids, allows interactions between polyphenols and the detected bioactivity could be directly associated to them and their interactions. Fruits and leaves polyphenolsenriched fractions presented IC50 values similar to the ones obtained for positive control tissues with recognized activity as anti-cancer and antioxidant agents. Therefore, it is important to confirm the potential of these enriched fractions as multitarget medicines with antiproliferative efficacy, using cell based assays such as proliferation and intracellular antioxidant tests.

Acknowledgements To FCT for financial support of Claudia Santos (SRFH/BPD/26562/2006) and Lucélia Tavares (SFRH/BD/37382/ 2007) and to Cost Office 863.

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N. Aftab and A. Vieira, Antioxidant activities of curcumin and combinations of this curcuminoid with other phytochemicals, Phytotherapy Research (2009), in press. M.L.C.M.M. Alarcao-E-Silva, A.E.B. Leitao, H.G. Azinheira and M.C.A. Leitao, The Arbutus berry: Studies on its color and chemical characteristics at two mature stages, Journal of Food Composition and Analysis 14 (2001), 27–35. F.A. Ayaz, M. Kucukislamoglu and M. Reunanen, Sugar, non-volatile and phenolic acids composition of strawberry tree (Arbutus unedo L. var ellipsoidea) fruits, Journal of Food Composition and Analysis 13 (2000), 171–177. M. Bnouham, F.Z. Merhfour, A. Legssyer, H. Mekhfi, S. Maallem and A. Ziyyat, Antihyperglycemic activity of Arbutus unedo, Ammoides pusilla and Thymelaea hirsuta, Pharmazie 62 (2007), 630–932. G. Cao, H.M. Alessio and R.G. Cutler, Oxygen-radical absorbance capacity assay for antioxidants, Free Radical Biology & Medicine 14 (1993), 303–311. E.J. Carcache-Blanco, M. Cuendet, E.J. Park, B.N. Su, J.F. Rivero-Cruz, N.R. Farnsworth and A.D. Kinghorn, Potential cancer chemopreventive agents from Arbutus unedo, Natural Product Research 20 (2006), 327–334. G.J. Dai, H.Y. Jin, Y.J. Ding, J.G. Xia, X.F. Liu, F. Liu, X.Z. Tan and J.X. Geng, Anticancer effects of tea polyphenols on colorectal cancer with microsatellite instability in nude mice, Zhong Xi Yi Jie He Xue Bao 6 (2008), 1263–1266. J. Dai, J.D. Patel and R.J. Mumper, Characterization of blackberry extract and its antiproliferative and anti-inflammatory properties, Journal of Medicinal Food 10 (2007), 258–265. S. Dore, Unique properties of polyphenol stilbenes in the brain: more than direct antioxidant actions; gene/protein regulatory activity, Neurosignals 14 (2005), 61–70. S.M. Fiuza, C. Gomes, L.J. Teixeira, M.T. Girao da Cruz, M.N. Cordeiro, N. Milhazes, F. Borges and M.P. Marques, Phenolic acid derivatives with potential anticancer properties-a structure-activity relationship study, Part 1: methyl, propyl and octyl esters of caffeic and gallic acids, Bioorganic & Medicinal Chemistry 12 (2004), 3581–3589. G. Galati and P.J. O’Brien, Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties, Free Radical Biology & Medicine 37 (2004), 287–303. Z.S. Galis and J.J. Khatri, Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad and the ugly, Circulation Research 90 (2002), 251–262. S. George, P. Brat, P. Alter and M.J. Amiot, rapid determination of polyphenols and vitamin C in plant-derived products, Journal of Agricultural and Food Chemistry 53 (2005), 1370–1373. P. Grundhofer, R. Niemetz, G. Schilling and G.G. Gross, Biosynthesis and subcellular distribution of hydrolyzable tannins, Phytochemistry 57 (2001), 915–927. D.H. Han, J.H. Jeong and J.H. Kim, Anti-proliferative and apoptosis induction activity of green tea polyphenols on human promyelocytic leukemia HL-60 cells, Anticancer Research 29 (2009), 1417–1421. S.P. Hehner, R. Breitkreutz, G. Shubinsky, H. Unsoeld, K. Schulze-Osthoff, M.L. Schmitz and W. Droge, Enhancement of T cell receptor signaling by a mild oxidative shift in the intracellular thiol pool, Journal of Immunology 165 (2000), 4319–4328. E. Klein and N. Weber, In vitro test for the effectiveness of antioxidants as inhibitors of thiyl radical-induced reactions with unsaturated fatty acids, Journal of Agricultural and Food Chemistry 49 (2001), 1224–1227. C.G. Knight, F. Willenbrock and G. Murphy, A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases, FEBS Letters 296 (1992), 263–266.

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L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues P.M. Kris-Etherton, K.D. Hecker, A. Bonanome, S.M. Coval, A.E. Binkoski, K.F. Hilpert, A.E. Griel and T.D. Etherton, Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer, American Journal of Medicine 113(Suppl 9B) (2002), 71S–88S. A. Legssyer, A. Ziyyat, H. Mekh, M. Bnouham, C. Herrenknecht, V. Roumy, C. Fourneau, A. Laurens, J. Hoerter and R. Fischmeister, Tannins and catechin gallate mediate the vasorelaxant effect of Arbutus unedo on the rat isolated aorta, Phytotherapy Research 18 (2004), 889–894. C. Locatelli, P.C. Leal, R.A. Yunes, R.J. Nunes and T.B. Creczynski-Pasa, Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death, Chemico-Biological Interactions 181 (2009), 175–184. Z. Males, M. Plazibat, V.B. Vundac and I. Zuntar, Qualitative and quantitative analysis of flavonoids of the strawberry tree-Arbutus unedo L. (Ericaceae), Acta Pharmaceutica 56 (2006), 245–250. S. Mandel and M.B. Youdim, Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases, Free Radical Biology & Medicine 37 (2004), 304–317. F. Mannello, Natural bio-drugs as matrix metalloproteinase inhibitors: new perspectives on the horizon? Recent Patents on Anticancer Drug Discovery 1 (2006), 91–103. L.J. Marnett, Lipid peroxidation-DNA damage by malondialdehyde, Mutation Research 424 (1999), 83–95. H. Mekhfi, M. ElHaouari, M. Bnouham, M. Aziz, A. Ziyyat and A. Legssyer, Effects of extracts and tannins from Arbutus unedo leaves on rat platelet aggregation, Phytotherapy Research 20 (2006), 135–139. H. Nagase and K. Brew, Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors, Biochemical Society Symposia (2003), 201–212. K. Pallauf, J.C. Rivas-Gonzalo, M.D. del Castillo, M.P. Cano and S. de Pascual-Teresa, Characterization of the antioxidant composition of strawberry tree (Arbutus unedo L.) fruits, Journal of Food Composition and Analysis 21 (2008), 273–281. A.M. Pawlowska, M. De Leo and A. Braca, Phenolics of Arbutus unedo L. (Ericaceae) fruits: identification of anthocyanins and gallic acid derivatives, Journal of Agricultural and Food Chemistry 54 (2006), 10234–10238. Phenomenex, SPE reference Manual & Users Guide, 2000. M. Popova, V. Bankova, D. Butovska, V. Petkov, B. Nikolova-Damyanova, A.G. Sabatini, G.L. Marcazzan and S. Bogdanov, Validated methods for the quantification of biologically active constituents of poplar-type propolis, Phytochemical Analysis 15 (2004), 235–240. H.A. Ross, G.J. McDougall and D. Stewart, Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts, Phytochemistry 68 (2007), 218–228. N.P. Seeram, L.S. Adams, M.L. Hardy and D. Heber, Total cranberry extract versus its phytochemical constituents: antiproliferative and synergistic effects against human tumor cell lines, Journal of Agricultural and Food Chemistry 52 (2004), 2512–2517. V.L. Singleton and J.A. Rossi, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, American Journal of Enology and Viticulture 16 (1965), 144–158. M. Suganuma, S. Okabe, Y. Kai, N. Sueoka, E. Sueoka and H. Fujiki, Synergistic effects of (–)-epigallocatechin gallate with (–)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancercell line PC-9, Cancer Research 59 (1999), 44–47. P. Tate, J. God, R. Bibb, Q. Lu and L.L. Larcom, Inhibition of metalloproteinase activity by fruit extracts, Cancer Letters 212 (2004), 153–158. M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes and J. Telser, Role of oxygen radicals in DNA damage and cancer incidence, Molecular and Cellular Biochemistry 266 (2004), 37–56. M. Valko, H. Morris, M. Mazur, P. Rapta and R.F. Bilton, Oxygen free radical generating mechanisms in the colon: do the semiquinones of vitamin K play a role in the aetiology of colon cancer? Biochimica et Biophysica Acta 1527 (2001), 161–166. M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic and M. Mazur, Free radicals, metals and antioxidants in oxidative stress-induced cancer, Chemico-Biological Interactions 160 (2006), 1–40. S.Y. Wang and H.S. Lin, Antioxidant activity in fruits and leaves of blackberry, raspberry and strawberry varies with cultivar and developmental stage, Journal of Agricultural and Food Chemistry 48 (2000), 140–146. J. Westermarck and V.M. Kahari, Regulation of matrix metalloproteinase expression in tumor invasion, FASEB Journal 13 (1999), 781–792. K.L. Wolfe, X. Kang, X. He, M. Dong, Q. Zhang and R.H. Liu, Cellular antioxidant activity of common fruits, Journal of Agricultural and Food Chemistry 56 (2008), 8418–8426. C.S. Yang, J. Hong, Z. Hou and S. Sang, Green tea polyphenols: antioxidative and prooxidative effects, Journal of Nutrition 134 (2004), 3181S. C.S. Yang, J.D. Lambert and S. Sang, Antioxidative and anti-carcinogenic activities of tea polyphenols, Archives of Toxicology 83 (2009), 11–21.

Journal of Berry Research 1 (2010) 1 DOI:10.3233/BR-2010-007 IOS Press

1

Editorial

I would like to begin by first giving a warm welcome to all the readers and contributors to the Journal of Berry Research. In the vast current scientific-editorial panorama it is quite understandable to ask oneself why, how and what will this new scientific journal be like. Why have a new journal? Because in spite of the many thousands of articles indexed in PubMed under “berries” (over 12,000 in 2008 and nearly 15,000 in 2009) there is no specialized journal which can be used for reference, collection, discussion and promotion of the many excellent studies carried out worldwide. In fact, most articles are scattered among a large variety of journals, ranging from chemistry of the soil to anti-tumour therapy, with obvious difficulties both on the part of reviewers who have to evaluate the contents in a rigorously scientific way and on the part of the readers. Moreover, many excellent articles are not given the visibility they deserve while others are “sacrificed” to a concept of necessary priority for journal management, but at the same time penalized if applied to productive studies and sectors in which the pharmaceutical or food industry still do not have a decisive position. How will the Journal of Berry Research function? The journal will be multidisciplinary, aimed at improving knowledge about the quality and production of berries to benefit consumers’ health; it will cover classical berries (strawberry, raspberry, blackberry, blueberry, cranberry, etc.) as well as grapes and small fruit in general (e.g., kiwifruit, tomato) as currently occurs, for example, for the “Berry Group” in the framework of the International Horticultural Society. JBR will publish research results covering all areas of plant breeding, including plant genetics, genomics, functional genomics, proteomics and metabolomics, plant physiology, plant pathology and plant development, as well as results dealing with the chemistry and biochemistry of bioactive compounds contained in such fruits and their possible role in human health. Contributions detailing possible pharmacological, medical or therapeutic use or dietary significance will be welcomed in addition to studies regarding biosafety issues of genetically modified plants. What will the Journal of Berry Research be like? This depends only partially on us (the Editorial Board) but above all on you: Our readers and authors. We on the Editorial Board will be committed to following the goals which we set and which are clearly described in this letter and in the “Aims” of the journal, and to running the journal with scientific rigour yet without any kind of prejudice. What JBR becomes depends above all on who reads and, we hope, appreciates it: It will depend on the quantity but above the quality of your scientific contributions and on how much these will be read, discussed and cited. The invitation and wish is therefore the following: Help us to make the Journal of Berry Research an important journal, your journal. Editor-in-Chief Dr. Maurizio Battino, PhD, MD (Hon) Department of Biochemistry, Biology & Genetics Faculty of Medicine Università Politecnica delle Marche Via Ranieri 60100 Ancona Italy Tel.: +39 071 2204646 Fax: +39 071 2204398 E-mail: [email protected] 1878-5093/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved


53

Nutritional quality of berries and bioactive compounds in the leaves of black currant (Ribes nigrum L.) cultivars evaluated in Estonia Piret Raudseppa,∗ , Hedi Kaldmäeb , Ave Kikasb , Asta-Virve Libekb and Tõnu Püssaa a Department b Polli

of Food Hygiene, Estonian University of Life Sciences, Tartu, Estonia Horticultural Research Centre, Viljandimaa, Estonia

Abstract. Polli Horticultural Research Centre (58◦ 7 N; 25◦ 32 E) in Estonia has focused on selecting cultivars with high productivity and suitable to use in local climatic conditions since 1945. Besides important agronomic characteristics, more attention has been recently paid to fruit quality and content of various bioactive compounds. The results of a biochemical analysis of 4 prospective black currant selections (10B, 2-96-51, 1-96-16, 4-96-1), 4 new cultivars (‘Karri’, ‘Almo’, ‘Ats’, ‘Elo’) from our own breeding program and 7 introduced cultivars (‘Öjebyn’, ‘Zagadka’, ‘Ben Sarek’, ‘Intercontinental’, ‘Pamyati Vavilova’, ‘Titania’ and ‘Pilenai’) are presented. In addition to the analysis of main biochemical characteristics, the anthocyanin content of the berries was determined using high performance liquid chromatography (HPLC). The total anthocyanin content of the berries varied in a wide range. The highest anthocyanin content was found in the cultivar ‘Almo’ (212 ± 9 mg/100 g) and the lowest in ‘Ben Sarek’ (83 ± 24 mg/100 g). The ascorbic acid content varied from 98 mg/100 g with ‘Ats’ to 209 mg/100 g with elite selection 4-96-1. The polyphenol composition of the black currant leaves was determined by HPLC, the compounds were identified using polyphenol commercial standards and/or compounds mass spectrometric (MS) characteristics. Keywords: Black currant, seedlings, fruit quality, leaves, poly phenol composition

1. Introduction Ascorbic acid and anthocyanins are the most highly valued health beneficial compounds in black currants contributing to the antioxidant capacity of the fruit [13]. Anthocyanins have also been associated with fruit colour and are, therefore, important contributors to the quality of the product, they appear to be very stable and remain active after a prolonged frozen storage, processing into juice, wine and jam [9, 13]. The change of focus to quality and nutritional factors in breeding programs in Europe stems from the retail price reduction due to the overproduction of black currants in Europe in 2003–2005 [1]. The focus on the nutritional aspects of the berries is an important sales argument for promoting black currants for fresh consumption. The processors’ main requirements are high levels of ascorbic acid and antioxidant properties together with a low acidity and improved sensory characteristics [2]. The anthocyanin content of 4 prospective black currant selections (10B, 2-96-51, 1-96-16, 4-96-1), 4 new cultivars (‘Karri’, ‘Almo’, ‘Ats’, ‘Elo’) from our own breeding program and 7 introduced cultivars (‘Öjebyn’, ‘Zagadka’, ‘Ben ∗ Corresponding author: P. Raudsepp, Department of Food Hygiene, Kreutzwaldi 58A, Tartu 51014, Estonia. Tel.: +372 7313432; Fax: +372 7313432; E-mail: [email protected].


54

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

Sarek’, ‘Intercontinental’, ‘Pamyati Vavilova’, ‘Titania’ and ‘Pilenai’) were analysed in this study, additionally to the main biochemical characteristics. The leaves of black currant have been used in folk medicine for their diaphoretic and diuretic properties as well as to relieve rheumatic pain [18]. The pharmacological effect other than anti-inflammatory effect of black currant leaves has not been scientifically proved [3, 4]. Their anti-inflammatory effect may be due to the polyphenol compounds that are originally synthesised by plants to protect themselves from pathogens [5]. Surveys have indicated that some of the polyphenol compounds may be exposed on the cuticle of leaves to give a quick response to the microbial attack [25]. Although black currant leaves are widely in use as tea material [17], there are not many scientific studies about the composition of the chemical compounds responsible for the health beneficial effect. We used a mixture of known polyphenol standard compounds, previously known to be present in the leaves of black currant [17, 18] and some additional standards that have been found to be present in other leaves of pharmacological value [6] to determine the composition of polyphenol compounds in the leaves.

2. Materials and methods 2.1. Materials The anthocyanidin standards and other chemicals, used in sample preparation or chromatographic separation were all with HPLC purity grade: catechin, kaempherol, quercetin-3-glucoside, myricetin, quercetin-3-galactoside from Sigma-Aldrich Inc.; cyanidin-chloride from Carl Roth 4545.1, Germany; delphinidin-chloride from Carl Roth 4537.1, Germany; formic acid from Fluka, USA; quercitrin, Alfa-Aesar; acetonitril, ethanol 96%, hydrochloric acid 37%, NaOH and methanol were with analytical grade; water, ultra pure made prior to analysis using Milli Q by Millipore Corporation. The chemicals used for biochemical analysis: potassium ferricyanide (III), sulfuric acid, lead subacetate, potassium iodate, NaOH, 2,6-dichloroindophenol, methylene blue and oxalic acid were of analytical grade. 2.2. Collecting of samples The research was carried out in 2005–2007 and 2009. 500 g of berry samples per cultivar were collected in the middle of the harvesting season from the cultivar evaluation test plots established in 2000. Four prospective selections (10B, 2-96-51, 1-96-16, 4-96-1) and 4 new cultivars (‘Karri’, ‘Almo’, ‘Ats’, ‘Elo’) [11] from our own breeding program and 7 introduced cultivars (‘Öjebyn’, ‘Zagadka’, ‘Ben Sarek’, ‘Intercontinental’, ‘Pamyati Vavilova’, ‘Titania’ and ‘Pilenai’) were analysed in three consecutive years 2005, 2006 and 2007. Fully developed leaves for the qualitative analysis of polyphenols were collected in August 2009 randomly from 6 different bushes of the same cultivar (‘Karri’) including equal proportions of leaves from different sides and inner and outer range of the bush. 2.3. Methods The representative samples were prepared from the field samples, 200 g of berries were homogenized using a kitchen blender and analysed on the same day for sugar, soluble solids and ascorbic acid content. The soluble solids content in the homogenized samples were recorded in a refractometer (Abbe WYA-1S, Optic Ivymen System) at 20◦ C, organic acids were determined by titration with 0.1 NaOH. Ascorbic acid was determined using the modified Tillman’s method. Ascorbic acid was titrated with 2,6-dichloroindophenol under acid conditions [16]. The ferricyanide method was used for the sugar content analysis [23]. The results are expressed in mg per 100 g of fresh berries. The average weight of berries was determined by weighing 20 berries per sample. The samples for the anthocyanin content determination were collected at the same time and frozen at 40◦ C for further preservation. The leaves were dried at 50◦ C for 24 h until air-dry and were ground prior to analysis.


55

2.4. Sample preparation for HPLC analysis of anthocyanins and leaf polyphenols For the anthocyanidin analysis, 200 g of fresh frozen berries were homogenized with a Heidolph DIAX 900, 2 g of homogenizate was weighed with an OHAUS analytical standard scale into a 50 ml centrifuge tube, 3 parallel samples of each. The samples were extracted for 30 min at room temperature with 20 ml ethanol/water/HCl (70:30:1), after which the samples were shaken on a minishaker IKA MS 1 by IKA-WORKS for 30 s twice after 5 min. The tubes were then centrifuged with an Eppendorf centrifuge 5810 R at 3220 g at 20◦ C for 10 min, the supernatant was removed and the samples were extracted once more with 20 ml ethanol/water/HCl (70:30:1) using the same procedure, and for the third time with 5 ml ethanol/water/HCl (70:30:1). The supernatants were combined and 1 ml of the final solution was filtered through a Spartan 13 mm regenerated cellulose membrane filter with a pore size of 0.2 ␮m (modified [26] and [24]). For the acid hydrolysis of the samples, the final filtrate was taken to 1 M HCl concentration and heated in the Binder oven at 90◦ C for 60 min [25]. For the polyphenol analysis of the leaves: 0.1 g of dried leaves were weighed into a 15 ml centrifuge tube, 10 ml of 40% ethanol was added, and 3 parallel samples were made. The samples were extracted at room temperature for 24 h. The extracts were centrifuged at 3220 g at 20◦ C for 10 min. The supernatant was filtered through Spartan 13 mm regenerated cellulose membrane filter with a pore size 0.2 ␮m 2.5. Chromatographic conditions For detection and quantification of anthocyanins, Agilent 1100 Series LC/MSD Trap-XCT with an ESI interface (m/z interval 50–1000 in positive ion mode) and UV-Vis diode array detector was used. HPLC reversed phase column Zorbax 300SB-C18 with dimensions of 2.1 × 150 mm with a particle size of 5 ␮m was used at 35◦ C, and the speed of the mobile phase was 0.3 ml min–1 . The elution was carried out with 1% formic acid in water (mobile phase A) and acetonitril (solvent B), a multistep mobile phase gradient was used as follows: 0 min 95:5 (A:B), linear gradient until 30 min 70:30 (A:B), by 40 min the ratio of A:B was 10:90 and maintained for 50 min, after what the concentration of mobile phase A was raised again to 95% while mobile phase B was dropped to 5% and the system was reequilibrated 10 min. For the quantification of the anthocyanins, the peak area at 510 nm was used. The detection limit for anthocyanins was 0.1 ␮g/ml and the quantification limit 0.2 ␮g/ml. The calibration curve was constructed using a standard mixture of delphinidin and cyanidin. The analysis of the polyphenol composition of the leaves was conducted on the same chromatographic system but in the negative ionisation mode. The elution gradient was set as follows: 0 min 90:10 [0.1% formic acid in water (A):acetonitril (B)], the linear gradient until 30 min 70:30 (A:B), by 40 min the ratio of A:B was 10:90 and maintained until 50 min and from 50.1 min the concentration of solvent A was raised again to 90% and the concentration of solvent B dropped to 10% and maintained until 60 min to re-equilibrate the system. The calibration curves were constructed using a standard mixture of catechin, chlorogenic acid, quercetin galactoside, quercetin glucoside, myricetin, quercetin rhamnoside also known as quercitrin, and kaempherol.

3. Results and discussion 3.1. Biochemical and morphological characteristics of fruit The average fruit weight was 1.25 g. The cultivar ‘Öjebyn’ had the smallest fruits (0.9 g) and ‘Karri’ the largest ones 1.7 g. The ascorbic acid content varied from 98 mg/100 g with ‘Ats’ to a very high level of 209 mg/100 g with elite selection 4-96-1 with average ascorbic acid content of 132.5 mg/100 g (Table 1). 130 mg has been set as the requirement by some producers to ensure the healthiness of the processed product [12]. The sugar content and sugar acid ratio, that are major factors affecting the sweetness of the taste of the berries, were highest with the cultivar ‘Pilenai’ (12.6% and 4.7, respectively) and lowest with ‘Zagadka’ (8.4% and 2.7 respectively). Above-the-average sugar acid ratio (3.7) was recorded for the cultivars ‘Pamyati Vavilova’ ‘Titania’ ‘Intercontinental’and elite selections 4-96-1 and 2-96-51. These selections both have the cultivar ‘Pamyati Vavilova’as

56

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds Table 1 The average biochemical and morphological characteristics of berries, measured in 2005 and 2007 Cultivar 10 B 1-96-16 2-96-51 4-96-1 Almo Ats Ben Sarek Elo Intercontinental Karri Pamyati Vavilova Pilenai Zagadka Titania Öjebyn

Soluble solids in juice (%)

Water (%)

Titratable acids (%)

Sugars (%)

Sugar acid ratio

Ascorbic acid (mg/100g)

Average fruit weight (g)

17.3 17.2 15.6 16.3 17.8 16.9 16.3 17 18.5 16.9 16.4 16 16 16 17.9

79 76 83 79 79 78 80 79 79 77 79 81 78 77

2.6 2.7 2.7 2.6 2.8 2.4 3.6 2.6 2.8 2.4 2.7 2.7 3.1 3.1 2.7

9.8 11 10.4 10.7 9.5 8.8 8.7 9.2 10.4 8.9 11.9 12.6 8.4 12.6 8.7

3.5 4.1 3.9 4.2 3.4 3.7 2.4 3.6 3.7 3.7 4.4 4.7 2.7 4.1 3.2

138 134 141 209 112 98 115 117 132 134 164 110 107 141 133

1.2 1 1.2 1.2 1.2 1.4 1.5 1.2 1.5 1.7 1.2 1.3 1.2 1.1 0.9

one of the crossing parents. Both selections have also high acorbic acid contents (209 mg and 141 mg/100 g) similar to their parental genotype ‘Pamyati Vavilova’(ascorbic acid content 164 mg/100 g). Comparison with our earlier research [11] including the 4 new cultivars from our breeding program and cultivar ‘Öjebyn’used as the standard cultivar, showed that the sugar content in the current study years was slightly higher than average and might have been influenced by the weather conditions and relatively modest yields (data not shown) in these years. The ascorbic acid levels obtained with the above listed cultivars are consistent with the average results obtained previously and show a similar variation between genotypes. 3.2. Analysis of the anthocyanin content of the berries Our analysis revealed that anthocyanins are present in form of cyanidin and delphinidin glycosides in black currant berries. The prevailing glycosides are delphinidin glucoside (1), delphinidin 3-O-rutinoside (2), cyanidin-glucoside (3), cyaniding 3-O-rutinoside (4) (Fig. 1). Previous studies have revealed similar results [10, 14, 19]. Some authors have discovered a total of 15 different anthocyanin glycosides in black currant berries [21, 22]. After the acid hydrolysis of the samples, it was revealed that cyanidin was slightly dominant over delphinidin (Table 2) in most of the studied cultivars. It is interesting to remark that at the beginning of the berry ripening in the example of ‘Pilenai’, the cyanidin was largely prevailing over delphinidin 73:23, but in the fully ripened berries, the ratio 57:43 was closer to equal (Table 2). The highest content of anthocyanins was found in cultivars ‘Titania’, ‘Almo’, ‘Öjebyn’, ‘Ats’, ‘Intercontinental’ and ‘Elo’ from which ‘Almo’, ‘Elo’ and ‘Ats’ are from our own breeding programme. Seedling 1-96-16 from our breeding programme showed also a high content of anthocyanins in three consecutive years (Table 2). It has to be mentioned that in the second year of the study (2006), the weather conditions were not favourable for the berry production, and the content of anthocyanins in the berries gathered approximately at the same time as in the two other years was lower than in the first (2005) and the third (2007) year. It may be the result of incomplete ripening by that time. Previous studies have revealed that anthocyanin content depends on the ripening stage of the berries, increasing during ripening and reaching the maximum when berries are fully ripened [7, 8, 15]. The anthocyanin content may also be influenced by weather conditions [12]. The overall crop yield of berries was likewise lower in the year 2006.


Intens. mAU

40

57

4

30 2

20

3 1

10

0 5

10

20

15

25

30

Time (min) Fig. 1. UV-chromatogram of fruit anthocyanin analysis at 510 nm. 1) delphinidin glucoside, 2) delphinidin 3-O-rutinoside, 3) cyanidin-glucoside, 4) cyaniding 3-O-rutinoside. Table 2 The average anthocyanin content of black currant berries in 3 consecutive years and the percentage of the main aglycones after acid hydrolysis

10B 1-96-16 1-96-51 4-96-1 Almo Ats Ben Sarek Elo Intercontinental Karri Pamjati Vavilova *Pilenai I *Pilenai II *Pilenai III *Pilenai IV Zagadka Titania Öjebyn

Anthocyanin content of berries (ug/ml)

Delphinidin (%)

Cyanidin (%)

158 ± 21 221 ± 29 150 ± 66 148 ± 33 213 ± 9 185 ± 65 84 ± 24 172 ± 21 177 ± 6.5 154 ± 89 143 ± 22 0 14 ± 4 111 ± 32 177 ± 93 179 ± 43 216 ± 29 194 ± 52

44 55 41 42 38 39 37 40 44 41 40 – 27 40 43 52 49 42

56 45 59 58 63 61 63 60 56 59 60 – 73 60 57 48 51 58

*The cultivar ‘Pilenai’ was measured in four different phases of ripening; stage I being green berries and IV stage the fully ripened stage, collected at the same time as the berries of the other cultivars and seedlings.

3.3. Polyphenol composition of leaves The results of the HPLC analysis of the leaves are presented in Fig. 2. The analysis was conducted using polyphenol standards (2-catechin, 3-chlorogenic acid, a-quercetin galactoside, 6-quercetin glucoside, b-myricetin, 8-quercitrin,

58


BPC of blackcurrant leaves 0.1g diluted in 10 ml of 40% ethanol (1/100 m/v) 8

6 7 1

6

4

10

3 2

8

Intens.

10 6

2

5

9

4

11

0

BPC of standard mixture with concentration 12.5 ug/ml 6 5 8 4

a d

6

3

b 2 2

c

3

1 0 10

20

30

40

50

Time (min) Fig. 2. The base peak chromatogram (BPC) of analysis of the leaves and standard mixture of the known leaf specific polyphenols. The polyphenols found in black currant leaves are marked with numbers repeating the peak numbers on leaf BPC and the standard compounds not found in black currant leaves are marked with letters. 1. gallocatechin; 2. epigallocatechin; 3. chlorogenic acid; 4. unidentified compound with [M-H]– = 383; 5. myricetin glucoside; 6. quercetin glucoside; 7. quercetin malonylhexoside; 8. mixture of kaempferol glucoside, quercetin rutinoside and quercetin acetylglucoside; 9. mixture of isorhamnetin rutinoside and an unidentified glucoside with [M-H]– = 373; 10. kaempferol malonylhexoside; 11. isorhamnetin glucoside.

c-quercetin, d-kaempherol) previously known as present in the black currant leaves [17, 18] or in the other leaves of pharmacological value [6]. Some of the standard compounds were present in the black currant leaves as well (peaks 2, 3, 6 and 8). The compounds that were not overlapping with any of the used standards were identified using their molecular weight and MS2 collision fragments. The quantitative analysis was performed using the peak area under MS extracted ion chromatograms. The analysis revealed that the concentration of catechin in the dried leaves was 786 mg/100 g; chlorogenic acid 1493 mg/100 g; quercetin glucoside 1947 mg/100 g and quercetin rutinoside 399 mg/100 g. The content of catechin was lower than found in the green tea by Rusak et al. (3330 mg/100 g), but the content of quercetin was significantly higher than found in green tea by Rusak et al. (28 mg/100 g) [20]. The health beneficial effect of black currant leaves compared to the green tea needs to be investigated further.


59

Acknowledgements We thank the Estonian Science Foundation (research grants no. 6801 and 7703) and Ministry of Education and Research of Estonia (target financing project no. 1092711s06) for financial support.

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The inhibitory effects of berry-derived flavonoids against neurodegenerative processes David Vauzour, Katerina Vafeiadou, Catarina Rendeiro, Giulia Corona and Jeremy P.E. Spencer∗ Molecular Nutrition Group, Department of Food and Nutritional Sciences, School of Chemistry, Food and Pharmacy, University of Reading, Reading, UK

Abstract. Evidence suggests that a combination of oxidative stress, neuroinflammation and the formation of endogenous neurotoxins contribute to the underlying neuronal death associated with neurodegenerative diseases. In this study we have investigated the ability of the berry-derived flavonoids to protect against neuronal damage induced by neuroinflammation and the neurotoxin 5-S-cysteinyl-dopamine. The flavanols (+)-catechin and (–)-epicatechin, but not the anthocyanidin pelargonidin, were observed to attenuate LPS/IFN-␥-induced TNF-␣ production in glial cells and associated neuronal injury. In contrast, pre-treatment of primary cortical neurons with pelargonidin, (+)-catechin and (–)-epicatechin (0.1 and 0.3 ␮M) resulted in concentration-dependant protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. Together these data suggest that berry-derived flavonoids may offer some protection against the neuronal injury relevant to the aetiology of the Parkinson’s and Alzheimer’s diseases. Keywords: Brain, neuroinflammation, flavonoids, Parkinson disease, neurotoxins

1. Introduction Abundant evidence exists to suggest that increased oxidative stress may contribute to the neuropathology of agerelated brain disorders such as Alzheimer’s and Parkinson’s disease [11, 21]. Although the precise mechanisms of neuronal death in these disorders remains uncertain, it is likely that a combination of elevated oxidative stress, the formation endogenous neurotoxic cysteinyl-catecholamine conjugates [26] and neuroinflammation [9] are believed to play a role in the underlying pathogenesis. For example, 5-S-cysteinyl-dopamine (CysDA) has been shown to possess strong neurotoxicity and may initiate a sustained increase in intracellular reactive oxygen species (ROS) in neurons leading to DNA oxidation, caspase-3 activation and delayed neuronal death [28]. Furthermore, glial cells may become activated by various inflammatory stimuli to produce cytokines such as TNF-␣ which contribute to neuronal injury [7]. Deleterious effects of TNF-␣ have been reported [19], and appear to be mediated by its binding to neuronal tumour necrosis factor receptor-1 (TNFR1) and the subsequent activation of downstream caspase-8 and caspase-3-mediated apoptotic pathways [29]. Recent studies have focused on the ability of dietary-derived phenolic compounds to protect against neuronal damage resulting from aging and other neurodegenerative processes [18, 32, 38]. Much attention has centred on the potential neuroprotective effects of flavonoids, which have been shown to protect against age-related cognitive decline [12, 39], against 6-hydroxydopamine neurotoxicity [8] and against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ∗ Corresponding author: J.P.E. Spencer, School of Chemistry, Food and Pharmacy, The University of Reading, PO Box 226, Whiteknights, Reading, RG2 6AP, UK. Tel.: +44 118 378 8724; Fax: +44 118 931 0080; E-mail: [email protected].


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D. Vauzour et al. / The inhibitory effects of berry-derived flavonoids against neurodegenerative processes

(MPTP) lesioning of the nigrostriatal tract [16]. For example, the consumption of flavonoid-rich blueberries and strawberries has been shown to reverse age-related cognitive and motor behavioural deficits [12, 27, 35, 39], whilst green tea and isolated EGCG protect against MPTP induced striatal dopamine depletion and neuronal loss [16] in animal models. Furthermore, flavonoids have also been shown to inhibit inflammatory processes mediated by glial cells [32], with the flavones wogonin and baicalein [5, 14], the flavonol quercetin [6], the isoflavone genistein [37] and the flavanols catechin and epigallocatechin gallate [10, 17] all expressing anti-inflammatory activity. In the present study, we provide evidence that both 5-S-cysteinyl-dopamine and LPS/IFN-␥-induced TNF-␣ release resulted in concentration-dependent neurotoxicity. However, pre-treating the neuronal cells with the berry-derived flavonoids pelargonidin, (+)-catechin and (–)-epicatechin significantly decreased the injury elicited by these endogenous neurotoxins at low micromolar concentrations. The same results were observed for LPS/IFN-␥-induced TNF-␣ release treated glial cells for the flavanols (+)-catechin and (–)-epicatechin, but not the anthocyanidin pelargonidin.

2. Materials and methods 2.1. Materials The following chemicals were obtained from Sigma Chemical Company (Poole, UK): (+)-catechin, (–)-epicatechin, bacterial endotoxin LPS (Escherichia Coli, serotype 0127:B8) and mouse interferon-␥ (IFN-␥)). Pelargonidin was purchased from Extrasynthese (Genay, France). The 5-S-CysDA was prepared as previously described [36]. All tissue culture reagents were purchased from Invitrogen (Paisley, UK). Ultrapure water (18.2 M.cm) passed through a purification system (Purite Ltd, Oxon, UK) was used for all purposes. 2.2. Cell culture Neuronal cells: Primary culture of neuronal cells were prepared as described previously [34]. Cells (106 /ml) were plated in a serum-free medium composed of a mixture of DMEM and F-12 nutrient (1:1 v/v) supplemented with glucose (33 mM), glutamine (2 mM), sodium bicarbonate (6.5 mM), HEPES (pH 7.4, 5 mM), streptomycin (100 ␮g/ml) and penicillin (100 UI/ml) (all Invitrogen, Paisley, UK). A mixture of hormones and salts composed of insulin (25 ␮g/ml), transferrin (100 ␮g/ml), putrescine (60 ␮g/ml), progesterone (20 nM) and sodium selenate (30 nM) (all from Sigma, Poole, Dorset, UK) was also added to the culture medium. Cells were cultured at 37◦ C in a humidified atmosphere of 95% air and 5% CO2 and after 5–6 days the vast majority of cells were neuronal (>98%) with 98% compared to vehicle, 100). Exposure of primary cortical neurons to 5-S-cysteinyl-dopamine (10–500 ␮M) led to concentration-dependent increases in neuronal injury (IC50 = 100.2 ± 2.1 ␮M) when assessed 24 h after exposure (Fig. 1). The strongest neuroprotective effect was observed when neurons were pre-treated with pelargonidin (0.1–3 ␮M). Indeed, concentrations ranging from 0.1 to 1 ␮M significantly protected neurons from injury induced by 5-S-CysDA (Fig. 2A), with 1 ␮M being the most potent (90.1 ± 4.2%, p < 0.001). Pelargonidin (3 ␮M) was also observed to be significantly protective, although to a lesser degree (34.2 ± 2.5%, p < 0.001). Significant protection against 5-S-CysDA-induced neuronal damage was also observed for (–)-epicatechin (0.1 to 3 ␮M), the greatest degree of protection apparent at the 1 ␮M concentration (46.7 ± 4.0%; p < 0.001) (Fig 2B). Finally, a small but significant protection was also afforded by (+)-catechin, with the 300 nM concentration being the most protective (19.0 ± 5.0%; p < 0.05) (Fig 2C).

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CysDA concentration (µM) Fig. 1. 5-S-cysteinyl-dopamine induces toxicity on primary cortical neurons. 5, 6 days in vitro primary neurons were exposed to 5-S-cysteinyldopamine (10–500 ␮M) or vehicle for 24 h. After 24 h, cell viability was determined by Alamar blue reduction. Results are expressed as mean ± SD of quadruplicate wells from single experiments repeated three times with similar results (***p < 0.001; compared with control, as analysed by one way ANOVA followed by post-hoc t-test).

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Fig. 2. Protective effects of flavonoids against 5-S-cysteinyl-dopamine-induced neuronal injury. Following pre-treatment with (A) pelargonidin, (B) (–)-epicatechin or (C) (+)-catechin (0.1–3 ␮M) for 18 h, 5–6 DIV primary neurons were exposed to 5-S-cysteinyl-dopamine (100 ␮M) for 24 h. Alamar Blue solution (10% v/v) was added to plates and they were returned to the incubator for 2–3 h, before fluorescence was measured (Ex: 540 nm; Em: 612 nm). Results are expressed as mean ± SD of quadruplicate wells from single experiments repeated three times with similar results. Data were analysed by one way ANOVA followed by post-hoc t-test. ***p < 0.001 respresent a significant decrease of neuronal viability relative to control; a = p < 0.001, b = p < 0.01, c = p < 0.05 represent a significant increase in neuronal viability relative CysDA treated cells.


49

3.2. Inhibitory effect of flavonoids on LPS/IFN-γ-induced TNF-a release Pelargonidin and the flavanols (+)-catechin and (–)-epicatechin (0.1–3.0 ␮M) did not induce glial cell injury as assessed by the Alamar blue assay 24 h post exposure. Treatment of glial cells with LPS (1 ␮g/mL) and IFN-␥ (10 ng/mL) for 24 h resulted in a significant increase in TNF-␣ production (p < 0.001, n = 6) (Fig. 3). However, preincubation of glial cells with either (+)-catechin or (–)-epicatechin (0.1–3 ␮M; 24 h), prior to LPS/IFN-␥ exposure, resulted in a significant decrease in medium TNF-␣ levels (Fig. 3A and B). In contrast, pre-treatment with pelargonidin (0.1–3 ␮M; 24 h) had no significant effect on LPS/IFN-␥-induced TNF-␣ release (Fig. 3C). 4. Discussion Abundant evidence exists to suggest that increased oxidative stress may contribute to the neuropathology of age-related brain disorders such as Alzheimer’s and Parkinson’s diseases [11, 21]. In this study, we show that 5-cysteinyl conjugates of dopamine possess a strong cytotoxicity towards primary cortical neurons. 5-S-CysDA, have been reported previously to be neurotoxic by inducing both oxidative DNA base modification and the activation of caspase-3 activity in neurons [20, 26]. Once formed, 5-S-CysDA may be further oxidised, yielding more complex conjugated species, such as DHBT-1 [24], an event which may be accelerated in the presence of peroxynitrite [33], a powerful oxidant and cytotoxic molecule formed from the reaction of nitric oxide (NO• ) with superoxide (O2 -) [13]. Presence of such oxidant molecules may be the results of activation of the iNOS enzyme, an event triggered under neuro-inflammatory processes. Indeed, under inflammatory conditions, excess release of cytokines, such as TNF-␣, is a central event in glial-induced neurotoxicity. Such detrimental actions of TNF-␣ induced neurotoxicity have had been reported in neuronal models [19], and are thought to be the result of the activation iNOS expression and the subsequent NO• release, thus enhancing its neurotoxic potential [15]. As a consequence, there has been much interest in the development of novel therapeutic agents that can selectively attenuate neurotoxins-induced toxicity. Recent evidence from human, animal and cell studies suggest that flavonoids may exert neuroprotective effects [3, 8, 25]. In the brain, the potential actions of flavonoids on neurons will be influenced by interactions with both astrocytes and microglia, which occupy the majority of the brain volume and play a key role in the maintenance of neuronal integrity. In this study, we show that specific berry-derived flavonoids are capable of inhibiting glial activation and of reducing cysteinyl-dopamine neurotoxicity at physiologically achievable plasma concentrations (nanomolar). Whilst the three flavonoids tested (pelargonidin, (+)-catechin and (–)-epicatechin) were able to decrease the neuronal cell death in primary neurons challenged with CysDA, only the flavanols (+)-catechin and (–)-epicatechin were able to inhibit TNF-␣, production in LPS/IFN-␥-activated glial cells. Such protective effects may be the results of an inhibition of the inducible nitric oxide synthase (iNOS) in glial cells as reported previously for the flavanone naringenin [30], or an activation of the detoxification enzymes such as the glutathione-S-transferase as observed previously in primary neurons in presence of (–)-epicatechin [4]. Action of such enzymes may decrease the neurotoxic potential of some molecules, as observed for quercetin where its neurotoxicity was significantly reduced by glial cell metabolism, notably by its conversion to 2 -glutathionyl quercetin [31]. Although flavonoids have been reported to exert strong antioxidant effects in vitro, accumulating evidence suggests that dietary polyphenols may cross the blood brain barrier [2, 40], accumulate in the brain at nanomolar concentrations [1, 2] and exert neuromodulatory effects through selective actions on different components of a number of protein kinase and lipid kinase signalling cascades, such as PI 3-kinase, Akt/PKB, tyrosine kinase, PKC and members of the MAP kinase family [23]. Modulation of these pathways may underlie the ability of berry-derived flavonoids to exert their protective effects, as previously reported for the flavan-3-ol, (–)-epicatechin, which has been observed to stimulate the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity [22]. Moreover, both hesperetin and 5-nitro-hesperetin were also effective at preventing neuronal apoptosis via a mechanism involving the activation of both Akt/PKB and ERK1/2 [34]. Further research is required in order to establish the underlying mechanisms involving the neuroprotective effects of the berry-derived flavonoids.

50


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Fig. 3. Flavonoids inhibit TNF-␣ production in LPS/IFN-␥ activated primary glial cells. Cells were treated with LPS (1 ␮g/mL) and IFN-␥ (10 ng/mL) for 24 h alone (LPS/IFN-␥ group) or pre-treated with naringenin (A), (–)-epicatechin (B), (+)-catechin or (C), pelargonidin (0.1–3 ␮mol/L) for 24 h before LPS/IFN-␥ activation for another 24 h. After incubation TNF-␣ concentrations were measured in the cell culture medium by enzyme-linked immunosorbent assay. Results are presented as means ± SD of three independent experiments. ***p < 0.001, indicates significant increase in TNF-␣ production relative to control; a = p < 0.001; b = p < 0.01; c = p < 0.05 indicates significant decrease in TNF-␣ production relative to LPS/IFN-␥ group.


51

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D. Vauzour et al. / The inhibitory effects of berry-derived flavonoids against neurodegenerative processes J.P. Spencer, P. Jenner, S.E. Daniel, A.J. Lees, D.C. Marsden and B. Halliwell, Conjugates of catecholamines with cysteine and GSH in Parkinson’s disease: Possible mechanisms of formation involving reactive oxygen species, Journal of Neurochemistry 71 (1998), 2112–2122. J.P. Spencer, D. Vauzour and C. Rendeiro, Flavonoids and cognition: The molecular mechanisms underlying their behavioural effects, Archives of Biochemistry and Biophysics 492 (2009), 1–9. J.P. Spencer, M. Whiteman, P. Jenner and B. Halliwell, 5-s-Cysteinyl-conjugates of catecholamines induce cell damage, extensive DNA base modification and increases in caspase-3 activity in neurons, Journal of Neurochemistry 81 (2002), 122–129. D.L. Taylor, F. Jones, E.S. Kubota and J.M. Pocock, Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand, Journal of Neuroscience 25 (2005), 2952–2964. K. Vafeiadou, D. Vauzour, H.Y. Lee, A. Rodriguez-Mateos, R.J. Williams and J.P. Spencer, The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury, Archives of Biochemistry and Biophysics 484 (2009), 100–109. K. Vafeiadou, D. Vauzour, A. Rodriguez-Mateos, M. Whiteman, R.J. Williams and J.P. Spencer, Glial metabolism of quercetin reduces its neurotoxic potential, Archives of Biochemistry and Biophysics 478 (2008), 195–200. K. Vafeiadou, D. Vauzour and J.P. Spencer, Neuroinflammation and its modulation by flavonoids, Endocrine, Metabolic & Immune Disorders – Drug Targets 7 (2007), 211–224. D. Vauzour, G. Ravaioli, K. Vafeiadou, A. Rodriguez-Mateos, C. Angeloni and J.P. Spencer, Peroxynitrite induced formation of the neurotoxins 5-S-cysteinyl-dopamine and DHBT-1: Implications for Parkinson’s disease and protection by polyphenols, Archives of Biochemistry and Biophysics 476 (2008), 145–151. D. Vauzour, K. Vafeiadou, C. Rice-Evans, R.J. Williams and J.P. Spencer, Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic effects of flavanones in cortical neurons, Journal of Neurochemistry 103 (2007), 1355–1367. D. Vauzour, K. Vafeiadou, A. Rodriguez-Mateos, C. Rendeiro and J.P. Spencer, The neuroprotective potential of flavonoids: A multiplicity of effects, Genes and Nutrition 3 (2008), 115–126. D. Vauzour, K. Vafeiadou and J.P. Spencer, Inhibition of the formation of the neurotoxin 5-S-cysteinyl-dopamine by polyphenols, Biochemical and Biophysical Research Communications 362 (2007), 340–346. X. Wang, S. Chen, G. Ma, M. Ye and G. Lu, Genistein protects dopaminergic neurons by inhibiting microglial activation, Neuroreport 16 (2005), 267–270. O. Weinreb, S. Mandel, T. Amit and M.B. Youdim, Neurological mechanisms of green tea polyphenols in Alzheimer’s and Parkinson’s diseases, Journal of Nutritional Biochemistry 15 (2004), 506–516. C.M. Williams, M.A. El Mohsen, D. Vauzour, C. Rendeiro, L.T. Butler, J.A. Ellis, M. Whiteman and J.P. Spencer, Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels, Free Radicals in Biology and Medicine 45 (2008), 295–305. K.A. Youdim, M.Z. Qaiser, D.J. Begley, C.A. Rice-Evans and N.J. Abbott, Flavonoid permeability across an in situ model of the blood-brain barrier, Free Radical Biology and Medicine 36 (2004), 592–604.


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Evaluating the health benefits of fruits for physical fitness: A research platform Birgit Schragea , David Stevensona , Robyn W. Wellsa , Kirsty Lyalla , Selena Holmesa , Dawei Denga and Roger D. Hurstb,∗ a Food

& Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Hamilton, New Zealand

b Food & Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Palmerston North, New Zealand

Abstract. To evaluate the health promoting attributes of fruits and their compounds the New Zealand Institute for Plant & Food Research Ltd (PFR) is using exercise as a model for oxidative stress and immune depression. Regular exercise has health benefits believed to be derived from adaptive responses to moderate oxidative stress. However, following exhaustive or unaccustomed exercise, excessive and prolonged oxidative stress and inflammation can be detrimental and the right balance of modulation from nutritional support via fruit phytochemicals (and vitamins) may prevent damage, aid recovery, and/or enhance muscular and immune function. We have developed a research platform to evaluate physical health, performance and recovery to position new fruit varieties in this area. Utilising compositional analysis of fruit extracts, in vitro screening of muscle cells, electrically stimulated muscle ex vivo, and animal and human intervention and exercise trials, we are evaluating the physical health-promoting effects of polyphenolic phytochemicals derived from fruit, particularly berry fruits. Our research demonstrates that certain fruits may complement the benefits of regular exercise through appropriate modulation of excessive oxidative stress and inflammation. Keywords: Berry fruit, kiwifruit, fruit, polyphenolics, phytochemicals, anthocyanins, oxidative stress, inflammation, exercise, skeletal muscle

1. Introduction The New Zealand Institute for Plant & Food Research Ltd (PFR) provides research and innovation for New Zealand’s horticultural sector. PFR has developed a research platform to evaluate physical performance and recovery. Regular exercise has health benefits derived from moderate oxidative stress. However, depending on the mode, intensity and duration, exercise can inflict high levels of mechanical and metabolic stress on muscle [28]. Dietary supplementation is an attractive choice for health-conscious individuals. However, the right balance of modulation from nutritional support needs to be established to prevent damage/injury, maximise recovery for enhanced muscle function, but also to optimise immune function. Utilising fruit varieties from breeding programmes at PFR with well-defined compositional data combined with a range of cell-based screening, ex vivo muscle experiments, and animal/human trials, we are evaluating the physical health-promoting effects of phytochemicals derived from some new fruit varieties, including berry fruits. The determination of biochemical markers of oxidative stress, inflammation and immune function in cell and tissue assays, and animal and human trials enables us to evaluate the effects of fruit consumption and to ultimately guide the breeding of health promoting fruit. ∗ Corresponding author: Roger D. Hurst, Food & Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Private Bag 11600, Palmerston North 4442, New Zealand. Tel.: +64 6 355 6203; Fax: +64 6 351 7050; E-mail: [email protected].


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B. Schrage et al. / Evaluating the health benefits of fruits for physical fitness: A research platform

2. Materials and methods 2.1. Fruit extract preparation and composition The ZESPRI® GOLD kiwifruit (Actinidia chinensis ‘Hort16A’) extract was prepared by homogenising freeze-dried fruit (20 g) with methanol (50 mL), followed by 30 min in an ultrasonic bath. Solvent was recovered by filtration and the residue extracted twice more in methanol. The extract was rotary evaporated, dissolved in the minimum volume of water and applied to a 10 g C-18 solid-phase extraction cartridge (Strata, Phenomenex, Torrance, CA). The cartridge was flushed with deionised water (30 mL) and eluted with methanol (40 mL). Evaporation of the methanol eluate yielded 0.24 g of extract which was prepared in buffer for the analysis of ex vivo muscle performance. The polyphenolic extract of blueberry fruit (Vaccinium corymbosum, ‘Reka’) was prepared from approximately 800 g of frozen fruit homogenised with acetone and the residue recovered by filtration. Following a further homogenisation and filtration with acetone/water (70:30), acetone extracts were combined and concentrated by rotary evaporation. Lipids were removed by partitioning the aqueous extract with heptane and the residual heptane in the aqueous layer removed by rotary evaporation. The polyphenolics were isolated from this extract by absorption, recovery, and elution (methanol) of XAD-7 (Sigma, Sydney, Australia) and dried by rotary evaporation to yield a friable powder. In all experiments, the blueberry fruit extracts were prepared from a stock in DMSO. DMSO concentrations in the final experimental conditions were below 1% and had no observable effect. Juices of various cultivars of blackcurrant (Ribes nigrum) were prepared by blending fruit (180 g) with a domestic hand blender, then treating with Pectinex (0.18 mL; Novozymes, Bagsvaerd, Denmark) at 48◦ C for 4 h. After centrifugation (4000 rpm, 10 min) the yield of juice was typically ∼50%. Blackcurrant juice samples were analysed by liquid chromatography-mass spectrometry (LC-MS) using a Shimadzu (Auckland, New Zealand) 20-Series UFLC system including an autosampler, column oven and photodiode-array detector, linked to an LCMS 2020 single-quadrupole mass spectrometer. The LC system was fitted with a 150 × 2 mm, 4␮ Synergi Fusion RP column (Phenomenex). For analysis, blackcurrant samples were diluted × 10 into 1% aqueous formic acid and kiwifruit extracts dissolved in water at ∼2 mg/mL. Samples of 10 ␮L were injected and eluted by the following solvent programme. Flow rate 0.6 mL/min, Solvent A: 2% formic acid, solvent B: methanol. The initial conditions were 8% B and ramped linearly to 10% at 2.5 min, 18% at 5 min, 38% at 8.5 min, 63% at 10.5 min, held at 70% between 11.5 and 12.5 min, then back to the starting conditions between 13 and 15 min. Column oven temperature was 50◦ C. The mass spectrometer was operated under standard tuning conditions, as specified by the manufacturer and half the LC flow was diverted to waste through a split valve. Identification for compounds of interest was achieved by comparison of a combination of UV-visible and mass spectra with standard compounds. The commercially available juices GHO “Natural Quenchers” (Good Health Organisation, Nelson, New Zealand) contained 65% of either ZESPRI® GOLD kiwifruit or blackcurrant juice. 2.2. Determination of muscle cell intracellular free radical levels The rat skeletal muscle cell line L6 (American Type Culture Collection, Manassas, USA) was cultured and differentiated as described elsewhere [13]. Muscle cells were plated at 5000 cells/well in 96 well plates and incubated with 2 ,7 -dichlorohydrofluorescein diacetate (DCFDA) (10 ␮M) for 60 min at room temperature. DCFDA is cleaved intracellularly into DCF, which produces an increase in fluorescence in the presence of reactive free radicals. Following a wash in D-PBS (2 ×), cells were equilibrated at room temperature for 10 min prior to incubation (simultaneously [time zero] and at the times indicated) with the blueberry fruit extract (50 ␮g/mL), followed by exposure to H2 O2 (0.5 mM). Fluorescence intensity was determined over time using a fluorescence platereader (BMG FluorStar Optima, Alphatech Systems Ltd) with excitation and emission wavelengths of 485 and 520 nm respectively. 2.3. Evaluation of muscle cell total glutathione (GSH) levels and glutathione peroxidase (GPx) activity The total amount of muscle cell GSH and GPx was determined colourimetrically using Bioxytech® assay kits (GSH-420TM and GPx-340TM , Sapphire Bioscience Pty. Ltd, Auckland). Differentiated L6 myotubes were incubated with the blueberry fruit extract both simultaneously and for 24 h before GSH and GPx determination. For GSH


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analysis, cells were plated at a density of 1 × 106 cells/well in 6 well plates and grown until confluent. Following extract exposure, cells were harvested using TrypleTM , washed in D-PBS, and permeabilised by sonication in the precipitation reagent provided by the kit. All other analysis steps were then as recommended by the supplier. To determine muscle cell GPx levels, cells were grown in T75 flasks until confluent. Following extract exposure, cells were harvested using a cell scraper and prepared into 3 × 106 cell aliquots before sonication in D-PBS containing 1 mM mercaptoethanol. All other analysis steps were as recommended by the supplier. 2.4. Animals Male Swiss mice, 8–12 weeks old, were used for the muscle bath and animal exercise experiments. Animals were cared for in accordance with the Code of Ethical Conduct, Regulations Act, 1987 (New Zealand) and all procedures were approved by our local Animal Ethics Committee. 2.5. Ex vivo model of muscle performance To investigate the effect of fruit extracts on muscle tissue, a model bath system was established in which isolated fresh muscle (Soleus) was exposed to extracts and subsequently electrically stimulated to examine the effects on contractile activity (both maximum force produced and fatigue profiles). Animals were killed by CO2 asphyxiation. Soleus muscles were isolated from both hindlimbs, leaving tendons attached. Muscles were placed in an organ bath in Krebs-Ringer solution (in mM: 118 NaCl, 4.75 KCl, 1.18 MgSO4 , 2.54 CaCl2 , 24.8 NaHCO3 , 1.18 KH2 PO4 , 10 glucose), bubbled continuously with 95% O2 – 5% CO2 to maintain a pH of 7.5 at 25◦ C. Muscles were attached to a force transducer (World Precision Instruments, Sarasota, USA) and positioned between electrodes. Using a micrometer, muscles were adjusted to their optimum length to yield maximal force at 120 Hz stimulation. The output of the force transducer was monitored using Data-Trax software (World Precision Instruments, Sarasota, USA). Baseline tetanic force production (3 millisecond pulses at 20 Hz over 14 s) in each individual muscle was measured in the absence of fruit extract exposure. After a recovery period of 10 min, the buffer was replaced with fresh KrebsRinger solution for control muscles, or Krebs-Ringer solution containing 5 ␮g/mL ZESPRI® GOLD kiwifruit extract or 2% GHO “Natural Quenchers” containing 65% of either ZESPRI® GOLD kiwifruit or blackcurrant juice. Muscles were incubated for 15 min before stimulating another tetanus response. Muscles were then removed and length and weight measured; the cross sectional area was estimated according to Mendez and Keys [19] and force expressed as mN/mm2 . Force measured during the fatigue protocols was assessed in relation to the peak force generated in each individual muscle’s baseline stimulation. 2.6. Animal exercise Mice were randomly assigned to the following five groups: no-exercise controls, exercising mice sacrificed at 0, 1, 3, or 24 h after fatiguing uphill running. All mice were acclimated to exercise conditions on the motorized treadmill (Muromachi Kikai, Tokyo, Japan) for five days, during which they walked/ran for 15 min at a speed of 0–15 metres/min and a maximum incline of +2◦ . The running adaptation was followed by at least five days of rest. During the fatiguing exercise, mice ran on the treadmill with a maximum speed of 36 metres/min and a maximum incline of +8◦ until the point of fatigue, at which they were no longer able to maintain pace despite gentle prodding and discharges of compressed air. Control mice were kept in the room with the treadmill to expose them to the same noise and handling as the exercising mice. After completion of the exercise, mice in the control and 0 hour group were sacrificed immediately, all other mice were sacrificed after 1, 3, or 24 h of recovery. Animals were killed by CO2 asphyxiation and blood was collected by heart puncture. Blood was centrifuged (9000g, 10 min) and plasma was stored at –80◦ C until further analysis. Plasma creatine kinase (CK) activity was analysed by Gribbles Veterinary Laboratory (Hamilton, New Zealand). Results are expressed as international units per litre of plasma (IU/L). Plasma with a haemolysis index greater than 60 was excluded from the analysis. Interleukin-6 (IL-6) concentration in plasma was analysed using the Quantikine Mouse IL-6 ELISA from R&D Systems (Minneapolis,

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USA) and was performed according to the manufacturer’s instructions. Results are expressed as pg IL-6 per mL plasma (pg/mL). 2.7. Statistical analysis Results are expressed as means ± standard errors of the mean (SEM) for at least four observations in each case. Statistical significance for the comparison of two groups was assessed by the Student’s t-test. A probability value (P) of less than 0.05 was considered significant.

3. Results and discussion 3.1. Anthocyanin content of fruit juices and effects of fruit on muscle cell oxidative stress Many fruits, and especially berries, contain bioactive compounds whose health-promoting properties are widely recognised [1, 27]. Utilising fruit varieties from breeding programmes at PFR with compositional analysis we are evaluating the physical health-promoting effects of phytochemicals derived from some new fruit varieties, including berry fruits. Breeding programmes at PFR have targeted high anthocyanin content as one of the possible desirable characteristics of new fruit cultivars for health benefits. A compositional survey of juices prepared from commercially grown non-New Zealand as well as New Zealand blackcurrant cultivars and new selections from the PFR breeding programmes demonstrates that the anthocyanin content of the New Zealand fruit juice is approximately 1.5 times that of the non-New Zealand and one new cultivar is particularly high in anthocyanins (Table 1). The fruits for the juices were grown under New Zealand conditions and the data suggest that New Zealand cultivars perform well when compared with those grown in North America [20]. The exceptionally high anthocyanin content makes some New Zealand blackcurrants cultivars unique and might provide potential strategies to counter/modulate the stress and damage associated with over-exercise, as well as to improve overall body wellness. Cellular studies can be used to determine the underlying mechanisms of action of active fruit extracts/compounds. We report here insights into the protective mechanism of action of a blueberry fruit extract. We analysed the antioxidant ability of skeletal muscle myotubes (differentiated from muscle myoblast cell lines) following exposure to the extract. Using DCFDA, which accumulates intracellularly and is cleaved to produce the radical sensitive product DCF, enabled the determination of intracellular free radical levels by monitoring of relative fluorescence intensities after exposure to H2 O2 . Figure 1 displays the antioxidant ability of muscle cells following different lengths of incubation exposure Table 1 Anthocyanin content of blackcurrant juice from New Zealand and non-New Zealand cultivars Non-New Zealand Cultivars* ‘Baldwin’ ‘Ben Dorain’ ‘Ben Lomond’ ‘Ojebyn’

Anthocyanins

New Zealand

Anthocyanins

(mg/100 mL)

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(mg/100 mL)

310 297 249 179

‘Ben Ard’ ‘Ben Rua’ ‘Blackadder’ ‘Magnus’ New Selection 1 New Selection 2

463 477 446 336 471 850

Juices were made from fruit harvested in the 2009 season from bushes on research plots in Canterbury, New Zealand. *A comparable range of anthocyanin content was obtained from the large study carried out on 32, mostly European, blackcurrant fruit cultivars grown in North America [20].

Relative Fluorescence (% from time zero)


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Fig. 1. Effect of incubation time on the intracellular free radical scavenging ability of muscle cells following exposure to a blueberry polyphenolic extract. Muscle cells were loaded with the reactive oxygen species indicator DCF as described in Materials and Methods and incubated (simultaneously [time zero] and at the times indicated on the figures) with a blueberry fruit (Vaccinium corymbosum ‘Reka’) polyphenolics extract (BP, 50 ␮g/mL) prior to challenge with H2 O2 and fluorescence intensity determination. Values are means ± SEM from at least four experiments and are expressed as relative fluorescence intensity (% from time zero). The percentage inhibition of H2 O2 -induced fluorescence by the blueberry extract is shown in parentheses.

(simultaneously [time zero] to 24 h) with the blueberry extract (50 ␮g/ml) before oxidative challenge. H2 O2 elicited a sustained, progressive, and large increase in muscle intracellular free radical levels. The magnitude of this response was reduced from 4 h onwards and was probably due to the leakage of the DCF from the cells over time, but this did not affect the determination of the intracellular antioxidant ability. With the simultaneous incubation of the muscle cells with the blueberry fruit extract, a marked increase in cellular antioxidant ability was observed, which was reflected by a 64.3% inhibition of fluorescence signal elicited by H2 O2 . This immediate antioxidant ability was further enhanced by longer extract pre-incubations (Fig. 1). After a 24-h extract pre-incubation, the antioxidant scavenging ability of the muscle cells had increased and was reflected by a 91.9% inhibition of the fluorescence signal elicited by H2 O2 . We further evaluated whether washing the blueberry extract from the cells prior to challenge with H2 O2 prevented the immediate and longer-term antioxidant effects. There was no significant difference between washed and non-washed cells (data not shown). In vitro models of oxidative stress using skeletal muscle cells have been used successfully in the past to monitor, under controlled conditions, the responses of muscle to oxidative stress that cannot be easily evaluated in human trials, and to provide a cost-effective method for the screening of beneficial substances. A useful and simple system is the use of skeletal muscle myotubes (differentiated from muscle myoblast cell lines) exposed to calcium ionophores [17]. We have previously demonstrated that the blueberry fruit (Vaccinium corymbosum ‘Reka’) polyphenolic extract mediated protection against muscle cell oxidative stress and damage [13]. The data presented here give insights into the potential process of action. We compared the functional activity of anthocyanin rich sub-extracts derived from the total extract with individual anthocyanin components and demonstrated that the most likely actives responsible

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for the protection against the oxidative stress were malvidin glycosides, particularly malvidin galactoside, and/or malvidin glucoside [13]. 3.2. Effects on muscle cell GSH and GPx activity The data in Fig. 1 suggest that while there is an immediate antioxidant ability of the fruit extract, the increase in antioxidant ability over time is not associated with the extract itself. Tissues and cells are equipped with efficient enzymatic and non-enzymatic antioxidant defence systems, which serve to counter the damaging action of oxidative stress [10]. Figure 2 shows the determination of muscle cell GSH and GPx activity following blueberry fruit extract exposure. While there were no changes in GPx activity, GSH levels after a 24-h exposure were significantly enhanced and suggest the induction of endogenous antioxidant pathways. These data support other reports that highlight that polyphenolics can modulate the levels of antioxidant defence enzymes such as superoxide dismutase, catalase, GSH and GPx [29]. Evidence suggests that these adaptive actions to ensure cell and tissue survival following oxidative stress may be mediated by the induction of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Nrf2 is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes, where it initiates the transcription of many cytoprotective proteins including endogenous antioxidant enzymes [15, 22]. More research is warranted to elucidate the significance of fruit polyphenolic-mediated induction of endogenous antioxidant pathways for modulation of unregulated oxidative stress and the potential long-term effects for muscle performance. 3.3. Effect of fruit on muscle function Exposure of isolated muscle tissue to some fruit extracts also modulates muscle tissue contractile activity and performance. Assessing force production and fatigue profile in electrically stimulated murine soleus muscles, we have previously reported [26] that a ZESPRI® GOLD kiwifruit extract mediated an increased maximum tetanic force in a similar manner to the antioxidant superoxide dismutase (1000 IU/mL). Here we report (Fig. 3) the contractile performance of soleus muscle following exposure to a ZESPRI® GOLD Kiwifruit extract and two commercially prepared ZESPRI® GOLD kiwifruit and blackcurrant juices (2% GHO

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Fig. 3. Effect of fruit extract and juices on fatigue in mouse soleus muscles induced by electrical stimulation. Muscles incubated with (A) ZESPRI® GOLD Kiwifruit (Actinidia chinensis ‘Hort16A’) phenolic extract (5 ␮g/ml, ⵧ control, 䊏 kiwifruit extract), or (B) GHO ZESPRI® GOLD Kiwifruit and blackcurrant juices (2%, ⵧ control, kiwifruit, 䊏 blackcurrant) were compared with buffer-only controls. The recorded force was divided by the cross sectional area of the muscle and normalized to the maximum force generated during baseline stimulation. A minimum of five muscles were analysed for each group. Values are means ± SEM. *Represents p < 0.05 statistical significance (paired Student t-test) between the area under the curve for muscles incubated with buffer-only and fruit extract.

“Natural Quenchers”). Mouse soleus muscles were electrically stimulated ex vivo after 15 min of incubation with the ZESPRI® GOLD Kiwifruit extract (5 ␮g/ml), or control buffer and tetanic force production was measured over 14 sec (Fig. 3A). Mean peak force in fruit extract exposed muscles was 86.1 ± 9.7%, 23.8% higher than the peak force in the controls (62.3 ± 14.2%). After the 14-sec tetanus stimulation the muscles demonstrated fatigue – a decline in muscle performance due to muscle activity. With kiwifruit extract exposure, muscle fatigue was reduced, with the mean force retaining 71.4% of the peak baseline force, whereas force in the controls dropped to 34.4%. Comparison of the area under the curves highlighted that the ZESPRI® GOLD Kiwifruit mediated protection against fatigue was statistically significant (kiwifruit 1108.5 ± 147.3, control 615.2 ± 151, p < 0.05). The commercially prepared ZESPRI® GOLD Kiwifruit and blackcurrant juices mediated a greater enhancement in muscle performance and protection against fatigue (Fig. 3B). Mean peak force in blackcurrant and kiwifruit juice-exposed muscles was 147.1 ± 20.6% and 148.9 ± 13.8% respectively, 85% higher than the peak force in controls. After the 14 sec tetanus stimulation, muscle fatigue was reduced by the blackcurrant and kiwifruit juice extracts with the mean force retaining 93.6% (blackcurrant) and 89.2% (kiwifruit) of the peak baseline force. Protection against fatigue, analysed as area under the curve, was statistically significant for both blackcurrant (1546.5 ± 225.8) and kiwifruit juices (1498.4 ± 170.1, p < 0.05). These data demonstrate that ZESPRI® GOLD Kiwifruit and blackcurrant fruit extracts and juices have an effect on muscle performance, which could be mediated by antioxidant scavenging of exercise-mediated reactive oxygen species (ROS). Active muscles produce excess ROS and it is widely accepted that they play a role in fatigue. During intensive exercise, muscle oxygen consumption increases up to 100-fold, and in parallel, superoxide anion production in mitochondria increases to a level where endogenous ROS scavengers are overwhelmed and hydrogen peroxide diffuses into the muscle cell cytoplasm. Some exogenous antioxidants like N-acetylcysteine (NAC), vitamins C and E, have been reported to reduce fatigue in isolated muscles, but only NAC has so far been shown to improve fatigue performance in vivo [23]. The fruit compound responsible for the enhanced force production and reduced fatigue in our isolated mouse soleus muscles remains to be elucidated, but it is possible that vitamin C and/or a variety of polyphenolics present in the fruits and extracts play a role. Indeed, Dorchies and colleagues showed that five weeks of feeding green tea extract and (–)-epigallocatechin gallate (EGCG) to muscular dystrophic mice protected the animals from fatigue and improved muscle force generation [11]. However, while many antioxidants have been shown to provide protection against oxidative stress and fatigue ex vivo, this has not always translated into the in vivo situation. With this in mind we have recently initiated the establishment of animal models of exercise at PFR. Figure 4 shows biochemical data in blood samples taken at various times from mice undertaking an exhaustive uphill running exercise. While not statistically significant, there was a trend towards plasma CK activity (an indicator of muscle tissue damage),

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slightly higher one hour after the exercise, compared with the activity in the control (non-exercised mice Fig. 4A). The release of IL-6 from muscle post exercise is known to be dependent upon both exercise intensity and duration [6, 12]. Figure 4B shows the plasma levels of IL-6 in exercising mice following our exercise regime. Immediately after the exercise (0 h), the mean plasma IL-6 concentration significantly increased (7-fold) from 0.45 ± 0.37 pg/mL in resting control mice to 3.11 ± 1.04 pg/mL in exercised mice (p < 0.05). After only 1 hour of recovery, plasma IL-6 concentrations returned to pre-exercise levels. While the exact role of the release of muscle tissue IL-6 is not clear, it is probably released in response to metabolic changes and to oxidative stress and/or tissue damage. It is suspected that IL-6 may be involved in activating aspects of the immune system to assist with tissue repair and recovery. These data also suggests that our uphill running protocol was inducing IL-6 response in exercising mice, without being strenuous or damaging enough to significantly increase CK levels. This is consistent with the findings of others in which no CK increase was observed with an uphill run, while downhill running, which mediates more tissue damage because of its eccentric nature, consistently increased CK in plasma [4, 18]. Others have utilised exercise routines in animals to evaluate the effects of nutrient supplementation on exercise-induced stress and performance. For example, curcumin [9], green tea extract [21], and Ginseng extract [3] have all been shown to decrease markers of damage, oxidative stress, and inflammation in exercising animals. Our preliminary data are therefore encouraging and in the near future we hope to be able to evaluate the benefits of fruits on stress and inflammation robustly using animal models of exercise. In humans, exercise can have benefits on the immune system including an ability to positively modulate the acute inflammatory response to a simulated bacterial infection [14]. Furthermore, appropriate fruit extract consumption may assist these health benefits. In a recent study we reported the effect of supplementation with a blackcurrant fruit extract on exercise-induced health benefits [16]. Consumption of the anthocyanin-rich blackcurrant extract prior to a moderate rowing exercise mediated an amelioration of plasma markers of oxidative stress and micro muscle damage, but also complemented the ability of exercise to stimulate an acute inflammatory response to a simulated bacterial infection in ex vivo experiments. These findings could be significant in the augmented activation (by fruit polyphenolics) of


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appropriate adaptive immune responses and of associated positive health benefits derived from moderate and regular exercise. Other studies have evaluated the potential of fruits and/or polyphenolic compound supplementation as antioxidants and reported potential protective effects and benefits to muscle and exercise performance [2, 7, 25]. In contrast however, some studies show that antioxidant supplementation may in fact neutralize the health benefits of regular exercise [5, 24] and that in some individuals undergoing long-term strenuous exercise regimes immune suppression can be observed [8]. Further studies are being undertaken at PFR in this area to elucidate the health potential of fruit polyphenolics as appropriate modulators of oxidative stress and inflammation.

4. Summary Our emerging evidence suggests that some fruits (and derived phenolic compounds) offer significant health and wellness potential. Our aim is to utilise unique New Zealand germplasm to unleash opportunities for the creation of premium functional foods (backed by science), to prevent muscle damage/injury, aid recovery, and/or enhance muscular and immune function.

Acknowledgements We are grateful for the preparation of the blueberry fruit phenolic extract and the blackcurrant juice by Drs Tony McGhie and Catherine Snelling of PFR. We are grateful for funding support from the New Zealand Foundation for Research, Science and Technology (contract C06X0807, cell and human work) and internal funding from PFR (animal studies).

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M. Battino, et al., Bioactive compounds in berries relevant to human health, Nutrition Reviews 67 (2009), S145–S150. F.P. Bonina, et al., Oxidative stress in handball players: effect of supplementation with a red orange extract, Nutrition Research 25 (2005), 917–924. A.C. Cabral de Oliveira, A.C. Perez, G. Merino, J.G. Prieto and A.I. Alvarez, Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise, Comparative Biochemistry and Physiology. Part C. Toxicology and Pharmacology 130C (2001), 369–377. M.D. Carmichael, et al., Recovery of running performance following muscle-damaging exercise: Relationship to brain IL-1 beta, Brain Behavior and Immunity 19 (2005), 445–452. A. Childs, C. Jacobs, T. Kaminski, B. Halliwell and C. Leeuwenburgh, Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise, Free Radical Biology and Medicine 31 (2001), 745–753. L.H. Colbert, J.M. Davis, D.A. Essig, A. Ghaffar and E.P. Mayer, Tissue expression and plasma concentrations of TNF alpha, IL-1 beta, and IL-6 following treadmill exercise in mice, International Journal of Sports Medicine 22 (2001), 261–267. D.A.J. Connolly, M.P. McHugh and O.I. Padilla-Zakour, Efficacy of a tart cherry juice blend in preventing the symptoms of muscle damage, British Journal of Sports Medicine 40 (2006), 679–683. D.M. Cooper, S. Radom-Aizik, C. Schwindt and F. Zaldivar, Dangerous exercise: lessons learned from dysregulated inflammatory responses to physical activity, Journal of Applied Physiology 103 (2007), 700–709. J.M. Davis, et al., Curcumin effects on inflammation and performance recovery following eccentric exercise-induced muscle damage, American Journal of Physiology-Regulatory Integrative and Comparative Physiology 292 (2007), R2168-R2173. V.B. Djordjevic, Free radicals in cell biology, in: International Review of Cytology – a Survey of Cell Biology, Vol. 237, edn, Elsevier Academic Press Inc, San Diego, 2004, pp. 57–89. O.M. Dorchies, S. Wagner, T.M. Buetler and U.T. Ruegg, Protection of dystrophic muscle cells with polyphenols from green tea correlates with improved glutathione balance and increased expression of 67LR, a receptor for (–)-epigallocatechin gallate, Biofactors 35 (2009), 279–294. C.P. Fischer, Interleukin-6 in acute exercise and training: what is the biological relevance? Exercise Immunology Review 12 (2006), 6–33.

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B. Schrage et al. / Evaluating the health benefits of fruits for physical fitness: A research platform R.D. Hurst, R.W. Wells, S.M. Hurst, T.K. McGhie, J.M. Cooney and D.J. Jensen, Blueberry fruit polyphenolics suppress oxidative stressinduced skeletal muscle cell damage in vitro, Molecular Nutrition & Food Research 53 (2009), 1–11. S.M. Hurst, K.A. Lyall, R.D. Hurst and L.M. Stevenson, Exercise-induced elevation in plasma oxidative generating capability augments the temporal inflammatory response stimulated by lipopolysaccharide, European Journal of Applied Physiology 107 (2009), 61–72. W.G. Li and A.N. Kong, Molecular mechanisms of Nrf2-mediated antioxidant response, Molecular Carcinogenesis 48 (2009), 91–104. K.A. Lyall, et al., Short-term blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharidestimulated inflammatory responses, American Journal of Physiology-Regulatory Integrative and Comparative Physiology 297 (2009), R70-R81. A.A. Maglara, A. Vasilaki, M.J. Jackson and A. McArdle, Damage to developing mouse skeletal muscle myotubes in culture: protective effect of heat shock proteins, Journal of Physiology-London 548 (2003), 837–846. C. Malm, et al., Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill or downhill running, Journal of Physiology-London 556 (2004), 983–1000. J. Mendez and A. Keys, Density and composition of mammalian muscle, Metabolism-Clinical and Experimental 9 (1960), 184–188. R.A. Moyer, K.E. Hummer, C.E. Finn, B. Frei and R.E. Wrolstad, Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: Vaccinium, Rubus, and Ribes, Journal of Agricultural and Food Chemistry 50 (2002), 519–525. T. Murase, S. Haramizu, A. Shimotoyodome, A. Nagasawa and I. Tokimitsu, Green tea extract improves endurance capacity and increases muscle lipid oxidation in mice, American Journal of Physiology-Regulatory Integrative and Comparative Physiology 288 (2005), R708R715. T. Nguyen, P. Nioi and C.B. Pickett, The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress, Journal of Biological Chemistry 284 (2009), 13291–13295. M.B. Reid, D.S. Stokic, S.M. Koch, F.A. Khawli and A.A. Leis, N-Acetylcysteine inhibits muscle fatigue in humans, Journal of Clinical Investigation 94 (1994), 2468–2474. M. Ristow, et al., Antioxidants prevent health-promoting effects of physical exercise in humans, Proceedings of the National Academy of Sciences of the United States of America 106 (2009), 8665–8670. A.L. Rossi, A. Blostein-Fujii and R.A. DiSilvestro, Soy beverage consumption by young men: increased plasma total antioxidant status and decreased acute, exercise-induced muscle damage, Journal of Nutraceuticals, Functional & Medical Foods 3 (2000), 33–44. M.A. Skinner, et al., Health benefits of Zespritm gold kiwifruit: effects on muscle performance, muscle fatigue and immune responses, Proceedings of the Nutrition Society of New Zealand 32 (2007), 49–59. A. Szajdek and E.J. Borowska, Bioactive compounds and health-promoting properties of berry fruits: A review, Plant Foods for Human Nutrition 63 (2008), 147–156. J.C. Tee, A.N. Bosch and M.I. Lambert, Metabolic consequences of exercise-induced muscle damage, Sports Medicine 37 (2007), 827–836. C. Zhan and J. Yang, Protective effects of isoliquiritigenin in transient middle cerebral artery occlusion-induced focal cerebral ischemia in rats, Pharmacological Research 53 (2006), 303–309.


25

Antioxidative efficiency of an anthocyanin rich bilberry extract in the human colon tumor cell lines Caco-2 and HT-29 Markus Schantz, Christiane Mohn, Matthias Baum and Elke Richling∗ Food Chemistry and Toxicology, Molecular Nutrition, University of Kaiserslautern, Kaiserslautern, Germany

Abstract. Bilberries (Vaccinium myrtillus L.) and its major polyphenolic constituents, the anthocyanins, are discussed to be preventive against diseases, such as colon cancer or inflammatory bowel diseases (e.g. Crohn’s disease, ulcerative colitis), are associated with oxidative stress. Therefore the gastrointestinal tract (GIT) might be a target for prevention of these diseases. In this study antioxidative efficiency of a commercially available anthocyanin rich bilberry extract (BE) was investigated in vitro in the human colon tumor cell lines Caco-2 and HT-29. The cell cytotoxicity of the BE was measured by alamar blue assay. Modulation of intracellular generated reactive oxygen species (ROS) levels was investigated by dichlorfluorescein assay (DCF). Oxidative DNA damage was monitored by single-cell gel electrophoresis (comet assay) with additional treatment of the DNA with formamido-pyrimidinglycosylase (FPG) to enhance sensitivity towards ROS induced DNA lesions. Modulation of the total glutathione (tGSH) level was assayed in a photometric kinetic assay. In a two step protocol cells were first treated with the protective extract (5–500 ␮g/ml; 1 and 24 h) and then with the redox-cycler menadione (Md) (HT-29: 20 ␮M and Caco-2: 6 ␮M) or the oxidant TBH (tert-butyl hydroperoxide) (250 ␮M, 40 min). Under all conditions tested BE was not cytotoxic in Caco-2 and HT-29 cells. The data achieved revealed that BE significantly reduce ROS level in HT-29 (250 ␮g/ml; 24 h, p < 0.05) and Caco-2 (50 ␮g/ml; 1 h, p < 0.05) cells. Significant decrease of induced DNA damage was detected in Caco-2 cells after BE treatment (5 ␮g/ml; 24 h; FPG, p < 0.05). Trend towards increase of tGSH was observed at concentrations of 50–500 ␮g/ml BE in Caco-2 cells after 24 h incubation. In total, the BE was shown to possess antioxidative activity under the used assay conditions towards prevention of oxidative DNA damage, reduction of intracellular ROS and cellular tGSH. Keywords: Anthocyanins, bilberries, antioxidants, DNA damage, glutathione, ROS, cytotoxicity

1. Introduction Anthocyanins (ACN) and their glycosides are polyphenols that play an important role in fruits, flowers and vegetables and are responsible for their bright colours like orange, blue and red, which help to attract animals [11, 27, 48] and prevent plants from oxidative stress due to their photoprotective functions such as light induced photooxidation or against UVB damage [7]. Polyphenols showed beneficial effects on degenerative diseases and are regarded to exhibit protective effects against cardiovascular diseases and cancer [33]. The main daily intake of anthocyanins averages 12.7 mg/person in USA and 82 mg/person in Finland [45]. Among dietary sources of anthocyanins, bilberry (vaccinium myrtillus L.) is one of the richest [23, 29], with varying anthocyanin patterns [34]. In contrast to other flavonoids, anthocyanidins consist of a flavylium ion whereas the structural variations are due ∗ Corresponding author: Prof. Dr. Elke Richling, Food Chemistry and Toxicology, Molecular Nutrition, University of Kaiserslautern, ErwinSchroedinger-Str. 52, 67663 Kaiserslautern, Germany. Tel.: +49 631 205 4061; Fax: +49 631 205 3085; E-mail: [email protected].


26

M. Schantz et al. / Antioxidative efficiency of anthocyanins in vitro

to differences in the number of hydroxyl and methoxyl groups at the B-ring. To the anthocyanidin aglycons sugar moieties (like glucose, galactose, arabinose, rhamnose and xylose [8, 12]) are attached to the phenolic molecule at position 3 and 7; and less frequently to 3 and 5 positions [11, 26]. Structures of the ACN found in bilberries are shown in Fig. 1. During the last decades, there has been intense interest in beneficial health effects of anthocyanins because of increasing evidence demonstrating potential biological effects after consumption of fruits and vegetables [3, 41]. A number of studies in vitro and in vivo have been reported that anthocyanins from bilberries are strong antioxidants [17, 38, 43], inhibit the growth of a range of tumor cells [16, 24, 31], induce apoptosis [14, 19] and have anticarcinogenic [6, 10] properties. Diseases of the gastrointestinal tract like inflammatory bowel diseases (crohn’s disease, ulcerative colitis) or colon cancer, are associated with an imbalance in the cellular redox system based on an increased level of reactive oxygen species (ROS) [36]. The antioxidant potential of flavonoids and other polyphenols is their ability to scavenge ROS and protect the organism against oxidative stress induced damage [41]. The specific object of this study was to investigate the in vitro antioxidant effectiveness of a BE. In vitro human adenocarcinoma cell lines HT-29 and Caco-2, representing characteristics of adult colon cells. As markers for antioxidant capacity the intracellular ROS level (DCF assay), oxidative DNA damage (comet assay), cellular tGSH level (kinetic assay) and cytotoxicity (alamar blue assay) was used.

2. Materials and methods 2.1. Chemicals, cells and media All chemicals and solvents were of analytical grade or complied with the standards needed for cell culture experiments. The BE was provided from Kaden Biochemicals (Hamburg, Germany) (BE, 600761 Bilberry Extract; consisted of 25% anthocyanins), formamido-pyrimidinglycosylase (FPG) was a gift of Prof. A.R. Collins (University Oslo, Norway), HT-29 and Caco-2 cells were obtained from DSMZ (Braunschweig, Germany), resazurin, tertbutyl hydroperoxide (TBH), menadione (Md), reduced/oxidized glutathione, glutathione reductase (GSR), catalase, potassium persulfate, 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), 5-sulfosalicylic acid (SSA), dimethylsulfoxide (DMSO) and ethidium bromide were purchased from Sigma Aldrich (Munich, Germany). NADPH and 2 ,7 dichlorfluorescein diacetat (DCFH-DA) were purchased from Fluka (Deisenhofen, Germany), low and normal melting agarose from Bio-Rad (Munich, Germany), bicinchoninic acid (BCA) protein quantification kit was from Interchim (Sankt Augustin-Buisdorf, Germany). Cell culture media (DMEM and DMEM/F-12 (1:1)) and reagents (fetal calf serum (FCS), penicillin/streptomycin, Trypsin 0.05%) were purchased from Invitrogen (Karlsruhe, Germany). Cell culture material (Petri dishes, well plates, flasks, etc.) were from Greiner Bio-One (Essen, Germany). 2.2. Bilberry extract (BE) The BE was made from European bilberry pomace (Vaccinium myrtillus L.) by extraction with methanol, filtration, evaporation and lyophilisation. It was stored dry and cold at –24◦ C. The anthocyanin content was adjusted at 25%.

Fig. 1. Chemical structure of the most common anthocyanins found in bilberry.


27

The BE also contains other polyphenols, tannins, carbohydrates and roughages. Basic characterisation of the anthocyanin profile was performed by Symrise GmbH & Co.KG. Control measurements of BE were performed by using a Jasco HPLC-UV system equipped with Jasco PU-2080 intelligent HPLC pump, Jasco LG-2080-02 ternary gradient unit, Jasco UV-2075 plus intelligent UV/VIS detector, Jasco DG-2080-53 3-line-degaser, AS-2055 plus intelligent sampler (Jasco, Gross-Umstadt, Germany). Separation of 15 anthocyanins was performed on a phenomenex Luna C18 (2) column (Phenomenex, Aschaffenburg, Germany) equipped with a security guard column; injection volume: 20 ␮l; flow rate: 0.5 ml/min; solvent A (acetonitrile/water/HCOOH; 87/3/10 v/v/v); solvent B (acetonitrile/water/HCOOH; 50/40/10 v/v/v); gradient elution: 0–20 min from 2–14% B, 20–40 m hold 14% B, 40–50 min to 15% B, 50–55 min to 19% B, 55–65 min to 20% B, 65–110 min washing and reequilibration, detection wavelength 520 nm. 2.3. Cell culture HT-29 cells were cultivated in 175 cm2 flasks in DMEM with addition of 10% FCS, Caco-2 cells in DMEM/F12 (1:1) with addition of 20% FCS. Both were supplemented with 100 U/ml penicillin and 100 ␮g/ml streptomycin in humidified atmosphere with 5% CO2 at 37◦ C. 2.4. Incubations with BE HT-29 and Caco-2 cells were seeded in Petri dishes (6 cm for HT-29, 9 cm for Caco-2; tGSH level and comet assay: for 1 h incubation 1.5 × 106 cells per dish and for 24 h incubation 106 cells per dish, respectively), cultivated for 24 h (cultures were semiconfluent and undifferentiated), washed with PBS and incubated in the first step with the BE in incubation medium containing 10% FCS for Caco-2 and 5% for HT-29 cells (dissolved in DMSO at a final concentration of 1%). In the second step, cells were treated with Md (HT-29: 20 ␮M and Caco-2: 6 ␮M). After incubation, cells were washed with PBS and isolated by Trypsin (0.05%) treatment. Cell suspensions were directly used for tGSH level analysis and comet assay. For DCF assay, cells were seeded in 96 well plates (HT-29: 3.2 × 104 and Caco-2: 2.0 × 104 ) and incubated with BE (dissolved in DMSO at a final concentration of 1%). The alamar blue assay was used to check for cytotoxicity, therefore cells were seeded in 48 well plates (HT-29: 6.0 × 104 and Caco-2: 5.5 × 104 ) and incubated with BE (dissolved in DMSO at a final concentration of 1%). All incubations were performed in the presence of catalase (100 U/ml) to inhibit formation of extracellular hydrogen peroxide (H2 O2 ), resulting from pro-oxidative interaction of phenolic compounds with cell media constituents like metal ions [13, 22]. Addition of catalase provides an opportunity to preclude effects resulted from extracellular generated H2 O2 . 2.5. Intracellular ROS level (DCF assay) DCF assay was performed according to Wang and Joseph [42] with modifications [5]. Briefly, after BE treated cells were incubated for 30 min with DCFH-DA dissolved in DMSO (50 ␮M in PBS; pH 7.0), washed and incubated with TBH (250 ␮M in PBS; pH 7.4) for another 40 min. After TBH treatment, formation of DCF was measured as increase of fluorescence intensity between 0 and 30 min in a microplate reader (MWG, Ebersberg, Germany) and stated as relative fluorescence intensity (FI) in percent control (without BE treatment). 2.6. Cellular GSH level (kinetic assay) The cellular GSH level was measured by photometric determination of 5-thio-2-nitrobenzoate (TNB), formed from DTNB [35]. After BE incubation, cells were treated with Md (HT-29: 20 ␮M and Caco-2: 6 ␮M for 1 h in serum free medium) suspension. The formation of TNB after 2 min reaction time was measured in 96 well plates by microplate reader (MWG, Ebersberg, Germany) at 412 nm. The GSH level was specified as nanomoles per milligram of protein and expressed as percent of Md-treated control. 2.7. Oxidative DNA damage (comet assay) After extract incubation, cells were incubated with Md (see above) and isolated by trypsin treatment. The detailed process of the alkine single cell gel electrophoresis was described previously [35]. Briefly, 70,000 cells per preparation

28


were centrifuged (10 min, 2000 rpm, 4◦ C) and the pellet was resuspended with low melting agarose, applied on a previously prepared microscope slide (per condition, 2 slides with and without FPG respectively), added to lysis and treated with FPG enzyme to enhance sensitivity towards induced DNA lesions. After DNA unwinding and gel electrophoresis, slides were neutralized, stained with ethidium bromide and analyzed by fluorescence microscope (Zeiss, Germany). Per slide 2 × 50 cells were scored, averaged and the DNA damage was calculated as tail intensity (TI%: DNA in the comet tail in percent of total DNA) [9, 37, 39]. Results were expressed as percent of Md-treated control. 2.8. Cytotoxicity (alamar blue assay) Metabolic activity results in the chemical reduction of resazurin (blue and nonfluorescent) to resorufin (pink and highly fluorescent) [30]. After extract incubation, cells were washed with PBS, treated with serum free medium (500 ␮l) containing 10% resazurin solution and measured by a microplate reader (MWG, Ebersberg, Germany) (excitation wavelength 530 nm, emission wavelength 590 nm, 37◦ C). Treatment with 0.1% saponin was used as positive control for cytotoxicity. Results were calculated as percent of control (without BE treatment). 2.9. Statistics Results of the performed assays are presented as mean ± SD of 3 to 6 independent experiments. Differences (p < 0.05) were determined by independent one-sided t-test by origin analysis 6.0 (MicrocalTM Origin® , Northampton, USA).

3. Results and discussion 3.1. Cytotoxity Resorufin formation from resazurin (alamar blue assay) was analysed as a measure of cytotoxicity in terms of mitochondrial integrity and cell growth [30]. Incubation time of 1 h was used to minimize confounding factors like instability and degradation of the anthocyanins as reported earlier [20, 44]. As a positive control resorufin formation was completely inhibited (0.1% saponin), whereas under all other treatment conditions resorufin formation reached median 95% of the negative control. Only in HT-29 cells a significant effect at the highest concentration of 500 ␮g/ml BE (Fig. 2) was detectable. Our data are in good agreement with a study by Yi et al., who reported that higher doses from 1 to 3 mg/ml crude blueberry extract inhibited cell growth in HT-29 and Caco-2 cells [47], while other studies investigating cytotoxicity of corresponding anthocyanidins (aglycons) at longer incubation times, cell growth was inhibited also at lower concentrations [16, 20, 24]. 3.2. Intracellular ROS level The TBH induced modulation of the intracellular ROS level in HT-29 and Caco-2 cells after BE incubation (1 and 24 h) was measured by DCF assay. Quercetin (30 ␮M), used as a positive control, reduced cellular ROS level in the range of 20% as compared to the control as also demonstrated earlier [35]. With the BE (1 to 250 ␮g/ml) a significant, concentration dependent decrease of intracellular ROS was observed at 50 ␮g/ml in Caco-2 and 250 ␮g/ml in HT-29 cells after 1 and 24 h incubation. Maximum reduction of ROS level in Caco-2 cells was detected at concentrations >100 ␮g/ml. The BE was less efficient in HT-29 cells, a significant reduction of ROS level was observed beginning at 250 ␮g/ml (24 h), respectively (Fig. 3). Our results are comparable to a study of Milbury et al. [28], demonstrating significant decreases of intracellular ROS level in ARPE-19 cells after bilberry extract treatment (0.01 ␮g/ml). With a fermented apple extract a significant decrease of ROS level was observed only at higher concentration ranges [4].


29

Caco-2: 1 h incubation Caco-2: 24 h incubation HT-29: 1 h incubation HT-29: 24 h incubation

120

rel. viability [% control]

100

80

60

40

20

0 Saponin 0 (control)

10

50

100

250

500

concentration [µg/ml BE] Fig. 2. Alamar blue assay to assess the cytotoxicity of an anthocyanin rich bilberry extract (BE) toward Caco-2 and HT-29 cells. For incubation, cells were treated with 10 to 500 ␮g/ml BE for 1 and 24 h and incubated with resazurin for another 1 h whereby the fluorescence of resorufin was measured. Saponin was used as positive control for cytotoxicity. Results were calculated as percent of DMSO treated control; n = 4 (mean ± SD). Differences were determined by independent one-sided t-test related to the DMSO treated control: *p < 0.05, ***p < 0.001.

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Caco-2: 1 h incubation Caco-2: 24 h incubation HT-29: 1 h incubation HT-29: 24 h incubation

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rel. FI [% control]

100

80

60

40

20 0

ue

tin rce

Q µM 30

l) BH tro con 0(

+T

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5

10

50

100

250

500

concentration [µg/ml BE] + TBH

Fig. 3. Modulation of tert-butyl hydroperoxide (TBH)-induced intracellular ROS level in Caco-2 and HT-29 cells after 1 and 24 h incubation with an anthocyanin rich bilberry extract (BE) (1 to 500 ␮g/ml). Results were calculated as percent of TBH treated control; n = 3–5 (mean ± SD). Differences were determined by independent one-sided t-test related to the TBH treated control: *p < 0.05, **p < 0.01, ***p < 0.001.

30


3.3. Oxidative DNA damage The comet assay was used to study the effects of the BE on the reduction of Md induced oxidative DNA damage in Caco-2 and HT-29 cells with and without FPG to enhance sensitivity towards ROS induced DNA lesions. Preventive effects of quercetin (positive control, 30 ␮M) reducing DNA damage in the range of 60% tail intensity (TI) compared to the control was demonstrated earlier in the literature [35]. After treatment of cells only with DMSO, a basal level of 24 to 31% TI was measured in Caco-2 cells. A slight decrease of DNA damage was obtained in a range from 5 to 100 ␮g/ml BE in Caco-2 cells. Concentration curves, resembling a “U-shape”, were observed reaching a minimum at 5 ␮g/ml. Significant DNA-protecting effects were shown after 24 h of incubation with 50 ␮g/ml BE and 5 ␮g/ml BE treated with FPG, respectively (Fig. 4). No reduction of Md induced DNA damages in HT-29 cells was observed (data not shown). The “U-shaped” form of concentration curve in the same test system was also observed earlier with apple juice extract [35]. In low BE (0.01 to 1 ␮g/ml) concentrations no effects and in high BE concentrations (250 to 500 ␮g/ml) a prooxidative effect was detectable. These data are also in agreement with the results of an intervention study with hemodialysis patients consuming daily 200 ml anthocyanin rich fruit juice, resulting in a significant decrease of oxidative DNA damage and a significant increase of GSH level in blood [38]. In another in vivo study with healthy volunteers who consumed an anthocyanin/polyphenolic-rich fruit juice, a decrease of oxidative DNA damage during the juice-uptake phase was observed [43]. 3.4. Cellular tGSH level In a photometric kinetic assay, the cellular tGSH level in Caco-2 and HT-29 cells after BE and Md treatment was measured. DMSO without further treatment as negative control, to quantify the basal level of tGSH in the cells, was used as a positive control, demonstrated tGSH levels in the range of 160% as compared to negative Md-control as published earlier [35]. After 24 h incubation, Caco-2 cells showed slight but insignificant tGSH increases in the highest used BE concentrations (50–500 ␮g/ml). In contrast, after 1 h incubation 500 ␮g/ml extract significantly reduced the 1 h incubation 1 h incubation + FPG 24 h incubation 24 h incubation +FPG

180

tail intensity [% control]

160 140 120 100 80 60 40 20 0 tin

30

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Q

ce uer

d l) + M ontro c 0(

1

0.0

0.1

1

5

10

50

100

250

500

concentration [µg/ml BE] + Md

Fig. 4. Modulation of Menadione (Md)-induced (HT-29: 20 ␮M; Caco-2: 6 ␮M; 1 h) oxidative DNA damage in Caco-2 cells after 1 and 24 h incubation with an anthocyanin rich bilberry extract (BE) (0.01 to 500 ␮g/ml); quercetin (30 ␮M) was used as positive control. Results were calculated as percent of Md treated control; n = 3–6 (mean ± SD). Differences were determined by independent one-sided t-test related to the TBH treated control: *p < 0.05, ***p < 0.001.


31

200 1 h incubation 24 h incubation

180

tGSH level [% control]

160 140 120 100 80 60 40 20 0 d

-M

DM

SO

l)

tro

con

0(

1

0.0

0.1

1

5

10

50

100

250

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concentration [µg/ml BE] + Md Fig. 5. Modulation of Md-induced total glutathione (tGSH) level in Caco-2 cells after 1 and 24 h incubation with an anthocyanin rich bilberry extract (BE) (0.01 to 500 ␮g/ml); quercetin (30 ␮M) was used as positive control. Results were calculated as percent of Md treated control; n = 3–6 (mean ± SD). Differences were determined by independent one-sided t-test related to the TBH treated control: *p < 0.05, **p < 0.01, ***p < 0.001.

tGSH level down to about 70% of the negative control. BE in a range between 0.01 to 10 ␮g/ml induced no effects after 1 and 24 h of incubation in comparison to the negative control (Fig. 5). This suggests that low anthocyanin concentrations used have no effect on the tGSH level in the used cell systems. In contrast to our in vitro data, in vivo studies, one with hemodialysis patients and the other with healthy volunteers which consumed an anthocyanin/polyphenolic-rich fruit juice, an increased level of tGSH in blood after juice uptake was detectable [38, 43]. Taken together, our data indicate that anthocyanins from berries (bilberries) exert antioxidant activity contributing to the health preventing properties of fruits and vegetables. The anthocyanin profile of the used BE corresponds to those of native bilberries. Anthocyanin content of 1 g of enriched BE is equivalent to 17 g of native bilberries [23]. In a study with ileostomy volunteers who consumed 300 g blueberries (with a total anthocyanin amount of 7834 mg/kg) up to 85% of the anthocyanins reached the colon [18]. He et al. quantified the anthocyanin uptake by small intestine tissue to be 7.5% of the administrated dose after 120 min in rats [15]. Due to these data concentrations in a range of 0.01 to 500 ␮g/ml BE were investigated in our cellular systems. As found by us, ROS level increases in Caco-2 cells significantly at 50 ␮g/ml BE. These data are comparable to a study in which anthocyanins showed significant positive effects on intracellular ROS and lipid peroxidation in rat pheochromocytoma cells (PC-12) after incubation with sweet potato anthocyanins (50 ␮g/ml) [46]. Further in vitro studies with anthocyanins demonstrated the high antioxidative capacity due to their ability to scavenge free radicals [17, 21, 40]. Due to these scavenging effects, anthocyanins (2.5 nm in media) were able to stabilize the DNA against hydroperoxide radicals, associated with a decrease in DNA damage [25]. Acquaviva et al. monitored a concentration dependent (100 to 500 ␮mol/l) reduction of plasmid DNA damage after cyanidin and cyanidin 3-Oglucoside treatment [1]. In agreement with these data our comet assay results showed significant DNA-protecting effects at 5 and 50 ␮g/ml BE in Caco-2 cells. In two in vivo studies with mice under oxidative stress, positive effects of anthocyanin rich extracts (concentration range 100 to 200 mg/kg KG) like increasing tGSH level and other oxidative stress markers were reported [2, 32]. In total, we suppose that the preventive properties of the anthocyanin rich bilberry extract to reduce oxidative DNA damage, lower ROS level and elevate tGSH in human colon tumor cell lines, is associated with protection of the colon from oxidative stress.

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Acknowledgements The authors gratefully acknowledge the gift of FPG enzyme by Prof. A.R. Collins (Institute for Nutrition Research, University of Oslo, Norway) and the Symrise GmbH & Co.KG for BE characterization. This work was supported by Bundesministerium für Wirtschaft und Technologie/AiF via the Forschungskreis der Ernährungsindustrie e.V. (FEI), Bonn, project No. 15614 N for financial support.

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Superoxide anion radical scavenging activity of bilberry (Vaccinium myrtillus L.) ˇ ´ Vesna Tumbasa,∗ , Jasna Canadanovi´ c-Bruneta , Lars Gilleb , Sonja Ðilasa and Gordana Cetkovi´ ca a Faculty

of Technology, University of Novi Sad, Novi Sad, Serbia Pharmacology and Toxicology Unit, Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria b Molecular

Abstract. Naturally occurring antioxidants present in bilberry were separated into three groups: vitamin C, phenolic acids and flavonoids using solid phase extraction (SPE). Chemical composition of bilberry extract fractions was obtained by spectrophotometry and high performance liquid chromatography (HPLC). The results of HPLC analysis point out the high content of vitamin C (1529 ␮g/g) in fraction Fr1, flavonoids (1328.58 ␮g/g) in fraction Fr2 and phenolic acids (494.31 ␮g/g) in fraction Fr3. The free radical scavenging activities of these antioxidant fractions on superoxide anion radicals and detection of free radical intermediates was studied using electron spin resonance (ESR) spectroscopy. Superoxide anion free radical assay demonstrated very potent free radical scavenging activity of bilberry extract fractions. The results of correlation analysis showed that the separated classes of antioxidants from bilberry (vitamin C, flavonoids and phenolic acids) are responsible for its antioxidant activity. Keywords: Bilberry, ESR spectroscopy, superoxide anion radicals, phenolic acids, flavonoids, vitamin C

1. Introduction Many of the human age-related degenerative diseases are associated with oxidative processes. Many edible plants are capable of producing natural chemopreventive compounds which have no synthetic counterparts and play a protective role in human health maintenance. It has been well established that many of the phytochemicals present in plant derived foods have antioxidant capacity, i.e. are able to remove damaging radical species, as shown by a range of in vitro assays. Fruits, vegetables and teas contain a wide range of antioxidant compounds, including phenolic compounds and vitamins [41]. Phenolic secondary metabolites play an important role in plant-derived food quality, as they affect quality characteristics such as appearance flavour and health-promoting properties. Edible berries have been a part of man’s diet for centuries. Berries are one of the most important dietary sources of fibre and essential vitamins, minerals and vast number of phytochemicals such as phenolic compounds, including anthocyanins, phenolic acids, flavonol glycosides and flavan-3-ols [26, 33]. It has been reported that berry extracts have cardioprotective effects and beneficial effects on platelet aggregation, they are very effective inhibitors of low density lipoprotein oxidation and inhibitors of the growth of cultured cancer cells [3]. Research at the Scottish Crop Research Institute and the University of Ulster has shown that berry extracts can inhibit the initiation, progression, and invasiveness of colon cancer cells [40]. Different berry components are responsible for the inhibition of ␣-glucosidase and ␣-amylase, which suggests considerable potentiation of effects on blood glucose levels [34]. Similar effects on lipid digestion have been noted [35]. ∗ Corresponding author: V. Tumbas, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia. Tel.: +381 21 485 3652; Fax: +381 21 450 413; E-mail: [email protected].


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V. Tumbas et al. / Radical scavenging activity of bilberry

Compounds present in the fruits of Vaccinium species are reported to have beneficial influence on human health [38, 44]. Bilberry (Vaccinium myrtillus L.) is a member of the Eriacaceae family and is also known as European blueberry, huckleberry, whortleberry or blueberry. Fruits of wild bilberry have a well-established role in pharmacognosy and it has been used as food for centuries, due to its high nutritive value [48]. Bilberry is a rich source of anthocyanins, and its extracts are extensively used in food/dietary supplements and pharmaceutical products. Biological properties of the bilberry fruit extract include antioxidant capacity, astringent and antiseptic properties, ability to decrease the permeability and fragility of capillaries, inhibition of platelet aggregation, inhibition of urinary tract infection and strengthening of collagen matrices via cross linkages [15]. Bilberry crude extracts are marketed as pharmaceutical preparations for the treatment of both ophthalmological diseases and blood vessel disorders. More recently, they have been used for the treatment of diarrhea, dysentery, and mouth and throat inflammations [48]. Bilberries may be eaten fresh or dried. Bilberry tea may also be made from fresh or dried berries, or from the leaves. Dried bilberries are high in tannin and pectin, which have an astringent action that controls the inflammation that causes diarrhea. Dried billberies are traditionally used as herbal tea remedy. Distribution and contents of antioxidant compounds (anthocyanins, flavonoids, phenolic acids) as well as antioxidant activity of fresh bilberries has already been confirmed in the literature [12, 19, 22, 37]. Laplaud et al. [28] reported that bilberry extract, characterised by 74.2 ± 4.9 mg of polyphenols/g, inhibited copperinduced oxidative modifcation of human LDL. In another study, Viljanen et al. [49] found that raspberry, bilberry, lingonberry and black currant extracts inhibited copper-induced protein and lipid oxidation in a lactalbumin-liposome oxidation system, bilberry extract being one of the most effective. However, contents of phenolic compounds and vitamin C, as well as antioxidant activity of dried brilberry fruits has not been evaluated. Yue and Hue [52] examined changes of anthocyanins, anthocyanidins, and antioxidant activity in fresh bilberry extract during dry heating at 80◦ C, 100◦ C and 125◦ C. Conjugated sugars of anthocyanins were cleaved from the anthocyanins to produce their corresponding anthocyanidins or aglycones during heating and the heated extracts had higher free radical scavenging capability than unheated extract. In this study we used dried bilberries as a potential source of naturally occuring antioxidants. Extract from dried bilberries was fractionated into three groups of naturally occuring antioxidants: vitamin C, neutral and acidic phenolics using solid phase extraction (SPE). Present study describes behaviour of obtained bilberry extract fractions in superoxide anion generating system – their free radical scavenging activities and free radical intermediates formed during this reaction. In addition, correlation matrix was conducted in order to evaluate the contribution of separated phytochemicals from bilberry and relationship with free radical scavenging activity.

2. Materials and methods 2.1. Plant material Dried bilberry fruits were obtained in a local drugstore. The air dried fruits were ground using a coffee mill and passed through a 0.36 mm sieve. 2.2. Preparation, purification and fractionation of bilberry extract 20 g of dried and ground bilberry fruits were macerated with 500 ml of 80% acetone during 24 hours. The macerat was filtered (Whatman No. 4) and the maceration was repeated once more. Two macerates were mixed and evaporeted to dryness under reduced pressure. In order to separate vitamin C from the phenolic antioxidants and to remove organic acids, residual sugars, amino acids, proteins and other hydrophilic compounds as well as to exchange solvents, a cleanup of the bilberry extract by solid phase extraction (SPE) was performed according to Rigo et al. [39], with Chromabond C-18 (1000 mg, J.T. Baker, Holland). The dry bilberry extract was redissolved in 5 ml of 0.5 M H2 SO4 , filtered through 0.45 ␮m (pore size) membrane filters (Millipore, Bedford, MA) and loaded on the Chromabond C-18 preconditioned with 2 ml of methanol followed by 5 ml of 5 mM H2 SO4 . The polar substances, including vitamin C, were removed with 2 ml of 5 mM H2 SO4 and this fraction was marked as Fr1. The phenolic compounds were eluted with 2 ml of methanol followed by 5 ml of distilled water and this solution was considered as purified


15

bilberry extract. Spectrophotometrical determiantion of total phenols, flavonoids and anthocyanins was conducted with purified bilberry extract. Further, extraction and fractionation of neutral and acidic phenolics was conducted according to Chen et al. [10]. The purified bilberry extract was adjusted to pH 7.0 with 2.0 M NaOH solution and loaded onto the Chromabond C-18 preconditioned for neutral phenolics with 8 ml of methanol followed by 4 ml of distilled-deionized water adjusted to pH 7.0. The column was washed with 10 ml of distilled-deionized water and eluted with 12 ml of methanol. Fraction eluted in this step contains neutral phenols and is used further as Fr2. The effluent portion was adjusted to pH 2.0 with 2.0 M HCl, passed through the preconditioned acidic column. For isolation of acidic phenolics, cartridge was preconditioned by passing 8 ml of methanol and 4 ml of 0.01 M HCl. Then the column was washed with 5 ml of 0.01 M HCl and the adsorbed fraction was eluted with 12 ml of methanol. This fraction contains acidic phenols and is labeled as Fr3. The obtained fractions of bilberry extract, Fr1, Fr2 and Fr3, were evaporated using a rotary evaporator until dryness at 35◦ C under reduced pressure.

2.3. Spectrophotometrical determination 2.3.1. Total phenol concentration in purified bilberry extract The amount of total soluble phenolics in extracts was determined spectrophotometrically according to the FolinCiocalteu method [45]. The reaction mixture was prepared by mixing 0.1 ml of water solution (concentration 1 mg/ml) of purified bilberry extract, 7.9 ml of distilled water, 0.5 ml of Folin-Ciocalteu’s reagent and 1.5 ml of 20% sodium carbonate. After 2 h, the absorbance at 750 nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan) was obtained against control that had been prepared in a similar manner, by replacing the extract with distilled water. The total phenolic content, expressed as mg of chlorogenic acid equivalents per g dry weight of purified bilberry extract, was determined using calibration curve of chlorogenic acid standard.

2.3.2. Total flavonoids in purified bilberry extract Total flavonoids (expressed as mg rutin per g dry weight) in purified extract were estimated spectrophotometrically according to Markham [30]. Flavonoids from purified bilberry extract (0.5 ml) were extracted with 1 ml of extraction medium (70% [v/v] methanol, 5% [v/v] acetic acid and 25% [v/v] distilled water) at room temperature for 60 min. The resulting solution was filtered trough Whatman paper No. 4 and filtrate volume was adjusted to 100 ml. The probes were prepared by mixing: 5 ml of dilluted extract, 1 ml of distilled water and 2.5 ml of AlCl3 solution (26.6 mg AlCl3 · 6H2 O and 80 mg CH3 COONa dissolved in 20 ml distilled water). A blank probe was prepared by replacing AlCl3 solution with distilled water. The absorbance of probes and blank probe were measured immediately at 430 nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan). Total flavonoid content, expressed as mg rutin per g dry weight of purified bilberry extract, was calculated from a calibration curve using rutin as standard.

2.3.3. Total and monomeric anthocyanins in purified bilberry extract Total anthocyanin content in purified extract was estimated spectrophotometrically using the pH single and differential method [9]. The spectrophotometric single pH method was used to determine total (monomeric plus polymerized) anthocyanins and differential pH method for determination of monomeric anthocyanins in the extract. The aqueous extract was diluted with two buffer solutions at pH 1 and 4.5. The absorbance of each dilution was measured at 510 and 700 nm against a distilled water control using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The total anthocyanin concentration was obtained from equation: Ctot (mg/l) = (Atot × MW × DF × 1000)/␧ × L while monomeric anthocyanin concentration was calculated from the equation: Cmon (mg/l) = (Amon × MW × DF × 1000)/␧ × l, where Atot is calculated as Atot = A510 –A700 , Amon is calculated as Amon = (A515 –A700 )pH 1 –(A515 –A700 )pH 4,5 , ␧ is cyanidin-3-glucoside molar absorbance coefficient (26900 l/(mol × cm)), MW is cyanidin-3-O-glucoside molecular weight (449.2 g/mol), DF is dilution factor and L is cell path length (1 cm). Total anthocyanin and monomeric anthocyanin content was expressed as mg cyanidin-3-Oglucoside equivalents per g of purified bilberry extract [1].

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2.4. HPLC analysis of bilberry extract fractions All analyte solutions and solvents were filtered prior to analysis through 0.45 ␮m (pore size) membrane filters (Millipore, Bedford, MA). Quantification of phenolics in Fr2 and Fr3 was done by HPLC analysis by a Waters Breeze chromatographic system (Waters, Milford, MA), which consisted of 1525 binary pumps, thermostat and 717+ autosampler connected to the Waters 2996 diode array detector (Waters, Milford, USA). Chromatograms were recorded in 3D mode. Separation was performed on a Symmetry C-18 RP column 125 × 4 mm with 5 ␮m particle size (Waters, Milford, USA) with an appropriate guard column. Two mobile phases, A (0.1% phosphoric acid) and B (acetonitrile), were used at flow rates of 1 ml/min with the following gradient profile: the first 20 m from 10 to 22% B; next 20 m of linear rise up to 40% B, and for last 10 m 55% of B, followed by 10 m reverse to initial 10% B with additional 5 m of equilibration time. The data acquisition and spectral evaluation for peak confirmation were carried out by the Waters Empower 2 Software (Waters, Milford, USA). Quantification of total phenolic compounds in bilberry fractions Fr2 and Fr3 were performed using quercetin and p-coumaric acid as secondary standards for flavonoids and phenolic acids, respectively. HPLC analysis of vitamin C content in Fr1 was performed on a liquid chromatograph “Agilent 1100”, USA, equiped with a ultraviolet diode array detector (UV-DAD). For separation a C-18 column with a 5 ␮m particle size was used at a flow rate of 0.4 ml/min and temperature 37◦ C. Reference substance (vitamin C) and samples were dissolved/extracted in solution of meta-phosphoric acid (3% w/w) in 8% acetic acid. Ammoniumacetate (0.1 mol/l, pH 5.1) was used as a mobile phase. The injected volume was 20 ␮l and the total running time was 6 min.

2.5. ESR measurements 2.5.1. Superoxide anion radical scavenging activity of bilberry extract fractions A solution containing superoxide anion radicals was prepared by dissolving KO2 /crown ether (10 mM/20 mM) in dry dimethylsulfoxide (DMSO) and 0.005 ml of this solution were added to 0.5 ml of dry DMSO and 0.005 ml of an DMSO spin trap solution (DMPO, 5,5-dimethyl-1-pyrroline-N-oxide, 2 M). The influence of extracts on the formation of DMPO/• OOH adducts was studied by adding the N,N-dimethylformamide (DMF) solution of bilberry extract fraction to the superoxide anion reaction system at a final concentration range of 0.001–1 mg/ml. Synthetic antioxidant, butylated hydroxyanisole (BHA), was used for comparison. Then the solution was transferred to a quartz flat cell ER-160FT and 2 m after mixing ESR spectra were recorded on an EMX spectrometer from Bruker (Rheinstetten, Germany) using a TE102 cavity. The following instrument settings were used: field modulation 100 kHz, modulation amplitude 4.00 G, receiver gain 1 × 104 , time constant 327.68 ms, conversion time 40.96 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23◦ C. The extent of scavenging by antioxidant fractions was expressed as scavenfing activity (SA) values. The SAo2 •– value of the extract was defined as: SAo2 •– (%) = 100 × (h0 –hx )/h0 , where h0 and hx are the hight of the second peak in the ESR spectrum of DMPO/• OOH spin adduct of the sample without and with antioxidant fractions, respectively.

2.5.2. Detection of antioxidant-derived radicals The antioxidant-derived radicals were determined in the reaction system containing 0.5 ml of DMSO solution of KO2 /crown ether (10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction (5 mg/ml). These solutions were aspirated from autosampler vial into the quartz flat cell, which was located in the TE102 -resonator of the EMX spectrometer (Bruker, Rheinstetten, Germany). The mixing of both solutions was performed in the lower part of the cell prior to reaching the active zone of the flat cell. ESR measurements were started 20 s after mixing. The following instrument settings were used: microwave frequency, 9.73 GHz; modulation frequency, 100 kHz; microwave power, 20 mW; center field, 3491.1 G; sweep, 25 G; modulation amplitude, 0.48 G; receiver gain, 8 × 105 ; scan rate, 35.77 G/min; time constant, 0.163 s; scans 5. ESR spectral files were imported into the WINSIM program [16] for the analysis of the hyperfine splitting constants.


17

2.6. Statistical analysis All analysis were run in triplicate and were expressed as means ± standard deviation (SD). Statistical analysis was done by using Statistica 8.0 software package (StatSoft Inc., 1984–2007). Significant differences were calculated by ANOVA test and then least significant difference (LSD) test (p < 0.05, unless noted otherwise).

3. Results 3.1. Chemical composition of bilberry The contents of total phenols, flavonoids, anthocyanins and monomers of anthocyanins in purified bilberry extract are given in Table 1. According to these results, flavonoids are the most abundant class of phenolic compounds present in bilberry extract (82.24%). Anthocyanins are one of the main flavonoid subgroups in fruits and berries and they are responsible for their red, violet, purple and blue colours. The anthocyanins are considered the most important of the pharmacologically active constituents. Anthocyanin concentration in the fresh bilberry fruit is approximately 0.1–0.5%, while concentrated bilberry extracts are usually standardized to 25% anthocyanins [24, 53]. Valentová et al. [48] detected cyanidin, delphinidin, malvidin, paeonidin and petunidin and its glycosides in bilberry extract, summary 25% of extract. Presented results show that 35% of flavonoids and less than 30% of total phenols in bilberry extract represent anthocyanins. The compounds of interest in this study were vitamin C, flavonoids and phenolic acids. Total phenolic content measured by the Folin-Ciocalteu method may give false and overestimated results because possible interference from other chemical components present in the extract (sugars, aromatic amines, sulfur dioxide, ascorbic acid, organic acids, Fe(II), and other nonphenolic organic substances that react with Folin-Ciocalteu reagent) [42, 45]. High performance liquid chromatography (HPLC) method was used for further quantification of total phenolic compounds, flavonoids in Fr2 and phenolic acids in Fr3, as well as vitamin C in Fr1. As presented in Table 2. bilberry extract contains two times higher levels of flavonoids than phenolic acids. Herrmann [23] and Wildanger and Herrmann [51] reported that the far predominant phenolic compound in bilberry is quercetin. Beside quercetin, the detectable levels of myricetin and kaempferol were found in several European bilberry varieties [46]. High contents of quercetin were confirmed also by Häkkinen et al. [20] where quercetin was abundant in 21.4% of total phenolic compounds detected in bilberry. In that study the main phenolic acid in bilberry was p-coumaric acid followed by ferulic and caffeic acids. Also, total contents of phenolic compounds obtained in this study were lower comparing to the values obtained in the study of Prior et al. [37] probably due to the variability of plant material used for extraction. Their data are based on fresh fruit flesh and we used dried plant material. According to the study of Häkkinen [21] quercetin content in bilberry decreased markedly during 9 months of storage, the lower values we observed seem to be reasonable. Also, the contents of vitamin C, as expected, were much lower than reported [2] due to the the sensitivity and instability of this vitamin during storage and processing

Table 1 Total contents of phenols, flavonoids and anthocyanins in purified bilberry extract Compounds phenolsa

Total Total flavonoidsb Total anthocyaninsc Monomeric anthocyaninsc a mg b mg c mg

chlorogenic acid/g. rutin/g. cyanidin-3-glycoside/g.

Total content mg/g dry weight

mg/g plant material

273.25 ± 10.69

136.29 ± 4.28

224.71 ± 7.34

112.08 ± 3.87

78.50 ± 3.34 58.45 ± 2.28

39.15 ± 1.34 29.15 ± 0.98

18

V. Tumbas et al. / Radical scavenging activity of bilberry Table 2 Contents of phenolic compounds and vitamin C in bilberry extract fractions Compounds

Vitamin C (Fr1) Flavonoids (Fr2) Phenolic acids (Fr3)

Total content Expressed as mg per 100 g of bilberry extract fraction

Expressed as mg per 100 g of bilberry extract

Expressed as mg per 100 g of bilberry sample extracted

152.90 ± 14.60 132.86 ± 0.05 49.43 ± 0.02

2.02 ± 0.19 0.77 ± 0.01 0.36 ± 0.00

1.01 ± 0.09 0.38 ± 0.01 0.18 ± 0.00

[17]. In general, vitamin C content of berries declines during storage by up to 56% depending on duration and temperature [4]. Finally, actual levels of antioxidants in plants depend greatly on the variety of plant analysed, the way in which it was cultivated (since synthesis of antioxidants can be a response to stress), and how it is harvested and stored [11]. 3.2. Superoxide anion radical scavenging activity of bilberry extract fractions Although the superoxide anion radical is a weak oxidant, it gives rise to the generation of more powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to the oxidative stress [29]. Superoxide anion radical can be produced enzymatically, in xanthine/xanthine oxidase system or chemically, as in this study. The advantage of the latter system for determination of superoxide anion radical scavenging activity is the simplicity in interpretation of results, since possible inhibition of the xanthine oxidase enzyme by antioxidant fractions is not a problem. The superoxide adduct of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) has been detected by ESR spectroscopy using KO2 solubilized in crown ether as a source of superoxide (Fig. 1). The ESR hyperfine splitting constants of the DMPO/• OOH adduct were: aN = 12.65 G; aH␤ = 10.4 G; aH␥ = 1.3G, which is in accordance with literature data [8].

Fig. 1. ESR spectrum of DMPO/• OOH spin adduct (blank) recorded 2 min in after mixing of 0.005 ml of KO2 /crown ether (10 mM/20 mM) dissolved in dry DMSO, 0.5 ml of dry DMSO and 0.005 ml of an DMSO spin trap solution (DMPO, 2 M).


Fig. 2. Superoxide anion radical scavenging activity of bilberry extract fractions: of three determinations.

Fr1;

Fr2;

19

Fr3. Each value is presented as mean ± SD

All three fractions of bilberry extract have efficiently scavenged superoxide anion radicals (Fig. 2). These fractions showed similar, not significantly different (p < 0.05) scavenging activity, when applied at lower concentrations (0.001–0.01 mg/ml). Complete elimination of superoxide anion radicals (SAo2 •– = 100%) was achieved with the same concentration, 0.75 mg/ml, of Fr2 and Fr3, and with 1 mg/ml of Fr1. The SAo2 •– of bilberry extract fractions decreased in the following order: Fr3 > Fr2 > Fr1. In the present study bilberry fractions scavenged superoxide anion radicals with an efficiancy higher than BHA. Calculated IC50 values for Fr1 (49 ␮g/ml), Fr2 (21 ␮g/ml) and Fr3 (15 ␮g/ml) were much lower than IC50 value for BHA (2680 ␮g/ml). IC50 values of bilberry extract fractions were comparable to the superoxide anion radical scavenging activity values of bilberry extract obtained in the study of Valentová et al. [48] (final concentration 47.6 ␮g/ml scavenged 25.7% of superoxide anion radicals formed) and Martin-Aragon et al. [32] (final concentration 50 ␮g/ml scavenged 63% of superoxide anion radicals formed). Difference in reactivity is due to the difference in the contents of active compounds, resulting from different extraction methods, and different experimental conditions, especially because in these studies superoxide anion radical was produced enzymatically. The scavenging properties of antioxidant compounds are often associated with their ability to form stable radicals. It is well known that aromatic compounds containing hydroxyl groups, especially those having ortho di- or trihydroxyfunction, can give rise to radicals stable enough to be directly detected by ESR spectroscopy [14]. Seyoum et al. [43] proposed a mechanism of antioxidant reaction of flavonoids, via hydrogen donation leading to formation of a flavonoid radical and termination of flavonoid radicals by further loss of a hydrogen atom via disproportionation or other subsequent reactions. An ESR signal assignable to a flavonoid semiquinone radical was found in the superoxide generating system in the presence of Fr2 and without adding the spin trap (Fig. 3). After simulation of the obtained ESR spectrum (Fig. 3) and comparing the obtained hyperfine constants (aH 2 = 1.7 G; aH 5 = 0.8 G and aH 6 = 2.8 G) 2 5 6 with the reports of Canadanovic-Brunet et al. [6] (aH = 1.5 G; aH = 0.7 G and aH = 2.7 G), it was determined that the observed radical was quercetin semiquinone radical. This is consistent with the findings of Herrmann [51] and Wildanger and Herrmann [46] who determined that the dominant bilberry flavonoid is quercetin. The stability of flavonoid, i.e. quercetin is proposed to be due to the large conjugated system providing delocalization of the unpaired electron. However, the reaction of superoxide anion radical with Fr3 without presence of a spin trap did not result in an ESR visible product, probably due to the low stability of the radicals of phenolic acids. This could be caused by the lack of stabilization by resonance due to the smaller conjugated system comparing to the flavonoid molecule. On the other hand, the free-radical product obtained by oxidation of Fr1 with superoxide anion radicals was characterized by ESR spectroscopy as a simple doublet showing coupling constants of aH = 1.84 G (Fig. 4). According to literature data and due to the high content of vitamin C confirmed by HPLC analysis (Table 2), this can be assigned to an ascorbyl radical [18, 36]. It has been reported that the reaction of ascorbic acid with more aggressive radicals results in the production of an intermediate radical, ascorbyl, of low reactivity. The lower activity comes from the ability of ascorbate to delocalize the radical electron through its π-system.

20


Experiment

Simulation

Fig. 3. ESR spectrum of antioxidant-derived radical obtained in the reaction system containing 0.5 ml of DMSO solution of KO2 /crown ether (10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction Fr2 (5 mg/ml) 20 s after mixing. Simulated spectrum was obtained using WINSIM program.

Experiment

Simulation

Fig. 4. ESR spectrum of antioxidant-derived radical obtained in the reaction system containing 0.5 ml of DMSO solution of KO2 /crown ether (10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction Fr1 (5 mg/ml) 20 s after mixing. Simulated spectrum was obtained using WINSIM program.

Berries of the genus Vaccinium have been reported to express antioxidative and anticarcinogenic effects in vitro, which are partly proposed to be due to phenolic compounds in these berries [2, 13, 20, 27]. In these studies berry extracts, including bilberry, had high total phenolic contents and high antioxidant activity when compared to other plant materials. Kay et al. [25] reported that ingestion of 100 g of freeze dried blueberries (100 g) and berry juices increases the antioxidant capacity of blood plasma by 14–30%. A commercial extract of bilberry scavenged superoxide anion and hydroxyl radicals, inhibited lipid peroxidation in rat liver microsomes and liver lipid peroxidation in vivo in mice [31].


21

Understanding the link between the antioxidant capacity of individual components and the bioactivities of different berries may direct the biotechnological improvement of new berry varieties [3]. Many researchers have found a strong correlation between antioxidant activity and total phenolics [7, 20, 50]. Beekwilder et al. [5] reported that about half of antioxidant activity of raspberries was due to ellagitannins, 20% due to vitamin C and 25% to anthocyanins, while Tulipani et al. [47] found greater contribution of vitamin C (30–35%) in some strawberry varieties. Positive linear correlation between SAo2 •– and total conents of vitamin C (r2 = 0.810), flavonoids (r2 = 0.707) and phenolic acids (r2 = 0.608) obtained in this study indicate that these phytochemicals are one of the main components responsible for antioxidant behaviour of bilberry.

4. Conclusion Analysis of bilberry extract fractions containing vitamin C (Fr1), neutral phenols (Fr2) and acidic phenols (Fr3) showed high superoxide anion radical scavenging activities, better than synthetic antioxidant BHA. The highest activity was achieved with Fr3. HPLC analysis confirmed significant levels of vitamin C, flavonoids and phenolic acids in investigated bilberry extract fractions. Correlation analysis proved the involvement of these compounds in antioxidative activity. Further research is focused in the investigation of exact and comlete polyphenol profile of bilberry extract, especially anthocyanin contents, and other biological activities contributing the well known beneficial health effects of bilberry.

Acknowledgements This research is part of the Project No. 142036 which is financially supported by the Ministry of Science and Technological Development of the Republic of Serbia. A part of this research was supported by a Grant from ÖAD Austria.

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