Acute Cardioprotective and Cardiotoxic Effects of Bilberry Anthocyanins in Ischemia–Reperfusion...

Acute Cardioprotective and Cardiotoxic Effects of BilberryAnthocyanins in Ischemia–Reperfusion Injury: BeyondConcentration-Dependent Antioxidant Activity

Lovro Ziberna • Mojca Lunder • Spela Moze •

Andreja Vanzo • Federica Tramer •

Sabina Passamonti • Gorazd Drevensek

Published online: 27 October 2010

� Springer Science+Business Media, LLC 2010

Abstract Despite being reported to reduce the risk of

cardiovascular diseases, little is known about acute direct

effects of bilberry anthocyanins on whole mammalian heart

under ischemia–reperfusion (I–R) conditions. Bilberry

anthocyanins were prepared from the ripe bilberries and

analyzed using HPLC–DAD. Their antioxidant activity was

evaluated by measuring the intrinsic free radical–scavenging

capacity and by cellular antioxidant assay (CAA) on endo-

thelial cells, where we quantified the intracellular capacity to

inhibit the formation of peroxyl radicals. Experiments on the

isolated rat hearts under I–R were carried out according to the

Langendorff method. Perfusion with low concentrations of

bilberry anthocyanins (0.01–1 mg/L) significantly attenu-

ated the extent of I–R injury as evidenced by decreasing the

release rate of LDH, increasing the postischemic coronary

flow, and by decreasing the incidence and duration of

reperfusion arrhythmias. High concentrations (5–50 mg/L)

diminished cardioprotection and show cardiotoxic activity

despite having their radical scavenging and intracellular

antioxidant capabilities increased in a concentration-

dependent manner. This study reveals the biphasic concen-

tration-dependent bioactivity of bilberry anthocyanins under

I–R, which results in strong cardioprotective activity in

low concentrations and cardiotoxic activity in high

concentrations.

Keywords Bilberry anthocyanins � Cellular antioxidant

activity � Endothelium � Ischemia–reperfusion injury �Isolated rat heart

Introduction

Flavonoid-rich diets, such as among the Finnish [1], are

associated with a decreased risk of cardiovascular dis-

eases. Bilberries (Vaccinium myrtillus L.) are one of the

richest dietary sources of anthocyanins, which are also

considered the most pharmacologically active constituents

[2, 3].

In cardiovascular system, bilberry anthocyanins exhibit

a wide range of biologic effects, including antioxidant [4,

5], anti-inflammatory [5], and vasodilatory actions [6],

among others. Interpretation of these results is, however,

complicated since different extraction procedures, oxida-

tion models, and corresponding experimental conditions

were applied.

Antioxidant activity of anthocyanins can be described

directly, in terms of their per se activity as free radical

scavengers, or indirectly as modulators of intracellular pro-

and anti-oxidant enzymes [7]. However, in recent years,

flavonoids were reported to have both antioxidant and pro-

oxidant activity depending upon certain experimental

conditions [8]. Often, these pro-oxidant effects involve

interactions of polyphenols with transition metal ions,

L. Ziberna (&) � M. Lunder � G. Drevensek

Institute of Pharmacology and Experimental Toxicology,

Faculty of Medicine, University of Ljubljana, Korytkova 2,

1000 Ljubljana, Slovenia

e-mail: [email protected]

S. Moze

Department of Food Science and Technology, Biotechnical

Faculty, University of Ljubljana, Jamnikarjeva 101,

1000 Ljubljana, Slovenia

A. Vanzo

Central Laboratories, Agricultural Institute of Slovenia,

Hacquetova 17, 1000 Ljubljana, Slovenia

F. Tramer � S. Passamonti

Department of Life Sciences, University of Trieste,

via L. Giorgieri 1, 34127 Trieste, Italy

Cardiovasc Toxicol (2010) 10:283–294

DOI 10.1007/s12012-010-9091-x

which can be ubiquitously found in biologic systems, or as

universal contaminants of biologic reagents [9].

The aim of our study was thus to examine concentration-

dependent acute effects of bilberry anthocyanins under

ischemia–reperfusion (I–R) conditions on the isolated rat

heart. We found that bilberry anthocyanins were cardio-

protective at low concentrations, but cardiotoxic at high

concentrations. To understand these observations, we

decided to measure the intrinsic free radical–scavenging

effect (in the absence of biologic structures) using the

DPPH method, and cellular antioxidant activity on endo-

thelial cells in culture using the CAA method developed by

Wolfe and Liu [10]. In both assays, anthocyanins acted

solely as antioxidants. Thus, we show here that the dis-

crepant bioactivity of anthocyanins can be revealed only in

highly complex experimental models, such as on the heart

under I–R conditions, rather than in simplified models,

such as cell cultures, or even cell-free systems.

Materials and Methods

Chemicals and Reagents

Methanol and formic acid, both HPLC grade, were

obtained from Merck (Darmstadt, Germany). Acetonitrile

(HPLC grade) was obtained from J. T. Baker (Deventer,

Netherlands). Dulbecco’s Modified Eagle’s Medium

(DMEM), 20,70-dichlorofluorescin diacetate (DCFH-DA),

2,20-azobis (2-amidinopropane) dihydrochloride (ABAP),

1,10-diphenyl-2-picrylhydrazyl (DPPH), and ammonium

formate were obtained from Sigma–Aldrich (Steinheim,

Germany). Fetal bovine serum (FBS), L-glutamine and

penicillin-streptomycin solution were obtained from

EuroClone (Milano, Italy). Phosphate Saline buffer (PBS)

was used in CAA assay: 200 mg/L KCl (Merck, Darms-

tadt, Germany); 200 mg/L KH2PO4 (Carlo Erba, Milano,

Italy); 8,000 mg/L NaCl; 1,150 mg/L Na2HPO4 (Carlo

Erba, Milan, Italy). Hanks’ Balanced Salt Solution (HBSS)

was used in CAA assay: 185 mg/L CaCl2 9 2H2O; 60 mg/L

KH2PO4; 350 mg/L NaHCO3; 8,000 mg/L NaCl; 47.88 mg/L

Na2HPO4 (Carlo Erba, Milano, Italy); 100 mg/L MgCl2 9

6H2O; 1,000 mg/L glucose (Sigma, Steinheim, Germany);

100 mg/L MgSO4 9 7H2O; 400 mg/L KCl (Merck,

Darmstadt, Germany). pH was adjusted to 7.4 using 0.1%

HCl (Merck, Darmstadt, Germany). Krebs-Henseleit (K-H)

solution was used in isolated heart experiments (composi-

tion in mM: 118.6 NaCl; 4.7 KCl; 11.1 glucose; 25

NaHCO3; 1.66 MgSO4; 1.2 NaH2PO4, and 2.52 CaCl2) (all

Merck Darmstadt, Germany). Aqueous solutions of all

aforementioned chemicals were prepared with Milli-Q

water (Millipore, Bedford, USA).

Preparation of Bilberry Extract

Handpicked bilberries (Vaccinium myrtillus L.) from the

natural location in subalpine forest (Smrecje, Slovenia)

were used and stored at -20�C until extraction. Fifty grams

of frozen bilberries were homogenized in 150 mL of ice-

cold, deoxygenated methanol. Homogenate was extracted

for 3 h by shaking in the dark at room temperature. The

extract was centrifuged at 32,700g for 5 min at 4�C, and the

supernatant was stored at -20�C. The sediment was

extracted again in 100 mL of deoxygenated methanol for

2 h in the dark at room temperature, and centrifuged as

above. Finally, supernatants were pooled and the volume

was adjusted with methanol to 250 mL. Final extract was

flushed with nitrogen and stored at -20�C until being used.

Bilberry Anthocyanins Extraction

Five mL of methanolic extract was evaporated to dryness

under reduced pressure at 38�C; the residue was dissolved

in 5 mL of Milli-Q water and loaded onto 1 g SEP-PAK

C18 cartridge (Waters, Miliford, USA), previously condi-

tioned with 3 mL of methanol and 5 mL of 5 mM H2SO4.

The cartridge was washed with 5 mL of 5 mM H2SO4, and

dried with nitrogen before anthocyanins were eluted with

4 mL of methanol. The eluate of bilberry anthocyanins was

evaporated to dryness, dissolved in 1 mL of initial HPLC

gradient mixture, filtered through a 13 mm 0.22 lm PVDF

filter (Millipore, Bedford, USA) into a HPLC vial and

immediately analyzed by HPLC–DAD.

HPLC–DAD Analysis of the Anthocyanins

To detect and quantify the contents of bilberry anthocyanins,

we used Agilent 1100 HPLC with DAD detector coupled to

an Agilent NDS ChemStation (Agilent Technologies, Palo

Alto, USA). Separation was performed according to the

modified method of Latti et al. [11] using a Gemini C18

column (150 9 4.60 mm, 5 lm) coupled with guard col-

umn Gemini C18 (4 9 3.0 mm, 5 lm) (Phenomenex,

Torrance, USA). The mobile phase consisted of 3 % formic

acid in water (A) and acetonitrile:methanol (85:15) (B).

The gradient conditions were linear: 2 min, 6% B;

2–4 min, 6–8% B; 4–12 min, 8–10% B; 12–13 min, 11% B;

13–25 min, 11–12% B; 25–40 min, 12–100% B. The col-

umn was equilibrated 6 min prior to the next analysis. The

flow rate was: 1.0 mL/min 0–4 min; 0.9 mL/min 4–13 min;

0.8 mL/min 13–40 min. The injection volume was 10 lL.

The UV–VIS spectra were recorded from 200 to 700 nm,

with the detection at 520 nm. Total anthocyanins in the

sample were quantified and expressed as equivalents of

cyanidin 3-glucoside.

284 Cardiovasc Toxicol (2010) 10:283–294

Preparation of Samples for Testing in the Antioxidant

Models

The aliquot of bilberry anthocyanins dissolved in methanol

was evaporated to dryness under reduced pressure at 38�C

and re-dissolved in Milli-Q water just prior experiments

were carried out. As such, bilberry anthocyanins were

applied in increasing concentrations, which were quantified

by comparison with a standard curve obtained using known

concentrations of cyanidin 3-glucoside.

Determination of Antioxidant Activity Using DPPH

Method

Free radical–scavenging activity of DPPH was measured

by the modified method of Blois [12]. Different dilutions of

bilberry extract (100 lL) were added to 2.9 mL of 100 lM

DPPH solutions, dissolved in methanol. DPPH is a stable

free radical with deep purple color in methanolic solution

and a typical absorbance at 517 nm, but becomes pale

yellow when trapped by an antioxidant. Following con-

centrations of the bilberry anthocyanins (expressed as

equivalents of cyanidin 3-glucoside) were tested (mg/L):

0.01, 0.1, 1, 5, 10, 25, and 50. The control consisted of

2.9 mL of 100 lM DPPH-methanol solution and 100 lL of

methanol. The decrease in absorbance of the resulting

solution was measured at 517 nm for half an hour. The

antioxidant activity of the bilberry anthocyanins was cal-

culated as the DPPH scavenging activity (%) as follows:

DPPH radical scavenging activity %ð Þ¼ Acontrol � Asample

� �= Acontrolð Þ

� �� 100

where Acontrol is the absorbance of DPPH radical and

methanol; Asample is the absorbance of DPPH radical and

sample of bilberry extract. All determinations were per-

formed in triplicates (n = 3).

Endothelial Cell Cultures

Human endothelial cell line (EA.hy926) was obtained from

the American Type Culture Collection (Rockville, USA).

EA.hy926 cell line was cultured in completed DMEM:

Dulbecco’s Modified Eagle’s Medium supplemented with

10% fetal bovine serum, 1 mM L-glutamine and 1 mM

penicillin–streptomycin solution. Cells were grown in an

incubator at 37�C in a humidified atmosphere (95% air and

5% of carbon dioxide).

Cellular Antioxidant Activity (CAA) Assay

The assay developed by Wolfe and Liu [10] was carried out

as previously described, with minor modifications. The

cells were seeded on 96-well plates (104 cells/well),

containing 100 lL complete DMEM and grown for 24 h.

Then, cells were incubated for 1 h with the bilberry antho-

cyanins dissolved in 100 lL of MGD solution (incomplete

DMEM, 1 mM L-glutamine, 50 lM DCFH-DA) at the fol-

lowing concentrations: 0 (control), 0.1, 1 lg/L, 0.01, 0.1,

0.5, 1, 5, 10, and 50 mg/L. After incubation, solution was

removed and washed twice with PBS. Then, cells were

treated with the peroxyl radical-generating reagent ABAP,

at 5 mM in 100 lL Hank’s buffered saline solution (HBSS).

Blank wells were filled with HBSS solution without ABAP.

Fluorescence was measured at 535 nm with excitation at

485 nm every 5 min for 1 h at 37�C on a microplate reader

(Bio-Tek Instruments, Winooski, VT, USA). To quantify the

cellular antioxidant activity, the CAA units (%) were cal-

culated as follows:

CAA units %ð Þ ¼ 100�Z

SA=

ZCA

� �� 100

where $SA is the integrated area under the sample fluo-

rescence readings, previously subtracted by blank, versus

time curve and $CA is the integrated area under the control

curve.

Animals

Adult male Wistar rats weighing 260–280 g were housed

under standard laboratory conditions in a temperature-

controlled environment (22 ± 1�C, 60% humidity) main-

tained on a 12-h light/dark cycle, and given ad libitum

access to food and water. All animal procedures and study

protocols were conducted in accordance with permission

issued by the Veterinary Administration of the Republic of

Slovenia (permit SI-No. 34401-23/2009/3), which con-

forms with the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health

(NIH Publication No. 85-23, revised 1996).

Perfused Heart Preparation

Rats were anaesthetized by intraperitoneal injection of

urethane (130 mg/100 g of body weight) (Sigma–Aldrich,

St. Louis, USA) mixed with heparin (1,000 U/animal)

(Krka, Novo mesto, Slovenia). Following ventrolateral

thoracotomy, a canula filled with cold (4�C) K–H solution

with heparin was introduced into the ascending aorta.

During preparation, the heart was washed with ice-cold

K–H solution to decrease its contractility. Hearts were

mounted on a Langendorff’s apparatus and retrogradely

perfused with K–H solution under constant pressure con-

ditions. The solution was bubbled continuously with 95%

O2 ? 5% CO2 (pH 7.4 at 37.5�C). A pressure catheter

(SPR-524, Millar, Houston, TX, USA) was introduced

through the left atrium and mitral valve to the left ventricle.

Cardiovasc Toxicol (2010) 10:283–294 285

An electrocardiogram (ECG) was recorded from the sur-

face of the heart by two AgCl electrodes (ITIS, Ljubljana,

Slovenia) placed in the direction of the electrical axis of the

heart, and signals were pre-amplified (preamplifier Paar,

Graz, Austria). Data of all parameters measured in isolated

hearts were acquired with Dewetron equipment (Graz,

Austria) and Dewesoft 6.0 software (Trbovlje, Slovenia)

and recorded in real-time on the computer hard drive for

off-line analysis. Hearts were protected with a glass coat

and Parafilm to maintain a constant temperature (37.5�C)

and humidity. The temperature of the experimental envi-

ronment was kept at 23–25�C. Isolated hearts with coro-

nary flow of less than 6 or more than 12 mL/min, or with

the appearance of arrhythmias at the beginning of the

experiment, were excluded from further experiments. Thus,

all hearts used in our study beat spontaneously at sinus

rhythm without being paced through coaxial stimulating

electrodes.

Experimental Protocols

All hearts were randomized into several groups, each

composed of 5–7 hearts, and subjected to perfusion pro-

tocols as shown in Scheme 1. The control group (CTRL)

was perfused with oxygenated K–H solution during the

first 30 min (perfusion phase), followed by 40 min of

global zero flow ischemia with complete flow cessation of

K–H solution to the isolated heart. Hearts were then

perfused with oxygenated K–H solution for 50 min

(reperfusion period). In the test groups (BA groups),

hearts were firstly perfused with oxygenated K–H for the

first 20 min only, followed by 10 min perfusion of the

corresponding BA solution, consisting of K–H solution

supplemented with bilberry anthocyanins (0.01, 0.1, 1, 5,

10, 25, and 50 mg/L, corresponding to cyanidin 3-glu-

coside equivalents). Then, 40 min of global zero flow

ischemia was applied, followed by 50 min of reperfusion

with BA solutions.

Measured Parameters on Isolated Heart Experiments

Coronary Flow Rate

Coronary flow rate (CF, mL/min) was measured by col-

lecting the coronary effluent into calibrated test tubes.

Samples thereof were further used for biochemical analysis.

Lactate Dehydrogenase Release Rate

Lactate dehydrogenase (LDH) release rate in the effluent

was determined by the modified Wroblewski-LaDue

method [13]. Results for LDH release rate activity were

expressed as lkat g-1 min-1.

Left Ventricular Pressure

Left ventricular pressure (LVP) was measured continu-

ously with a Millar pressure catheter-transducer (model

SPR-524, size 3.5F = 1.15 mm). The catheter was intro-

duced through dissection of the left atrium and through a

mitral valve to the left ventricle. The pressure signal was

registered with a sampling rate of 500 Hz. Signals were

converted with an analogue–digital converter and stored on

a computer hard disk. Diastolic and systolic pressures were

registered. LVP was expressed as the difference between

systolic and diastolic pressures.

Heart Rate, Incidence, and Duration of Arrhythmias Using

ECG Analysis

Heart rate (HR), in beats per min, was obtained from

oscillations detected in the ECG aligned to ventricular

pressure values. The ECG was recorded with a 2.5 kHz

sample rate to obtain data for precise time interval analysis.

Electrodes were positioned on the heart surface to allow the

detection of the P wave during the whole experiment.

The ECG was analyzed for the incidence and duration of

different types of arrhythmias, in accordance with the

Lambeth Conventions [14]: Heart arrest or asystole is

represented by a flat ECG signal for which the rate is zero.

Ventricular tachycardia is defined as a run of four or more

consecutive ventricular premature complexes; it is an

abnormally rapid ventricular rhythm with wide QRS

complexes, in rat heart usually in excess of 500/600 beats

per minute. It is generated within the ventricle, below the

bundle of His. Ventricular fibrillation is indicated by a

signal for which individual QRS deflections can no longer

be distinguished from one another and for which a rate can

no longer be measured. Ventricular premature complexes

or extrasystoles are premature contractions of the ventricle

defined as discrete and premature QRS complexes in

relation to the P wave.Scheme 1 Schematic experimental protocols of isolated rat hearts

exposed to ischemia–reperfusion


Statistical Analysis

All values are expressed as means ± SEM. All statistical

analyses and graphical presentations of results were per-

formed with GraphPad Prism version 5.00 (GraphPad

Software, San Diego California, USA). One-way analysis

of variance (ANOVA) with post-Dunnet test was used to

compare studied groups to the control group. A value of

P \ 0.05 was considered significant.

Results

Chemical Identification of Bilberry Anthocyanins

The HPLC–DAD method for the separation of anthocya-

nins enabled us to identify individual anthocyanins on the

basis of their UV–VIS spectra and retention times (Fig. 1).

Quantification was based on peak areas at A520, and con-

centrations were expressed as cyanidin 3-glucoside equiv-

alents (Table 1). A twelve-point standard calibration curve

was linear in the range 50–500 mg/L (r2 = 0.999).

DPPH Radical-Scavenging Activity of Bilberry

Anthocyanins

The antioxidant activities of bilberry anthocyanins were

evaluated by measuring their DPPH radical-scavenging

activities. In this part of research, DPPH scavenging

activity was strongly correlated to the bilberry anthocya-

nins levels, since all of the tested samples exhibited anti-

oxidant activities in a concentration-dependent manner

(Fig. 2). Under the tested conditions, we observed no

saturable activity of bilberry anthocyanins in their free

radical–scavenging activity.

Cellular Antioxidant Activity of Bilberry Anthocyanins

Intracellular antioxidant activity of bilberry anthocyanins

was assessed in EA.hy926 cells, a model of vascular

endothelium, by the CAA assay. Given the critical role

played by the endothelium in influencing the whole

Fig. 1 HPLC–DAD chromatogram at A520 of the bilberry extract:

1 delphinidin 3-galactoside, 2 delphinidin 3-glucoside, 3 cyanidin

3-galactoside, 4 delphinidin 3-arabinoside, 5 cyanidin 3-glucoside,

6 petunidin 3-galactoside, 7 cyanidin 3-arabinoside, 8 petunidin

3-glucoside, 9 peonidin 3-galactoside, 10 petunidin 3-arabinoside, 11peonidin 3-glucoside, 12 malvidin 3-galactoside, 13 peonidin

3-arabinoside, 14 malvidin 3-glucoside, 15 malvidin 3-arabinoside

Table 1 Qualitative and quantitative composition of bilberry

(Vaccinium myrtillus L.) anthocyanins used in our study

Anthocyanins mg/100 g rel. ab. %

1 Delphinidin 3-galactoside 138.57 14.3

2 Delphinidin 3-glucoside 135.30 14.0

3 Cyanidin 3-galactoside 89.34 9.2

4 Delphinidin 3-arabinoside 117.25 12.1

5 Cyanidin 3-glucoside 97.40 10.1

6 Petunidin 3-galactoside 38.32 4.0

7 Cyanidin 3-arabinoside 74.27 7.7

8 Petunidin 3-glucoside 85.52 8.8

9 Peonidin 3-galactoside 10.27 1.1

10 Petunidin 3-arabinoside 24.87 2.6

11 Peonidin 3-glucoside 36.18 3.7

12 Malvidin 3-galactoside 24.61 2.5

13 Peonidin 3-arabinoside 4.88 0.5

14 Malvidin 3-glucoside 76.40 7.9

15 Malvidin 3-arabinoside 14.60 1.5

Total anthocyanins 967.77 100

Individual anthocyanins are expressed as cyanidin 3-glucoside

equivalents (mg/100 g of bilberries), and are also presented in terms

of their corresponding relative abundances (in %)

Fig. 2 Radical scavenging (DPPH-test) of bilberry anthocyanins

(expressed as equivalents of cyanidin 3-glucoside). Measurements

were performed in triplicates (n = 3) and compared to the group with

the highest concentration of BA (50 mg/L). All results are expressed

as mean ± SEM. ** P \ 0.01; *** P \ 0.001


vascular and cardiac function, its protection against oxi-

dative stress by bilberry anthocyanins was worth being

investigated. Figure 3a shows the kinetics of the intracel-

lular fluorescence increase triggered by the radical initiator

ABAP. Bilberry anthocyanins can attenuate the formation

of intracellular fluorescence, and thus indicate protection

against the acute intracellular oxidative stress. This effect

was quantified as CAA values (percentage of the antioxi-

dant activity) against the corresponding concentrations of

bilberry anthocyanins, as shown in Fig. 3b. Bilberry

anthocyanins were strongly active in the low concentration

range (\0.5 mg/L); a saturable effect was observed above

0.5 mg/L and up to 50 mg/L.

Ischemia–Reperfusion Model on Isolated Rat Heart

Coronary Flow (CF) Values

Figure 4 shows the coronary flow under normal condi-

tions and in the absence of BA (during the first 20’). No

large inter-group and inter-individual differences in cor-

onary function were found. The addition of bilberry

anthocyanins to the K–H solution (at 20 min) during

preischemic period caused a biphasic response in coro-

nary flow, depending on BA concentration (Fig. 4),

namely: (1) vasodilation at BA B 1 mg/L); (2) vasocon-

striction at BA C 10 mg/L and up to 50 mg/L. With

respect to the control, CF values increased by up to 1.3-

fold (in BA 0.1) (P \ 0.05) but it decreased to 0.56-fold

(P \ 0.001) and 0.44-fold (P \ 0.001) in BA 25 and BA

50, respectively. After the ischemic period, similar

biphasic responses were observed in CF and were most

pronounced in the first 10 min of reperfusion. In the BA

0.1 and BA 1 groups, CF was increased by up to 2.3-fold

(P \ 0.001) and 1.9-fold (P \ 0.001) compared to the

control group, respectively. In contrast, CF was signifi-

cantly decreased in the BA 25 and BA 50 groups

throughout the reperfusion period.

Lactate Dehydrogenase (LDH) Release Rate

To determine the extent of myocardial injury, release of

LDH was measured in the coronary effluent (Fig. 5).

During the preischemic perfusion, no changes in LDH

release rate were observed in any of the studied groups,

showing that the hearts were intact. However, reperfusion

after global ischemia markedly increased the release of

LDH in the control group. Bilberry anthocyanins dis-

played a concentration-dependent biphasic effect: in low

concentrations they significantly attenuated the I–R-

induced increase in release of LDH, while their effect

was opposite in high concentrations. The most pro-

nounced anti-ischemic activity was observed in BA 1 and

BA 0.1 groups, where the endpoint LDH release rate was

decreased down to 0.14-fold (P \ 0.001) and 0.28-fold

(P \ 0.001) of the control (CTRL) group values,

respectively. In BA 0.01 and BA 5 groups, LDH release

rate was decreased down to 0.77-fold (P \ 0.05) and

0.57-fold (P \ 0.01) of the control values, respectively.

Interestingly, we observed no marked effect in the BA 10

group, whereas in BA 25 and BA 50 groups LDH release

rate was increased up to 1.5-fold (P \ 0.001) and 2.2-

fold (P \ 0.001) of the control group values, respec-

tively. This indicates that high concentration of bilberry

anthocyanins facilitate the I–R injury-induced increase in

LDH.

Fig. 3 Cellular antioxidant activity of bilberry anthocyanins in

endothelial cell line EA.hy926 a Time course of peroxyl radical-

induced oxidation of DCFH in EA.hy 926 cells. Cells were exposed to

bilberry anthocyanins for 60 min, which were removed before adding

the radical initiator. Concentrations (expressed as equivalents of

cyanidin 3-glucoside) were the following: none (filled circle), 0.1 lg/L

(open square), 1 lg/L (filled triangle), 10 lg/L (open invertedtriangle), 0.1 mg/L (filled diamond), 1 mg/L (open circle), and

10 mg/L (filled diamond); to improve the clarity, tests done with 0.5,

5, and 50 mg/L are not displayed. b Calculated values for cellular

antioxidant activity (CAA) of bilberry anthocyanins in EA.hy926

cells. The calculated CAA values from all experimental groups were

compared to the group with the highest concentration of bilberry

anthocyanins (50 mg/L). All results are expressed as mean ± SEM

(n = 5). *** P \ 0.001


Heart Rate (HR)

During the preischemic perfusion with bilberry anthocya-

nins, HR tended to increase (Fig. 6), though without attain-

ing statistical significance. In the postischemic reperfusion

period, HRs, measured after the normal heart rhythm (sinus

rhythm) was restored, reached their lowest values and then

steadily increased, as usually observed in similar experi-

mental models. However, no significant differences were

observed between the studied groups and control in the HR

Fig. 4 Coronary flow (CF)

values of isolated rat hearts in

experiments with 40-min

ischemia, followed by

reperfusion. In the control group

(CTRL, n = 7), hearts were

perfused with K–H alone, while

in BA groups perfusion was

done with bilberry anthocyanins

(expressed as equivalents of

cyanidin 3-glucoside) (mg/L):

0.01, 0.1, 1, 5, 10, 25, and 50

(n = 5). All data are

means ± SEM. * P \ 0.05;

** P \ 0.01; *** P \ 0.001

versus control

Fig. 5 Lactate dehydrogenase

(LDH) release rate values of

isolated rat hearts in

experiments with 40-min

ischemia. In the control group







0.01, 0.1, 1, 5, 10, 25, and 50

(n = 5). All data are

means ± SEM. * P \ 0.05;

** P \ 0.01; *** P \ 0.001

versus control


during reperfusion period, except in the BA 1 group where

HR was lower at the end of the experiment.

Left Ventricular Pressure (LVP) Values

Bilberry anthocyanins caused concentration-dependent

biphasic inotropic effects in the preischemic (normoxic)

perfusion (Fig. 7), shown by: (1) positive inotropic effect

(increase in LVP) in low concentrations (BA \ 5 mg/L);

and (2) negative inotropic effect (decrease in LVP) in high

concentrations (BA [ 10 mg/L). LVP increased up to

1.44-fold (P \ 0.05) and 1.61-fold (P \ 0.01) of the con-

trol values, in BA 0.1 and BA 1, respectively. On the

contrary, it decreased in the BE 25 and BE 50 groups down

to 0.56-fold (P \ 0.001) and 0.28-fold (P \ 0.001) of the

control, respectively. However, during the postischemic

reperfusion period, a modest and not significant decrease in

LVP was observed in all studied groups.

Fig. 6 Heart rate (beats/min)

values of isolated rat hearts

in experiments with 40-min

ischemia. In the control group







0.01, 0.1, 1, 5, 10, 25, and 50

(n = 5). Data in the

postischemic period are

presented after the normal heart

rhythm was restored. All data

are means ± SEM. * P \ 0.05;

** P \ 0.01; *** P \ 0.001

versus control

Fig. 7 Left ventricular pressure

(mmHg) values of isolated rat

hearts in experiments with

40-min ischemia. In the control

group (CTRL, n = 7), hearts

were perfused with K–H alone,

while in BA groups perfusion

was done with bilberry

anthocyanins (expressed as

equivalents of cyanidin

3-glucoside) (mg/L): 0.01, 0.1,

1, 5, 10, 25, and 50 (n = 5).

Data in the postischemic period

are presented after the normal

heart rhythm was restored. All

data are means ± SEM.

* P \ 0.05; ** P \ 0.01;

*** P \ 0.001 versus control


Duration and Characterization of Arrhythmias

We studied four distinct types of reperfusion arrhythmias

that appeared in all studied hearts in the postischemic

reperfusion period (Table 2): heart arrest (HA), ventricular

fibrillation (VF), ventricular tachycardia (VT), and ven-

tricular premature complexes (VPC). Bilberry anthocya-

nins caused concentration-dependent biphasic response in

the duration of reperfusion arrhythmias when compared to

the control groups (Fig. 8), namely: (1) anti-arrhythmic

effects (BA 0.1, BA 1, and BA 5 groups), and (2) no anti-

arrhythmic activity or pro-arrhythmic effects (BA 0.01, BA

25, and BA 50 groups). The longest duration of all types of

arrhythmias was observed in the control group (CTRL) and

in the BA 50 group. However, the most pronounced anti-

arrhythmic activity was observed in BA 0.1 and BA 1

groups, where duration of arrhythmias was maximally

shortened to 0.03-fold (P \ 0.001) and 0.04-fold

(P \ 0.001) of the control group values, respectively.

Interestingly, low anthocyanins concentrations protected

against the most malignant types of arrhythmias: in BA 0.1

and BA 1 groups, not only was the heart arrest completely

abolished, but also ventricular fibrillation had the lowest

duration.

To detect potential arrhythmic activity of bilberry

anthocyanins per se in the preischemic perfusion period,

we also analyzed ECG signal parameters throughout this

period. However, the addition of bilberry anthocyanins

before ischemia did not affect the sinus rhythm, or pro-

duced any evident changes in ECG signal parameters.

Discussion

Presently, there is little information about the acute effects

of anthocyanins on a whole mammalian heart, since most

of the reports were focused on either chronic studies or

other flavonoid subclasses. Here, we demonstrate that

bilberry anthocyanins display concentration-dependent

biphasic effects in the isolated rat heart, under both

normoxic and ischemia–reperfusion (I–R) conditions.

These can be described as cardioprotective activities in low

concentrations and as cardiotoxic activities in high con-

centrations. It has been unknown up to date that high

concentrations of anthocyanins exert deleterious effects in

I–R injury on the isolated heart.

To characterize the activity of bilberry anthocyanins

during normoxia, we analyzed all studied cardiac param-

eters during preischemic perfusion period. Under nor-

moxia, two parameters were found to be sensitive to

anthocyanins, i.e. left ventricular pressure (LVP) and cor-

onary flow (CF). We observed that bilberry anthocyanins

induced a biphasic inotropism: positive at lower concen-

trations and negative at higher concentrations. This effect

is in agreement with a similar study done with quercetin

[15]. The coronary flow also increased in low concentration

Table 2 Characterization and the average duration of separate types of reperfusion arrhythmias in the isolated rat hearts

HA (s) VF (s) VT (s) VPC (s)

CTRL 225.0 ± 58.8 1070.1 ± 183.8 919.1 ± 74.6 266.6 ± 149.9

BA 0.01 40.0 ± 20.0 957.5 ± 129.5 704.0 ± 485.0 704.8 ± 310.1

BA 0.1 0.0 – 0.0*** 3.3 – 3.0*** 13.8 – 10.0*** 62.5 ± 9.2

BA 1 0.0 – 0.0*** 2.5 – 2.0*** 13.7 – 9.0*** 87.2 ± 23.1

BA 5 5.8 – 3.8** 630.5 – 66.7* 737.3 ± 267.5 53.8 ± 18.1

BA 10 75.0 ± 15.0 1040.0 ± 206.9 899.0 ± 190.1 116.0 ± 32.0

BA 25 103.3 ± 83.5 1104.1 ± 476.0 1073.0 ± 297.2 163.3 ± 40.3

BA 50 147.7 ± 89.7 1365.5 ± 183.0 1147.0 ± 99.5 212.5 ± 34.4

In the control group (CTRL, n = 7), hearts were perfused with K–H alone, while in BA groups hearts were perfused with bilberry anthocyanins

(expressed as equivalents of cyanidin 3-glucoside) (mg/L): 0.01, 0.1, 1, 5, 10, 25, and 50 (n = 5). Abbreviations used for different types of

arrhythmias: HA heart arrest, VF ventricular fibrillation, VT ventricular tachycardia, VPC ventricular premature complexes. All data are

presented as means ± SEM. * P \ 0.05; ** P \ 0.01; *** P \ 0.001 versus control

Fig. 8 The average duration of reperfusion arrhythmias in isolated

rat hearts. In the control group (CTRL, n = 7), hearts were perfused

with K–H alone, while in BA groups perfusion was done with bilberry

anthocyanins (expressed as equivalents of cyanidin 3-glucoside) (mg/L):

0.01, 0.1, 1, 5, 10, 25, and 50 (n = 5). All data are means ± SEM.

* P \ 0.05; *** P \ 0.001 versus control


and decreased in high concentrations of tested bilberry

anthocyanins. This was unexpected since bilberry antho-

cyanins produce endothelium-dependent vasorelaxation

in porcine coronary arteries by activating eNOS [6].

However, NO could be scavenged by pro-oxidant radicals

potentially generated by high concentrations of anthocya-

nins, which could ultimately lead to vasoconstriction. This

phenomenon could be even further emphasized during the

post-reperfusion period, where production of ROS exceeds

the available antioxidant defence systems. In addition,

there is evidence that persisting oxidative stress will render

eNOS dysfunctional (uncoupled eNOS) such that it no

longer produces NO, but O2-� [16].

In addition, direct effects on vascular smooth muscle

cells might be envisaged. In a study with epigallocatechin-

3-gallate, a transient endothelium-independent contraction

was observed in the rat aorta as a consequence of increasing

smooth vascular cell membrane permeability to calcium

ions [17]. Likewise, quercetin was shown to be activator of

L-type calcium channels on artery smooth muscle cells,

even though it induced endothelium-dependent vasorelax-

ation in isolated blood vessels [18]. Indeed, under certain

anthocyanin concentrations, vasodilation might be accom-

panied by an increase in capillary permeability, which could

ease the diffusion of anthocyanins from the capillary lumen

in the interstitium of the muscle layer of both vessels and

the myocardium itself. Thus, under certain conditions,

anthocyanins might be directly available to the myocytes.

Taken together, this shows that some flavonoids have an

ambivalent role on vasomotor response, combining domi-

nating vasorelaxation and less evident vasoconstriction. In

our experiments in normoxic condition, low concentrations

of bilberry anthocyanins increased both CF and LVP, so

acting in favor of cardioprotection. By contrast, higher

concentrations of anthocyanins caused opposite effects on

CF and LVP, whereas LDH release rate was unaltered, and

no arrhythmias were detected. These results show that under

normoxic conditions, no evident cardiovascular tissue

damage was observed.

Under I–R conditions, CF is markedly reduced, but

again sensitive to anthocyanins in a concentration-depen-

dent biphasic manner. As a consequence of the drastic

decrease in CF during reperfusion, myocardial damage is

recorded as LDH release. Cardioprotection, observed at

low BA concentrations (0.01–1 mg/L), was evidenced as

improved postischemic coronary flow, decreased LDH

release rate, and decreased duration of reperfusion

arrhythmias. These findings are in line with strong bene-

ficial effects by cyanidin 3-glucoside against I–R injuries

on the isolated rat heart [19]. In addition, cardioprotection

against I–R was also seen in experiments using non-alco-

holic extracts of red wine enriched in polymeric proanth-

ocyanidins and anthocyanins, but not in other flavonoid

subclasses [20, 21]. Interestingly, bilberry anthocyanins,

tested in the in vivo model of I–R on the hamster cheek

pouch microcirculation, reduced microvascular impair-

ments with preservation of endothelium, attenuated leu-

kocyte adhesion, and improved capillary perfusion [22].

On the other hand, we observed unexpected results in

experiments with high concentrations of bilberry anthocya-

nins, as represented by diminished cardioprotective activity

(5–10 mg/L), or even cardiotoxic activity (25–50 mg/L) in

I–R. This was evidenced by markedly decreased coronary

flow, increased LDH release rate, and absence of anti-

arrhythmic activity when compared to the lower concen-

tration of anthocyanins. These findings suggest that some

mechanisms counteract the demonstrated radical scavenging

properties. First, anthocyanins can act as pro-oxidants in

systems containing redox-active metals and oxygen, leading

toward increased formation of reactive oxygen species and

phenoxyl radicals that damage biologic molecules [8].

Under reperfusion conditions, high concentration of antho-

cyanins could further increase the oxidative stress, thereby

aggravating myocardial injury. Second, anthocyanins could

affect the membrane fluidity by influencing the appearance

and development of lipid rafts cell membrane [23]. It is well

established that lipid rafts serve as a platform for gathering of

signaling and trafficking proteins [24], and are, thus, relevant

for cell–cell communication, cell signaling, and metabolism

regulation, among others. Consistent with this, anthocyanins

in high concentrations can have an impact on membrane-

dependent functions in myocardial or endothelial cells,

especially under high oxidative stress during reperfusion.

Under latter conditions, anaerobic metabolism induces the

decrease of intracellular pH, which can further influence the

radical scavenging properties of anthocyanins [25].

To examine the antioxidant properties of bilberry

anthocyanins in the absence of biologic structures, we

employed the DPPH radical scavenging method, which is

considered a standard method for assessing the in vitro

antioxidant activity. We observed strong concentration-

dependent correlation with the free radical–scavenging

capacity throughout the tested concentration range. These

observations are in agreement with many similar studies,

where total anthocyanins concentrations of different berries

were tested for their corresponding antioxidant activities by

using DPPH test [26]. To translate these observations to the

cellular level, we employed the novel CAA assay on the

endothelial EA.hy926 cell line. This assay uses radical

initiator ABAP, who spontaneously decomposes to form

peroxyl radicals, and thus simulates deleterious effects of

increased oxidative and nitrosative stress in I–R in vivo

conditions. In contrast to the results of the DPPH test, here

we obtained a saturable effect in the antioxidant activity of

bilberry anthocyanins. Thus, the concentration-dependent

increase in antioxidant activity was seen only in low


concentration range (\0.5 mg/L), while higher concentra-

tions ([0.5 mg/L) kept antioxidant activity constant, and

still partial. Indeed, this saturable activity of flavonoids was

already suggested by another study employing CAA assay,

where dose–response curves for blueberry extracts had

hyperbolic shape with apparent saturation in higher con-

centrations [10]. In addition, similar experiments on the

smooth muscle cell line A7r5, from the rat aorta, showed

less cellular anti-oxidant activity compared to endothelial

cells and similar concentration-dependent effect with an

apparent saturation (data not shown here). Nevertheless,

the highest tested concentration of bilberry anthocyanins

showed neither pro-oxidant activity nor reduced antioxi-

dant activity. Overall, these results showed that endothelial

cells have a limited intracellular capacity for antioxidant

protection induced by anthocyanins.

The possible mechanisms by which anthocyanins could

exert antioxidant activities include direct free radical–

scavenging capacities due to hydrogen (electron) donating

ability of a flavonoid molecule [27, 28]; or indirectly by

increasing the capacity of endogenous antioxidant defenses,

thereby modulating the cellular redox state. These include

numerous suggested mechanisms, such as increasing the

production of antioxidant NO by activation of endothelial

nitric oxide synthase [29, 30]; increasing the concentration

of thiol-containing tripeptide glutathione and small thiol-

containing proteins, such as thioredoxin, glutaredoxin, and

peroxiredoxin [31]; activation of antioxidative enzymes

such as superoxide dismutase, peroxidase, and catalase

[32]; reducing the formation of endogenous ROS by inhi-

bition of NADPH oxidase and xanthine oxidase [33], or by

modifying mitochondrial respiration and arachidonic acid

metabolism [34]; and many others. Paradoxically, protec-

tive activity of flavonoids on a local level can be even

considered in terms of their pro-oxidant properties, as

suggested by previous studies [8], since responses to a mild

degree of oxidative stress may activate compensatory

mechanisms that confer protection against repeated expo-

sure, thereby resulting in an overall net antioxidant activity.

Thus, the cellular levels of antioxidant defenses, and

xenobiotic-metabolising enzymes can be raised, thereby

providing overall cytoprotection [8, 35].

Even though anthocyanins have low bioavailability, the

observed potent cardioprotective properties are still in the

range of reported post-absorption plasma levels of antho-

cyanins. After oral administration of cyanidin 3-glucoside

in rats, the peak plasma concentration was 0.14 mg/L at

30 min and began to fall from 60 min [36]. In another

study, after a single oral administration of bilberry antho-

cyanins, the plasma concentrations reached peak levels

(2–3 mg/L) after 15 min, and then rapidly declined within

2 h [37]. These show that anthocyanins are absorbed and

rapidly enter the circulatory system, but they are prone to

extensive metabolism. However, peak concentrations of

total anthocyanins (parent and metabolites) found in plasma

of human subjects are in nanomolar to low micromolar

range [38], thereby providing rationale for using anthocy-

anins as nutraceuticals.

As a regnant belief, cardioprotective activity of flavonoids

in I–R has been traditionally attributed solely to their anti-

oxidant activity. However, recently novel protective mech-

anisms are arising, e.g. inhibition of signal transducer and

activator of transcription 1 (STAT1) by anthocyanidin del-

phinidin [39], and others. Thus, modulation of heart func-

tioning under conditions of increased oxidative stress must

be ascribed to multilevel biologic activities of flavonoids,

incorporating intracellular signal transduction cascades.

In conclusion, this study demonstrated that bilberry antho-

cyanins exert concentration-dependent biphasic responses

on isolated heart under I–R conditions. Low concentrations

conferred strong cardioprotective activity, while high con-

centrations aggravated I–R injury. Contrary to in vitro

studies on antioxidant activity of anthocyanins, which

showed strong concentration-dependent correlation, obser-

vations on a more complex biologic system, such as isolated

heart, are not well associated. Thus, in recent years, we are

observing the shift from studying the antioxidant properties

of flavonoids toward discovering novel biochemical path-

ways, thereby offering new unique therapeutic options in

heart disease prevention and health promotion. Further

investigations are required to determine the potential toxic

effects linked with prospective usage of anthocyanins in

specific clinical settings, e.g. during ischemic events,

whereas anthocyanins as dietary supplements are considered

safe.

Acknowledgments We thank the reviewers for their suggestions

that improved this paper. We are grateful for the financial support by

the Slovenian Research Agency [research projects J3-0024 and

Z4-2280]; grant for international mobility of students (Ad Futura,

Slovenia); Universita di Trieste (Italy); Fondazione Cassa di Rispar-

mio di Trieste and Ministero degli Affari Esteri (Cooperazione

scientifica e tecnologica Italia-Slovenia 2006-2009).

Conflict of interest None declared.

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