Pharmacological Research 113 (2016) 808–815

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Pharmacological Research
journal homepage:www.elsevier.com/locate/yphrs

Review

Anthocyanins in cardioprotection: A path through mitochondria

∗
Julius Liobikas, Kristina Skemiene, Sonata Trumbeckaite, Vilmante Borutaite
Neuroscience Institute, Lithuanian University of Health Sciences, Eiveniu str. 4, LT-50009 Kaunas, Lithuania

article info abstract

Article history: Constantly growing experimental data from in vitro, in vivo and epidemiological studies show the great potential of
Received 15 February 2016 anthocyanin-containing fruit and berry extracts or pure individual anthocyanins as cardioprotective food components or
Received in revised form 24 March 2016 pharmacological compounds. In general it is regarded that the car-dioprotective activity of anthocyanins is related to their
Accepted 29 March 2016 Available online 30
antioxidant properties. However there are recent reports that certain anthocyanins may protect the heart against
March 2016
ischemia/reperfusion-induced injury by activating signal transduction pathways and sustaining mitochondrial functions
instead of acting solely as antioxidants. In this review, we summarize the proposed mechanisms of direct or indirect actions of
Chemical compounds studied in this article:
anthocyanins within cardiac cells with the special emphasis on recently discovered their pharmacological effects on
Cyanidin (PubChem CID: 128861) Cyanidin 3-
O-glucoside (PubChem CID: 197081)
mitochondria in cardioprotection: reduction of cytosolic cytochrome c preventing apopto-sis and sustainment of electron
transfer between NADH dehydrogenase and cytochrome c supporting oxidative phosphorylation in ischemia-damaged
Cyanidin 3-O-galactoside (PubChem CID: mitochondria.
44256700)
Delphinidin (PubChem CID: 128853) © 2016 Elsevier Ltd. All rights reserved.
Delphinidin 3-O-glucoside (PubChem CID:
165558)
Petunidin 3-O-glucoside (PubChem CID
443651): Peonidin 3-O-glucoside (PubChem
CID: 443654)
Malvidin 3-O-glucoside (PubChem CID:
443652)
Malvidin 3-O-galactoside (PubChem CID:
94409)
Malvidin 3-O-arabinoside (PubChem CID:
91810654)
Gallic acid (PubChem CID: 370)
Protocatechuic acid (PubChem CID: 72)

Keywords:
Anthocyanin
Heart ischemia
Antioxidant
Signaling pathway
Mitochondrion
Oxidative phosphorylation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809
2. Heart ischemia/reperfusion-induced injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809

Abbreviations: Akt, serine/threonine specific protein kinase B; Cy, cyanidin; Cy3G, cyanidin 3-O-glucoside; Dp, delphinidin; Dp3G, delphinidin 3-O-glucoside; eNOS, endothelial NO synthase;
ERK1/2, extracellular-signal-regulated protein kinase 1 and 2; GSK3ˇ, glycogen synthase kinase 3 beta; JAK, Janus kinase; Mv, malvidin; Mv3G, malvidin 3-O-glucoside; Pg, pelargonidin; Pg3G,
pelargonidin 3-O-glucoside; PKC, protein kinase C; PKG, protein kinase G; PI3K, phosphatidylinositol 3-kinase; Pim-1, Pim-1 proto-oncogene serine/threonine protein kinase; Pn, peonidin; Pn3G,
peonidin 3-O-glucoside; Pt, petunidin; Pt3G, petunidin 3-O-glucoside; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription.

∗
Corresponding author.
E-mail address: vilmante.borutaite@lsmuni.lt (V. Borutaite).

http://dx.doi.org/10.1016/j.phrs.2016.03.036
1043-6618/© 2016 Elsevier Ltd. All rights reserved.
 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815 809

3. Chemical structure and natural sources of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809

4. Possible cardioprotective activities of anthocyanins and anthocyanin-rich extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810
4.1. Antioxidant effects of anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810
4.2. Effects of anthocyanins on intracellular signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
4.3. Effects on mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813 References . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813

1. Introduction

It is widely accepted that diets rich in fruits and vegetables pro-vide

beneficial effects on human health. Most of these effects are thought to be
related to flavonoids—polyphenolic compounds of coloured fruits, berries
and vegetables, which may act as strong antioxidants and prevent
inflammatory reactions leading to var-ious chronic diseases. Chronic
inflammation is considered as one of the main factors in development of
cardiovascular patholo-gies such as hypertension, atherosclerosis, endothelial
dysfuncion leading to heart ischemia, myocardial infarction and heart fail-ure.
Therefore search for new pharmacological means, particularly from natural
sources, for controlling such processes is of great value, and it is not
surprising that antioxidant and antiinflam-matory functions of flavonoids are
widely investigated. However in recent years accumulated evidence showed
that cardioprotec-tive mechanisms triggered by various flavonoids may
extend far beyond antioxidant actions of these conpounds (see reviews Refs.
[1–7]). Some of such effects, like modulation of intracellular sig-naling
pathways, have been also attributed to anthocyanins and their derivatives (i.e., Fig. 1. Scheme of ischemia/reperfusion targets in heart mitochondria leading to cell death.
Ischemia/reperfusion in the heart leads to inhibition of Complex I, ATP synthase, opening of
chemically different substances from other subgroups of flavonoids) which
mitochondrial permeability transition pore and subsequent release of cytochrome c, ATP
may affect regulation of cellular metabolism and survival under pathological depletion, apoptosis and cell death (for more details see text).
conditions including ischemia/reperfusion. Thus, in this short review we will
discuss recent experimental findings on effects of anthocyanins in protec-tion
against ischemic heart damage with particular emphasis on three important
activates procaspase-9 and initiates caspase cascade resulting in apoptotic cell
effects on mitochondria regulating cell death and survival during myocardial
death [17,18]. Though the loss of cytochrome c from mitochondria is usually
ischemia/reperfusion: (1) anthocyanins serving as substrates for Complex I of
considered as «a point of no return» after which cells become comited to
electron transfer system, (2) anthocyanins as mild uncouplers of oxidative
death by apoptosis or necrosis, there is experimental evidence suggesting that
phosphorylation, and (3) anthocyanins in reduction of cytosolic cytochrome c.
the execution phase of apoptosis can be regulated by the redox state of
cytochrome c, so that reduced form of cytochrome c has been shown to be less
potent in activating caspases than oxidized in cultured cells [19–21] or
isolated perfused hearts [22].

Anthocyanins, according to the recently reported in vitro and in vivo

2. Heart ischemia/reperfusion-induced injuries experiments with animals as well as several epidemiological or clinical studies
in humans, have been demon-strated to be effective in protection of the heart
Heart diseases, such as myocardial infarction or ishemic heart failure, are against ischemia/reperfusion-induced injury and in reduction of mortal-ity
the major causes of death in the developed countries, and unfortunately the related to cardiovascular diseases [23–29]. Putative molecular mechanisms of
mortality rate from these diseases is still increasing [8,9]. During ischemia, cardioprotective action of various anthocyanins will be discussed in further
the supply of oxygen and nutri-ents to the heart is impaired, and mitochondria sections.
in cardiomyocytes undergo rapid structural and functional changes:
suppression of oxidative phosphorylation mainly due to the inhibition of
Com-plex I of the electron transport system and/or ATP synthase, loss of 3. Chemical structure and natural sources of anthocyanins
mitochondrial membrane integrity due to opening of mitochon-drial
permeability transition pore and release of cytochrome c from mitochondria Anthocyanins are polyphenolic water soluble pigments provid-ing the
[10–13] (see Fig. 1). Furthermore, intracellular sig-naling pathways that blue, purple and red colour for various parts of plants especially in edible
promote apoptosis and necrosis are activated [14]. Inhibition of Complex I berries [30,31]. To date, more than 600 different anthocyanins have been
activity caused by short periods of ischemia usually can be reversed during reported [32] and that number constantly grows. Chemically, anthocyanins are
reperfusion, however pro-tracted ischemic periods cause severe irreversible the glycosylated forms of agly-cones (anthocyanidins), which are
inhibition of Complex I and subsequent cessation of ATP production by polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium salts
oxida-tive phosphorylation [15]. One of the earliest irreversible events in (Fig. 2).
ischemic heart damage—the liberation of cytochrome c from mito-chondria There are 30 different anthocyanidins identified and six of them, namely
may lead to cardiomyocyte apoptosis (reviewed in Ref. [16]). Cytochrome c delphinidin (Dp), cyanidin (Cy), malvidin (Mv), pelargoni-din (Pg), petunidin
when in cytosol binds to apoptotic protease-activating factor-1. This leads to (Pt), and peonidin (Pn), comprise aproximately 90% of all anthocyanidin
the formation of apoptosome that derivatives found in nature [30,31,33]. Interestingly, these six anthocyanidins
differ only in the presence of hydroxyl and methoxyl groups in the 3 and 5
positions in B ring
810 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815
Fig. 2. The basic structure of six most common anthocyanidins.
(see Fig. 2). Most anthocyanins are mono-, di-, or tri-glycosylated in C ring at
3 position or in A ring at 5 and 7 positions. Carbohydrate molecules bind to
anthocyanidins usually through an O-glycosidic bond forming mono-, di- or
tri- saccharide forms [34,35]. Glucose is the most common monosaccharide,
found in 90% of known antho-cyanins, the rest include galactose, arabinose,
rhamnose, and xylose residues in their structures [36]. Anthocyanidin 3-O-
glycosides are the most common forms, as they occur about twice as often as
3,5-O-diglycosides [33]. More than 65% of the reported anthocyanins are
acylated. The sugar units of anthocyanins may be acylated with aliphatic acyl
groups like malonic acid (which is the most frequent aliphatic acyl group),
acetic, malic, succinic, tartaric and oxalic acids, and/or aromatic acyl groups
(p-coumaric, caffeic, fer-ulic, sinapic, gallic and p-hydroxybenzoic acids)
[36]. Thus, it is generally accepted that the number and position of hydroxyl
side chains in the phenolic ring and even the extent of glycosylation of
aglycon lead to different biological effects of anthocyanins espe-cially their
oxidation-reduction properties [33,37,38].
Anthocyanins are found in certain cereal species, vegetables (eggplants,
red cabbage, red beans, blue onion, red radish), but are the most abundant in
fruits, especially berries (blueberries, cranberries, blackcurrants, raspberries,
elderberries, cherries and etc.). For example, it has been estimated that up to
1480 g of anthocyanins can be extracted from 100 g of fresh weight aro-nia
berries, 1380 g from the same amount of elderberries, while from black
raspberries—687 mg of anthocyanins [39], from fresh blueberries—588 mg
[40], and from red cabbage only 322 mg of anthocyanins [39]. It is also
known that anthocyanin content in fruits depends not only on the plant
cultivar or species but also on the climate and growing conditions, fruit
ripeness, storage and processing.
Bioavailability of anthocyanins and their metabolites is still under
investigation. It is known that certain anthocyanins, like Cy3G and Pg3G, are
absorbed into the gastrointestinal wall in their intact form. Part of the parent
compounds are metabo-lized into their corresponding phenolic acids or other
derivatives and can enter the systemic circulation. In serum, low micromo-lar
concentrations (up to 5,97 ± 2,14 M) of anthocyanins and their metabolites
can be reached [6,41–43]. In hearts and other tissues the highest reported
concentrations were in the range 0.06–2.37 nmol Cy3G equivalents/g tissue
[2]. More details on bioavailability of anthocyanins can be found in recently
published comprehensive reviews in Refs. [6,41,42].
4. Possible cardioprotective activities of anthocyanins and
anthocyanin-rich extracts
4.1. Antioxidant effects of anthocyanins
Oxidative stress plays an important role in the development of ischemic
heart diseases [44], and in vitro and in vivo studies suggest
the ability of anthocyanins or their metabolites to limit oxidative stress
[4,45,46]. Anthocyanins can remove ROS by various mecha-nisms: (a) direct
scavenging of ROS such as superoxide, hydroxyl radical, hydrogen peroxide,
and peroxynitrite [45], (b) induction of enzymes responsible for ROS removal
(for instance, increasing superoxide dismutase, catalase activity [4]), (c)
chelatation of metal ions needed to generate ROS [45], (d) modulation of ROS
form-ing enzymes such as xanthine oxidase, NADPH oxidase and others
[47,48].
The antioxidant activity of anthocyanins is largerly dependent on their
basic structure such as ring orientation, which determines the capacity of the
anthocyanin molecule to donate a hydrogen atom from a hydroxyl group to a
free radical [49], the number and position of hydroxyl groups around the
pyrone ring (the greater the number of OH groups—the greater is antioxidant
activity), presence of acyl groups [45], the position and degree of methoxyl
groups [38], the number of sugar residues and their position [45]. Among
several investigated anthocyanidins, Dp (for structure see Fig. 2) followed by
Cy and Pg have been found to be the most effec-tive in scavenging superoxide
anion, whereas Pg was the most powerful against the hydroxyl radical
[45,50,51]. Another study
[46] showed that potency of anthocyanins towards the superox-ide radical,
regardless of the sugar attached, was in the following order: Dp > Pt > Mv ≈
Cy > Pn > Pg. The peroxynitrite radical scav-enging activity was found to be
in a similar order [46].
Anthocyanins can also reduce oxidative stress in cells by modifying
mitochondrial respiration. The study on isolated heart mitochondria exposed
to anthocyanin-rich bilberry extract showed a significant decrease in
mitochondrial H2O2 production, which was associated with slight uncoupling
of oxidative phosphoryla-tion [52]. It is well documented that mild
uncoupling attenuates mitochondrial ROS generation due to decrease in
mitochondrial membrane potential [53–55]. The uncoupling effect seems to be
exerted mainly by Dp (containing three hydroxyl groups in B ring, Fig. 2) and
its glycosides (e.g. delphinidin 3-O-glucoside (Dp3G)) as other tested
anthocyanins such as cyanidin 3-O-glucoside (Cy3G; containing two hydroxyl
groups in B ring, Fig. 2) and pelargoni-din 3-O-glucoside (Pg3G; with one
hydroxyl group in B ring, Fig. 2) do not possess uncoupling activity [56].
There are no experimen-tal data published on the uncoupling effects of other
anthocyanins or their derivatives in relation to antioxidant activities in the
cells. Therefore at present only Dp and its glycosides and possibly Cy and Cy
3-O-rutinoside at higher concentrations (our unpublished data, see Section 4.3
below) may be considered as multifunctional antioxidants exerting ROS-
scavenging and ROS formation prevent-ing effects through mitochondrial
pathway.
In order to be effective antioxidants in tissues and organs, antho-cyanins
should be accumulated in the target tissues at appropriate intracellular
concentrations. However, some studies state that in vivo the measured
for ROS quenching.
concentrations of anthocyanins are too low
According to study by Kay and colleagues, con-
 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815
centrations of anthocyanins and their derivatives found in plasma of humans
is in nanomolar range [49]. On the other hand, such con-centrations may be
adequate to affect signal transduction and gene expression pathways leading
to changes in levels of antioxidant enzymes [57]. Such indirect antioxidant
actions of anthocyanins include activation of endothelial NO synthase (eNOS)
leading to increased production of NO which is effective scavenger of super-
oxide [58,59]. Certain anthocyanins can increase concentrations of thiol-
containing glutathione, glutarredoxin and thioredoxin [60], can cause
activation of catalase, peroxidase, superoxide dismutase
[61] or inhibition of xanthine oxidase and NADPH oxidase [47,48]. It has
been demonstrated that hearts of rats fed with anthocyanin-rich diet exhibited
higher resistance against ischemia/reperfusion insult, which correlated with
increased myocardial glutathione levels, suggesting that anthocyanins might
increase antioxidant defense capacity in cells as one of the possible
cardioprotective mechanisms [27].
On the other hand, polyphenols (including anthocyanins) can act as pro-
oxidants, especially when administered at high doses, and this may lead to
cellular dysfunction and cell death [62]. Study by Wang and colleagues
showed that acylated Pg deriva-tives extracted from red radish possess both
antioxidant and pro-oxidant activities depending on the reaction conditions
[63]. Ziberna and colleagues demonstrated that bilberry anthocyanins,
depending on concentration, exhibited biphasic response on iso-lated rat heart
under ischemia/reperfusion: at low concentrations (0.01–1 mg/L) they exerted
cardioprotection, whereas at high con-centration (25–50 mg/L) anthocyanins
were cardiotoxic [62].
4.2. Effects of anthocyanins on intracellular signaling pathways
According to a recent comprehensive rewiev by Penna and colleagues
[64] certain intracellular signaling molecules and path-ways are involved in
ishemia/reperfusion injury, and therefore, can be regarded as potential targets
for cardioprotection. These targets include: (a) activation of both the
phosphatidylinositol 3-kinase (PI3K)/serine/threonine specific protein kinase
B (Akt) pathway and the extracellular-signal-regulated protein kinase 1 and 2
(ERK1/2), (b) activation of Janus kinase (JAK) and modulation of signal
transducer and activator of transcription (STAT) proteins,
(c) activation of Pim-1 proto-oncogene serine/threonine protein kinases
(Pim-1) as well as (d) modulation of cell death/survival genes (Bax, Bak, Bcl-
2) (summarised in Fig. 3). Notably, it has been proposed that activation of the
above-mentioned kinases projects onto downstream targets, including protein
kinase G (PKG), pro-tein kinase C (PKC) or glycogen synthase kinase 3 beta
(GSK3 ), and ultimately to the mitochondrion to affect cell survival [64–67].
However, despite the extensive studies on the cardioprotective effects of
various flavonoids [1,2,4,68–71] the knowledge about anthocyanin-mediated
protection against ischemia/reperfusion injury at the cell-signaling level is
very scarce.
The increased expression of phosphorylated Akt with a con-comitant
elevation of eNOS activity, and therefore the attenuated oxidative stress, have
been shown in global ischemia/reperfusion-affected rat hearts after treatment
with nonalcoholic extract of Colombian blueberry (Vaccinium meridionale
Swartz, Ericaceae) containing Dp3G, Cy3G and petunidin 3-O-glucoside
(Pt3G) [72]. It has been also shown that long-term feeding of rats with
alcohol-free red wine extract rich in Dp3G, Pt3G, peonidin 3-O-glucoside
(Pn3G), malvidin 3-O-glucoside (Mv3G) and gallic acid (a metabolic product
of Dp3G) can prevent postinfarction cardiac remodeling by repressing PKC /
II phosphorylation and by activating Akt [73]. Recently it has been confirmed
that Mv3G-dependent cardiac effects involved the NO–cGMP–PKG pathway
and were associated with phosphorylation of PI3K, Akt, ERK1/2, and GSK3
[74]. Thus,
811
in the latter case the expected outcome would be a recruitment of
mitochondrial KATP channels to elicit a cardioprotection.
Ischemia/reperfusion injury also leads to cardiomyocyte necro-sis and
apoptosis with concomitant activation of JAK and STAT proteins, namely
STAT1, STAT3 and STAT5 [75]. While the activa-tion of STAT1 is associated
with enhanced cell death, the activation of STAT3 and STAT5 is essential for
cardioprotection—the decrease of necrosis and apoptosis [65,76]. Noteworthy,
STAT proteins are located not only in cytosols and nuclei of cardiomyocytes
but also in mitochondria where STAT1 and STAT3 have been recently found
to reside [77]. It has been shown that perfusion of isolated rat hearts with an
anthocyanidin Dp before regional ischemia/reperfusion resulted in both—the
inhibition of STAT1 activation without an effect on STAT3 phosphorylation
and in the reduced number of apoptotic and necrotic cells, suggesting that Dp
might block the STAT1-dependent extrinsic apoptotic pathway [26]. This also
sug-gests that Dp does not promote or hinder the cardioprotection via the
STAT3-mediated signal transduction from the plasma mem-brane to both the
nucleus and to the mitochondria [66,77].
Since the Bcl-family proteins are amongst the downstream effectors of
STAT proteins, Pim-1 kinase and PI3K/Akt pathway [67,75,78,79], one may
consider them as potential targets for anthocyanin-mediated cardioprotection
as well. Theoretically acti-vation or upregulation of anti-apoptotic proteins
(Bcl-2, Bcl-xL), suppression or downregulation of pro-apoptotic proteins
(Bak, Bax) and regulators of anti-apoptotic ones (Bad, Bim) would be a
desirable effect of anthocyanins leading to the preserved mitochon-drial
functionality and integrity, and halted cell death. However to the best of our
knowledge there are only indirect evidence on possible cardioprotective
effects of anthocyanins at Bcl-family protein levels. Thus, in a rat model of
cardiac injury, intake of blueberry extracts enriched in malvidin 3-O-
galactoside, malvidin 3-O-arabinoside, Mv3G, petunidin 3-O-galactoside,
Pt3G, Pn3G, cyanidin 3-O-galactoside, Dp3G and acetylated Mv
anthocyanins resulted in decreased Bax protein expression, and increased Bcl-
2 protein expression in cardiomyocytes [80]. In a model of type 1 diabetes
using streptozotocin-induced rats, administration of pro-tocatechuic acid, a
major metabolite of Cy, in combination with insulin significantly improved
cardiac mitochondrial function and increased Bcl-2 expression [81]. However,
causative relationships between application of anthocyanins, expression of
Bcl-2 proteins and cardioprotective mechanisms in these studies were not
fully elucidated.
Although data mentioned above indicate that anthocyanins and their
metabolites trigger the intracellular effectors (see Fig. 3), fur-ther experiments
are required to reveal how they are activated. It is suggested that anthocyanin-
dependent cardiac modulation can occur via a receptor–ligand mechanism
[74], as it is proposed for flavonols quercetin and myricetin [82]. Moreover,
the direct interaction between anthocyanins and intracellular signaling path-
ways (e.g., JAK/STAT) could not be ruled out as well. For example, molecule
docking simulations predicted good binding affinities for Dp and two putative
sites at the SH2 domain on the STAT1 therefore suggesting an anthocyanin-
mediated STAT1-specific mechanism of inhibition [26]. However, due to the
lack of evidence a question whether the effectors, like Bcl-family proteins,
acting on mitochon-dria as the final target could be directly or indirectly
affected by anthocyanins or their metabolites remains open. The situation is
further complicated due to the possible interplay of intracellular pathways
involved in ischemia/reperfusion injury suggesting the complex nature of
treatment specificity.
4.3. Effects on mitochondria
Mitochondrial damage during ischemia/reperfusion is consid-ered as a key
event leading to cardiomyocyte dysfunction and cell
812 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815

Fig. 3. Scheme of possible interactions between anthocyanins and intracellular targets involved in cardioprotection. Anthocyanins can act as ROS scavengers or prevent ROS generation by
mitochondria and other enzymes described in the text (Section 4.1). Anthocyanins interact with indicated signaling pathways (see text in Section 4.2). Anthocyanins can serve as electron transfering
molecules between Complex I (I) of the mitochondrial electron transport system and cytochrome c (CytC) supporting ATP synthesis by oxidative phosphorylation. Anthocyanins can reduce
cytochrome c which is released into cytosol preventing caspase activation (for detailed discussion see the text Section 4.3). Inhibitory effects are shown by (-|), other effects—by arrowheads (→).

death. This makes mitochondria attractive target for pharmaco-logical
interventions directed to prevent ischemic heart injuries. In this respect,
effects of various flavonoids (usually in relation to their antioxidant
properties) on mitochondrial functions have been widely investigated
(reviewed in Refs. [69,83–85]). In con-trast, experimental data on possible
direct interactions between anthocyanins or anthocyanidins and mitochondria
are scarce and incomplete. Relatively low interest of investigators on such
effects may be due, in part, to the fact that bioavailability of dietary antho-
cyanins was thought to be low [6]. Absorbtion and distribution of these
compounds inside cells is underinvestigated. Situation started changing after
discovery that certain flavonoids such as quercetin and epigallocatechin-3-
galate, which have some struc-tural similarities with anthocyanins,
accumulate in large amounts in mitochondria [86,87]. This raises possibility
that similar pattern of intracellular distribution may occur with other
flavonoids includ-ing anthocyanins and this needs further detailed
investigation. Recently it has been reported that bioavailability of
anthocyanins is much higher than previously thought and in vivo
concentrations of anthocyanins may be comparable to that of other flavonoid
sub-classes, such as flavan-3-ols (e.g., epigallocatechin-3-galate) [41].
Though there are more than 600 various anthocyanins, yet only very few
of them have been tested for their interactions with mitochondria and
oxidative phosphorylation system. These include glycosides of Dp, Cy, Mv,
Pg and some other. Anthocyanins that were tested at physiologically or
pharmacologically relevant concentrations (up to low micromolar) had no
direct effect on phos-phorylating respiration in state 3 or non-phosphorylating
(or leak) respiration in state 4 in mitochondria isolated from heart, liver or
brain [52,88,89]. It is important to note that Dp3G (in contrast to Cy3G and
Pg3G) at 40 M and higher concentrations caused a slight uncoupling of
oxidative phosphorylation in heart mito-chondria [88]. Our unpublished data
also demonstrate that Cy and Cy 3-O-rutinoside at higher than 50 M
concentrations increased
non-phosphorylating respiration in state 4 (substrates pyruvate plus malate),
whereas Cy3G had no effect. These results suggest that the observed
variations in activities of anthocyanins on mito-chondrial respiratory rates
might be attributed to the presence of different glycosyl groups in the
anthocyanin molecule. However, more detailed analysis is necessary to clarify
this mechanism.
In addition, Dp3G and Dp 3-O-rutinoside at 40 M and higher
concentration caused inhibition of state 3 respiration rate in iso-lated heart
mitochondria (unpublished data). Such an effect may be due to pro-oxidant
properties of anthocyanins and may be responsible for cardiotoxicity observed
when higher concentra-tions of bilberry anthocyanins were used in
ischemia/reperfusion model as described in study by Ziberna et al. [62].
Nevertheless, there is a substantial line of evidence that at low or moderate
(nanomolar and low micromolar) concentrations anthocyanins or anthocyanin-
rich extracts of fruits and berries can protect against mitochondrial
dysfunction caused by oxidative stress or mito-chondrial poisons. Serraino
and colleagues have reported that blackberry juice containing 14.5–80 ppm
Cy3G or chemically pure Cy3G chloride at 8.5–85 nM concentrations reduced
peroxynitrite-induced inhibition of mitochondrial respiration in human
umbilical vein endothelial cells and protected against endothelial dysfunc-tion
[90]. This effect was attributed to peroxynitrite-scavenging properties of
anthocyanin, though other explanations could be also possible as peroxynitrite
may cause inhibition of Complex I of the mitochondrial electron transport
system [91] (explained below). In later studies, it has been shown that extracts
of berries rich in anthocyanins and proanthocyanidins (but not in other
flavonoids) stimulated mitochondrial respiration in cultured neuronal cells
exposed to a specific inhibitor of the mitochondrial electron trans-fer system
—rotenone, and protected against rotenone-induced cell death [92].
Inhibition of Complex I of the electron transport sys-tem is
one of the main injuries occuring during prolonged heart
ischemia/reperfusion causing supression of ATP production in
 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815
oxidative phosphorylation system [16]. Therefore we extended this line of
research and showed that in the absence of coenzyme Q or in ischemia-
damaged heart mitochondria, Cy3G and Dp3G can serve as electron acceptors
from Complex I stimulating NADH oxidation by NADH dehydrogenase [89]
(see Fig. 3). Cy3G and Dp3G also stimulated state 3 respiration rate and ATP
synthesis in ischemia-damaged mitochondria with pyruvate, malate and
glutamate as respiratory substrates, but had no effect on Complex II-
dependent mitochondrial respiration [89]. Interestingly, such an effect was
observed only with certain anthocyanins (Cy3G and Dp3G) but not with other
such as Pg3G. The main difference between these sub-groups of anthocyanins
(‘good’ and ‘poor’ Complex I substrates) seems to be related to their reductive
capacity [88]. Capacity of anthocyanins to transfer electrons from
mitochondrial NADH dehydrogenase to cytochrome c may be important in
aleviating ischemia-induced inhibition of Complex I and preventing deple-
tion of ATP in ischemic/reperfused myocardium, thus preventing heart
dysfunction and necrotic cell death.
Anthocyanins are redox active compounds and, as such, they possess an
ability to reduce cytochrome c–a component of the mitochondrial eletron
transport system. It has been demonstrated that the highest cytochrome c
reducing activity was observed with Dp3G and Cy3G whereas other
anthocyanins such as Mv3G, Pn3G and Pg3G exhibited much lower reducing
activities [88]. Cytochrome c reducing activities of anthocyanins correlate
with their cardioprotective effects, so that Cy3G but not Pg3G have been
shown to protect against heart ischemia-induced caspase activa-tion and
cardiomyocyte apoptosis and necrosis [56,88]. However as shown in [88], no
one of these anthocyanins protected against ischemia-induced loss of
cytochrome c from mitochondria and sub-sequent inhibition of mitochondrial
respiration with succinate as substrate (which has been shown to be related to
cytochrome c deficiency in ischemia-damaged mitochondria [93]). These
find-ings support the hypothesis (reviewed in Ref. [94]) that reduction of
cytosolic cytochrome c by such compounds as Cy3G and Dp3G can prevent
caspase activation in ischemic myocardium and protect against ischemia-
induced cell death.
Taken together experimental data suggest that certain antho-cyanins such
as Cy3G and Dp3G can form an additional electron transport chain in
ischemia-damaged heart mitochondria serv-ing as electron acceptors at
Complex I and directly reducing cytochrome c, which then stimulates
respiration and ATP produc-tion. In addition, anthocyanins with high
reductive capacity can reduce cytosolic cytochrome c, preventing caspase
activation and cell death in ischemic/reperfused myocardium (see Fig. 3).
There are studies demonstrating that a number of flavonoids can exert
effects on mitochondrial ion channels, calcium accu-mulation and
mitochondrial permeability transition pore [69]. However, anthocyanins are
not investigated yet in this aspect. There is some indirect evidence that
anthocyanins do not protect against ischemia-induced opening of
mitochondrial permeability transition pore as they do not prevent ischemia-
induced loss of cytochrome c [88] which has been previously shown to be
caused by permeability transtition pore [93].
5. Conclusions
Anthocyanins were found to exert multiple effects on cel-lular metabolic
processes, intracellular signaling and oxidative stress which may be important
for cardiomyocyte survival after ischemia/reperfusion. These effects also
include direct actions of anthocyanins on mitochondria suporting oxidative
phosphory-lation and ATP production in ischemia-damaged mitochondria,
mild uncoupling preventing ROS generation and reduction of cytochrome c
preventing apoptosis. The latter mechanism, reduc-
813
tion of cytosolic cytochrome c, suggests that anthocyanins could be protective
at reperfusion, and this may have important clini-cal implications.
Anthocyanins or anthocyanin-based compounds can be applied as additives at
reperfusion of infarcted myocardium or after heart bypass surgery preventing
ischemia/reperfusion-induced apoptosis and supporting energy supply to
recovering myocardium. Anthocyanins and their derivatives can be used in
organ preservation solutions in heart transplantations. Further studies on
application of anthocyanins at post-ischemic periods are necessary.
Anthocyanins are relatively unstable compounds under physiological
conditions and their accumulation in cells is not well investigated. This
prompts to further investigations of pharmacoki-netic and pharmacodynamic
properties of various anthocyanins and their derivatives, possibilities to
chemically modify the com-pounds to improve their beneficial, health-
promoting properties. There are also un-answered questions regarding effects
of antho-cyanins in ischemic pathologies in other organs such as the brain or
application in preservation solutions during transplantation of organs.
Functions of certain anthocyanins as alternative elec-tron carriers in the
mitochondrial electron transport system may have important implications in
treatment or prevention of diseases related to dysfunction of Complex I
(Parkinson‘s or other neurode-generative diseases).
Acknowledgments
The research was supported by the Lithuanian Science and Stud-ies
Foundation grant awarded to S.T. and J.L (No. N-07015) and by the Research
Council of Lithuania (J.L.; No. SVE-11008). V.B. and J.L. were supported by
the COST Action FA0602 «Bioactive food com-ponents, mitochondrial
function and health». V.B. and K.S. were supported by European Social Fund
under the Global grant measure (project No. VP1-3.1-SMM-07-K-01-130).
References
[1] M. Akhlaghi, B. Bandy, Mechanisms of flavonoid protection against myocardial ischemia-
reperfusion injury, J. Mol. Cell Cardiol. 46 (2009) 309–317.
[2] S. de Pascual-Teresa, D.A. Moreno, C. Garcia-Viguera, Flavanols and anthocyanins in
cardiovascular health: a review of current evidence, Int. J. Mol. Sci. 11 (2010) 1679–
1703.
[3] M. Yadav, S. Jain, A. Bhardwaj, R. Nagpal, M. Puniya, R. Tomar, V. Singh, O. Parkash,
G.B.K.S. Prasad, F. Marrotta, H. Yadav, Biological and medicinal properties of grapes
and their bioactive constituents: an update, J. Med. Food 12 (2009) 473–484.
[4] M.J. Kruger, N. Davies, K.H. Myburgh, S. Lecour, Proanthocyanidins, anthocyanins
and cardiovascular diseases, Food Res. Int. 59 (2014) 41–52.
[5] L.M. McCune, C. Kubota, N.R. Stendel-Hollis, A. Cynthia, Cherries and health: a review,
Crit. Rev. Food Sci. Nutr. 51 (2011) 1–12.
[6] A. Speciale, F. Cimino, A. Saija, R. Canali, F. Virgili, Bioavailability and molecular
activities of anthocyanins as modulators of endothelial function, Genes Nutr. 9 (2014)
404.
[7] T. Tsuda, Dietary anthocyanin-rich plants: biochemical basis and recent progress in
health benefits studies, Mol. Nutr. Food Res. 56 (2012) 159–170.
[8] D. McCartney, P. Scarborough, P. Webster, M. Rayner, Trends in social inequalities for
premature coronary heart disease mortality in Great Britain, a time trend ecological study,
BMJ Open 2 (3) (2012) 1994–2008.
[9] A.M. Minino, S.L. Murphy, J. Xu, K.D. Kochanek, Deaths: final data for 2008, Natl.
Vital Stat. Rep. 59 (2011) 1–126.
[10] Q. Chen, S. Moghaddas, C.I. Hoppel, E.J. Lesnefsky, Ischemic defects in the electron
transport chain increase the production of reactive oxygen species from isolated rat
heart mitochondria, Am. J. Cell Physiol. 294 (2008) C460–C466.
[11] E.J. Lesnefsky, C.L. Hoppel, Cardiolipin as an oxidative target in cardiac mitochondria in
the aged rat, Biochim. Biophys. Acta 1777 (2008) 1020–1027.
[12] P. Pasdois, J.E. Parker, E.J. Griffiths, A.P. Halestrap, The role of oxidized cytochrome c in
regulating mitochondrial reactive oxygen species production and its perturbation in
ischaemia, Biochem. J. 436 (2011) 493–505.
[13] S.B. Ong, P. Samangouei, C.B. Kalkhoran, D.J. Hausenloy, The mitochondrial
permeability transition pore and its role in myocardial ischemia reperfusion injury, J.
Mol. Cell Cardiol. 78 (2015) 23–34.
[14] S. Kumphune, S. Surinkaew, S.C. Chattipakorn, N. Chattipakorn, Inhibition of p38
MAPK activation protects cardiac mitochondria from ischemia/reperfusion injury,
Pharm. Biol. 53 (2015) 1831–1841.
814 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815
[15] E.J. Lesnefsky, Q. Chen, T.J. Slabe, M.S. Stoll, P.E. Minkler, M.O. Hassan, B. Tandler,
C.L. Hoppel, Ischemia, rather than reperfusion, inhibits respiration through
cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin, Am. J.
Physiol. Heart Circ. Physiol. 287 (2004) 258–267.
[16] V. Borutaite, A. Toleikis, G.C. Brown, In the eye of the storm: mitochondrial damage
during heart and brain ischaemia, FEBS J. 280 (2013) 4999–5014.
[17] J. Yang, X. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J. Cai, T.I. Peng, D.P. Jones, X.
Wang, Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria
blocked, Science 275 (1997) 1129–1132.
[18] P. Li, D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, X. Wang,
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an
apoptotic protease cascade, Cell 91 (1997) 479–489.
[19] V. Borutaite, G.C. Brown, Mitochondrial regulation of caspase activation by
cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c
redox state, J. Biol. Chem. 282 (2007) 31124–31130.
[20] Z. Pan, D.W. Voehringer, R.E. Meyn, Analysis of redox regulation of cytochrome c-
induced apoptosis in a cell-free system, Cell Death Differ. 6 (1999) 683–688.
[21] D. Suto, K. Sato, Y. Ohba, T. Yoshimura, J. Fujii, Suppression of the pro-apoptotic
function of cytochrome c by singlet oxygen via a haem redox state-independent
mechanism, Biochem. J. 392 (2005) 399–406.
[22] J. Barauskaite, R. Grybauskiene, R. Morkuniene, V. Borutaite, G.C. Brown,
Tetramethylphenylenediamine protects the isolated heart against ischaemia-induced
apoptosis and reperfusion-induced necrosis, Br. J. Pharmacol. 162 (2011) 1136–
1142.
[23] A. Cassidy, K.J. Mukamal, L. Liu, M. Franz, A.H. Eliassen, E.B. Rimm, High
anthocyanin intake is associated with a reduced risk of myocardial infarction in young
and middle-aged women, Circulation 127 (2013) 188–196.
[24] A. Cassidy, G. Rogers, J.J. Peterson, J.T. Dwyer, H. Lin, P.F. Jacques, Higher dietary
anthocyanin and flavonol intakes are associated with anti-inflammatory effects in a
population of US adults, Am. J. Clin. Nutr. 102 (2015) 172–181.
[25] P.J. Mink, C.G. Scrafford, L.M. Barraj, L. Harnack, C.P. Hong, J.A. Nettleton, D.R.
Jacobs, Flavonoid intake and cardiovascular disease mortality: a prospective study in
postmenopausal women, Am. J. Clin. Nutr. 85 (2007) 895–909.
[26] T.M. Scarabelli, S. Mariotto, S. Abdel-Azeim, K. Shoji, E. Darra, A. Stephanou, C. Chen-
Scarabelli, J.D. Marechal, R. Knight, A. Ciampa, L. Saravolatz, A.C. de Prati, Z. Yuan, E.
Cavalieri, M. Menegazzi, D. Latchman, C. Pizza, D. Perahia, H. Suzuki, Targeting STAT1
by myricetin and delphinidin provides efficient protection of the heart from
ischemia/reperfusion-induced injury, FEBS Lett. 583 (2009) 531–541.
[27] M.C. Toufektsian, L.M. de, N. Nagy, P. Salen, M.B. Donati, L. Giordano, H.P. Mock, S.
Peterek, A. Matros, K. Petroni, R. Pilu, D. Rotilio, C. Tonelli, L.J. de, F. Boucher, C.
Martin, Chronic dietary intake of plant-derived anthocyanins protects the rat heart against
ischemia-reperfusion injury, J. Nutr. 138 (2008) 747–752.
[28] X. Wang, Y.Y. Ouyang, J. Liu, G. Zhao, Flavonoid intake and risk of CVD: a systematic
review and meta-analysis of prospective cohort studies, Br. J. Nutr. 111 (2014) 1–11.
[29] T.L. Zern, R.J. Wood, C. Greene, K.L. West, Y. Liu, D. Aggawal, N.S. Shachter, M.L.
Fernandez, Grape polyphenols exert a cardioprotective effect in pre- and postmenopausal
women by lowering plasma lipids and reducing oxidative stress, J. Nutr. 135 (2005) 1911–
1917.
[30] J. Fleschhut, F. Kratzer, G. Rechkemmer, S.E. Kulling, Stability and
biotransformation of various dietary anthocyanins in vitro, Eur. J. Nutr. 45 (2006) 7–
18.
[31] J.M. Kong, L.S. Chia, N.K. Goh, T.F. Chia, R. Brouillard, Analysis and biological
activities of anthocyanins, Phytochemistry 64 (2003) 923–933.
[32] J. He, M.M. Giusti, Anthocyanins: natural colourants with health promoting properties,
Annu. Rev. Food Sci. Technol. 1 (2010) 163–187.
[33] R.L. Prior, X. Wu, Anthocyanins: structural characteristics that result in unique metabolic
patterns and biological activities, Free Radic. Res. 40 (2006) 1014–1028.
[34] O. Bjoroy, T. Fossen, O.M. Andersen, Anthocyanin 3-galactosides from Cornus alba
‘Sibirica’ with glucosidation of the B-ring, Phytochemistry 68 (2007) 640–645.
[35] C.A. Williams, R.J. Grayer, Anthocyanins and other flavonoids, Nat. Prod. Rep. 21
(2004) 539–573.
[36] M.N. Clifford, Anthocyanins—nature, occurance and dietary, J. Sci. Food Agric. 80
(2000) 1063–1072.
[37] Y. Yao, A. Vieira, Protective activities of Vaccinium antioxidants with potential relevance
to mitochondrial dysfunction and neurotoxicity, Neurotoxicology 28 (2007) 93–100.
[38] J. Muselik, M. Garcia-Alonso, M.P. Martin-Lopez, M. Zemlicka, J.C. Rivas-Gonzalo,
Measurement of antioxidant activity of wine catechins, procyanidins, anthocyanins and
pyranoanthocyanins, Int. J. Mol. Sci. 8 (2007) 97–109.
[39] X. Wu, G.R. Beecher, J.M. Holden, D.B. Haytowitz, S.E. Gebhardt, R.L. Prior,
Concentrations of anthocyanins in common foods in the United States and estimation of
normal consumption, J. Agric. Food Chem. 54 (2006) 4069–4075.
[40] N.A. Nyman, J.T. Kumpulainen, Determination of anthocyanidins in berries and red
wine by high-performance liquid chromatography, J. Agric. Food Chem. 49 (2001)
4183–4187.
[41] C. Czank, A. Cassidy, Q. Zhang, D.J. Morrison, T. Preston, P.A. Kroon, N.P. Botting,
C.D. Kay, Human metabolism and elimination of the anthocyanin,
cyanidin-3-glucoside: a (13)C-tracer study, Am. J. Clin. Nutr. 97 (2013)
995–1003.
[42] J. Fang, Bioavailability of anthocyanins, Drug Metab. Rev. 46 (2014) 508–520.
[43] I. Fernandes, A. Faria, C. Calhau, V. de Freitas, N. Mateus, Bioavailability of
anthocyanins and derivatives, J. Funct. Foods 7 (2014) 54–66.
[44] J. Dai, R.J. Mumper, Plant phenolics: extraction, analysis and their antioxidant and
anticancer properties, Molecules 15 (2010) 7313–7352.
[45] M.G. Miguel, Anthocyanins: antioxidant and/or anti-inflammatory activities, J. Appl.
Pharm. Sci. 01 (2011) 07–15.
[46] M.M. Rahman, T. Ichiyanagi, T. Komiyama, Y. Hatano, T. Konishi, Superoxide radical-
and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and
their synergism, Free Radic. Res. 40 (2006) 993–1002.
[47] M. Braunlich, R. Slimestad, H. Wangensteen, C. Brede, K.E. Malterud, H. Barsett,
Extracts, anthocyanins and procyanidins from aronia melanocarpa as radical scavengers
and enzyme inhibitors, Nutrients 5 (2013) 663–678.
[48] T.G. Lim, S.K. Jung, E.J. Kim, Y. Kim, H.J. Lee, T.S. Jang, K.W. Lee, NADPH oxidase is
a novel target of delphinidin for the inhibition of UVB-induced MMP-1 expression in
human dermal fibroblasts, Exp. Dermatol. 22 (2013) 417–437.
[49] C.D. Kay, G. Mazza, B.J. Holub, J. Wang, Anthocyanin metabolites in human urine
and serum, Br. J. Nutr. 91 (2004) 933–942.
[50] D.S. Antal, G. Garban, Z. Garban, The anthocyanins: biologically-active
substances of food and pharmaceutic interest, Food Technol. 6 (2003) 106–115.
[51] T. Tsuda, F. Horio, K. Uchida, H. Aoki, T. Osawa, Dietary cyanidin
3-O-beta-d-glucoside-rich purple corn color prevents obesity and ameliorates
hyperglycemia in mice, J. Nutr. 133 (2003) 2125–2130.
[52] S. Trumbeckaite, D. Burdulis, L. Raudone, J. Liobikas, A. Toleikis, V. Janulis, Direct
effects of Vaccinium myrtillus L: fruit extracts on rat heart mitochondrial functions,
Phytoter. Res. 27 (2013) 499–506.
[53] S. Miwa, M.D. Brand, Mitochondrial matrix reactive oxygen species production is
very sensitive to mild uncoupling, Biochem. Soc. Trans. 31 (2003) 1300–1301.
[54] M.N. Sack, Mitochondrial depolarization and the role of uncoupling proteins in ischemia
tolerance, Cardiovasc. Res. 72 (2006) 210–219.
[55] V.P. Skulachev, Uncoupling: new approaches to an old problem of
bioenergetics, Biochim. Biophys. Acta 1363 (1998) 100–124.
[56] K. Skemiene, G. Jablonskiene, J. Liobikas, V. Borutaite, Protecting the heart against
ischemia/reperfusion-induced necrosis and apoptosis: the effect of anthocyanins,
Medicina (Kaunas.) 49 (2013) 84–88.
[57] P.E. Milbury, J.A. Vita, J.B. Blumberg, Anthocyanins are bioavailable in humans
following an acute dose of cranberry juice, J. Nutr. 140 (2010) 1099–1104.
[58] M.C. Lazze, R. Pizzala, P. Perucca, O. Cazzalini, M. Savio, L. Forti, V. Vannini, L.
Bianchi, Anthocyanidins decrease endothelin-1 production and increase endothelial nitric
oxide synthase in human endothelial cells, Mol. Nutr. Food Res. 50 (2006) 44–51.
[59] C.A. Schmitt, V.M. Dirsch, Modulation of endothelial nitric oxide by plant-
derived products, Nitric Oxide 21 (2009) 77–91.
[60] J.O. Moskaug, H. Carlse, M.C. Myhrstad, R. Blomhoff, Polyphenols and
glutathione synthesis regulation, Am. J. Clin. Nutr. 81 (2005) 277S–283S.
[61] K. Han, M. Sekikawa, K. Shimada, M. Hashimoto, N. Hashimoto, T. Noda, H. Tanaka, M.
Fukushima, Anthocyanin-rich purple potato flake extract has antioxidant capacity and
improves antioxidant potential in rats, Br. J. Nutr. 96 (2006) 1125–1133.
[62] L. Ziberna, M. Lunder, S. Moze, A. Vanzo, F. Tramer, S. Passamonti, G. Drevensek,
Acute cardioprotective and cardiotoxic effects of bilberry anthocyanins in ischemia-
reperfusion injury: beyond concentration-dependent antioxidant activity, Cardiovasc.
Toxicol. 10 (2010) 283–294.
[63] L.S. Wang, X.D. Sun, L. Wang, F.J. Li, Y.F. Wang, Antioxidant and pro-oxidant
properties of acylated pelargonidin derivatives extracted from red radish (Raphanus
sativus var. niger, Brassicaceae), Food. Chem. Toxicol. 48 (2010) 2712–2718.
[64] C. Penna, M. Perrelli, P. Pagliaro, Mitochondrial pathways, permeability transition
pore, and redox signaling in cardioprotection: therapeutic implications, Antioxid.
Redox. Signal. 18 (2013) 556–599.
[65] K. Boengler, D. Hilfiker-Kleiner, H. Drexler, G. Heusch, R. Schulz, The myocardial
JAK/STAT pathway: from protection to failure, Pharmacol. Ther. 120 (2008) 172–185.
[66] D.J. Hausenloy, S. Lecour, D.M. Yellon, Reperfusion injury salvage kinase and survivor
activating factor enhancement prosurvival signaling pathways in ischemic
postconditioning: two sides of the same coin, Antioxid. Redox. Signal. 14 (2011) 893–
907.
[67] K. Samse, N. Hariharan, M.A. Sussman, Personalizing cardiac regenerative therapy:
at the heart of Pim1 kinase, Pharmacol. Res. 103 (2016) 13–16.
[68] A. Crozier, I.B. Jaganath, M.N. Clifford, Dietary phenolics: chemistry, bioavailability
and effects on health, Nat. Prod. Rep. 26 (2009) 1001–1043.
[69] L. Testai, Flavonoids and mitochondrial pharmacology: a new paradigm for
cardioprotection, Life Sci. 135 (2015) 68–76.
[70] C. Chen, H. He, Y. Luo, M. Zhou, D. Yin, M. He, Involvement of Bcl-2 signal pathway in
the protective effects of apigenin on anoxia/reoxygenation-induced myocardium injury, J.
Cardiovasc. Pharmacol. 67 (2016) 152–163.
[71] J. Huang, X. Zhang, F. Qin, Y. Li, X. Duan, J. Jian, Y. Li, J. Chen, R. Huang,
Protective effects of Millettia pulchra flavonoids on myocardial ischemia in vitro
and in vivo, Cell Physiol. Biochem. 35 (2015) 516–528.
 J. Liobikas et al. / Pharmacological Research 113 (2016) 808–815
[72] Y.E. Lopera, J. Fantinelli, L.F. Gonzilez Arbeliez, B. Rojano, J.L. Rios, G. Schinella,
S. Mosca, Antioxidant activity and cardioprotective effect of a nonalcoholic extract of
Vaccinium meridionale swartz during ischemia-reperfusion in rats, Evid.-Based
Complement. Altern. Med. (2013) 1–10.
[73] A. Palfi, E. Bartha, L. Copf, L. Mark, F. Gallyas Jr., B. Veres, E. Kalman, L. Pajor, K.
Toth, R. Ohmacht, B. Sumegi, Alcohol-free red wine inhibits isoproterenol-induced
cardiac remodeling in rats by the regulation of Akt1 and protein kinase C alpha/beta II, J.
Nutr. Biochem. 20 (2009) 418–425.
[74] A.M. Quintieri, N. Baldino, E. Filice, L. Seta, A. Vitetti, B. Tota, B. De Cindio, M.C.
Cerra, T. Angelone, Malvidin, a red wine polyphenol, modulates mammalian myocardial
and coronary performance and protects the heart against ischemia/reperfusion injury, J.
Nutr. Biochem. 24 (2012) 1221–1231.
[75] S. Lecour, Activation of the Protective Survivor Activating Factor Enhancement
(SAFE) pathway against reperfusion injury: does it go beyond the RISK pathway? J.
Mol. Cell Cardiol. 47 (2009) 32–40.
[76] G. Heusch, J. Musiolik, E. Kottenberg, J. Peters, H. Jakob, M. Thielmann, STAT5
activation and cardioprotection by remote ischemic preconditioning in humans: short
communication, Circ. Res. 110 (2012) 111–115.
[77] K. Boengler, D. Hilfiker-Kleiner, G. Heusch, R. Schulz, Inhibition of permeability
transition pore opening by mitochondrial STAT3 and its role in myocardial
ischemia/reperfusion, Basic Res. Cardiol. 105 (2010) 771–785.
[78] X. Meng, H. Pei, C. Lan, Icariin exerts protective effect against myocardial
ischemia/reperfusion injury in rats, Cell Biochem. Biophys. 73 (2015) 229–235.
[79] J. Wang, S.Y. Ji, S.Z. Liu, R. Jing, W.J. Lou, Cardioprotective effect of breviscapine:
inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway
following simulated ischemia/reperfusion injury, Pharmazie 70 (2015) 593–597.
[80] Y. Liu, D. Tan, L. Shi, Y. Zhang, C. Tong, D. Song, M. Hou, Blueberry
anthocyanins-enriched extracts attenuate cyclophosphamide-induced cardiac
injury, PLoS One 10 (2015).
[81] Y. Semaming, S. Kumfu, P. Pennangpetch, S.C. Chattipakorn, N. Chattipakorn,
Protocatechuic acid exerts a cardioprotective effect in type 1 diabetic rats, J. Endocrinol.
223 (2014) 13–23.
[82] T. Angelone, T. Pasqua, D. Di Majo, A.M. Quintieri, E. Filice, N. Amodio, B. Tota,
M. Giammanco, M.C. Cerra, Distinct signalling mechanisms are involved in the
dissimilar myocardial and coronary effects elicited by quercetin and myricetin, two red
wine flavonols, Nutr. Metab. Cardiovasc. Dis. 21 (2011) 362–371.
[83] M.R. De Oliveira, S.M. Nabavi, N. Braidy, W.N. Setzer, T. Ahmed, S.F. Nabavi,
Quercetin and the mitochondria: a mechanistic view, Biotecnol. Adv. (2015), in press.
815
[84] M.R. De Oliveira, S.F. Nabavi, M. Daglia, L. Rastrelli, S.M. Nabavi,
Epigallocatechin gallate and mitochondria—a story of life and death,
Pharmacol. Res. 104 (2015) 70–85.
[85] C. Sandoval-Acuna, J. Ferreira, H. Speisky, Polyphenols and mitochondria: an update on
their increasingly emerging ROS-scavenging independent actions, Arch. Biochem.
Biophys. 559 (2014) 75–90.
[86] M. Fiorani, A. Guidarelli, M. Blasa, C. Azzolini, M. Candiracci, E. Piatti, O. Cantoni,
Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial
damage and release upon oxidation of the extramitochondrial fraction of the flavonoid, J.
Nutr. Biochem. 21 (2010) 397–404.
[87] E.K. Schroeder, N.A. Kelsey, J. Doyle, E. Breed, R.J. Bouchard, F.A. Loucks, R.A.
Harbison, D.A. Linseman, Green tea epigallocatechin 3-gallate accumulates in
mitochondria and displays a selective antiapoptotic effect against inducers of
mitochondrial oxidative stress in neurons, Antioxid. Redox. Signal. 11 (2009) 469–480.
[88] K. Skemiene, G. Rakauskaite, S. Trumbeckaite, J. Liobikas, G.C. Brown, V. Borutaite,
Anthocyanins block ischemia-induced apoptosis in the perfused heart and support
mitochondrial respiration potentially by reducing cytosolic cytochrome c, Int. J. Biochem.
Cell Biol. 45 (2013) 23–29.
[89] K. Skemiene, J. Liobikas, V. Borutaite, Anthocyanis as substrates for mitochondrial
complex I—protective effect against heart ischemic injury, FEBS J. 282 (2015) 963–
971.
[90] I. Serraino, L. Dugo, P. Dugo, L. Mondello, E. Mazzon, G. Dugo, A.P. Caputi, S.
Cuzzocrea, Protective effects of cyanidin-3-O-glucoside from blackberry extract
against peroxynitrite-induced endothelial dysfunction and vascular failure, Life Sci. 18
(2003) 1097–1114.
[91] N.A. Riobo, E. Clementi, M. Melani, A. Boveris, E. Cadenas, S. Moncada, J.J.
Poderoso, Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity
through peroxynitrite formation, Biochem. J. 359 (2001) 139–145.
[92] K.E. Strathearn, G.G. Yousef, M.H. Grace, S.L. Roy, M.A. Tambe, M.G. Ferruzzi, Q.L.
Wu, J.E. Simon, M.A. Lila, J.C. Rochet, Neuroprotective effects of anthocyanin- and
proanthocyanidin-rich extracts in cellular models of Parkinsons disease, Brain. Res. 25
(2014) 60–77.
[93] V. Borutaite, A. Jekabsone, R. Morkuniene, G.C. Brown, Inhibition of mitochondrial
permeability transition prevents mitochondrial dysfunction, cytochrome c release and
apoptosis induced by heart ischemia, J. Mol. Cell Cardiol. 35 (2003) 357–366.
[94] G.C. Brown, V. Borutaite, Regulation of apoptosis by the redox state of
cytochrome c, Biochim. Biophys. Acta 1777 (2008) 877–881.