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Medicinal Plants as Antioxidant Agents: Understanding Their Mechanism of Action and Therapeutic
Efficacy, 2012: 27-57 ISBN: 978-81-308-0509-2 Editor: Anna Capasso
3. Anthocyanins: Mechanism of action and
therapeutic efficacy
Simona Lucioli
Centro di Ricerca per la Frutticoltura, CRA-Consiglio per la Ricerca e la Sperimentazione in
Agricoltura, Via di Fioranello, 52 - 00134 Roma, Italy
Abstract. The current status of research on anthocyanins, their
biological effects in vitro and in vivo and their potential application
in human therapy are reviewed. Anthocyanins are a class of
compounds belonging to the larger flavonoids class which
comprises a subset of the polyphenol class of compounds.
Anthocyanins are almost exclusively found in higher plants. Major
sources of anthocyanins are blueberries, cherries, raspberries,
strawberries, black currants, purple grapes and red wine.
Anthocyanins are included in the list of natural compounds known
to work as powerful antioxidants. The properties of anthocyanins
for human health are due to their peculiar chemical structure, as
these substances are very reactive towards reactive oxygen species
(ROS). Anthocianins have been reported to exert positive effects in
the treatment of various diseases and are prescribed as medicines in
several countries for thousands of years. The available scientific
evidence indicates that anthocyanins contained in a diet rich in
fruits and vegetables are associated with a decreased risk of
inflammation-related chronic diseases and that anthocyanins
display a wide range of biological activities including antimicrobial,
anti-carcinogenic and proapoptotic activities; improvement of vision
Correspondence/Reprint request: Dr. Simona Lucioli, Centro di Ricerca per la Frutticoltura, CRA-Consiglio per la
Ricerca e la Sperimentazione in Agricoltura, Via di Fioranello, 52 - 00134 Roma, Italy
E-mail: [email protected]
Simona Lucioli 28
and neuroprotective effects. In addition, anthocyanins display a variety of effects on
blood vessels and platelets. The present review summarizes our knowledge on the
bioavailability, antioxidant activity and health enhancing components of anthocyanin-
rich foods and extracts, with a focus on the role of anthocyanins in obesity, diabetes,
cardiovascular diseases, visual and brain functions and cancer protection. Since most
health benefits are produced by chemical properties beyond the antioxidant capacity
of the molecules, much remains to be elucidated before a comprehensive
understanding of the effects of anthocyanins emerges.
Introduction
Anthocyanins are a class of compounds belonging to the larger
flavonoids class which comprises a subset of the polyphenol class of
compounds. The polyphenol class of compounds includes all molecules with
more than one hydroxyl group on an aromatic ring. Flavonoids compounds
share a common framework consisting of two aromatic rings (A and B), that
are bound together by three carbon atoms forming an oxygenated heterocycle
(ring C). Flavonoids are subsequently divided into several groups differing in
the oxidation state of the heterocyclic pyran ring C. The subclasses consist of
flavanols, flavanones, flavones, isoflavones, flavonols, and anthocyanins
(listed in ascending order of oxidation) and within each of these subclasses,
individual compounds are characterized by specific hydroxylation and
conjugation patterns [1]. Most flavonoids are present in nature as the
glycosidic form, with the exception of flavanols, and this contributes to their
complexity and the large number of individual molecules that have been
identified [2]. The group of anthocyanins represents the largest class of
polyphenols. Anthocyanins are the most oxidized flavonoids with the C ring
fully unsaturated and a hydroxyl at position 3. The basic structure is an
aglycone, or anthocyanidin, with one or more sugars attached at most often
C3, C5, or C7 and possibly esterification on the sugars. Anthocyanins are
predominantly found in nature as water soluble glycosides of polyhydroxy
and polymethoxy derivatives of 2-phenyl-benzopyryliurn or flavylium salts.
Individual members are differentiated by the number of hydroxyl and
methoxyl groups of the B-ring, by the number of sugars attached to the
aglycon and the position of attachment, and by the nature and number of
aliphatic or aromatic acids attached to the sugar residues. Currently, there are
19 naturally occurring anthocyanidins. The six most common anthocyanidins
found in edible plants include pelargonidin, peonidin, cyanidin, malvidin,
petunidin, and delphinidin [3]. The prevalence of sugar occurrence in natural
anthocyanins is glucose, rhamnose, xylose, galactose, arabinose, and
fructose. Many anthocyanins have been found to be acylated by aliphatic or
Anthocyanins 29
aromatic acids, the most commonly seen acyl groups being coumaric, caffeic,
ferulic, p-hydroxy benzoic, synapic, malonic, acetic, succinic, oxalic, and
malic acids. Considering all these factors, the number of probable
anthocyanin compounds is quite large, leading to over 600 having been
identified from natural sources [4, 5]. Anthocyanins are responsible for much
of the red, blue, and purple colors of fruits, vegetables, grains, flowers, and
herbs, which explains their name, in Greek, anthos means flower and kyanos
means blue. Anthocyanins are almost exclusively found in higher plants,
although a few have been found in lower plants such as mosses and ferns
[4, 6]. In general, the anthocyanins in most of the fruits and vegetables are
observed in concentrations from 0.1% up to 1.0% dry weight [4, 7].
Anthocyanins are included in the list of natural compounds known to work as
powerful antioxidants. In the last years, great attention was given to the
possible protection exerted by natural antioxidants present in dietary plants,
towards tissue injury mediated by reactive oxygen species (ROS). The
anthocyanin-health properties are due to their peculiar chemical structure, as
they are very reactive towards ROS because of their electron deficiency.
They have been reported to have positive effects in the treatment of various
diseases and are prescribed as medicines in any countries.
Bioavailability and adsorption of anthocyanins
The daily intake of anthocyanins in humans has been estimated at 180-
215 mg/d in USA [8]. This value is considerably higher than the intake of
other flavonoids such as flavones and flavonols as estimated in the Dutch diet
(23 mg/d, measured as aglycones) [9]. Major sources of anthocyanins are
blueberries, cherries, raspberries, strawberries, black currants, purple grapes
and red wine. The early methods for testing anthocyanin absorption consisted
of measuring the presence of red pigments in urine after oral administration.
The evolution of testing methods has lead to observing anthocyanin
absorption in plasma and urine to determine both location of absorption and
rate of excretion. For many years, studies reported very low numbers for
absorption of anthocyanins after oral administration, on the order of 0.004%
to 0.1% of the intake, and indicated a rapid absorption and excretion with
time to the maximum concentration to be 1.5h for plasma and 2.5 h for urine
[10]. The metabolites persist in the urine for up to 24 h and may retain their
basic anthocyanin structure [11]. However, most of the analyses were
performed with UV-Vis detection after acidification, under the assumption all
the anthocyanins would be converted into the colored flavylium form, and it
is possible that some forms existing at neutral pH could not be colored due to
chemical reactions within the plasma or urine. Also, the common technique
Simona Lucioli 30
involved freezing and storing the urine and plasma samples before analysis
but chromatograms of samples immediately after collection showed
additional peaks that had degraded in the chromatograms of frozen samples
[12]. Evidence from several laboratory indicates that anthocyanins are rapidly
absorbed from both the stomach [13, 14] and small intestine [15], and
appear in blood circulation and urine as intact, methylated, glucuronide
derivatives and/or sulfoconjugated forms [12, 16-23]. In rats fed an
anthocyanin- rich diet for 15 days, anthocyanins have been found in several
organs, including stomach, small intestine (jejunum), liver, kidney and
brain. In the brain, total anthocyanin content (blackberry anthocyanins and
peonidin 3-O-glucoside) reached 0.25 ± 0.05 nmol/g of tissue [24].
Pharmacokinetic evidence suggests that the concentration of the parent
glycosides and their glucuronide derivatives are prominent in early blood
samples (0-5 h), with increasing methylation occurring over time (6-24 h).
This evidence suggests that anthocyanins bioactivity is likely altered over
time as a result of metabolic transformation post consumption.
As anthocyanins are rapidly degraded by intestinal microflora, this
additional metabolism could account for the unrecovered anthocyanins,
suggesting the possibility that large concentrations of anthocyanin
derived compounds might be present in the gastrointestinal trait (GIT).
Anthocyanidin glycosides are hydrolyzed by the microflora with cleavage of
the protective 3-glycosidic linkage. The released aglycones are very unstable
molecules under any condition, but in the neutral pH of the physiological
conditions, they are spontaneously degraded into monomeric phenolic acids
and aldehydes, specifically protocatechuic acid (3,4-dihydroxybenzoic acid),
syringic acid, vanillic acid and phloroglucinol aldehyde, [25-29]. Support
for this view was provided by Tsuda and colleagues [30], who found a
plasma concentration of protocatechuic acids eight times greater than that of
cyanidin-3-glucoside. The fate of anthocyanins was studied by González-
Barrio and coworkers [31] following the consumption of 300 g of raspberries
by healthy human volunteers and subjects with an ileostomy. Postingestion
plasma and urine from the former and ileal fluid and urine from the latter
group were collected and analyzed by HPLC-PDA-MS. Plasma from the
healthy volunteers did not contain detectable quantities of either the native
raspberry polyphenolics or their metabolites. The three main raspberry
anthocyanins were excreted in urine in both healthy and ileostomy volunteers
0-7 h after ingestion, in quantities corresponding to <0.1% of intake. This
indicates a low level of absorption in the small intestine. In subjects with an
intact functioning colon, these compounds would pass to the large intestine.
In vitro anaerobic incubation of raspberry anthocyanins with fecal
suspensions demonstrated conversion to phenolic acids [32]. In vivo
Anthocyanins 31
urinary excretion of phenolic acids after ingestion of raspberries indicates
that after formation in the colon some phenolic acids undergo phase II
metabolism, resulting in the formation of products that do not accumulate
when anthocyanins are degraded in fecal suspensions. Even with the
observation of these anthocyanin metabolites, the majority of ingested
anthocyanins are not recovered and therefore continued investigation is
necessary to determine the fate of the compounds in the body.
The limited data that are available indicate that simultaneous intakes with
foods can affect the absorption and excretion of flavonoids. It has been
shown in a study with both rats and human subjects that phytic acid (myo-
inositol hexaphosphate), a component of hulls of nuts, seeds and grain [33],
increases the bioavailability of blackcurrant anthocyanins [34].
Urinary recovery of the anthocyanins from rats was enhanced 5·8- fold by co-
ingestion with a 1% solution of phytic acid, which reduced gastrointestinal
mobility and slowed the passage of the anthocyanins through the stomach,
duodenum and jejunum, presumably thereby providing a longer time frame
for the absorption of anthocyanins. Human plasma and urinary anthocyanin
levels were also enhanced by phytic acid. The peak excretion was delayed
until 4–8 h post-ingestion, and the recovery of anthocyanins increased 4·5-
fold.
Anthocyanin biological activities
Numerous studies have shown that anthocyanins display a wide range of
biological activities [35, 36] including antioxidant [16, 37-39], anti-
inflammatory [40, 41], antimicrobial [42] anti-carcinogenic and proapoptotic
activities [43-45], improvement of vision [46, 47] and neuroprotective effects
[48, 49]. In addition, anthocyanins display a variety of effects on blood
vessels [50, 51] and platelets [52, 53] that may reduce the risk of coronary
heart disease [54].
Antioxidant activity
Anthocyanins are potent antioxidant superior to classical antioxidants
such as butylated hydroxyanisole (BHA) [55, 56], butylated hydroxytoulene
(BHT), α-tocopherol [37, 39] 6-hydroxy-2,5,7,8-tetramethychromane-2-
carboxylic acid (Trolox), catechin and quercetin [57]. Glycosylation of an
anthocyanin decreases radical scavenger activity compared with the aglycone,
as it reduces the ability of the anthocyanin radical to delocalize electrons. In
accordance with this, Fukumoto and Mazza [39] reported increased
antioxidant activity with increase in the hydroxyl groups and decreased
Simona Lucioli 32
antioxidant activity with glycosylation of anthocyanidins. Since cyanidin and
its glycosides represent one of the major groups of naturally occurring
anthocyanins their antioxidant and biological properties have been deeply
investigated. Particularly, cyanidin 3-glucoside, alone or together with other
cyanidin 3-glycosides and with the aglicone form, has been widely
investigated with the aim to establish its antioxidant activity in different
experimental conditions.
In vitro studies
Acquaviva [58] showed that cyanidin 3- glucoside and cyanidin had a
protective effect on DNA cleavage, a dose-dependent free radical scavenging
activity and significant inhibition of xanthine oxidase activity. Cyanidin 3-
glucoside has been showed to be a scavenger of peroxynitrite and to be able
to exert a protective effect against in vitro endothelial dysfunction and
vascular failure induced by peroxynitrite tested on human umbilical vein
endothelial cells (HUVEC) [59]. Duthie [60] found that cyanidin 3- glucoside
protected against oxidative DNA damage in human colonocytes. Guerra [61]
showed the ability of cyanidin 3-glucoside to reduce the production of ROS,
and the inhibition of protein and DNA synthesis caused by aflatoxin B1 and
ochratoxin A in a human hepatoma cell line (Hep G2) and a human colonic
adenocarcinoma cell line (CaCo-2). Cyanidin 3-glucoside significantly
reduced free radical species production and prevented genomic DNA damage
due to ochratoxin A on human fibroblasts [62]. Cyanidin 3-glucoside has
been showed to be able to modulate hepatic stellate cells proliferation and
type I collagen synthesis induced by a ferric nitrilotriacetate complex as pro-
oxidant agent, thus suggesting a potential role for this antioxidant
compound in the prevention of fibrosis in chronic liver diseases [63]. In a cell
culture study in which neuronal cells (PC 12) were exposed to a variety of
sweet and tart cherry phenolic compounds, total phenolics, and
predominantly anthocyanins, demonstrated a dose-dependent reduction in
oxidant stress [64]. Cyanidin 3-glucoside from mulberry fruit extract [65]
have been assumed as neuroprotective constituent on the PC12 cells
exposed to cell-damaging oxidative stress.
Exposure to UV-A radiation is known to induce discrete lesions in DNA
and the generation of free radicals that lead to a wide array of skin disease.s
Two in vitro studies on human keratinocytes (HaCaT) concluded that
cyanidin 3-glucoside could successfully be employed as a skin
photoprotective agent against, respectively, ultraviolet-A and B [66, 67]
(UVA and UVB) radiations. Tarozzi particularly, showed that UVA
induced apoptosis and DNA fragmentation caused by the generation of ROS
Anthocyanins 33
has been counteracted by cyanidin 3-glucoside by the inhibition of hydrogen
peroxide (H2O2) release after UVA irradiation and by the enhancement of
the resistance to the apoptotic effects of both H2O2 and the superoxide anion
(O2-). The authors also suggested that cyanidin 3-glucoside protective effects
could be attributed to the high membrane levels of incorporation. Giampieri
and colleagues [68] very recently analyzed methanolic extracts from the
strawberry Sveva cultivar for anthocyanin content and for their ability to
protect human dermal fibroblasts against UV-A radiation. Five anthocyanin
pigments were identified using high-performance liquid chromatography-
diode array detection-electrospray ionization/mass spectrometry. Moreover,
the strawberry extract showed a photoprotective activity in fibroblasts exposed
to UV-A radiation, increasing cellular viability, and diminishing DNA damage.
The antioxidant activity exerted in a liposomal membrane system by
different cyaniding glycosides (arabinoside, rutinoside, galactoside and
glucoside) has been found to be higher than that of trolox in the case of
Fe(II)-induced liposome oxidation and to be comparable with the action of
trolox (a water-soluble tocopherol derivative) in the case of UV- and AA
PH (2,2’-azobis[2-amidinopropane] dihydrochloride)-induced liposome
membrane oxidation. The inhibitory effects of lipid peroxidation exerted by
cyanidin 3-glucoside, cyanidin and cyanidin 3-galactoside have been showed
and quantificated by Adhikari [69] who also found a good effect respect to
commercial anti-oxidants butylated hydoxyanisole, butylated
hydroxytoluene, and tert-butylhydroxyquinone As regards other cyaniding
glycosides, cyaniding 3-galactoside showed a stronger activity respect to that
of other flavonoids as well as vitamin E or Trolox from cranberries’
extracts in two antioxidant assays consisting in the evaluation of 1,1-
diphenyl-2-picrylhydrazyl radical-scavenging activity and in the ability to
inhibit low-density lipoprotein oxidation [70]. Cyaniding also showed its
antioxidant activity. It was, infact, the stronger superoxide radical scavenger
among the polyphenols of different varieties of plums [71] and exerted a
protective effect against the toxicity induced by linoleic acid hydroperoxide
(LOOH) in cultured human fetal lung fibroblasts [72]. Similarly, cyaniding
inhibited malonaldehyde formation in oxidized calf thymus DNA oxidized by
Fenton’s reagent [73]. Lazze [74] showed that cyaniding was able to
protect rat smooth muscle and hepatoma cell lines against cytotoxicity, DNA
SSB formation and lipid peroxidation induced by tert-butyl-hydroperoxide
(TBHP) whereas its glycoside and rutinoside derivatives did not work. The
antioxidant activity exerted by cyaniding and by some glycosidic forms has
been confirmed in three lipid containing models (human low-density
lipoprotein – LDL – and bulk and emulsified methyl linoleate) by Kahkonen
[57] who also linked the differences in the antioxidant power with the
Simona Lucioli 34
different glycosylation patterns. The delphinidin and cyanidin anthocyanidins
were found to be the most active inhibitors followed by malvidin, peonidin,
pelargonidin and petunidin. Overall, the glucosylated forms or anthocyanins
were less active than the free forms or anthocyanidins in the prevention of
LDL oxidation, except for malvidin 3, 5-diglycoside. LDL does not form
atherosclerotic plaques unless it is oxidised which may lead to a build up of
plaque in the arteries. Therefore, the consumption of dietary antioxidants is
beneficial in preventing cardiovascular diseases, particularly atherosclerosis
[75]. In a recent study by Abdel-Aal and colleagues, the concentration of
conjugated dienes (CD) formed due to LDL oxidation in vitro dropped
significantly to varying extents depending upon the type of anthocyanin. The
cyanidin-containing anthocyanins possessed a lower inhibition capacity
against LDL oxidation compared to the delphinidin-based anthocyanins. The
differences in the inhibition capacity between cyanidin and delphinidin could
be due to the extra hydroxyl group at the C5-position in the delphinidin
structure. The additional hydroxyl group would change the release of
hydrogen ions and the hydration constant (pKH), thereby increasing
inhibition effects against copper-induced human LDL cholesterol oxidation
[55]. This findings further emphasize the degree to which antioxidant
properties of anthocyanins can be influenced by their structure, that is, the
site of glucosylation as well as type and degree of acylation with acid
residues such as cinnamic and aliphatic acids. The mechanisms by which
anthocyanins/anthocyanidins inhibit LDL oxidation in vitro remain unclear. It
has been speculated that the protecting effects of phenolics and anthocyanins
on LDL oxidation may be due to multiple factors such as scavenging of
various radical species in the aqueous phase, interaction with peroxy radicals
at the LDL surface and terminating chain-reactions of lipid peroxidation by
scavenging lipid radicals and regenerating endogenous alpha-tocopherol back
to its active antioxidative form [57].
Protocatechuic acid was able to suppresses (1-methyl-4-
phenylpyridinium)þ-induced mitochondrial dysfunction and apoptotic cell
death in PC12 cells, showing a potential clinical application to
counteract neurodegeneration such as in Parkinson’s disease [76]. In cultured
neural stem cells, protocatechuic acid decreased apoptosis, reducing the ROS
and significantly suppressing the caspase cascade [77].
In vivo studies
Following consumption of anthocyanins, serum antioxidant status is
significantly increased after 4 to 24 hr post-consumption [16, 48].
Antioxidant capacity in human serum has also been measured using various
Anthocyanins 35
methods following consumption of strawberries (240 g), spinach (294 g), red
wine (300 mL) or vitamin C (1250 mg) in eight elderly women [78]. Total
antioxidant capacity of serum increased significantly by 7-25% during 4 hr
period after consumption of red wine, strawberries, vitamin C or spinach. The
total antioxidant capacity of urine determined by ORAC also increased for all
the treatments except red wine, with the largest increase for vitamin C
(44.9%). Further studies report positive effects in older human subjects.
Thirty-six grams of a lyophilised red grape powder was able to significantly
reduce systemic oxidative stress as measured by urinary F2-isoprostanes in
twenty postmenopausal women [79]. The supplement was also able to
positively alter lipoprotein metabolism and inflammatory markers (TNFα),
therefore supposedly reducing recognised cardiovascular risk factors.
Consumption of tart cherry juice for 2 weeks was able to reduce the
ischaemia/reperfusion-induced F2-isoprostane response in plasma and
urinary excretion of oxidised nucleic acids, a measure of DNA oxidative
damage, in twelve volunteers aged 69 years [80].
DNA was significantly protected against oxidative insult in twenty-one
haemodialysis patients in a pilot intervention study by 200 ml/d intake for a
4-week period of an anthocyanin-rich red fruit juice [81]. Reduced DNA
oxidation damage was accompanied by decreased protein and lipid
peroxidation and an increase in glutathione. In twenty-six patients receiving
haemodialysis and fifteen healthy subjects, daily consumption of 100 ml of
red grape juice for a period of 14 d increased the antioxidant capacity of
plasma, without affecting concentrations of uric acid or vitamin C, reduced
oxidised LDL and increased the level of cholesterol-standardised
a-tocopherol [82].
Finally, anthocyanins shows systemic or central antioxidant protection,
as observed by Shih [83] who fed senescence-accelerated mice with a basal
diet supplemented with a 0·18 and 0·9% mulberry extract for 12 weeks.
Treated animals showed a higher antioxidant enzyme activity and reduced
lipid oxidation in both the brain and liver.
Eye health
In vitro studies
The beneficial health effect that anthocyanins have on vision was one of
the first reported health properties [84]. Jang [85] determined the ability of
bilberry extract to modulate adverse effects of A2E on retinal pigment
epithelial cells in vitro. A2E is an auto-fluorescence pigment that
Simona Lucioli 36
accumulates in retinal pigment epithelial cells with age and can mediate a
detergent like perturbation of cell membranes and light-induced damage to
the cell. This is significant since it is generally accepted that age-related
macular degeneration begins with the death of retinal pigment epithelial cells,
the degeneration of photoreceptor cells followed soon after by the loss of
vision. The results showed that the bilberry extracts were able to suppress the
photooxidation of pyridinium disretinoid A2E by quenching singlet oxygen.
Additionally, cells that had taken up anthocyanins also exhibited a resistance
to the membrane permeabilisation that occurs because of the detergent- like
action of A2E.
In vivo studies
The role of anthocyanins on vision has also been demonstrated in a few
animal studies. In a study by Kalt [86] using a pig model, the distribution of
anthocyanins in tissues such as the liver, eye and brain tissue was
investigated. The results suggest that anthocyanins can accumulate in tissues,
even beyond the blood-brain barrier. The highest amount of anthocyanins
was found in the eye tissue with maximum concentration of 700 pg of
anthocyanins/g of fresh weight. Anthocyanins from blackcurrant, which only
contains 4 anthocyanins (delphinidin-3- glucoside; delphinidin-3-rutinoside;
cyanidin-3-glucoside and cyanidin-3-rutinoside) have extensively been
examined for their effects on vision. Matsumoto investigated the ocular
absorption, distribution and elimination of blackcurrant anthocyanins in rats
after oral and intraperitoneal administration and in rabbits after intravenous
administration. Blackcurrant anthocyanins are absorbed and distributed in
ocular tissues as intact forms and pass through the blood-aqueous barriers and
blood-retinal barriers in both rats and rabbits [20]. Nakaishi [87] report that
an oral intake of 12.5, 20 or 50 mg of blackcurrant anthocyanins was
significant to decrease the dark adaptation threshold in a dose-dependant
manner. To examine the influence of the black currant anthocyanins on the
disease progression of open-angle glaucoma (OAG), a randomized, placebo-
controlled, double-masked trial was made in 38 patients with OAG treated
by antiglaucoma drops [88]. Black currant anthocyanins (50 mg/day) or their
placebos were orally administered once daily for a 24-month period.
Systemic blood pressure, pulse rates, intraocular pressure, ocular blood
circulation by laser-speckle flowgraphy, and Humphrey visual field mean
deviation (MD) were measured during the 24-month period. A
statistically significant difference was observed between the treatment groups
in mean change from baseline in MD 24 months after therapy.
Upon administration of black currant anthocyanins, the ocular blood flows
Anthocyanins 37
during the 24-month observational period increased in comparison with
placebo-treated patients. In his study, Miyake and colleagues [89] generated
a mouse model of endotoxin-induced uveitis (EIU) that shows retinal
inflammation, as well as uveitis, by injecting lipopolysaccharide. He
pretreated the mice with anthocyanin-rich bilberry extract and analyzed the
effect on the retina. Anthocyanin-rich bilberry extract prevented the
impairment of photoreceptor cell function, as measured by electroretinogram.
At the cellular level, he found that the EIU-associated rhodopsin decreased
and the shortening of outer segments in photoreceptor cells were suppressed
in the bilberry-extract-treated animals. Moreover, the extract prevented both
STAT3 activation, which induces inflammation-related rhodopsin decrease,
and the increase in interleukin-6 expression, which activates STAT3. In
addition to its anti-inflammatory effect, the anthocyanin-rich bilberry extract
ameliorated the intracellular elevation of ROS and activated NF-κB, a redox-
sensitive transcription factor, in the inflamed retina. In a randomized double-
blind placebo-controlled study, Lee [90] investigated the effect of purified
high-dose anthocyanin oligomer administration on nocturnal visual function
and clinical systems in low to moderate myopia subjects. There was a
significant improvement in the anthocyanin group (73.3% improved
symptoms) compared to the placebo group. In addition, the anthocyanin
group showed improved contrast sensitivity levels compared to the
placebo group.
This findings strongly suggest that anthocyanins are absorbed and display
several physiological activities and ocular health benefits. Oral administration
of black currant anthocyanins may be a safe and promising supplement for
patients with OAG in addition to antiglaucoma medication, while
anthocyanin-rich bilberry extract has a protective effect on visual function
during retinal inflammation.
Obesity
The inhibitory effects of anthocyanins on body fat accumulation were
first reported by Tsuda in 2003 [91]. In C57BL/6J mice, a cyanidin
3-glucoside-containing diet (2 g/kg) was found to significantly reduce body
fat accumulation induced by highfat meals (60% of energy), when compared
with controls. This effect was probably due to suppression of lipid synthesis
in the liver and in white adipose tissue. In addition, a cyanidin 3-glucoside-
containing diet also significantly reduced plasma glucose concentration,
which was elevated by highfat meals. Anthocyanins may act on adipocytes
and modulate the expression levels of adipocytokines. cyanidin 3-glucoside
(or its agylcone cyanidin) was reported to upregulate the expression of
Simona Lucioli 38
adiponectin, which can increase insulin sensitivity in human adipocytes
[92-94]. When anthocyanin extract from blueberries or whole
blueberry powder was added as a supplement to high-fat meals (45% of
energy) in C57BL/6 mice, intake of anthocyanin extract significantly
inhibited weight gain and body fat accumulation [95]. In a similar study
with C57BL/6 mice by DeFuria, intake of whole blueberry powder
supplemented to high-fat meals (60% of energy) [96] showed inhibitory
effects on obesity-induced inflammation in white adipose tissue in this study.
Specifically, mRNA levels of tumor necrosis factor-a (TNFα) and monocyte
chemoattractant protein-1 were upregulated in the high-fat meal control group,
although expression was significantly reduced in the blueberry group.
Additionally, intake of blueberries restored glutathione peroxidase 3 levels,
which were otherwise significantly reduced by high-fat meals as in the
control group. Studies have reported that intake of mulberry extract in an
aqueous phase significantly inhibited body weight gain [97], while intake of
tart-cherry powder extract inhibited body weight gain, reduced
retroperitoneal fat amount, suppressed proinflammatory cytokine (IL-6, TNFα)
expression, and upregulated mRNA expression of peroxisome proliferator- activated receptor (PPAR) a and g in Zucker fatty rats [98]. For non-berry
species, intake of blood orange (Moro orange), which contains anthocyanins,
reportedly inhibited body weight gain and body fat accumulation [99].
Diabetes
Diabetes mellitus (DM) is a metabolic disorder in the endocrine system
resulting from a defect in insulin secretion, insulin action or both of them.
Wedick and coworkers [100] evaluated whether dietary intakes of
major flavonoid subclasses (ie, flavonols, flavones, flavanones, flavan-3-ols,
and anthocyanins) are associated with the risk of type 2 diabetes in US adults.
They followed up a total of 70,359 women in the Nurses' Health Study (NHS;
1984-2008), 89,201 women in the NHS II (1991-2007), and 41,334 men in
the Health Professionals Follow-Up Study (1986-2006) who were free of diabetes, cardiovascular disease, and cancer at baseline. During 3,645,585
person-years of follow-up, the authors documented 12,611 incident cases of
type 2 diabetes. Higher intakes of anthocyanins were significantly associated
with a lower risk of type 2 diabetes. Consumption of anthocyanin-rich foods,
particularly blueberries and apples/pears, was also associated with a lower
risk of type 2 diabetes. No significant associations were found for
total flavonoid intake or other flavonoid subclasses.
The purpose of Lachin study [101] is to evaluate the effect of ethanolic
extract of cherry fruit on alloxan induced diabetic rats. In this study 36 Male
Anthocyanins 39
Wistar rats, body weight of 150-200gr were divided into 6 groups. Diabetes
was induced by intra peritoneal injection of 120 mg/kg Alloxan. The duration
of the cherries treatment was 30 days in which single dose of extracts
(200mg/kg) were oral administered to diabetic rats. Blood glucose levels
were estimated with glucometer before treatment, 2h and 1- 4 weeks after
administration of extracts. Treatment with extracts of the cherries resulted in
a significant reduction in blood glucose and urinary microalbumin and an
increase in the creatinine secretion level in urea. Single anthocyanins have
been tested for their potential antidiabetic activity. In a study with male
streptozotocin-induced diabetic Wistar rats, intraperitoneal injection of
pelargonidin normalised elevated glycaemia and improved serum insulin levels
in diabetic rats. The typical biochemical symptoms of induced diabetes, such
as lower serum levels of superoxide dismutase and catalase, increased
concentrations of malondialdehyde and fructosamine, were effectively reverted
to normal values after pelargonidin administration [102]. One possible
mechanism is linked to the anthocyanin counteracting Hb glycation,
consequent Fe release from the prosthetic group and Fe-mediated
oxidative damage.
In a type 2 diabetes mutant mouse (KK-Ay) model, intake of
anthocyanins was found to inhibit elevation of blood glucose levels and
improve insulin sensitivity via downregulation of retinol-binding protein
4 (RBP4). This was achieved by intake of high-purity anthocyanin cyanidin
3-glucoside [91, 103] or bilberry extract anthocyanin [104], which contains a
diverse variety of anthocyanins. Cyanidin 3-glucoside intake upregulated
glucose transporter 4 (Glut4) expression, which in turn led to downregulated
RBP4 expression. Consequently, this process constitutes an inhibitory effect
on the reduction of insulin sensitivity in peripheral tissue and glucose release
following excessive gluconeogenesis. However, because only a low level of
cyanidin 3-glucoside is contained in bilberry extract, the antidiabetic effects
of bilberry extract (which contains multiple anthocyanins) cannot be
established on the basis of downregulated RBP4 expression. Likewise,
dietary bilberry extract activates AMPK in white adipose tissue, skeletal
muscle, and liver. In white adipose tissue and skeletal muscle, activation of
AMPK induces upregulation of Glut4, which results in enhanced glucose
uptake and utilization in these tissues. In the liver, dietary bilberry extract
clearly reduces glucose production via AMPK activation. This reduction
efficiently ameliorates hyperglycemia in type 2 diabetic mice. Furthermore,
bilberry extract-induced AMPK activation in the liver results in significantly
decreased liver and serum lipid content via upregulation of PPARa and
acylCoA oxidase. Upregulation of carnitine palmitoyltransferase-1A by
dietary bilberry extract enhances the decrease in lipid content via
Simona Lucioli 40
enhancement of fatty acid oxidation. Such changes may also contribute to the
antidiabetic effect of bilberry extract [104]. It is important to further clarify
how complex formulations (such as bilberry extract) containing multiple
anthocyanins can exhibit antidiabetic effects through the activation of AMPK
pathways. In addition to the above-mentioned RBP4 and AMPK pathways, it
has been suggested that anthocyanins may also work as antidiabetes food
factors via inhibition of α-glucosidase activity in the small intestinal
endothelium. α-Glucosidase is the key enzyme catalyzing the final step in the
digestive process of carbohydrates. Hence, α-glucosidase inhibitors can retard
the liberation of d-glucose from dietary complex carbohydrates and delay
glucose absorption, resulting in reduced postprandial plasma glucose levels
and suppression of postprandial hyperglycemia. The inhibitory effects
of anthocyanins on α-glucosidase activity vary with the molecular structure
of drugs. Among the anthocyanins, glycosides such as that of cyanidin
3-glucoside were found to be very weak inhibitors of a-glucosidase [105,
106]. The IC50 value of cyanidin-3-galactoside was 0.50 ± 0.05 mM against
intestinal α-glucosidase sucrase [107]. A low dose of cyanidin-3-galactoside
showed a synergistic inhibition on intestinal α-glucosidases (maltase and
sucrase) when combined with α-glucosidase inhibitor acarbose [108]. Matsui
and colleagues established a unique assessment method for testing
the inhibition of α-glucosidase activity, with which they examined the
inhibition of α-glucosidase by acylated anthocyanins. In their results, purple
potato-derived acylated anthocyanins were shown to inhibit
α-glucosidase activity [109, 110]. This same group also studied the inhibitory
effects acylated anthocyanins on the elevation of blood glucose levels in rats
[111], and found that the molecular structure responsible for α-glucosidase
inhibition was not anthocyanidin itself, but caffeoylsophorose, which is a
component of the acylated anthocyanin molecule [112-114].
Cardiovascular disease (CVD)
Cardiovascular disease or heart disease is a class of diseases that involve
the heart or blood vessels (arteries and veins). While the term technically
refers to any disease that affects the cardiovascular system, it is often used to
refer to those related to atherosclerosis (AS) and/or hypertension. The causes,
mechanisms, and treatments of these conditions often overlap.
Cardiovascular diseases remain the biggest cause of deaths worldwide.
Cyaniding 3-glucoside exerts a protective effect against peroxynitrite-
induced endothelial dysfunction and vascular failure [59] acting as
efficacious scavenger of peroxynitrite. However, its ability is not limited to
the antioxidant activity but also results in the regulation of enzymes involved
Anthocyanins 41
in the NO synthesis. Indeed, the reduction of the levels of inducible nitric
oxide synthase (iNOS) expression has been recorded in different
experiments. The amelioration of endothelial dysfunction and the
vasoprotective effects exerted by the regulation of the vascular tension
regulator endothelial NO synthase (eNOS), a protective enzyme in the
cardiovascular system, has been recorded in different conditions.
In vitro studies
Cyaniding 3-glucoside from blackberry extract showed an anti-
inflammatory activity in J774 cells. At least some part of this activity is due
to the suppression of NO production; iNOS expression was inhibited through
the attenuation of NF-κB and/or MAPK activation [115]. In an in vitro test on
bovine artery endothelial cells, cyanidin 3-glucoside enhanced eNOS
expression and escalated NO production via an Src-ERK1/2-Sp1 signaling
pathway [116] and enhanced eNOS activity by regulating its phosphorylation
[117]. In a study on human endothelial cells Sorrenti [118], besides
confirming that 3-glucoside upregulated e-NOS, also demonstrated that
cyanidin 3-glucoside conferred an additional cytoprotective effect consisting
in the induction of the stress protein heme oxygenase-1 (HO-1), that exerts an
important cellular protective mechanism against oxidative injury. Cyanidin
showed its vasoprotective effect in an in vitro experiment performed on
cultured human umbilical vein endothelial cells (HUVECs) [119]. Cyanidin
from red wine increased human eNOS in human endothelial cells [120];
moreover, inhibitory effect of cyanidin on TNFα-induced apoptosis has
involved multiple pathways, such as eNOS and thioredoxin expression and
Akt activation [121]. In a very recent study from Chen, [122] the effect of
delphinidin on adhesion of monocytes to endothelial cells induced by ox-
LDL was investigated. The results showed that the pre-treatment with
delphinidin (50, 100, or 200 μM) dose-dependently decreased the ox-LDL- induced up-regulation of the expression of ICAM-1 and P-selectin, and the
enhanced adhesion and transmigration of monocytes. Delphinidin attenuated
ox-LDL-induced generation of ROS, p38MAPK protein expression, NF-κB
transcription activity and protein expression, IκB-α degradation, NADPH
oxidase subunit (Nox2 and p22phox) protein and mRNA expression in
endothelial cells in a dose-dependent manner. These results suggest that
delphinidin attenuates ox-LDL induced expression of adhesion molecules (P-
selectin and ICAM-1) and the adhesion of monocytes to endothelial cells by
inhibiting ROS/p38MAPK/NF-κB pathway.
Simona Lucioli 42
In vivo studies
Anthocyanins are thought to exert their cardiovascular protective effects
through anti-inflammation and antiplatelet coagulability [54, 123], the latter
which is reported to mediate anthocyanins or their putative colonic
metabolites [124]. Anthocyanins significantly inhibit TNFα- induced
inflammation through monocyte chemoattractant protein-1 in human
endothelium [125], suppress myocardial ischemia-reperfusion injury by
delphinidin through the inhibition of signal transducers and activators of
transcription 1 activity in cardiac muscle [126] and, via inhibition of
inflammation, inhibit atherosclerosis progression by cyanidin-3-O-
bglucoside with the aid of its metabolite protocatechuic acid [127]; cyanidin
3-glucoside orally administered in rats suppressed the zymosan-induced
inflammatory response in the peritoneal exudate cells [128]. Other
mechanisms such as inhibition of lipoprotein oxidation, free radical
scavenging and modulation of eicosanoid metabolism [129-132] are also
thought to play a role in the reduction of atherosclerosis.
The Kuopio ischemic heart disease risk factor study demonstrated that a
group of people who had consumed a large amount of berries rich in
anthocyanins (>408 g/day) had a significantly lower risk of CVD-related
death than those in the low-consumption group (133 g/day) [133].
A significant association between strawberry consumption and mortality due
to CVD was also revealed in the Iowa women’s health study involving
postmenopausal women [134]. With regard to human intervention trials [124,
134, 135], consumption of anthocyanin-containing plant foods, such
as blackcurrants, bilberries, and blueberries, reduced LDL cholesterol levels
and increased plasma antioxidant capacity.
Furthermore, the inhibitory effects of fruits containing anthocyanins
on atherosclerosis were reported in elderly men [136]. Ahmet [137] reported
that a blueberry enriched diet protected the myocardium of young
male Fischer-344 rats against induced ischaemic damage and demonstrated
the potential to attenuate the development of post-myocardial infarction
chronic heart failure. The effects of raspberry, strawberry and bilberry juices
on early atherosclerosis in hamsters have been investigated with the animals
receiving a daily dose corresponding to the consumption of 275 ml of juice
by a 70 kg human [138]. After 12 weeks on atherogenic diet, the berry juices
inhibited aortic lipid deposition by approximately 90% and triggered reduced
activity of hepatic antioxidant enzymes, which was not accompanied by
lowered plasma cholesterol. The features and progression of the lesions
observed in the hamster model of atherosclerosis are morphologically
similar to atheromatous lesions observed in human subjects [139].
Anthocyanins 43
In a study using tart cherry seed extract, rat hearts were subjected to
ischemic injury (which generally results in irregular and rapid heart beats and
possibly heart attack) and exposed to cherry extract at variable doses. Extract
at moderate doses was associated with reduced incidence of irregular and
rapid heart rates as well significantly less cardiac damage as a result of heart
attacks that did occur [140].
The intake of red grape juice rich in anthocyanin reduced the
concentrations of oxidized LDL and the activity of NADPH oxidase in
dialysis patients [141]. The association between grape phenolics and
coronary heart disease has been ascribed in part to the presence of
anthocyanins in red wine [142, 143]. In addition, several epidemiological
studies have shown that coronary heart disease mortality can be decreased by
moderate consumption of red wine [144, 145]. A study of the relationships
between vasodilation capacity, antioxidant activity and phenolic content
of 16 red wines reported that total phenol content correlated well with
vasodilation capacity and antioxidant activity of the wines, but only
anthocyanins were correlated with vasodilation capacity [146].
Hassellund and colleagues [147] assessed whether a purified
anthocyanin supplement improves cardiovascular metabolic risk factors and
markers of inflammation and oxidative stress in prehypertensive participants,
and whether plasma polyphenols are increased 1-3h following intake. In all,
31 men between 35-51 years with screening blood pressure >140/90mmHg
without anti-hypertensive or lipid-lowering medication, were randomized in a
double-blinded crossover study to placebo versus 640mg anthocyanins daily.
Treatment durations were 4 weeks with a 4-week washout. High-density
lipoprotein (HDL)-cholesterol and blood glucose were significantly higher
after anthocyanin versus placebo treatment. Several plasma polyphenols
increased significantly 1-3h following anthocyanin intake. This study
strengthens the evidence that anthocyanins may increase HDL-cholesterol
levels [145, 148], and this is demonstrated for the first time in
prehypertensive and non-dyslipidemic men.
Toufektsian [149] fed male Wistar rats an anthocyanin-rich diet for a
period of 8 weeks. The hearts of these rats were more resistant to regional
ischaemia and reperfusion insult ex vivo. With an in vivo model of coronary
occlusion and reperfusion, infarct size was reduced compared to the
anthocyanin-free diet. A parallel increase in myocardial glutathione levels
indicates that anthocyanins or anthocyanin-derived products may modulate
cardiac antioxidant defences. In male Sprague– Dawley rats, an anthocyanin-
rich supplement significantly reduced brain infarct volume after focal
cerebral ischaemic injury, and a putative mechanism was related to
Simona Lucioli 44
interaction of phenolic compounds with phospho-c-Jun N-terminal kinase
and the p53 signalling pathway [150].
This features provide a basis for the design of potent antiatherosclerotic
and antiypertensive agents that will have therapeutic potential in the
prevention and treatment of AS and CVD.
Brain health
In their study in humans, aged 76.275.2 years, Krikorian and colleagues
reported that intake of blueberry juice for 12 wk improved memory
performance [151]. This same group also reported that concord grape juice
produced similar effects on brain function [152, 153]. In animal models,
studies have suggested that intake of freeze-dried fruits or anthocyanin fruit
extracts (plum and blackberry) delays the onset of decline of neural functions
and improves cognitive and motor performance [154 155]. The effects of
anthocyanins might be mediated through inhibition of neuroinflammation.
For example, anthocyanins reportedly blocked age-related upregulation of
nuclear factor-kB (NF-kB) expression in Fischer rats [156]. Intake of
blueberries inhibited cognitive and motor impairments induced by kainic acid
challenge, as evidenced by the suppression of expression of IL-1b, TNFα,
and nuclear factor-kB in the hippocampus [157]. Moreover, production of
nitric oxide, IL-1b, and TNFα in microglia was reported to be inhibited
following blueberry intake [158], Williams showed that intake of blueberries
led to activation of cyclic AMP-response element-binding protein (CREB)
and upregulation of brain-derived neurotrophic factor [159]. Within the
hippocampus, activation of cyclic AMP-response element-binding protein
may be mediated through extracellular signal-related kinase1/2 signaling
rather than calcium calmodulin kinase II and IV or protein kinase A
pathways. Based on evidence from these studies, berry-derived effects of
anthocyanins on improvement of brain function might involve the inhibition
of neuroinflammation and modulation of neural signaling, while a collateral
effect on the improvement of cerebral blood flow may well be another
plausible factor [160]. Neuroprotective effects against cerebral ischemic
damage in vivo has also been performed by Kang [161] using a mouse brain-
injury model with a transient middle cerebral artery occlusion. Anthocyanins
are able to improve learning and memory of ovariectomised rats [162].
Ovariectomy caused oestrogen deficit in the animals, which in turn is
associated with mental health disorders, emotional difficulties, memory
impairment and other cognitive failures; the supplementation with red grape
anthocyanins resulted in memory-enhancing effects. Therefore, another
possible mechanism involved in cognitive-related effects of anthocyanins
Anthocyanins 45
could be their phyto-oestrogenic effects, with clear implications for human
subjects. Finally, it is interesting to note that berry-derived polyphenols may
be active in different and specific brain regions. Morris water maze tests
showed that strawberry supplementation to high-energy and charge particles
irradiated – rats partially overcame spatial deficits as animals were better able
to retain place information – behaviour associated with the a hippocampus. A
blueberry supplementation, however, seemed to improve reversal learning, a
behaviour more dependent on intact striatal function [163].
Anticarcinogenic activity
In vitro studies
Cancer-protective effects of cyanidin glucosides have been demonstrated
in studies employing cancer cell lines [164] including apoptotic effects via G2/M growth cycle arrest. Anthocyanin-rich extracts from berries and grapes and several pure anthocyanins and anthocyanidins have exhibited proapoptotic effects in multiple cell types in vitro [165-167]. Cell cycle arrest and apoptosis have been demonstrated in mutated cells exposed to cherry anthocyanins [168, 169] and in human oral (KB, CAL27), colon (HT-29,
HCT116, SW480, SW620), and prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines treated with cranberry extracts [170]. The anthocyanidins inhibited proliferation of human cancer cell lines AGS (stomach), HCT-116 (colon), MCF-7 (breast), NCI H460 (lung), and SF-268 (central nervous system) [171]. Malvidin exhibited a potent antiproliferative effect on AGS cells. The malvidin- induced inhibition of proliferation was accompanied by the arrest
of AGS cells at the G0/G1 phase. The occurrence of apoptosis induced by malvidin was confirmed by morphological and biochemical features, including apoptotic body formation, loss of mitochondrial membrane potential, elevation of the Bax: Bcl-2 ratio, caspase 3 activation, and PARP proteolysis [169]. Delphinidin showed G2/M phase cell cycle arrest, apoptosis, and inhibition of NF-κB signaling in 22Rnu1 cells. Delphinidin
treatment of human prostate cancer LNCaP, C4-2, 22Rnu1, and PC3 cells resulted in a dose-dependent inhibition of cell growth without showing any substantial effect on normal human prostate epithelial cells. The induction of apoptosis by delphinidin was mediated via activation of caspases [172]. Delphinidin also inhibited VEGF-stimulated human umbilical endothelial cell migration and proliferation and neovascularisation in vivo in a chorioallantoic
membrane model [173]. Finally, 3,4-dihydroxybenzoic acid was reported to be a strong apoptosis inducer in gastric adenocarcinoma cells [174]. Cyanidin may also promote cellular differentiation and thus reduce the risk for malignant transformation [175].
Simona Lucioli 46
Cancer metastasis refers to the spread of cancer cells from the primary
neoplasm to distant sites, where secondary tumors are formed, and is the
major cause of death from cancer. The metastatic cascade includes cell-cell
attachment, tissue-barrier degradation, migration, invasion, cell-matrix
adhesion and angiogenesis. The available scientific evidence indicates that
anthocyanin exert extensive in vitro anti-invasive and in vivo anti-metastatic
activities. In lung cancer, the treatment of cyanidin 3-glucoside and
cyanidin 3-rutinoside, which are isolated from mulberry, dose
dependently inhibited the migration and invasion of A549 cells and also
decreased MMP-2 and uPA and enhanced TIMP-2 and PAI. Moreover, the
transcription factors NFkB and AP-1 were also repressed [176]. By
attenuating the phosphorylation of ERK and the activation of AP-1,
peonidin 3-glucoside significantly inhibited the invasion, motility, and
expression of MMP-2/MMP-9 and uPA in H1299 cells [177].
Anthocyanins inhibit metastasis through regulation of MMP-2 and MMP-9
also in B16-F1 cells, and it modulates the expression levels of Ras, PI3K,
phospho-Akt, and NF-κB [178]. In breast cancer, treatment of HGF-
stimulated MCF-10A cells with delphinidin decreased the expression of
Met and the phosphorylation of FAK and Src; induction of the paxillin, Gab-
1, GRB-2, Ras–ERK MAPKs, and PI3K/Akt/ mTOR/p70S6K pathways was
also inactivated by delphinidin. Moreover, the blockage of HGF-mediated
activation of NF-kB and STAT3 and translocation of PKC were
also observed in delphinidin-treated MCF-10A cells [179]. Cyanidin 3-
glucoside attenuated ethanol-induced migration, invasion, and cell–matrix
adhesion and inhibited ethanol stimulated phosphorylation of ErbB2, cSrc,
FAK, and p130Cas of high ErbB2-expressing breast cancer BT474, MDA-
MB-231, and MCF-7ErbB2 cells [180]. In prostate cancer, anthocyanin-
enriched fractions from blueberry (Vaccinium angustifolium) downregulate
MMP-2/MMP-9 and upregulate TIMP-1/TIMP-2 in DU145 cells by
modulating PKC and MAPK pathways [181, 182]. The inhibitory effects of
anthocyanins on motility and invasion of HCT-116 human colon carcinoma
cells were associated with the suppression of claudin (a tight junction-
related protein) and the inhibition of MMP-2/MMP-9 through p38MAPK and
PI3K/Akt pathways [183]. In oral and cervical cancer, the invasion of SCC-4
cells and HeLa cells were diminished by the treatment of peonidin
3-glucoside and cyaniding 3-glucoside [184]. In fibrosarcoma, delphinidin
slightly inhibited the activities of MMP-2/MMP-9, which might
responsible, in part, for the inhibition of invasion in HT-1080 cells [185]. In
glioblastoma, Lamy [186] revealed that the aglycons of the most abundant
anthocyanins in fruits, including cyanidin, delphinidin, and petunidin, act as
potent inhibitors for the migration of glioblastoma U-87 cells.
Anthocyanins 47
In vivo studies
Anthocyanin-rich extracts from bilberry, chokeberry and grape were fed
for 14 weeks to male rats treated with a colon carcinogen, azoxymethane
[187]. The number and multiplicity of colonic aberrant crypt foci, colonic cell
proliferation, urinary levels of oxidative DNA damage and expression
of cyclo-oxygenase genes were measured as biomarkers of colon cancer. The
lower levels of these specific biomarkers in treated rats with respect to
controls suggest a protective role of berry extracts in colon carcinogenesis
and indicate multiple mechanisms of action. Black raspberries were also able
to suppress the development of N-nitrosomethylbenzylamine- induced
tumours in the rat oesophagus when administered as either a 5% freeze-dried
powder, an anthocyanin-rich fraction or an ethanol-based organic solvent-
soluble extract [188]. Grape juice inhibited mammary adenocarcinoma
multiplicity compared with that in controls through the inhibition of
DNA synthesis [189]. It was reported that anthocyanin-rich red grape extract
containing oenocyanin interferes with intestinal adenoma development in the
Apc(Min) mouse. The development of adenoma was found to be reduced by
oenocyanin-induced modulation of Akt in small intestinal adenomas [190].
Using a mouse model of colorectal cancer, a multiple regime feeding trial
was conducted to assess the role of cherry bioactive food components in
reducing cancer risk. Mice were fed one of the following: 1) a cherry diet, 2)
anthocyanins, 3) cyanidin, 4) control diet or 5) control diet with added
sulindac (an anti-inflammatory agent) to determine their effects on tumor
development [45]. Results suggested that mice assigned to any of the
three test diets showed significantly fewer and smaller volume cecal tumors,
but not colonic tumors, than control or sulindac supplemented mice,
suggesting that the bioactivity of cyanidin may be responsible for the site
specific inhibition of cecal tumors.
Conclusions
Anthocyanins assumed in a diet rich in fruits and vegetables are
associated with a decreased risk of inflammation-related chronic diseases.
However, evidence that anthocyanins are safe, multitargeted, efficacious, and
affordable demands further investigations. It remains unknown what amount
of anthocyanins are needed and for how long time and whether it is better to
consume food with anthocyanins or if supplements will suffice as well. Most
pharmacologic effects are presumed following preclinical investigation, and
more additional clinical trials are needed to further strengthen hypothesis for
Simona Lucioli 48
therapeutic efficacy. Most chronic diseases incubate for 20–30 years before
they manifest, therefore structuring such clinical trials will be difficult.
Anthocyanin from fruits and vegetables are absorbed to varying degrees
in the human body. As the evidence of therapeutic effects of anthocyanins
continues to accumulate, it is becoming more important to understand the
nature of absorption and metabolism in vivo. Conventionally, the
bioavailability of anthocyanins was thought to be very low. Several studies
hypothesize a potential for substantial absorption, but absorption is
hypothesized as disappearance of anthocyanin from the test system.
Conversely, it is possible that anthocyanin is transformed into
molecular structures that are not detected by the current analytical methods,
rather than being absorbed. More research is needed to fully understand
which molecules are effective and what mechanisms are involved in
their action. There is a large number of compounds that potentially could be
formed from the degradation of anthocyanins in the GIT. A comprehensive
study of anthocyanin transformation products present in the GIT would be
a valuable contribution towards the understanding of anthocyanin bioactivity.
Thanks to advancement in analytical technologies, the metabolic pathways
of anthocyanins can now be properly mapped to provide information on the
roles and significance of metabolites. These products may contribute to the
health effect of anthocyanins either directly in the GIT or after
absorption from the colon. As a result of the biotransformation by intestinal
microflora, some of these compounds can reach mM concentrations in faecal
water. Since microbial catabolites may be present at many sites of the body in
higher concentration than the parent compound, it is proposed that at least a
part of the biological activities ascribed to anthocyanins are due to their
colonic catabolites. The amount of anthocyanin metabolites formed in the
gastrointestinal tract may therefore be explored as a measure to evaluate
anthocyanin efficacy. However, the link between functional doses
of anthocyanin intake and metabolite concentrations remains poorly
understood.
Although anthocyanins are typically consumed as part of a meal, there is
little information on how their bioavailability is affected by other
components in the diet. In all likelihood, it is the synergy among bioactive
fruits and vegetable components (ascorbic acid, carotenoids, and other
flavonoids) that results in the health-promoting effects realized from
consuming the whole fruit. To obtain a better understanding of effects of
anthocyanins as drugs, it is necessary to characterize their properties at the
molecular level, and it is necessary to take into account the effects of co-
existing compound species in extracts. It is critical that the mechanistic
research findings be further substantiated through the implementation of well
Anthocyanins 49
designed human feeding studies using fruits produced, harvested, stored, and
distributed under standardized conditions as both pre-harvest and post-
harvest conditions can significantly affect the concentrations of bioactive
food component. Greater understanding of these processes will enable the
development of new food products, both fresh and manufactured, with
greater therapeutic efficacy.
So far, the potential health benefits of anthocyanins have been articulated
in the contexts of their antioxidant properties. Recent efforts have been
directed toward elucidating the molecular mechanisms underlying some of
anthocyanins’ novel health benefits. Such novel functions are not necessarily
dependent on the antioxidant effects of anthocyanins, but are produced by
currently unestablished chemical properties beyond the antioxidant capacity
of the molecules. The molecular basis for anthocyans pharmacological
activity includes the regulation of plethora of mechanisms mainly involved
in: (1) suppression of the inflammatory response through targeting the
PI3K/Akt and NF-κB pathways, (2) reduction of diabetes incidence through
modulation of insulin sensitivity and glucose utilization, (3) protection from
cardiovascular diseases by exerting (i) antihypertensive and endothelium-
protective activity through targeting the Akt/eNOS and ACE pathways, (ii)
antiatherogenic activity through targeting NF-κB mediated ICAM expression;
(4) neuroprotection through amelioration of oxidative stress and
neuroinflammation, (5) growth/differentiation control and tumor suppression
by exerting (i) cell cycle arrest and induction of apoptosis through the
JNK/p38 MAPK mediated caspase activation, (ii) anticancerogenic activity
through targeting the HGF signaling pathways, (iii) tumor anti-invasive
activity through targeting the VEGF signaling pathway. The estrogen-like
activity of anthocyans could be utilized in cancer and hormone-replacement
therapy.
The collected data provide a concise insight into molecular mechanisms
of protective and therapeutic activities of anthocyans in various pathological
conditions. Activities may not be attributed solely to their antioxidant activity
but also to direct blockage of signaling pathways. Structure-activity analysis
reveals that the number of hydroxyl groups and presence of sugar moiety are
crucial for their specific modulatory actions. Nevertheless, much remains to
be elucidated before a comprehensive understanding of the effects of
anthocyanins and related functions emerges.
In conclusion, with the aim to establish whether these compounds are
really capable to influence positively the incidence and progression of many
chronic diseases, a great deal of work in several areas is still necessary. This
includes: 1) epidemiological studies to evaluate the relationship between
anthocyanins rich food consumption and incidence of given pathologies; 2)
Simona Lucioli 50
feeding studies of anthocyanins to rats and to human healthy subjects to
unravel their main catabolites, to clarify their fate within the body and to
understand the main sites of absorption; 3) studies to understand the
interaction between anthocyanins and colon microflora; 4) analysis of factors
affecting bioavailability, including interaction with other dietary compounds;
5) identifying anthocyanins catabolites able to cross the blood–brain barrier;
6) studies to analyze metabolites at low concentrations by means of more
sensitive high-resolution analytical techniques such as mass spectrometry
analysis or immunoassays; 7) in vitro studies to fully characterise the
bioactivity of anthocyanins and anthocyanins catabolites generated in vivo.
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