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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: simona.lucioli@entecra.it

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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