Neurochem Res (2008) 33:24162426
DOI 10.1007/s11064-008-9697-6
REVIEW ARTICLE
Role of Nitric Oxide Synthases in Parkinsons Disease: A Review
on the Antioxidant and Anti-inflammatory Activity of Polyphenols
Katia Aquilano Sara Baldelli Giuseppe Rotilio
Maria Rosa Ciriolo
Accepted: 1 April 2008 / Published online: 16 April 2008
Springer Science+Business Media, LLC 2008
Abstract Natural polyphenols can exert protective action
on a number of pathological conditions including neuro-
degenerative disorders. The neuroprotective effects of
many polyphenols rely on their ability to permeate brain
barrier and here directly scavenge pathological concentra-
tion of reactive oxygen and nitrogen species and chelate
transition metal ions. Importantly, polyphenols modulate
neuroinflammation by inhibiting the expression of inflam-
matory genes and the level of intracellular antioxidants.
Parkinsons disease (PD) is a neurodegenerative disorder
characterized by several abnormalities including inflam-
mation, mitochondrial dysfunction, iron accumulation and
oxidative stress. There is considerable evidence showing
that cellular oxidative damage occurring in PD might result
also from the actions of altered production of nitric oxide
(NO). Indeed, high levels of neuronal and inducible NO
synthase (NOS) were found in substantia nigra of patients
and animal models of PD. Here, we evaluate the involve-
ment of NOS/NO in PD and explore the neuroprotective
activity of natural polyphenol compounds in terms of anti-
inflammatory and antioxidant action.
Keywords Nitric oxide
Parkinsons disease

Polyphenols
Inflammation
Anti-oxidants

Oxidative stress
Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.
K. Aquilano
S. Baldelli
G. Rotilio
M. R. Ciriolo (&)
Department of Biology, University of Rome Tor Vergata,
Via della Ricerca Scientifica, 00133 Rome, Italy
e-mail: ciriolo@bio.uniroma2.it
K. Aquilano
G. Rotilio
M. R. Ciriolo
Research Center IRCCS San Raffaele, Via dei Bonacolsi,
00165 Rome, Italy
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Introduction
Besides traditional nutrients (e.g., carbohydrates, amino
acids, vitamins, minerals, etc.), food contains many com-
pounds having the capacity to alter enzymatic and chemical
reactions, and therefore may impact human health both
positively and negatively [1]. These compounds are named
phytochemicals, phytonutrients, or non-traditional nutri-
ents. Among these are included polyphenols, which are
virtually present in all plants, fruits and vegetables. Notable
sources of polyphenols are apples, berries, nuts, tea, beer,
grapes and wine, cocoa and chocolate. This class, num-
bering more than 5,000 distinct species, is characterized by
the presence of more than one phenolic group per molecule
and is generally divided into flavonoids and non-flavo-
noids. Flavonoids are divided into anthocyanins and
anthoxantins, the latter subdivided in flavonols, flavanones,
flavones, isoflavones and flavonols [2]. Together with
flavonoids, tannins (condensed tannins, derived tannins and
hydrolyzable tannins) represent the most abundant group of
polyphenols and are polymerized large molecules, deriving
either by the plants themselves or as a result of food
processing.
Modulation of many biological systems by flavonoids,
tannins and other phytonutrients has been demonstrated by
a plethora of investigators. A variety of experimental and
epidemiological data have documented that selected natu-
ral polyphenols can exert protective action on a number of
pathological conditions including cardiovascular diseases,
cancer, infections, and neurodegenerative disorders (e.g.
Parkinsons and Alzheimers diseases, amyotrophic lateral
sclerosis, multiple sclerosis) as well [36]. The wide
spectrum of beneficial actions of polyphenols on human
health relies on their complicate chemistry, which may
result in pro-oxidant or anti-oxidant function. In fact,
Neurochem Res (2008) 33:24162426
polyphenols have been reported to undergo oxidation and
generate reactive oxygen species (ROS) [7], especially at
high concentration and/or in the presence of transition
metals and to be effective in arresting proliferation in a
number of tumor cells via redox-dependent pathways [8,
9]. On the contrary, at lower concentration (nanomolar
range) some polyphenols can function as direct or indirect
antioxidants both in in vivo and in vitro [1012]. However,
some polyphenols exhibit pro-oxidant activity and poten-
tial carcinogenicity, therefore dietary supplementation with
high concentration of a single antioxidant may be harmful
to human health and must be tightly controlled [9].
The brain most often suffers from the long-term impact
of the increase of ROS and reactive nitrogen species
(RNS), which play a major role in eliciting neuronal cell
death through irreversible oxidative/nitrosative injury to
neuronal macromolecules [13]. The susceptibility of brain
to oxidative stress derives from its scarce ability to coun-
teract the damaging effects of these reactive species.
Actually, brain is not endowed with an efficient antioxidant
defense system, having low levels of glutathione and
moderate activity of the antioxidant enzymes catalase,
superoxide dismutases (SODs) and glutathione peroxidase.
On the contrary, brain possesses high concentration of
transition metals (iron, copper), high aerobic metabolism
and elevated levels of ascorbic acid, overall contributing to
production of ROS/RNS and oxidized products. Finally,
many neurotransmitters, including NO, are autooxidized to
generate ROS and RNS. A main cause of oxidative stress in
neurodegenerative diseases is inflammation caused by
microglia activation [14]. Particularly, beside the induction
of immunocytes migration across the blood-brain barrier
and the engagement of astrocytes and endothelial cells, the
observed activation of microglia affects neuronal viability
through a persistent ROS and RNS generation. The rele-
vance of neuroinflammation in neurodegenerative disorders
derives from the evidence that it is detectable years before
significant loss of neurons occurs.
In vivo studies have demonstrated conjugation and
metabolism of dietary polyphenols in the small intestine
and liver, generating circulating active forms that reach the
tissues in glucuronidated, methylated or sulfated forms at a
nanomolar range after oral administration [15]. However,
the notion of polyphenols bioavailability in tissues inte-
grates several variables such as intestinal absorption and
excretion as well as cellular and tissue uptake, which are
specific for the polyphenol considered [15]. The uptake of
polyphenols and its metabolites is quite more complicated
in the brain in which an additional variable has to be
considered that is the blood-brain barrier, a regulatory
interface selectively limiting passage of most small polar
molecules and macromolecules from the cerebrovascular
circulation to the brain. Nevertheless, it has been
2417
demonstrated that many polyphenolic compounds are able
to permeate brain barrier and to exert their antioxidant
action directly in the brain [1618].
The beneficial effects of polyphenols on neurodegener-
ative disorders are likely to involve their ability to
modulate the level of intracellular antioxidants and many
biological processes including inflammation and cellular
signal transduction [1921]. Moreover, they have direct
antioxidant capacity both against ROS and RNS [10, 22].
Their anti-inflammatory action, free radical scavenging
activity and the ability to activate key antioxidant enzymes
in the brain allows breaking the vicious cycle of oxidative
stress and tissue damage [23, 24].
Parkinsons disease (PD) is a neurological disorder
characterized by degeneration of dopaminergic neurons in
the substantia nigra zona compacta. The cause of sporadic
PD is still unknown although increasing evidence indicates
that it may represent the final outcome of a complex set of
interactions, over decades of time, among factors that
include genetic predisposition, innate characteristics of the
nigrostriatal dopaminergic system of the brain, and expo-
sure to environmental toxins [25, 26]. Oxidative stress is
related to potentially toxic reactions such as elevation of
iron, mitochondrial function impairment and alteration of
brain antioxidants pool [14, 27]. In recent years, an
involvement of altered activity of NO synthases (NOSs)
(mainly the neuronal and inducible forms) has emerged. In
fact, high levels of neuronal NOS (nNOS) and inducible
NOS (iNOS) expression were observed in the nigrostriatal
region and basal ganglia in the post mortem PD brains. NO
has been also proposed to have a role in the inflammatory
processes occurring in PD [28]. In dependence on their
biological activity, polyphenols may have cytotoxic or
therapeutical effects in PD. A number of groups have
reported that administration of polyphenol herbicides such
as rotenone and paraquat induces parkinsonism in rodents
[2931]. On the contrary, molecules such as cathechines
and other polyphenols have been proposed as active factors
in mitigating or preventing PD [5, 3235], as they have an
anti-inflammatory and antioxidant activity. Here, we con-
sider the recent advances in the involvement of NO in PD.
Moreover, we also delineate the antioxidant and anti-
inflammatory action of some polyphenolic compounds and
the beneficial impact that these natural molecules may have
in the prevention and treatment of PD.
Nitric Oxide and Nitric Oxide Synthase in PD
PD is the most common age-related neurodegenerative
disease after Alzheimers disease and is grouped among
motor system disorders. PD is a slow and progressive
disease, mainly characterized by resting tremor, slowness
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of movement (bradykinesia), stiffness (rigidity), and poor
balance (postural instability). Beside mitochondrial dys-
functions, inflammation, apoptosis, oxidative and
nitrosative stress, mutated genes were also found in
familial PD. These include genes encoding for mitochon-
drial proteins such as Parkin, PTEN-induced putative
kinase 1 (PINK1), DJ-1, mitochondrial polymerase gamma
1 (POLG1), and non-mitochondrial proteins such as
a-synuclein, leucine-rich repeat kinase 2 (LRRK2). A
significant number of researchers found an association
between the expression of such mutated proteins and the
occurrence of redox status and/or mitochondrial homeo-
stasis alteration in PD models, thus reinforcing the
relevance of oxidative stress and mitochondrial dysfunc-
tion in the pathogenesis of familial and sporadic forms of
the disease [3638].
The role of NO in the pathogenesis of PD is still con-
troversial. Some research groups reported data showing no
correlation between PD and NO [39, 40]. On the contrary, a
large number of groups suggest that NO could be regarded
as one of the factors contributing to oxidative stress and
oxidative damage evidenced in post mortem studies,
in vitro and animal experimental models [41, 42]. NO is a
free radical playing an important messenger role in a wide
range of physiologic processes including vasodilatation,
immune response, neurotransmission [43]. This molecule is
synthesized from L-arginine by cytosolic NO synthases
(NOSs) using NADPH and molecular oxygen. To date,
three isoforms of NOS have been identified: nNOS,
endothelial NOS (eNOS) and iNOS [43]. While nNOS and
eNOS are constitutively expressed with their activity
depending on intracellular calcium levels, the activity of
iNOS is induced during cell inflammatory response. nNOS
is the predominant isoform in neurons, however, in central
nervous system, also eNOS and iNOS are expressed. In
particular, eNOS is present in cerebral vascular endothelial
cells and in motor neurons [44]; iNOS is expressed in
astrocytes and microglia when these cells respond to
inflammatory stimuli [45]. Recently, an additional isoform
of NOS has been identified in mitochondria (mtNOS),
whose nature and function is still a debated matter of
research [46]. In fact, it has been proposed that it may
represent a portion of iNOS, eNOS or nNOS translocating
into mitochondria where it likely participates in the regu-
lation of electron transfer chain [47, 48].
The implication of NO in PD has been firstly proposed
when high levels of nNOS and iNOS were found in the
nigrostriatal region and basal ganglia of the post mortem
PD brains [49, 50]. Immunoreactivity for nNOS and down-
stream effector guanylate cyclase were also found consis-
tently increased in the substantia nigra after treatment with
PD-inducing drugs [51, 52]. It has been suggested that a
significant portion of the increased NO production derives
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Neurochem Res (2008) 33:24162426
from the induction of iNOS as consequence of the
inflammatory processes due to microglia activation [28].
In this complex scenario, where the interaction of sev-
eral pathogenic pathways concurs to PD, NO seems to
represent an important downstream mediator and enhancer
of molecular events leading to dopaminergic neurons
death. As below reviewed, NO overproduction appears to
be an event that significantly contributes to death of
dopaminergic neurons via oxidative damage on cellular
lipids, proteins and DNA. This hypothesis is supported by
studies reporting that NOSs inhibitors or NOSs gene
knock-out significantly mitigate nigral cell loss in animal
experimental models [5355].
Neuronal Nitric Oxide Synthase in PD
When the production of NO is abundant and uncontrolled,
it results in damaging effects mainly mediated by its
reactive species. In PD, NO increase is caused either by
overexpression of NOSs or by other mechanisms including
glutamate excitotoxicity. The latter event causes the raise
of intracellular calcium levels, which in turn increases
nNOS dephosphorylation and its enzymatic activity. NO
reacts with superoxide anion formed during dopamine
metabolism thus generating peroxynitrite that is considered
one of the main damaging molecule in dopaminergic
neuronal cells [56].
Multiple lines of evidence indicate that NO increase is
associated with DNA damage, protein modifications and
citotoxicity, which are common pathogenic mechanisms
involved in PD and other neurodegenerative diseases.
Unpublished data from our laboratory support the idea that
NO plays a negative role in PD progression. In fact,
dopaminergic SH-SY5Y neuroblastoma cells overexpress-
ing nNOS are more sensitive to apoptosis elicited by the
PD-inducing drug rotenone (Fig. 1). The DNA damage is
mediated by direct reaction of RNS with DNA, through
inhibition of repair processes and by increasing the pro-
duction of genotoxic species such as lipid peroxidation
products (mainly 4-hydroxynonenal) and hydrogen perox-
ide. RNS also inhibit ribonucleotide reductase resulting in
blockage of DNA synthesis and induces over-single-strand
DNA breaks [57]. Overall, these DNA damages trigger
secondary effects including up-regulation of the tumor
suppressor p53 and increase of nuclear enzyme poly ADP-
ribose polymerase (PARP-1), which might promote apop-
tosis in PD animal models [58].
In addition to DNA damage, NO may also cause protein
modifications such as nitrosylation and nitration. Protein
nitration, generally, adds a nitro (NO2) group onto one of
the two carbons in position 3 of the aromatic ring of
tyrosine residues to form nitrotyrosine. Increased nitroty-
rosine were detected in substantia nigra in in vivo models
Neurochem Res (2008) 33:24162426
Fig. 1 nNOS overexpression enhances susceptibility of SH-SY5Y
neuroblastoma cells to rotenone treatment. SH-SY5Y neuroblastoma
cells were transiently transfected with a PME18s vector (kinfly gift of
Dr. Y. Watanabe, Kagawa University, Miki-Cho, Kida-gun, Japan)
containing rat nNOS cDNA (nNOS cells) or with a PME18 s empty
vector (Mock cells). About 48 h after transfection, Mock and nNOS
cells were treated with 5 lM rotenone. After 24 h, apoptosis was
evaluated by cytofluorimetric analysis upon staining of nuclei with
propidium iodide. Data are expressed as means SD (n = 4,
*P \ 0.01)
of PD [59, 60]. Particularly, an increased nitrotyrosine
immunostaining was found in the core of Lewy bodies
where a specific nitrated form of a-synuclein was also
present [61, 62]. This protein is highly expressed in the
substantia nigra and is present in the presynaptic terminals.
a-synuclein has a still debated function and belongs to the
family of SNARE proteins that are involved in the fusion
of cellular transport vesicles with target compartments.
Post-translational modifications such as phosphorylation or
nitration alter its conformational structure and lead to
abnormal aggregation [63].
S-nitrosylation occurs when proteins can be modified by
NO through their reactive cysteine thiols [64]. Several pro-
teins can be physiologically S-nitrosylated and this
modification may modulate their activity. These proteins
include the N-methyl-D-aspartate receptor (NMDAR),
p21ras, caspase 3 and 9, Nuclear Factor kB (NF-jB), and
many other proteins that are important to cell structure,
function, survival and death [65]. Recent findings demon-
strated that increased level of NO in both sporadic and
familial PD leads to specific S-nitrosylation of protein-di-
sulphide isomerase (PDI), which in endoplasmic reticulum
(ER) catalyses thiol-disulphide exchange, thus facilitating
disulphide bond formation and rearrangement reactions. The
up-regulation of PDI represents an adaptive response to
protect neuronal cells. S-nitrosylation of PDI inhibits its
enzymatic activity, leads to the accumulation of poly-
2419
ubiquitinated proteins, and elicits neuronal cell death via ER
stress [66]. Another molecule whose S-nitrosylation can lead
to abnormal protein accumulation is the E3 ubiquitin ligase,
parkin, which contributes to the pathogenesis of PD [67].
S-nitrosylated parkin initially stimulates ubiquitin E3 ligase
activity, resulting in enhanced ubiquitination as observed in
Lewy bodies, followed by a decrease in enzyme activity,
producing an inhibition of protein clearance by ubiquitin/
proteasome pathway [67, 68]. The generation of S-nitrosy-
lated parkin appears also to compromise its neuroprotective
effect [69].
Another possible mechanism for NO toxicity is due to its
high affinity for heme. This property makes NO a physio-
logic regulator of mitochondrial electron transfer and ATP
synthesis by inhibiting cytochrome c oxidase activity [70].
Nitrosylation or oxidations of protein thiols and subsequent
removal of iron from ironsulphur clusters in the cyto-
chrome complexes by NO also inhibit ATP synthesis [71].
Furthermore, it is suggested that NO could be the primary
agent involved in preferential complex I inhibition in
dopaminergic neurons in PD [37, 47]. The damaging effects
of NO on mitochondria could also derive from the increase
of NO production within mitochondria themselves,
although NOSs are virtually cytosolic enzymes. This phe-
nomenon could be due to translocation of iNOS, eNOS or
nNOS, or to the activation/expression of the mitochondrial
NOS (mtNOS). Recent reports have shown that mtNOS is
located on inner mitochondrial membrane [72], and shares
homology with nNOS, presumably representing one of its
splicing variants [73]. However, the exact nature of mtNOS
is still debated [46]. mtNOS has been proposed to be
important in the modulation of oxygen uptake and cell
signalling [47, 74], and to have a role in modulating
respiratory function and the release of cytochrome c into
cytosol, which represents an essential event in the apoptosis
triggering [48, 72]. In SH-SY5Y dopaminergic neuroblas-
toma cells, we found that a small but significant fraction of
the overexpressed nNOS targeted into mitochondria.
Although preliminary, our data suggest that the major sus-
ceptibility to rotenone of nNOS-overexpressing SH-SY5Y
cells may at least in part derive from the increased NO
production into mitochondria. In fact, being rotenone a
specific inhibitor of complex I and inducer of superoxide
generation, the augmented mitochondrial NO production
could result in cytotoxic peroxynitrite formation (Fig. 1).
Inducible Nitric Oxide Synthase in PD-Associated
Inflammation
Inflammation is elicited by immune system in response to
infection or irritation. It is characterized by the movement
of leukocytes and blood plasma from blood vessels to the
inflamed tissue due to the increased capillary permeability
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and to the expression of adhesion molecules and chemo-
attractant factors from endothelial and immune cells.
Importantly, inflammation is not per se healthy or unheal-
thy, because it helps the immune system to counteract
pathological states such as infection, but it can also lead to
progressive tissue damaging if unbalanced or prolonged.
This sort of paradox is particularly evident in the aetiology
of PD. Here the physiological inflammatory functions of
immune system in terms of cytokines and ROS/RNS pro-
duction by activated immune cells and complement cascade
launch are protracted and uncontrolled, leading to the fea-
tures of neurodegeneration [28]. The main cell type
involved in inflammation and consequent neurodegenera-
tion within PD is represented by microglia, which are
phagocytic cells participating in the physiological immune
control of the central nervous system [65]. Activation of
microglia can be triggered by different molecules such as
lipopolysaccharide from Gram-negative bacteria and by
PD-inducing agents both in humans and experimental
models of PD. In particular, microglia activation is trig-
gered using rotenone from pesticides and herbicides [75],
neurotoxins such as 6-hydroxydopamine (6-OHDA) [76]
or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
[77]. Such activation implies microglia to up-regulate the
expression of molecules involved in antigen presentation
(e.g. CD1, MHCII), as well as cell adhesion molecules.
Subsequently, microglial cells adhere to neurons, driven by
the recognition of chemoattractant proteins expressed by
neurons themselves and by ligation of activated comple-
ment. If the activating stimulus does not decrease, microglia
definitively assumes phagocytic functionality, leading to
neuronal cell death and consequent chemoattractants
release, which in turn induce a greater microglia infiltration
and enhanced neuroinflammation.
The inflammation developed within neuronal tissue has
been observed in several studies involving PD patients,
which primarily show increased number of activated
microglial cells [28]. However, the most reasonable evi-
dence of neuroinflammation in PD patients is the elevated
level of inflammatory cytokines, such as tumour necrosis
factor a (TNFa) and IL-6 [78]. Importantly, the up-regula-
tion of the inflammatory genes encoding for cyclooxygenase
2 (COX-2) and iNOS was observed in a population of
amoeboid microglial cells from PD patients [45, 79]. COX-2
can provoke neuronal cell death by producing prostaglan-
dins such as PGE2, which can exert a direct effect upon
dopamine-containing neurons inducing intraneuronal tox-
icity [80]. iNOS up-regulation in microglia seems to play an
important role in the onset of inflammatory processes in PD
and acts synergistically with other inflammatory mediators.
PD-inducing drugs such as rotenone and MPTP or dopamine
overexposure provoke activation of iNOS expression [39,
81, 82]. iNOS and COX-2 expression is induced by NF-jB
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Neurochem Res (2008) 33:24162426
signaling pathway, which involves phosphorylation of the
I-jB inhibitory subunit and its detachment from NF-jB,
allowing NF-jB translocation into the nucleus where acti-
vates transcription of these genes [43]. iNOS displays a
relative slow turnover and produce NO at very high levels
for long periods. During inflammation, the concomitant
activation of iNOS, NADPH oxidase and COX-2 produce
high levels of ROS and RNS. Given that microglia is
particularly abundant in the substantia nigra and that
dopaminergic neurons are highly susceptible to oxidative/
nitrosative stress, the resulting ROS/RNS overproduction
severely damage their cellular structures and macromole-
cules mainly as consequence of peroxynitrite formation. The
iNOS-derived NO is also implicated in the release of iron
from transferrin, thus enhancing Fenton reaction leading to
production of highly reactive hydroxyl radicals [83].
Polyphenols in PD Treatment
Overall these findings suggest that a therapeutical approach
to PD could be the employ of nNOS or iNOS inhibitors. In
fact, the toxic role of NO in PD was also demonstrated by
the use of NOS inhibitors, which protect mice and monkey
models from PD [55, 84]. Apart NOS isoforms, mtNOS is
emerging as a new target for therapeutics in the diseases
associated with mitochondrial dysfunction and neurode-
generative disorders. Moreover, another way to ameliorate
the effects of excessive NO production such as abnormal
S-nitrosylations and nitrotyrosine formation, could be to
inhibit NMDAR, whose altered activity is able to elicit
NO-mediated excitotoxicity. In fact, moderate blockade of
NMDAR activity by memantine can protect neurons from
this type of injury and death [67]. However, among the
novel therapeutic strategies proposed for achieving neuro-
protection in PD are included ROS/RNS scavengers, metal
chelators and anti-inflammatory molecules. Due to their
antioxidant and anti-inflammatory actions, polyphenols
represent novel promising tools for PD treatment (Fig. 2).
Antioxidant Function
Numerous studies have demonstrated that specific poly-
phenols are effective direct scavengers of physiologically
relevant ROS and RNS in vitro, including superoxide,
peroxyl radicals, singlet oxygen, peroxynitrite, and hypo-
chlorous acid [12, 8589]. Oxidative damage-induced
apoptosis in neuronal cells could be protected by naturally
occurring polyphenols [90]. The green tea flavonoid cate-
chins have been found to be the most efficient antioxidants;
however, other polyphenols such as oxyresveratrol, resve-
ratrol and quercetin are able to protect against NO
cytotoxicity as well [22, 89, 91]. Direct free radical
Neurochem Res (2008) 33:24162426 2421

Fig. 2 Chemical structures of

polyphenols suggested for
potential therapy of PD

scavenging capacity of polyphenols is primarily attributed

to the high reactivity of hydroxyl substituents. Inhibition of
RNS by polyphenols involves the same basic structure-
activity relationships as inhibition of ROS. For instance,
catechins are known to directly counteract RNS toxicity by
scavenging peroxynitrite and its progenitor, superoxide. It
has been reported that the most significant determinant of
activity against peroxynitrite is the 30 ,40 -catechol group in
the B-ring and there was an apparent positive correlation
between the number of hydroxyl groups, particularly of the
B-ring, and the anti-radical activity [11]. To our knowl-
edge, a direct anti-RNS action of polyphenols in vivo has
not been deeply investigated, however, a study from
Yokozawa demonstrated that green tea catechins protect
from peroxynitrite-mediated damage in a mice model of
ischemia-reperfusion by preventing 3-nitrotyrosine forma-
tion. In particular, these flavonoids exert stronger
protective activity against peroxynitrite-induced oxidative
damage than the peroxynitrite scavenger ebselen [92]. The
potential strong therapeutic role of catechins in PD also
derives from their ability to chelate iron. This property
contributes to their antioxidant activity by preventing
redox-active transition metal from catalyzing free radicals
formation. Moreover, the antioxidant function is also
related to the induction of the expression of antioxidant and
detoxifying enzymes particularly in the brain, which is not
sufficiently endowed of a well-organized antioxidant
defense system. The neuroprotective function of green tea
flavonoids and their possible employ in the treatment of
neurodegenerative disorders such as PD and Alzheimers
disease has been largely studied and adequately reviewed
during the last years [35, 93].
Other polyphenolic compounds have been explored as
possible tools to counteract oxidative stress in PD. A
neuroprotective action of resveratrol has been demon-
strated in some neuronal experimental systems: it inhibits
hippocampal cell death, induced by trauma or ischemia,
and intracellular ROS formation [9496]; it protects
against excitotoxicity brain damage [97]; it protects rat
primary neurons against apoptosis inhibiting expression of
caspases 3 and 12 and calcium overload [98]. Resveratrol
has been demonstrated to prevent apoptosis induced by
dopamine treatment in dopaminergic SH-SY5Y neuro-
blastoma cells. Dopamine acts directly as a pro-oxidant
agent as it can oxidize spontaneously to form ROS and
quinones and may contribute to the degeneration of
Fig. 3 Resveratrol protects SH-SY5Y neuroblastoma cells from
cytotoxicity mediated by rotenone. SH-SY5Y neuroblastoma cells
were treated with 5 lM rotenone for 24 h. Treatment with resveratrol
was performed at concentration of 0.5 lM, because higher concen-
trations were citotoxic. Resveratrol was added 1 h prior rotenone
treatment and maintained throughout the experiment. After treat-
ments, the viable cells were counted by optical microscope on
hemocytometer, after Trypan blue staining. Data are expressed as
means SD (n = 4, *P \ 0.001)
dopaminergic neurons in PD. Resveratrol inhibits the
dopamine-mediated activation of caspases and strongly
enhances the expression of the anti-apoptotic protein Bcl-2
[32]. Unpublished data from our laboratory demonstrated
that pre-treatment of SH-SY5Y cells with low doses of
resveratrol (0.5 lM) is also able to reduce cell death elic-
ited by rotenone added both at concentrations in a nM (data
not shown) and lM range (Fig. 3). Rotenone is widely used
as inducer of PD phenotypes within both in in vivo and
in vitro models and acts primarily inhibiting the electron
transport chain at the complex I, leading to impaired
energetic metabolism and high generation of superoxide
radical. These findings strongly suggest that resveratrol
may have a protective role in counteracting ROS cytotox-
icity at both cytosolic and mitochondrial level.
Resveratrol has been demonstrated to protect healthy
rats against lipid peroxidation and to promote enhance-
ment of antioxidant enzymes activity. Particularly, it has
been demonstrated that intraperitonelly administration of
resveratrol significantly enhances in a dose-dependent
manner the level of superoxide dismutase (SOD), catalase

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and peroxidases [18]. The enhancement of SOD activity
by resveratrol could be effective against NO toxicity as,
by scavenging superoxide, it counteracts the formation of
peroxynitrite [99]. Among the antioxidant genes modu-
lated by resveratrol, there is heme oxygenase (HO), which
is responsible for degradation of heme into biliverdin/
bilirubin, iron and carbon monoxide. Being heme a pro-
oxidant molecule, while biliverdin and bilirubin are anti-
oxidants, induction of high HO level by resveratrol is
antiapoptotic and cytoprotective against ROS/RNS dam-
age [33].
Mangiferin, a polyphenol extracted from mango, is
another recently proposed antioxidant for PD treatment
[100]. It is able to cross the blood-barrier [101], displays
direct antioxidant capacity and is a good iron chelator,
thus preventing ROS production through the Fenton and
HaberWeiss reactions [102]. Mangiferin has been dem-
onstrated to protect against cytotoxicity mediated by the
PD-inducing toxin MPTP and its active metabolite
MPP + in murine N2A neuroblastoma cells [100]. Par-
ticularly, mangiferin is able to revert the oxidative stress
most probably through its scavenging activity against
ROS.
Although mainly suggested as promising therapeutic
agent in Alzheimers disease [5], the polyphenol curcumin
has a protective role also against MPTP toxicity [103].
Indeed, in murine dopaminergic neuroblastoma cells, cur-
cumin induces overexpression of the anti-apoptotic Bcl-2,
reduces the loss of mitochondrial membrane potential and
the increase of ROS.
Anti-inflammatory Activity
Referring to the above reported observations, it appears
that inhibition of any step in the inflammation pathway
can be useful for the attenuation of its harmful conse-
quences in the progression of PD. Therefore, anti-
inflammatory agents that suppress microglial activation or
interfere with the production of neurotoxic factors by
activated microglia are candidates for therapeutic inter-
vention in PD. The usually employed drugs, such as
steroidal and non-steroidal anti-inflammatory molecules,
produce their effect in the treatment of acute inflammatory
diseases, whereas they are not resolutive when adminis-
tered in course of chronic disorders, such as rheumatoid
arthritis, atopic dermatitis or PD itself. Additionally, the
raising of unpleasing side effects during the prolonged
treatment with such drugs evidences the need for new safe
and effective anti-inflammatory molecules, which often
are recruited among natural compounds. Flavonoids have
been proposed as anti-inflammatory agents and their
activity is exerted by intervening in a wide range of
molecular and cellular mechanisms ascribable to
123
Neurochem Res (2008) 33:24162426
inflammation pathways [21], most of which are typical
features of chronic diseases such as PD.
The most evident effect of flavonoids is the inhibition of
inflammatory cytokines production, previously described
as principal actors in the development of neuronal injury
within PD tissues. On this ground, genistein has been
reported to inhibit IL-1b, TNFa and IL-6 production by
LPS-activated human monocytes [104]. In addition to
direct cytokine inhibition, flavonoids interfere with their
synthesis by blocking various components of the signal
transduction that leads to inflammatory genes activation.
For instance, flavonols inhibit protein kinase C (PKC)
[105] and mitogen activated protein kinases (MAPK). In
particular, quercetin reduces the activity of p38 MAPK and
Jun N-terminal kinase/stress-activated protein kinase, thus
inhibiting the binding of activator protein 1 (AP1) to the
DNA [106]. Quercetin and wogonin interfere with the
activation of extracellular signal-related kinase 1/2 leading
to NF-jB inhibition [22]. NF-jB is also inhibited by sev-
eral other flavonoid compounds, such as genistein,
kaempferol, amentoflavone [21]. These evidences show
that flavonoids, by counteracting the activation of several
factors including NF-jB and AP-1, can inhibit the syn-
thesis and the activity of several inflammation-involved
cytokines, which are important events during the patho-
genesis of PD.
Another important target of flavonoids for their anti-
inflammatory effect is COX. Particularly, COX-2 is the
most represented in inflammation-related immune cells,
such as macrophages, and it is typically induced through
LPS stimulation. Some flavonoids, i.e. luteolin or galangin
were firstly noted as COX inhibitors [107]. Following this
observation, a huge amount of papers have been published
on this topic showing other flavonoids to inhibit COX. In
particular, flavone, kaempferol and quercetin were
observed to be efficient COX inhibitors [21]. Additionally,
flavonoids can exert an inhibitory effect upon NF-jB
activation [22]. Since COX-2 is synthesized through an
NF-jB-mediated pathway, the effect of flavonoids upon
COX-2 activity is exerted also at expression level. In
particular, genistein, apigenin and kaempferol counteract
the activity of inhibitor-jB (I-jB) kinase, thus preventing
NF-jB to address the DNA [108, 109]. The evidences of
flavonoids as COX inhibitors enforce the idea of their
employ in the treatment of inflammatory pathologies,
including PD.
As above mentioned, the inflammatory process and the
consequent PD-related dopaminergic neuronal damage
found in PD is due to the production of NO from L-arginine
by iNOS. The isolation of some compounds that selectively
inhibit NO synthesis by iNOS without affecting eNOS or
nNOS is attractive. Nevertheless, none of the studies
reported so far showed a significant effect of flavonoids
Neurochem Res (2008) 33:24162426 2423

Fig. 4 Diagram of the NO and Polyphenols in PD

involvement of NO and the anti-
inflammatory and anti-oxidant
Increase of iNOS activity Increase of nNOS activity
activity of polyphenols in PD
Increase of NF-B activity - NMDAR stimulation (Ca2+ overload)
(I-B degradation, - nNOS overexpression
NF-B translocation into nucleus) - nNOS dephosphorylation

ROS/RNS scavenging
RNS Induction of cellular antioxidants
Polyphenols Inhibition of iNOS expression

Cellular nitrosative/oxidative stress

Inflammation
- DNA damage, lipid peroxidation
- Alteration of protein activity via S-nitrosylation, nitration
(-synuclein, parkin, PDI, cytochrome c oxidase, complex I)
- Mitochondria impairment (failure of energetic metabolism, apoptosis)

upon iNOS activity perhaps due to the high level of NO
produced by this NOS isoforms. To our knowledge, the
only flavonoid compound which showed a relevant activity
in iNOS inhibition was echinoisosophoranone, being
effective at a reasonable concentration [110]. On the other
hand, several flavonoids have been reported to inhibit
iNOS expression. Liang and colleagues firstly found that
apigenin, genistein and kaempferol inhibited NO produc-
tion by down-regulating iNOS synthesis [108]. Following
these findings, many other flavonoids were discovered to
be potent inhibitors of iNOS expression, including oroxylin
A, quercetin, resveratrol, bilobetin and ginkgetin via inhi-
bition of NF-jB signaling pathway [21, 111]. Altogether,
these data demonstrate that some flavonoids are inhibitors
of iNOS expression but not of its activity. Anyway, since
there is the possibility of a non-selective NOS inhibition,
the role of these compounds in counteracting inflamma-
tory-related NO production is still far from being
elucidated.
Conclusions
In recent years, NO and its reactive metabolites have been
proposed as important actors in the processes leading to
neuronal cell death in PD both in terms of pro-oxidants and
mediators of inflammatory responses. Therefore, the
blockage of NO synthesis or the scavenging of RNS could
represent an efficient tool against PD progression. In this
context, polyphenols are promising natural antioxidant and
anti-inflammatory molecules that are proposed to be
employed in neurodegenerative diseases associated with
oxidative/nitrosative stress.
The potential therapeutic function of polyphenols is
strictly related to their capacity to cross blood-brain barrier
and exert their beneficial activity directly [112, 113]. The
antioxidant function likely depends on the scavenging
activity of free radicals among which is included NO-derived
reactive species. Moreover, many polyphenols display the
ability to induce the expression of intracellular antioxidants
enzymes. The anti-inflammatory activity of polyphenols
depends on the inhibition of the expression of inflammatory
genes such as eicosanoid generating enzymes and iNOS,
mainly through interfering with the activation of the
upstream NF-jB transcription factor. Moreover, some
polyphenols may inhibit the release of pro-inflammatory
molecules (IL-1b, TNFa and IL-6). Being PD a multifactor
disease in which NO, inflammation and oxidative stress
play important roles, the employ of compounds such as
polyphenols, having poly-pharmacological activities, is
attractive. The schematic model reported in Fig. 4 summa-
rizes the mechanisms by which polyphenolic compounds can
modulate NO toxicity in PD.
Overall the findings reported in literature strongly sug-
gest that anti-oxidant and anti-inflammatory agents based
on polyphenols molecule could be proposed for treatment
of PD.
Acknowledgements This work was partially supported by Minis-
tero della Salute, MIUR and FIRB Idee Progettuali. We thank Dr.
Giovanni Auricchio for his advice in the matter of PD inflammatory
processes and helpful critical reading of the manuscript.

123
2424
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