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Current Medicinal Chemistry, 2012, 19, 2901-2917 2901
1875-533X/12 $58.00+.00 © 2012 Bentham Science Publishers
Protocatechuic Acid and Human Disease Prevention: Biological Activities and
Molecular Mechanisms
R. Masella*,1, C. Santangelo1, M. D’Archivio1, G. LiVolti2,3, C. Giovannini1 and F. Galvano2,3
1Dept. Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy
2Dept. of Drug Science, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
3Dept. of Cardiac Surgery, IRCCS Policlinico “San Donato”, San Donato Milanese, Milan, Italy
Abstract: Epidemiological evidence has shown that a high dietary intake of vegetables and fruit rich in polyphenols is associated with a
reduction of cancer incidence and mortality from coronary heart disease. The healthy effects associated with polyphenol consumption
have made the study of the mechanisms of action a matter of great importance. In particular, the hydroxybenzoic acid protocatechuic acid
(PCA) has been eliciting a growing interest for several reasons. Firstly, PCA is one of the main metabolites of complex polypheno ls s uch
as anthocyanins and procyanidins that are normally found at high concentrations in vegetables and fruit, and are absorbed by animals and
humans. Since the daily intake of anthocyanins has been estimated to be much higher than that of other polyphenols, the nutritional value
of PCA is increasingly recognized. Secondly, a growing body of evidence supports the concept that PCA can exert a variety of biological
effects by acting on different molecular targets. It has been shown that PCA possesses antioxidant, anti-inflammatory as well as anti-
hyperglycemic and neuroprotective activities. Furthermore, PCA seems to have chemopreventive potential because it inhibits the in vitro
chemical carcinogenesis and exerts pro-apoptotic and anti-proliferative effects in different tissues. This review is aimed at providing an
up-dated and comprehensive report on PCA giving a special emphasis on its biological activities and the molecular mechanisms of action
most likely responsible for a beneficial role in human disease prevention.
Keywords: Antioxidants, apoptosis, atherosclerosis, bioavailability, cancer, inflammation, phenolic acids, polyphenols, protocatechuic acid,
signaling pathways, type 2 diabetes.
INTRODUCTION
Over the last years great interest has b een growing in the effects
that lifestyle strategies may have on the risk of developing chronic-
degenerative diseases such as cardiovascular diseases, diabetes,
neurodegenerative diseases and cancer. The consumption of a diet
rich in vegetables and fruit seems to exert beneficial healthy effects
because of the high content in fibers, mineral salts, vitamins, and a
large number of non-nutrient phytochemicals such as polyphenols
[1,2]. Nowadays, daily consumption of five portions of fruit and
vegetables is recommended, although there is not guidance on the
specific type of fruit and vegetables to consume [3]. Thus, to date
there is not convincing evidence for the identification of those
vegetable foods that could significantly contribute to chronic-
degenerative disease prevention. The picture is further complicated
by the high number of potentially protective components, e.g.
polyphenols, contained in fruit and vegetables. Dietary polyphenols
comprise a great diversity of compounds among which flavonoids
and several classes of non-flavonoids are distinguished (Fig. 1) [4].
Notably, evidence has been growing on the potential biological
activities that individual compounds can exert, not necessarily
related either to the complexity of the structure or to the abundance
in food sources[5]. In this regard, protocatechuic acid, a catechol-
type o-diphenol phenolic acid (PCA, 3,4-dihydroxybenzoic acid),
has elicited the interest of researchers in th e last few years because
of the antioxidant activity and the additional biological functions it
is able to exert. The aim of this work is to review what is known
about PCA, including distribution in food, metabolic formation, and
bioavailability. Particular attention will be paid to the mechanisms
of action by which PCA might exert potential protective/therapeutic
effects. We will focus on the capability to modulate oxidative
stress, inflammation, and apoptosis processes that, often in
combination one another, play a main role in the pathogenesis of
chronic-degenerative diseases.
*Address correspondence to this author at the Istituto Superiore di Sanità, Viale Regina
Elena 299, 00161 Rome, Italy; Tel: +390649902544; Fax: +390649387101;
E-mail: roberta.masella@iss.it
NUTRITIONAL VALUE OF PCA
PCA is contained in fruit and vegetables, but also in plant-
derived beverages such as tea, white grape wine and in herbal
medicine [6-30], and its content varies considerably depending on
the type of food. In Table 1 are shown the main dietary sources of
PCA. It is important to underscore that numerous factors, such as
ripeness at the time of harvest, environmental factors, and storage,
may affect the polyphenol content of plants (for details see [31-
33]).
Because of its low concentrations in fruit and vegetables, until a
few years ago little attention has been paid to the beneficial effects
of PCA on health. However, recent data have clarified that PCA
concentration in vivo could be higher than the simple quantity
ingested because it is a main metabolite of complex polyphenols,
e.g. anthocyanins (ACNs) [34]. Polyphenols can undergo a great
deal of structural transformation both in vivo and in vitro that will
undoubtedly alter their activity. This is particularly apparent for the
flavonoid subclass ACNs. ACNs are water-soluble pigments
dissolved in the vacuolar sap of the ep idermal tissue of flowers and
fruit [35] to which they impart the peculiar red-orange to blue-
violet colors [36]. ACNs are widely distributed in the human diet:
they are mainly found in fruits, especially those of the berry family,
pomegranate and pigmented varieties of oranges (Tarocco, Mo ro ,
and Sanguinello). For these reasons, their daily intake is much
higher (180-250 mg/d) [37,38] than that of other polyphenols
(estimated at 20-30 mg/d) [39,40]. ACNs are absorbed in animals
and humans as both intact glycosides and metabolite [41-45].
Indeed, they are rapidly metabolized ultimately leading to the
formation of phenolic acids and aldehydes [45]. In particular, at
physiological pH, ACNs easily convert to PCA [46], which is also
abundantly formed and absorbed in the large intestine because of
microbial metabolization [34]. Consequently, PCA deserves great
nutritional interest as main human ACN metabolite that might reach
tissues in such an amount to exert biological healthy effects [47].
ABSORPTION, METABOLISM, AND BIOAVAILABILITY
The absorption of some, but not all, dietary polyphenols occurs
in the small intestine. It is widely accepted that two possible
2902 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
Fig. (1). Principal classes of polyphenols.
Table 1. PCA Food Content
Dietary sources PCA maximum concentration found in foo d References
Mushrooms (Ramaria botrytis) 34.27 mg/100g dry weight Barros L. et al. [16]
Green Chicory 30.18 mg/100g fresh weight Rossetto M. et al. [235]
Red Chicory 25.71 mg/100g fresh weight Rossetto M. et al. [235]
Black olives 21.00 mg/100g fresh weight Boskou, G. et al. [236]
Black raspberry 8.35 mg/100g dry weight Wu X. et al. [18]
Date, dried 8.34 mg/100g dry weight Al-Farsi M. et al. [237]
Date, fresh 4.49 mg/100g fresh weight Al-Farsi M. et al. [237]
Green olives 2.00 mg/100g fresh weight Boskou G. et al. [236]
Apple Juice 1.64 mg/100ml Gokmen V. et al. [238]
Vinegar 1.62 mg/100ml Natera R. et al. [239]
White wine 1.30 mg/100 ml Ping L. et al. [240]
Blackberries 1.01mg/100g dry weight Zadernowski R. et al. [241]
Red wine 0.72 mg/100 ml Minussi R.C. et al. [242]
Black currant 0.71mg/100g, dry weight Zadernowski R et al. [241]
Grape, raisin 0.68 mg/100g fresh weight Karadeniz F. et al. [243]
Beer (alcohol free) 0.51 mg/100ml Garcia A. et al. [244]
Cauliflower 0.45 mg/100g fresh weight Ping, L. et al. [240]
Almond 0.44 mg/100g fresh weight Milbury P.E. et al. [245]
Virgin olive oil 0.24 mg/100g fresh weight Liberatore L. et al. [246]
Pomegranate, pure juice 0.20 mg/100 ml Poyrazoglu E. et al. [247]
Lentils 0.14 mg/100g fresh weight Duenas M. et al. [248]
mechanisms exist to hydrolysed glycosides. The first mechanism
involves the lactase phloridizin hydrolase (LPH) that is present in
the brush-border of the small intestine epithelial cells [48]. The
released aglycones can enter th e epithelial cell by passive diffusion
as a result of their increased lipophilicity. The second mechanism
involves the cytosolic -glucosidase (CBG) that is present within
Lignans
Proanthocyanidins
POLYPHENOLS
Phenolic acids
Flavones lsoflavones
Flavonols Flavanones
Flavanols/Catechins
Stilbenes
Lignins
Hydroxy benzoic
acids
(R1=R2=OH, R3=H: Protocatechuic acid)
Hydroxy cinnamic
acids
Anthocyanins
(R3’=OH, R5’=H, R3= -O-glycoside: (Cyanidin 3 Glucoside)
R3
R5’
R3’
Flavonoids
Protocatechuic Acid and Human Disease Prevention Current Medicinal Chemistry, 2012 Vol. 19, No. 18 2903
the epithelial cells where th e polar glycosides are transported
through the active sodium-dependent glucose transporter SGLT1
[49].
The polyphenols that are not absorbed in the small intestine
reach the colon where they undergo substantial structural
modifications. In fact the colonic microflora hydrolyze glycosides
into aglycones and degrade them to simple phenolic acids [50, 51].
This is of great importance because specific active metabolites are
produced. Once absorbed, and p rior to the passage into the
bloodstream, the polyphenols, now simple aglycones, undergo
addition al structural modifications through the conjugation process.
The conjugation, mainly methylation, sulfation, and glucuroni-
dation, represents a metabolic detoxication process that facilitates
the biliary and urinary excretion of the polyphenols by increasing
their hydrophilicity (Fig. 2). Therefore, any single polyphenol
generates several metabolites, and the compounds that reach cells
and tissues are chemically, biologically and, in many cases
functionally different from the original dietary form [52]. These
modifications can affect the polyphenol bioavailability and
consequently their biological activity. The term ‘bioav ailability’
means the fraction or compound of an ingested nutrient that reaches
the systemic circulation and the specific sites where it can exert its
biological action [53]. Even though a compound has strong
antioxidant or other biological activities in vitro, it would have little
biological activity in vivo if insufficient or none of the compound
reaches the target tissues. The most abundant polyphenols in our
diet are not necessarily those that have the best bioavailability
profile. Only a small part of the dietary ACNs are absorbed, an d
large amounts of the ingested compounds are likely to enter the
colon. Until now, ACNs have been thought to have a very low
bioavailability, in fact human pharmacokinetic studies generally
identified less than 1% of the ingested parent ACNs in biological
fluids, despite the consumption of doses exceeding 500 mg [54].
This percentage is lower than that found for other flavonoids [55].
However, it is to underline that spontaneous degradation of ACNs
to phenolic acids and aldehydes has been reported to occur under
experimental [56] and biological conditions [34, 50]. The
incongruity between bioavailability and reported bioactivity has
undoubtedly hindered the field of ACN research, and future
research into ACNs bioactivity should include their degradation
products. In particular, it has been shown that, at physiological pH
such as in the bloodstream, Cyanidin-3-Glucoside (C3G), the most
abundant ACN in food, easily degrades to cyanidin, that rapidly and
spontaneously degrades to PCA, which can be further metabolized
to glucuronide and sulphate conjugates [47]. Furthermore, Wang et
al. [57] have demonstrated that C3G is intensively transformed into
PCA in apoE-deficient mice, and that the plasma maximal level of
C3G is 3.7-fold lower than that of PCA. In human, PCA accounts
for 44.4% of the ingested C3G in 6-h post-consumption
bloodstream [34]. A mean concentration of PCA of 2.0 nmol/g has
been found in fecal samples collected the day after pigmented
orange juice consumption, thus suggesting that the in vivo
production of PCA by human colon microflora occurs [34, 58].
Finally, studies carried out in rats and in perfused rat heart have
shown that PCA may further be converted to vanillic acid [59].
However, to establish conclusive evidence for the effectiveness
of a dietary compound in disease prevention and human health
improvement, it is essential to know its bioavailability but also to
evaluate its biological activity in target tissues.
In a recent study [60] seventy-two human subjects consuming
moderate amounts of berry for 8 weeks in a randomized dietary
intervention trial, have shown increased plasma PCA levels (+21%)
with respect to the control group. In another randomized, cross-over
study [61], 375 ml of Champagne wine or a control matched
beverage have been administered to 15 healthy human volunteers.
The consumption of Champagne wine induced an increase in
urinary excretion of a number of phenolic metabolite, among which
PCA, compared with the control intervention. Finally, an interesting
study has demonstrated that male Balb/cA mice fed a standard diet
supplemented with PCA for 12 weeks showed increased PCA
deposit in plasma and tissues such as brain, heart, liver, and kidneys
[62].
ANTIOXIDANT ACTIVITY
Oxidative stress plays a pivotal role in the pathogenesis of
several degenerative diseases such as atherosclerosis,
cardiovascular diseases, type 2 diabetes, neurodegenerative
diseases, and cancer. Furthermore, the ‘free radical theory of
ageing’ casts lights on understanding the process of ageing, and in
Fig. (2). Gastrointestinal metabolism and absorpt ion of polyphenols. In the stomach oligomeric polyphenols dissociate into monomers. In the small
intestine a few of the polyphenol glycosides are hydrolysed to aglycons. In the colon polyphenols undergo substantial structural modifications by colonic
microflora resulting in the formation of metabolites such as phenolic acids. Most of the conjugation process takes place in the liver. The conjugation, that
mainly includes methylation, sulfation, and glucuronidation, represents a metabolic detoxication process that facilitates the biliary and urinary excretion of the
polyphenols by increasing their hydrophilicity.
Monomeric
units
Oligomeric
polyphenols
Stomach
Small Intestine
jejunum
ileum
Colon
Liver
Phenolicacids
glucuronides
Kidney
Urine
Omethylated
Sulphates
Portal
vein
Further
metabolism
Renal excretion
ofglucuronides
Oligomers
cleaved cells
SKINAND
BRAIN
Gutmicroflora
Flavonoids
Polyphenols
glucuronides
2904 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
finding effective anti-ageing agents [63-67]. The reactive oxygen
species (ROS) are continuously produced within the cell,
particularly during mitochondrial electron transport chain, and in
peroxisomes [68]. Furthermore, ROS can be generated as a
consequence of foreign compounds, toxins, drugs and foods, or
exposure to environmental factors such as pollutants, heavy metals
or ultraviolet radiations [69]. Notably, at low physiological levels,
ROS function as ‘redox messengers’ in intracellular signaling and
regulation [70], whereas excess of ROS induces oxidative
modification of cellular macromolecules, which leads to aberrant
cell functions causing membrane and DNA damage, enzyme
inactivation and, ultimately, cell death [71]. Controlling the
intracellular redox state by means of efficient antioxidant
machinery represents thus a crucial event in the prevention of cell
damage, and it is easily understandable why humans have
developed sophisticated mechanisms to cope with an excess of free
radical production. Endogenous enzymatic and non-enzymatic
antioxidant defenses, and exogenous antioxidants supplied by the
diet play a pivotal role in such protective mechanisms.
A central role in the endogenous defense system is played by
glutathione (GSH), a ubiquitous cysteine-containing tripeptide that
participates in redox reactions by the reversible oxidation of its
active thiol group (GSSG) [72]. GSH scavenges directly free
radicals or act as an enzymatic substrate for antioxidant/detoxifying
enzymes including glutathione peroxidase (GPx) and glutathione-S-
transferases (GST), which are involved in the detoxification/
reduction of H2O2, lipid hydroperoxides and electrophilic
compounds. In the presence of oxidative stress the ratio
GSH/GSSG rapidly decreases. The produced GSSG can be reduced
by glutathione reductase (GR) [73]. In addition, GSH can be de
novo synthesized by the glutathione synthase complex [74]. The
efficiency of the entire GSH cycle depends on the functionality of
GSH-related enzymes and the availability of amino acids (Fig. 3).
Among the endogenous non-enzymatic antioxidant defenses, it
should be mentioned uric acid which is the most concentrated
antioxidant in human blood [75]. Uric acid is an intermediate
product of purine metabolism. It cannot scavenge superoxide, but
does act against peroxynitrite, peroxides, and hypoclorous acid
[76]. However, the presence of ascorbic acid in plasma is required
for the antioxidant effects of uric acid [77].
Nutritional antioxidants, among which plant polyphenols, have
been considered pivotal in determining the risk of disease because
of their capability of counteracting oxidative stress. Plant
polyphenols are multifunctional as they act as reducing agents,
hydrogen donating antioxidants, and singlet oxygen quenchers. In
some cases metal chelation properties have been demonstrated. The
chemical properties of polyphenols, in terms of availability of the
phenolic hydrogens as hydrogen donating radical scavengers,
predict their antioxidant activity in vitro. Actually, the effective in
vivo antioxidant potential of dietary polyphenols depends on several
factors such as quantities consumed, absorbed and/or metabolized,
plasma and/or tissue concentrations, type and content of single
polyphenol, and, finally, synergistic effects. However, the chemical
structure of phenolic compounds differently affects all the factors
described above. For instance, a glycosylated anthocyanin shows
lower radical-scavenging activity than the correspondent aglycone,
as the glycosylation reduces the ability of the anthocyanin radical to
delocalize electrons. Fukumoto & Mazza [78] have reported that
the antioxidant activity of anthocyanidins increase with the number
of hydroxyl groups in the molecule, and decrease with
glycosylation process.
Furthermore, strong evidence has been provided for an indirect
antioxidant activity of polyphenols exerted by activating the
endogenous defense system. In this regard, a number of studies
have shown that the consumption of food reach in polyphenols,
such as red wine, spinach, strawberries, black and green tea, and
wheat bran, is associated with a significant plasma antioxidant
capacity (PAC) enhancement [79-81], which seems to be associated
mainly to the increase of uric and ascorbic acids in plasm a [82, 83].
However, it has recently been demonstrated th at ascorbic acid an d
Fig. (3). GSH and related enzymes. Reduced glutathione (GSH) is the substrate for glutathione peroxidases (GPx) and glutathione transferases (GST) during
the detoxification of hydrogen peroxide, lipid hydroperoxides, and electrophilic compounds. During GST-mediated reactions, GSH is conjugated with various
electrophiles, and the GSH adducts formed are actively secreted by the cell. During GPx mediated reaction oxidized glutathione (GSSG) is formed. Since
GSSG is highly toxic for the cell, it is in part extruded from the cells and in part back-reduced to GSH by glutathione reductase (GR) utilizing NADPH as
reductant. The resulting depletion of cellular GSH is replaced by de novo synthesis through two sequential ATP-dependent reactions catalyzed by -
glutamylcysteine synthetase (-GCS),– the rate-limiting enzyme – and glutathione synthetase.
Glutamate Cysteine Glycine
J
-GCS
GSH H2O2
Hydroperoxides
H2O
Hydroxy compounds
GSSG
GPx
GR
NADP+
NADPH
Toxic
compounds
GST
GSH-adducts
out
in
Protocatechuic Acid and Human Disease Prevention Current Medicinal Chemistry, 2012 Vol. 19, No. 18 2905
urate levels do not significantly change from the baseline after
polyphenol consumption suggesting that the enhanced PAC is most
likely caused by phenolics [84]. In addition, several experimental
data indicate a tight interrelation between GSH and its related
enzymes, and dietary polyphenols. Actually, the strengthening of
the cellular antioxidant defense system by specific nutrients can
represent a central event in determining the fate of the cells since
the depletion of host antioxidant defenses contributes to sub-
cellular damage and cytotoxicity. In general, the increased
vulnerability of th e cells to the assault of free radical in an
inflammatory environment results in mitochondrial and DNA
damage until apoptosis in several tissues, which might lead to
multiple o rgan failure.
Antioxidant Activity of PCA
Hydroxybenzoic acid derivatives exhibit potent antioxidant
activities. In particular, catechol-type o-diphenols such as PCA and
its esters, show high antiradical activity towards 2,2-diphenyl-1-
picrylhydrazyl (DPPH) in vitro [85,86], as they are readily
converted to the corresponding o-quinones and further complex
products [87,88] (Fig. 4). The molecular structure of PCA indicates
that it possesses potent antioxidant and anti-radical activities [85]
due to the number of phenolic hydroxyl group present in PCA
molecule. However, PCA esters have higher antioxidant activity
than PCA likely because of the higher lipophilicity, which is a main
factor in counteracting lipoperoxidation [89, 90]. It has been
demonstrated that the radical scavenging activities of PCA and its
esters depend on the solvent used [91, 92]. In non-alcoholic acetone
or acetonitrile PCA and its esters consume two radicals and are
converted to the corresponding quinones. In contrast, in methanol
or ethanol PCA esters rapidly scavenge more than four radicals
with a concomitant conversion to the corresponding quinone-3-
hemiacetals and the production of alcohol adducts at C2 (Fig. 4)
[93]. In particular, the regeneration of catechol structures by the
nucleophilic addition of a solvent alcohol molecule on o-quinones
is a key reaction responsible for the higher radical scavenging
activity of PCA esters in alcoholic solvents than in non-alcoholic
solvents [91]. Notably, the radical scavenging activity is greatly
affected by the C-1 substituent on the catechol ring [94]. Studies
carried out in several cell models have confirmed the effectiveness
of PCA in counteracting lipoperoxidation and oxidative stress
mediated by hydrogen peroxides and oxidized lipids [95-100]. For
the sake of information it has to be taken in account that high
concentrations of PCA, as for other antioxidants, could exert pro-
oxidant activities, thus favoring toxic effects and tissue damages. A
few studies have been specifically addressed to clarify this aspect
showing that PCA (500mg/Kg) intraperitoneal administered to mice
induces depletion of GSH content and, consequently, liver, kidney,
and skin damages [101]. A growing body of evidence has
demonstrated a close relationship between PCA and the
endogenous antioxidant defenses that might be the principal
responsible for the antioxidant activity of PCA in biological
systems. It is worthy of note that the efficiency of the antioxidant
defense system is reduced in degenerative diseases and ageing
process; for instance, the activities of SOD, catalase, and the
enzymes of GSH cycle have been demon strated to be reduced in
plasma of patients with ageing brain disorders [102]. Interestingly,
PCA purified from Alpinia oxyphylla extracts dose-dependently
balances the endogenous antioxidant system by increasing the
activity of GPx, SOD, and catalase, consequently reducing lipid
peroxidative damage in rat pheochromocytoma cell line PC12
[103], as well as in spleen and liver of aged rats injected intra-
peritoneal with different doses of PCA for 7 days [104]. Notably,
PCA administered to aged rats improves rat cognitive function by
attenuating ageing alterations [96].
Many studies in different cell models have demonstrated that
PCA prevents the d ecrease in GSH levels in the presence of an
oxidant such as oxidized LDL (oxLDL), and increases the basal
level of intracellular GSH [100, 105]. This might be the result of
GSH sparing due to the antioxidant activity of PCA that substitutes
GSH in scavenging radicals. However, it might be related to the
ability of PCA in strengthening the activity of the entire GSH-cycle
by improving the efficiency of GSH-related enzymes. At this
regard, it has been shown that PCA can directly activate mRNA
transcription and activity of antioxidant/detoxifying enzymes such
as GPx and GR in murine macrophages [105].
It is well known that the transcriptional activation of
antioxidant/detoxifying enzyme genes is regulated by common
upstream regulatory elements called AREs, or antioxidant response
elements, present in the promoter region of those genes [106].
There is a fair amount of evidence that ARE sequences regulate the
cellular defense system, being in turn strictly regulated by
transcrip tional factors such as the nuclear factor erythroid 2 (NF-
E2)-related factor 2 (Nrf2), a member of the cap'n'collar family of
basic leucine zipper transcription factors ubiquitously expressed
[107]. Under homeostatic conditions, Kelch lik e ECH-associated
protein 1 (Keap1) sequesters Nrf2 in the cytoplasm by forming
heterodimers [108]. The intracellular level of ROS regulates gene
expression by modulating various signaling pathways and
transcription factors including Nrf2 [107, 109, 110]. A major
mechanism for Nrf2 activation is phosphorylation due to several
kinases such as mitogen-activated protein kinases (MAPKs),
protein kinase C (PKC) and phosphatidylinositol 3-kinases (PI3K)
[111-113]. Phosphorylated Nrf2 is more stable and has a reduced
binding affinity toward Keap 1; consequently it can translocate into
the nucleus where it binds to the ARE sequences of target genes
inducing the expression of the majority of detoxifying/antioxidant
enzymes [114]. ARE sequences have recently been demonstrated in
the promoter region of GPx and GR [115,116]. Polyphenols have
been demonstrated to induce ARE-dependent antioxidant genes by
activating Nrf2 (Fig. 5) [72, 117]. Recently, we have provided
scientific evidence of the molecular mechanism responsible for the
modulation of mRNA expression of antioxidant enzymes by PCA
in macrophages [118]. PCA induces the increase of Nrf2 mRNA
expression and, mainly, a strong increase in Nrf2 phosphorylation
mediated by JNK, which allows the transcription factor to escape
degradation, translocate into the nucleus, and bind the specific ARE
sequences present in GPx and GR promoters. Taken together these
findings support the concept that PCA, as well as several other
polyphenols, improves antioxidant cellular defenses in critical
CO
2
R
OH
OH
-2H
+
-2 .
(DPPH)
CO
2
R
O
O
MeOH
CO
2
R
O
OH
OMe
R=H Protocatechuic acid
R=Me Methyl protacatechuate
R=H Protocatechuic quinone
R=Me Protocatechuic quinone methyl ester
R=H Protocatechuic quinone methyl hemiacetal
R=Me Protocatechuic quinone methyl hemiacetal methyl ester
Fig. (4). Chemical structures of protocatechuic acid and its derivatives.
2906 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
target cells, such as macrophages, whose dysfunction has been
implicated in many patho-physiological processes that entail
inflammation and atherogenesis [119-120].
Fig. (5). PCA up-regulates antioxidant/detoxifying enzyme expressions.
PCA directly activates mRNA transcription of antioxidant/detoxifying
enzymes through antioxidant responsive elements (AREs). PCA might
influence the pathways that regulate ARE activation by i) modifying the
capability of Kelch like ECH-associated protein 1(Keap1) in sequestering
nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), ii) activating
Mitogen-activated protein kinases (MAPK) involved in Nrf2 stabilization,
and/or iii) directly affecting Nrf2 binding to AREs.
ANTI-INFLAMMATORY ACTIVITY
Oxidative stress and inflammatory processes seem to jointly
contribute to the pathophysiology of almost all chronic-
degenerative diseases. The generation of ROS as well as of reactive
nitrogen species (RNS) is able to activate redox sensitive
transcription factors that induce the expression of pro-inflammatory
molecules leading to a state of chronic inflammation. On the other
hand, inflammation itself might contribute to ROS and RNS
generation amplifying the damages in target cells and organs.
Inflammatory process represents a protective response occurring in
tissues as a consequence of any insult with the aim of destroying or
diluting the cause of cell injury with no detriment to the host [121].
As described by Celsus, rubor (redness), tumor (swelling), dolor
(pain), and calor (heat) are the symptomatic manifestations of
complex tissue responses to harmful stimuli. Briefly, inflammation
is a reaction of the microcirculation that is characterized by the
movement of serum proteins and leukocytes (neutrophils,
eosinophils and macrophages) from the blood to the extra-vascular
tissue, and by the activation of cell- and plasma-derived
inflammatory mediators. The inflammato ry process is regulated by
coordinated activation of different signaling pathways that
modulate the expression of both pro- and anti-inflammatory
mediators in resident tissue cells (such as fibroblasts, endothelial
cells, tissue macrophages, and mast cells), and recruit leucocytes
[122,123]. From a more actual point of view, inflammation
represents an important process for the maintenance of biological
homeostasis [121]. However, uncontrolled inflammation might be
potentially harmful and may represent a main pathogenetic
mechanism for many diseases [123]. Indeed, the inability to self-
limiting inflammatory response leads to prolonged activation of
pro-inflammatory mediators responsible for ongoing tissue injury
and organ dysfunction. As a consequence, chronic inflammation
plays a critical role in the pathophysiology of the major chronic
diseases including obesity, cardiovascular disease (CVD), type 2
diabetes (T2D), Alzheimer’s disease (AD) and many types of
cancer [121,124,125].
PCA and Inflammation
A relationship between nutrition and the immune system does
exist, as well as a link between nutrition and chronic diseases [126].
It is thus not surprising that huge efforts and resources have been
focused on the understanding of how dietary nutrients might impact
pharmacological targets of inflammation. In addition, since several
synthetic drugs provide unknown side effects, there has been a need
for new and safe anti-inflammatory agents. Various phyto-
chemicals, including phenolic compounds, have shown anti-
inflammatory activity in vitro and in vivo. Many data have
suggested that polyphenols can work as modifiers of signal
transduction pathways to elicit their beneficial effects [127-129].
Targeting these inflammatory regulators by using food components
may be a useful strategy to prevent or to ameliorate the
development of chronic inflammation-related diseases; PCA
appears to be a good candidate since recent studies have suggested
that ACNs and PCA might exert important anti-inflammatory
activities [98,130,131].
A large number of mediators, including nitric oxide, lipid
mediators, cytokines/chemokines, adhesion molecules, and matrix
metalloproteinases (MMPs), are involved in the initiation,
maintenance, and resolution of inflammatory process [132,133].
PCA and Nitric Oxide
Mediators such as histamine and nitric oxide (NO) are potent
vasodilatators that propagate and amplify the inflammatory
response. NO is ex-novo synthesized by endothelial (eNOS),
neuronal (nNOS), and inducible (iNOS) nitric oxide synthases.
Both lack and excess of NO production appear to have important
implications in chronic diseases [134-136]. In fact, while a
significant increase in NO -iNOS-produced- participates in
provoking inflammatory process [137], a low NO bioavailability –
eNOS produced- is central to the development and maintenance of
hypertension and related endothelial dysfunction [138].
Consequently, the inhibition of excessive inducible NO production
may be an anti-inflammatory therapeutic target, and the protection
against a decrease in constitutive NO production in the vasculature
may prevent the development of atherosclerosis and endothelial
dysfunction [135, 139]. PCA appears to significantly reduce iNOS
protein expression and NO production both in vitro and in vivo.
Specifically, PCA reduces NO production and iNOS expression in a
dose-dependent manner in LPS-treated RAW 264.7 cells [130]. The
PCA pre-treatment of CCl(4)-treated rat results in the reduction of
iNOS expression in liver, and NO levels in plasma [98]. Finally, the
topical application of PCA on mouse epidermis, counteracts the
increase in iNOS protein level and the nitrite production induced by
12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin [140].
PCA and Lipid Mediators
Lipid mediators such as arachidonic acid-derived prostanoids
and leukotrienes, as well as oxidized phospholipids and platelet-
activating factors (PAF), are potent pro-inflammatory mediators
[141]. Prostaglandin E2 (PGE2), prostaglandin I2 , and tromboxane
A2 are produced by cyclooxygenases (COXs) which exist in two
major isoforms (COX-1 and COX-2) and one variant (COX-3).
COX-1 is constitutively expressed in many tissues, whereas COX-2
is expressed in inflammation-related cells (i.e., macrophages,
monocytes, neutrophils, fibroblasts) in response to various stimuli
ARE/EpRE
Nucleus
Nrf 2
P
Keap1
Nrf 2
P
Nrf 2 Keap1
PCA
PCA
PCA
Nrf 2
MAPK
cascade
MAPK cascade
Protocatechuic Acid and Human Disease Prevention Current Medicinal Chemistry, 2012 Vol. 19, No. 18 2907
[142]. Lipoxygenases are the enzyme responsible for generating
hydroxyl acids, and leukotrienes B4 and C4.
Oxidized phospholipids (OxPLs) are generated when LDL and
cellular phospholipids containing polyunsaturated fatty acids
(PUFA) undergo oxidative attack. Accumulating data demonstrate
that OxPLs are formed in vivo in a variety of pathological
situations, and play an important role in atherosclerosis. However,
the large number of different OxPLs identified makes difficult to
elucidate their contribution to the pathology [141]. PAF is one of
the most potent phospholipid with atherogenic effects being
implicated in many inflammatory actions including monocytes
activation and infiltration [143,144]. The inhibition of lipid
mediator generating enzymes is one of the PCA anti-inflammatory
mechanisms. PCA inhibits COX-2 and PGE2 expression both in
vitro in LPS-treated RAW264.7 cells and in vivo in mice treated
with carrageenan, a polysaccharide that induces inflammation
[130]. The over-expression of COX-2 in Sprague-Dawley treated
with CCl4 is significantly reduced by the pre-treatment with PCA
[98]. Similarly, pre-treatment w ith PCA diminishes the dramatic
increase of COX-2 protein expression and activity, induced by TPA
in mouse skin, by approximately 50% [140].
Furthermore, in vitro experiments have demonstrated that PCA,
as well as other polyphenols, is able to inhibit LDL oxidation
[145,146].
PCA and Cytokines/Chemokines
Cytokines, produced by both resident and migrating cells, are
the major mediators of intercellular communications required for an
integrated response to a variety of stimuli in immune and
inflammatory processes [147,148]. A distinct group of cytokines,
called chemokines, which include IL-8, monocyte chemoattractant
protein (MCP)-1, and macrophage inflammatory proteins (MIP),
has shown the ability to recruit and activate leukocytes at the site of
inflammation [149]. Thus, the imbalance between pro-
inflammatory (i.e. IL-1, IL-2, TNF, Il-6, IL-8 and IFN-) and
anti-inflammatory cytokines (i.e. IL-10, IL-4, TGF) might
determine the outcome of disease, both on the short- and long-term
basis [127,148]. In addition, TNF and IL-1 interact with
endothelial cells to produce adhesion molecules, such as
intracellular adhesion molecule (ICAM)-1, vascular adhesion
molecule (VCAM)-1, and E-selectin, that allow inflammatio n
progression and exacerbate the severity of clinical disorders [149-
153]. Notably, IL-1 has recently b een shown to exert auto crine
action on monocytes and to influence redox balance and glutathione
levels [144]. Targeting cytokine/chemokine system with plant-
derived compounds represents thus an important and alternative
approach for the treatment of inflammatory diseases [127, 154].
Notably, the pre-treatment of mouse aortic endothelial cells with
PCA inhibited the cytokine-induced expression of ICAM-1 and
VCAM-1 [131].
PCA and the Complement System
The complement system orchestrates immunological and
inflammatory processes, extending far beyond simple elimination
of danger. It consists of a number of small proteins found in the
blood, normally circulating as inactive precursors that initiate and
amplify a cascade of further activations when stimulated by one of
several triggers [155]. The effects of complement are normally
beneficial to the host, but they can also cause adverse effects
depending on the site, extent, and duration of activation. AD is
associated with neuroinflammation, and up-to-date works provide
evidence of complement activation, localized with both fibrillar
amyloid- (A) plaques and tangles [124, 156]. Thus it appears
very hopeful a recent report demonstrating that compounds, among
which PCA extracted from black colored rice bran can exert anti-
complement activity against the classical complement pathway
[157].
PCA, Nuclear Factor-
B, and MAPKs
Among a huge number of pro-inflammatory mediators nuclear
factor-B (NF-B) and MAPKs are the principal effectors that,
directly or indirectly, regulate inflammatory responses [158-160].
In particular, the inhibitor B (IB) kinase (IKK)/NF-B signaling
pathway regulates genes involved in many aspects of the
inflammatory response, and has been implicated in the pathogenesis
of several inflammatory diseases [161]. Increased activation of NF-
B has often been detected in both immune and non-immune cells
in tissues affected by chronic inflammation, where it is believed to
exert detrimental functions by inducing the expression of pro-
inflammatory mediators that orchestrate and sustain the
inflammation [162].
NF-B is a dimer that classically consists of a p50 subunit and a
trans-activating subunit p65 (or relA). In un-stimulated cells, NF-
B is sequestered in the cytoplasm as an inactive non-DNA-binding
form associated with the inhibitory proteins IBs [163]. Upon cell
stimulation with various NF-B inducers, IBs are rapidly
phosphorylated by IKK complex on two serine residues, which
targets th e inhibitor protein for ubiquitination and subsequent
degradation by the ubiquitin-proteasome pathway. As a
consequence, the translocation of NF-B to the nucleus is
facilitated, as well as the binding to specific DN A sites and the
induction of a wide range of genes encoding pro-inflammatory
cytokines (e.g., IL-1, IL-2, IL-6, and TNF), chemokin es (e.g., IL-
8, MIP-1 and MCP-1), adhesion molecules (e.g., ICAM, VCAM,
and E-selectin), acute-phase proteins, immuno-receptors, growth
factors, and inducible enzymes such as COX-2, MMPs, and iNOS
[159]. The critical role of NF-B in pro-inflammatory gene
expression has generated an enormous effort to develop specific
inhibitors for the treatment of chronic inflammation [164, 165]. It is
worthy of note that PCA treatment inhibits, in a dose-dependent
manner, the TNF-induced expression and activation of NF-B
(p65) in mouse aortic endothelial cells [131]. A similar effect has
been described for PCA extracted from black rice and used to treat
LPS-induced RAW 264.7 cells. Specifically, this agent inhibits
LPS-induced IB d egradation and NF-B activation as well as the
translocation of p65 subunit into the nucleus [130]. The pre-
treatmen t with PCA has b een described to decrease NF-B p65-
DNA binding and to inhibit the TPA-induced IKK activity,
consequently reducing COX-2 activity and iNOS expression, in
mouse skin [140]. Interestingly, it has been demonstrated that PCA
specifically inhibits the nuclear translocation of NF-B, but not of
other nuclear transcription factors such as AP-1, in human gastric
carcinoma cells [166] (Fig. 6).
The MAPK cascades are intracellular signal transduction
pathways that respond to changes in the cellular environment, and
constitutive upstream regulators of transcription factor activities
that control a large number of fundamental cellular processes
including inflammation, cell growth, proliferation, death and
differentiation [158]. Mammals express at least four distinctly
regulated groups of MAPKs, namely the extracellular signal-related
kinases (ERK)-1/2, the c-Jun amino-terminal kinases (JNK1/2/3),
the p38 kinases (, , , and ) and the ERK5. The MAPKs are
activated by specific MAPK kinases (MAPKK s) that, in turn, can
be activated by different MAPKK kinases (MAPKKKs), increasing
the complexity of the MAPK pathways. MAPKs are activated in
response to diverse arrays of extracellular stimuli, and mediate
signal transduction from the cell surface to the nucleus. Mitogens
and growth factors frequently activate the ERK1/2 route, while
stress and inflammation constitute the main triggers for the JNK
and p38 cascades, sometimes referred as ‘stress activated protein
kinases’ [167]. Increased activity of MAPKs and their involvement
in the regulation of the synthesis of inflammation mediators make
them potential targets for novel anti-inflammatory therapeutics
[127, 167, 168]. Notably, PCA might exert potent anti-
inflammatory effects by regulating MAPK activation (Fig. 6). A
2908 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
recent work has shown that PCA counteracts the LPS-stimulated
phosphorylation of ERK1/2, JNK1/2, and p38 MAPKs in RAW
264.7 cells. In fact, LPS stimulates the activation of all three
MAPK pathways whereas PCA inhibits the activation of p38, ERK
and JNK pathways [130].
In conclusion, few studies have demonstrated that PCA presents
important anti-inflammatory actions because of the ability to inhibit
the synthesis and/or activity of most inflammatory mediators and
regulatory pathways. However, it is worth of note that, in vivo
studies, besides those in vitro, have suggested the potential utility of
PCA in reducing the severity of inflammatory response that takes
part in many chronic diseases. In particular, PCA supplied to
streptozotocin (STZ)-induced diabetic mice substantially decreases
inflammatory cytokine levels in kidney and heart, and consequently
plasma levels of C reactive protein (CRP) and von Willebrand
factor. In addition, in the same organs, PCA markedly d ecreases
MCP-1 levels. These findings strongly suggest that PCA might be a
potent agent against diabetic-asso ciated cardiac and renal
inflammatory injuries [169]. The same suggestion comes from two
other studies. One of these has shown that PCA pre-treatment
couples the inhibitory action on plasma TNF levels with the
enhancement of IL-10 release in LPS/D-galactosamine-treated
mouse [170]. The other one has showed that PCA diminishes
inflammatory stress by lowering the level of IL-1, IL -6, TNF,
and MCP-1 in the liver of mice fed a trans-fatty acid-rich diet, and
thus it could be considered a potent agent for alleviating hepatic
steatosis as well [171].
Min et al. have found that oral administration of black rice,
C3G, or PCA once a day for three days before carrageenan
injection into subcutaneous air pouches, protects male BALB/c
mice from in flammation. In fact, the carrageenan-induced increases
in inflam matory markers, such as TNF, IL-1, and PGE2, as well
as in COX-2 expression, and NF-B and MAPK activation, are
potently inhibited by all the tested polyphenols, being PCA the
most potent inhibitor [130].
However, for the completeness of information, we have to point
out that other studies have reported opposite effects of PCA [172];
this discrepancy might be due to the dosage of PCA and the cell
type used.
PCA, Atherosclerosis and CVD
It is well known that atherosclerosis is a multistep, chronic,
inflammatory process associated with a disorder of lipid
metabolism. Taken together the alterations in both lipid metabolism
and immune responses in macrophages play a significant role in
promoting the development of atherosclerotic lesions [173]. It has
been demonstrated that PCA is able to influence various events
involved in the pathogenesis of atherosclerosis, especially because
of its antioxidant and anti-inflammatory properties. First of all,
PCA exerted a powerful protective activity against vessel damage
by counteracting a major initiating event, i.e. the oxidation of LDL
by macrophage cells, considered to be responsible for early
vascular damages [105]. PCA inhibited LDL oxidation induced by
the macrophage-like cell line J774 A.1likely by the up-regulation of
GPx and GR expressions and activities, thus ameliorating the entire
functionality of GSH system [174]. A typical sign of the early stage
of atherosclerosis is the infiltration of circulating monocytes into
the vascular wall [175]; this process is modulated by a number of
chemokines and their receptors [176]. Notably, PCA treatment, at
physiological order of concentrations, attenuates the expression of
CCR2 in PBMC isolated from apo-E-deficient mice, which leads to
a functional consequence. More importantly, orally administered
PCA reduces macrophage infiltration into the abdominal cavity of
the mice [57]. Furthermore, PCA administration in apo-E-deficient
mice is able to reduce aortic and plasma VCAM-1 and ICAM-1
expressions, as well as NF-B activity, consequently inhibiting the
formation of the early atherosclerotic lesions. In addition, in the
same animal model, PCA supplementation reduces cholesterol
accumulation in aortas by 50%, without modifying the plasma lipid
profile [131]. This finding might indicate that the beneficial effect
of PCA on the cardiovascular system is mainly due to its anti-
inflammatory prop erties. A crucial event in the progression of
atherosclerotic plaque, as well as in the increase of inflammatory
process, is the occurrence of apoptosis (see below). In particular, in
Fig. (6). Schematic representation of potential points of action of PCA within the infla mmatory cascade. PCA might exert anti-inflammatory activities
by acting at different levels of the inflammatory process. PCA a) counteracts inflammatory mediators, b) inhibits the progression of inflammation by
modulating different signal transduction pathways, c) modifies the activity of transcription factors and, consequently, the expression of specific genes.
Mitogen-activated protein kinases (MAPK), MAPK-kinase (MAPKK); MAPK kinase kinase (MAPKKK); p50-RelA (NF-B); nuclear factor erythroid 2 (NF-
E2)-related factor 2 (Nrf2). Inhibition =; activation = .
inflammatory cytokines,
stress, aging, oxidative stress,
genotoxic agents
Maf
RelA
p50 Nrf2
transcription
Cytokines, transcription factors,
adhesion molecules
Nucleus
ARE system and cellular
defense enzymes
MAPKKKs
MAPKKs
MAPKs
PCA
a
b
b
b
c
c
Protocatechuic Acid and Human Disease Prevention Current Medicinal Chemistry, 2012 Vol. 19, No. 18 2909
advanced atherosclerotic plaques, up to 50% of the apoptotic cells
are macrophages, which may promote core expansion and plaque
instability. In advanced human atheroma the defective phagocytic
clearance of dead macrophages leads to plaque necrosis, which
triggers acute atherothrombotic vascular events [177]. We have
studied the properties of PCA to counteract oxLDL mediated
cytotoxic and pro-apoptotic activity in murine macrophages. Worth
of note, the treatment with PCA inhibits the apoptosis occurrence in
macrophages not only by counteracting oxidative stress, but also by
specifically modulating intracellular signaling pathways responsible
for caspase activation [178]. Furthermore, anti-thrombotic activity
of ACNs and colonic metabolites, mainly exerted through the
inhibition of platelet functions, has been demonstrated [179].
Anti-Hyp erglicemic Activity o f PCA
Maintenance of glucose homeostasis by strict hormonal control
is of utmost importance to human physiology. Failure of this
control, with defects in both insulin action and insulin secretion,
can result in the metabolic syndrome, a multi-symptom disorder of
energy homeostasis. The disturbance of glucose metabolism is often
related to the increase of fat mass, especially in the abdominal area,
which, in turn, results in inflammation, exacerbated oxidative stress
at the whole body level with increased circulating oxLDL levels
and malfunction in several organs including adipose tissue [180,
181]. Insulin resistance seems to underlie the early stages of the
development of the metabolic syndrome, and thus approaches to
improve insulin action have been, and remain, key targets for
slowing or ultimately preventing type 2 diabetes [182, 183]. The
potential of polyphenols in controlling glycemia is currently under
intensive study, and indications for positive effects of AC Ns on
glucose homeostasis have been obtained in vitro and in an imal
studies [184-187]. In particular, some compelling studies have
reported that ACNs improve insulin sensitivity and glucose uptake
in diabetic rats [186]. Notably, Harini et al. [188], have
demonstrated that PCA adm inistered orally to STZ-diabetic rats for
45 days prevents the increase in plasma glucose and glycosylated
hemoglobin, and the decrease in plasma insulin and hemoglobin. In
addition, PCA normalizes the activities of gluconeogenic enzymes
like glucose 6-phosphatase and fructose 1,6-bisphosphatase, as well
as that of the glycolytic enzyme glucokinase. Worth of note, PCA
does not induce significant effects in normal rats. Thus, PCA might
exert a potential antihyperglycemic effect that is comparable with
that obtained with classical antidiabetic drugs such as
glibenclamide. In addition, PCA reverts the alterations of lipid
profile associated with STZ-induced diabetes in rats [189]. In STZ-
diabetic mice, Lin et al. [189] have demonstrated that dietary
supplementation with PCA improves glycemic control and
attenuates homeostatic disorder. Furthermore, they have found that
orally administered PCA reduces the hyperglycemia-induced
advanced glycation end-products (AGEs) both in plasma and in
organs such as kidney [62]. This is of great importance since the
accumulation of AGEs leads to organ deterioration, and favors the
development of diabetic complications [190]. This antiglycative
activity of PCA might be related to the attenuation of oxidative and
inflammatory stresses [169]; however, the authors have also found
that PCA diminishes the renal activity and expression of aldose
reductase and sorbitol dehydrogenase, both involved in AGE
production. Interestingly, the authors have provided possible
molecular mechanisms for PCA action. First of all they have found
that PCA diminishes the mRNA expression and activity of PKC-
and PKC-, and, consequently, of TGF1 that, in turn, promotes
ECM formation and tissue fibrosis. Secondly, PCA reverts the
down-regulation of PPAR and, particularly, of PPAR, that is a
principal metabolic regulator of glucose and lipid metabolism, the
most extensively studied and clinically validated gene for
therapeutic utility in type 2 diabetes, and a main target for many
antidiabetic drugs such as thiazolidinediones (TZDs) [191, 192].
Indeed P PAR is a target of insulin activity, and regulates the
expression and activity of key players in the maintenance of
glucose transport machinery efficiency, such as glucose transporter
(GLUT) 4 and adiponectin. Very recently we have demonstrated
that PCA is able to increase the glucose uptake and to enhance
GLUT4 translocation and adiponectin secretion in human primary
omental adipocytes [193]. This insulin-like activity, probably
caused by the increased activity of PPAR induced by PCA, might
offer interesting possibility in diabetes care. In fact, new ligands for
PPAR that do not procure the unwanted side-effects shown by
many insulin-sensitizing agents, e.g. TZDs, are being sought.
ANTIAPOPTOTIC/PROAPOPTOTIC ACTIVITY
Polyphenols can improve cell survival and protect against
cytotoxicity by inhibiting apoptosis, but also they can induce
apoptosis and prevent tumor growth [194, 195]; these opposite
effects are mainly due to the control of cell redox state. In fact,
acting as antioxidants or pro-oxidants, these compounds behave as
anti-apoptotic or pro-apoptotic agents, respectively. Since the
modulation of cell apoptosis is one of the main mechanisms
responsible for the protective action against degenerative
pathologies, polyphenols have been proposed as therapeutic agents
against cancer, neurodegenerativ e diseases, and cardiovascular
diseases [196, 197].
Apoptosis is a genetically controlled and evolutionarily
conserved form of cell death of critical importance for the normal
embryonic development and for the maintenance of tissue
homeostasis in the adult organism. Apoptosis is mediated by two
main pathways that, although strictly intertwined, represent
different ways to reach the same result, i.e. the death of a cell via
caspase activation (Fig. 7).
The extrinsic, or death receptor, pathway is activated when a
specific ligand, such as Fas ligand, binds its corresponding death
receptor, such as Fas receptor, on the cell surface. This induces a
cascade of events leading to the formation of a supramolecular
complex called death-inducing signaling complex (DISC), which
results in the cleav age of inactive pro caspases-8 to the active
caspase-8. Caspase-8, in turn, triggers a series of reaction
eventually affecting the mitochondrial membrane potential, thus
interacting with the intrinsic pathway [195].
The intrinsic, or mitochondrial, pathway is activated by
different agents which all induce ROS overproduction and the onset
of oxidative stress. Activation of the intrinsic pathway is
accompanied by mitochondria membrane potential collapse and
cytochrome c translocation from the mitochondrial inter-membrane
space into the cytoplasm. Cytochrome c, and several pro-apoptotic
factors (Apaf-1, dATP), bind procaspase-9 to form the
‘apoptosome’ complex that activates caspase-9 through
autocatalysis. Both caspase-9 and caspase-8 cleave pro caspase-3
generating the active form of caspase-3 that, in turn, activates other
executor caspases, cleaves cyto skeletal, and activates specific
DNase. The activation of caspases is regulated by several factors
[195] including MAPKs and the tumor suppressor protein p53. The
activation of p53 is affected by ROS, and regulates, positively or
negatively, the transcription of dozen downstream effector genes
such as BAX, a member of the Bcl-2 family of proteins, and p66Sh,
an oxidative stress sensor protein, that promote the opening of the
mitochondrial permeability transition pore and trigger apoptosis
[198]. Furthermore, p53 modulates the balance between pro- and
anti-apoptotic proteins belonging to Bcl-2 family. MAPKs and the
isoform of PKC control the cellular level and nuclear
accumulation of p53 [95, 103, 199-201]. Among MAPKs, ERKs
have been associated with the regulation of cell proliferation. In
contrast, the activation of JNK and p38 kinases is controlled by
stress signaling and has been associated with the induction of
apoptosis.
2910 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
In conclusion, apoptosis represents a very complex process
whose modulation depends on many players differently regulating
crucial intracellular events. Consequently, these facto rs represent
suitable targets for focused interventions.
Several in vitro and in vivo studies have demonstrated that
PCA, as other polyphenols, can exert pro-oxidant/anti-oxidant and
pro-apoptotic/anti-apoptotic effects as well.
Anti-Apoptotic Effects of PCA
It has been found by Zhou-Stache [201] that PCA, isolated from
Salvia mitorrhizam, inhibits TNF-induced cell death of human
umbilical vein endothelial cells (HUVECs) and Jurkat cells. PCA,
isolated from th e kernels of Alpinia oxyphylla, stimulates cell
proliferation and markedly attenuates mitochondrial dysfunction by
inhibiting the loss of mitochondrial membrane potential, the
formation of ROS, the GSH depletion, the activation of caspase-3,
and the down-regulation of Bcl-2 [95,103,202,203]. In another
study carried out in PC12 cells, the protective effects of PCA on
H2O2-induced has been investigated. Authors showed that PCA
increases PC12 viability and markedly reduces apoptotic cell death
in a dose-dependent manner. In these cells, the levels of GSH and
the activity of catalase increase, while GPx activity remains
unchanged, suggesting that PCA acts mainly as antioxidant [204].
This hypothesis has been confirmed by Tarozzi et al. [205] that
have demonstrated the ability of cyanidin and PCA, but not of C3G,
to inhibit mitochondrial function loss and DNA fragmentation. The
potential protective effects of compounds generated during in vivo
metabolism of ACNs has been the topic of comparative studies on
the effects of different compounds in the human neuroblastoma cell
line SH-SY5Y. C3G, cyanidin, and PCA inhibit H2O2-induced ROS
formation, but at different cellular levels: C3G at membrane level,
PCA at cytosolic level, and cyanidin at both levels. In addition,
cyanidin shows a higher antioxidant activity at membrane and
cytosolic level than C3G and PCA, respectively.
Taken in account all these findings that demonstrate the
anticytotoxic and anti-apoptotic activities of PCA, it becomes clear
that PCA might have interesting application in the prevention and
treatment of chronic-degenerative diseases. The effects of PCA in
preventing neurodegenerative diseases, such as Parkinson’s disease,
have been extensively studied in PC12 cell model. Parkinson's
disease is characterized by the progressive degeneration of
dopaminergic neurons in substantia nigra with the presence of -
synuclein inclusions termed Lewy bodies. It has been demonstrated
in vitro that PCA inhibits cytotoxicity, apoptotic morphology,
reduction of tyrosine hydroxylase expression, and abnormal
oligomerization of -synuclein in PC12 cells treated with1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [206]. In an in vivo
study, the same group has demonstrated that PCA inhibits the
reduction of the latent periods in a rotarod test, and the contents of
dopamine and its metabolites in striatum as well. Furthermore, PCA
counteracts the decreased expression of tyrosine hydroxylase in
substantia nigra of C57BL/6J mice induced by MPTP [207].
The A peptide aggregation into oligomeric and fibrillar species
affects neuronal viability, having a causal role in the development
of AD. PCA isolated from Smilacis chinae rhizome has been found
to prevent A (25-35)-induced neurotoxicity in cultured rat cortical
Fig. (7). Apoptosis pathways. The extrinsic pathway is triggered by members of the death receptor superfamily such as Fas. Binding of Fas ligand (Fas-L) to
Fas induces trimerization of the receptor, recruitment of specific adaptor proteins (FADD) and procaspase 8 molecules. The multi-molecular complex (DISC)
results in the activation of caspase-8. Active caspase-8 can, in turn, activate Bid, a pro-apoptotic member of Bcl-2 family of proteins.
Oxidants, toxicants, drugs or ionizing radiation, which all induce ROS overproduction, can activate the intrinsic pathway (right). The intrinsic or mitochondrial
pathway is also triggered by DNA damage and ROS overproduction via p53 activities. The death stimuli result in loss of mitochon drial membrane integrity
and release of cythocrome c, apoptotic protease activating factor 1 (Apaf-1) and other pro-apoptotic factors in the cytoplasm. Maintenance or perturbation of
mitochondrial membrane potential depends on the ratio between pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) members of Bcl-2 family, causing or
preventing cythocrome c release. Multiple molecules of cythocrome c, Apaf-1, dATP and procaspase-9 associate to form a supramolecular complex termed
apoptosome, that activates caspase-9 through autocatalysis. Both the activated caspase-9 and caspase-8 cleave procaspase-3 generating the active caspase-3
that, in turn, activates other executor caspases and cleaves cellular targets. Procaspase cleavage and caspase activities are tightly controlled by several factors
such as the Inhibitors of Apoptosis Protein (IAPs) family.
D
D
ROS
Oxidants, Toxicants,
Drugs, Ionizing radiations
DNA DAMAGE
FasL
Fas/CD95
FADD
Cytochrome C
Cellular
Tar ge t s
Cellular
Tar ge t s
Procaspase 8
DISC
IAPs
c-FLIP
Bcl-2
D
BID
Bax
p53
p66Shc
Death Receptor pathway Mitochondrial pathway
Procaspase 3
Apaf-1
Procaspase 9
Apoptosome
Apaf-1
Target genes
Caspase 9
Caspase 3
Caspase 8
Protocatechuic Acid and Human Disease Prevention Current Medicinal Chemistry, 2012 Vol. 19, No. 18 2911
neurons by interfering with the increase of [Ca(2+)], and by
inhibiting glutamate release, generation of ROS and caspase-3
activity [208]. However, C3G, but not PCA, might protect and
rescue the neuronal cells from toxicity induced by A (1-42), by
inhibiting its spontaneous aggregation in human neuronal SH-
SY5Y cells [205]. A recent study has been aimed at investigating
the protective effects exerted by PCA against apoptosis induced by
oxidized lipids in intestinal Caco-2 cells [100]. We have found that
PCA fully counteracts the oxidized lipid-induced redox imbalance
by inhibiting ROS overproduction, GSH depletion, and the
impairment of SOD, catalase and GPx. Furthermore, the oxidized
lipids induce a dramatic increase in p66Shc expression that
irreversibly lead to cells apoptosis by mitochondrial depolarization,
and caspase-9 and caspase-3 activation. These effects are fully
counteracted by PCA that blocks the cascade of intracellular signals
responsible for the over-expression and rearrangement of p66Shc.
These results suggest that the anti-apoptotic effect of dietary
polyphenols might represent an intestinal protecting mechanism
able to neutralize dietary oxidant-induced intestinal damage. In fact,
although the bioavailability of the polyphenols is a limiting factor
in the majority of the body districts, dietary polyphenols
concentrate in th eir active forms in the intestinal lumen because of
the enzymatic activities of enterocytes and colon bacteria. This
could result in a more pronounced protective activity on the
intestinal mucosa, and, thus, in a highly effective prevention of
damages induced by diet oxidant.
PCA as Growth Inducer
The protective activity exerted by PCA against cytotoxicity
might be due not only to the anti-apoptotic activity, but also to the
capability of stimulating cell proliferation and growth. Recently, it
has been reported that PC A is able to increase cultu red neural stem
cells (NSC) viability and to stimulate cell proliferation in a dose-
and time-dependent manner [203]. This finding might strongly
support the hypothesis that PCA is a good candidate for the
treatment of neurodegenerative diseases. Indeed, NCS isolated from
embryonic brain are a heterogeneous population of multipotential
cells recently reported to exist also in the adult nervous system, in
particular in the hippocampus and in the subventricular zone. Worth
of note, it has been shown that NCS from hippocampus have the
potential for differentiating to functional neurons, which indicates
them as candidate targets for stimulating neurogenesis in
neurodegenerative diseases [209]. Notably, pharmacokinetics
studies have demonstrated that PCA readily permeates the blood
brain barrier after oral administration in rats [210].
Similar results have been obtained in human adipose tissue-
derived stromal cells [211]. The authors have demonstrated that the
cell cycle progresses from the G0/G1 phase to the S phase after
treatment with PCA, and the cells retain their functional
characteristics of multipotential mesenchymal progenitors.
Interestingly, the PCA-induced cell proliferation depends on the
over-expression of cyclin D1.
Pro-Apoptotic Activity of PCA
Besides the anti-apoptotic effects reported above, PCA has been
demonstrated to exert also a strong pro-apoptotic activity, which
might be relevant to some pathological conditions such as cancer.
This opposite behavior is likely related to environmental conditions.
Indeed, although polyphenols have been considered as antioxidant
agents, they can be pro-oxidant in certain circumstances such as in
cancer cells. Cancer cells constitutively generate large, but
tolerable, amounts of ROS [212]. As described above, polyphenols,
by acting as anti-oxidants, can contribute to counteract oxidative
stress occurrence, consequently suppressing the oxidative stress-
responsive genes and the proliferation of cancer cells. On the other
hand, polyphenols can induce ROS hyper-production determining
an intolerable lev el of oxidative stress in cancer cells that are thus
irreparably damaged and undergo apoptosis. Since apoptosis
eliminates genetically damaged cells or cells that may be
inappropriately induced to proliferate, it represents a protective
mechanism against neoplastic transformation and development of
tumors.
This dual-effect of PCA and other polyphenols has been
investigated in the human leukemia cells HL-60 [213],
demonstrating that PCA, apigenin, or bisabolol show an anti-
genotoxic effect by counteracting H2O2 and also exhibit tumoricidal
activity by inducing apoptosis. However, the pro-apoptotic activity
could be due to the ability to modulate oxidative stress as well as to
a direct action on regulatory proteins and signaling pathways, that
exert their regulatory effect in determining cell fate [214]. In the
study of Tseng et al. [215] PCA, isolated from the dried flower of
Hibiscus sabdariffa L. (Malvaceae), has been found to inhibit the
survival of human promyelocytic leukemia HL-60 cells in a
concentration- and time-dependent manner. The study has revealed
that HL-60 cells undergo internucleosomal DNA fragmentation and
morphological changes characteristic of apoptosis after a 9-hr
treatment with 2 mM PCA. Moreover, PCA treatment causes an
increase in the level of hypo-phosphorylated retinoblastoma protein
(RB) and, on the contrary, a decline in hyper-phosphorylated RB. A
rapid loss of RB has been observed when the treatment period is
extended. Furthermore, PCA application dramatically reduces Bcl-2
and increases BAX expressions. The authors conclude that the
reduction of RB phosphorylation and Bcl-2 protein may play a
crucial role in the pro-apoptotic induction exerted by PCA.
It has been demonstrated that antioxidant and antitumo r
activities of Rhus Verniciflua Stokes (RVS), used as a food additive
and a traditional herbal medicine, is closely associated with th e
polyphenol content. The purified polyphenol extract strongly
suppresses the proliferative capability of B lymphoma cells by
inducing apoptosis with DNA fragmentation, low fluorescence
intensity in the nuclei after propidium iodide staining, and
appearance of DNA laddering. In particular, the authors have
identified PCA, fustin, fisetin, sulfuretin, and butein, as the main
active compounds responsible for the antioxidant and
antiproliferative activities of RVS extract [216]. The same purified
extract inhibits growth and induces apoptosis in human
osteosarcoma (HOS) cells by activating caspase-8, modifying the
balance between BAX and Bcl-2, and inducing the release of
cytochrome c. Furthermore, PARP cleavage is closely associated
with the induction of apoptosis in HOS cells [217].
An interesting study by Stagos et al. [218] has been addressed
to investigate th e mechanisms by which g rape ex tracts and
individual polyphenols contained in those extract (caffeic acid,
ferulic acid, gallic acid, PCA, and rutin) exert their
chemopreventive and antitumor activities in cancer cells. The grape
extracts, the polyphenol-rich fractions, and individual polyphenols,
among which PCA, have been shown to be potent inhibitors of
topoisomerase I (topo I), indicating that the inhibition of this
enzyme might be one of the mechanisms accounting for the
anticancer and chemopreventive activity. Moreover, the grape
extracts inhibit the mitomycin C-induced DNA strand breakage
suggesting that they could prevent ROS-mediated DNA damage.
On the contrary, the polyphenol-rich fractions and the individual
polyphenols enhance the mitomycin C-induced DNA strand
breakage indicating a pro-oxidant activity that lead to the
enhancement of DNA damage and might account for the cytotoxic
and pro-apoptotic properties of these compounds towards cancer
cells. The anticancer activity of PCA has also been studied in
human gastric adenocarcinoma (AGS) cells, by analyzing in detail
the signaling pathways involved [219]. Molecular data have shown
that the effects of PCA in these cells might be mediated via
sustained phosphorylation and activation of JNK and p38 MAPK,
but not of ERK. In fact, either the treatment with pharmacological
2912 Current Medicinal Chemistry, 2012 Vol. 19, No. 18 Masella et al.
inhibitors or the transfection with the mutant p38 or/and JNK
expression vectors reduces the JNK/p38 MAPK-related protein
phosphorylation and expression, and eventually the PCA-mediated
apoptosis. Similar results have been obtained in HepG2 cells where
PCA, but not chlorogenic acid or luteolin, dose-dependently and
specifically stimulate JNK and p38 activities [220].
Szaefer et al. [221] have demonstrated that PCA, as well as
other phenolic acids, may affect TPA-induced tumor promotion and
inflammation in female Swiss mice by inhibiting specific isoforms
of PKC, which is considered a major intracellular recepto r for th e
mouse skin tumor promoter TPA. Pre-incubation with Nok-1
monoclonal antibody, which is inhibitory to Fas signaling,
interferes with PCA-induced cleavage of pro-caspase and
sensitization to anti-Fas receptor-induced apoptosis. These results
suggest that the multiple signaling pathways from the MAPK to the
subsequent mitochondria- and/or Fas-mediated caspase activation
might be potential requirements for PCA-induced apoptosis. Yin,
M.C. et al. [222] have examined the apoptotic effects of PCA at
micromolar concentrations on human breast cancer MCF7, lung
cancer A549, HepG2, cervix HeLa, and prostate cancer LNCaP
cells. PCA treatment causes strong apoptotic effects in these cancer
cells by increasing DNA fragmentation, decreasing mitochondrial
membrane potential, lowering Na
+
-K
+
-ATPase activity, and
elevating caspase-3 and caspase-8 activities.
Finally, it should be highlighted that PCA exerts cytotoxic
effects ag ainst hepatocellular carcinoma cells, whereas it protects
primary hepatocytes against cytotoxicity [220,223]. These findings
further support the hypothesis that PCA is likely to be a useful
chemotherapeutic agent, without harmful effects on normal cells,
since it shows a higher selectivity towards cancerous cells than to
normal human cells.
PCA, and Metastasis Development
It has recently been described the potential ability of PCA to
interfere with the metastasis development. The metastatic p rocess
consists of different steps such as tumor cell detachment, lo cal
invasion, migration, angiogenesis, vessel invasion, and adhesion to
endothelial cells, extravasation, and growth in other organs. When
cancer cells invade normal tissues, degradation of the extracellular
matrix (ECM), migration and proliferation of malignant cells occur.
These ev ents are accompanied by several cell alteration s, including
the over-expression of proteolytic enzyme activities, such as
MMPs, especially MMP-2 and MMP-9. In this regard, phenolic
acids, including PCA, have been shown to exert strong anticancer
activity by inhibiting cell migration and reducing metastatic power
of AGS cells, most likely because of the down-regulation of
PI3K/AkT/small GTPase signals that inhibit NF-B, MMP-2, and
MMP-9 activities [224]. Furthermore, in different cancer cell lines,
PCA also inhibits the production of the vascular endothelial growth
factor (VEGF), a signal protein that contribute to cancer growth and
dissemination when over-expressed [222].
Lin et al. [166] have used B16/F10 mouse melanoma cells,
widely used as model system in studying metastasis, to inject
C57/BL6 mice orally treated with PCA for 6 weeks. Interestingly,
the treatment with PCA reduces the number of metastatic nodules,
the volume of hepatocellular tumors, and the weight of liver
compared with the untreated animals. The authors also
demonstrated that the down-regulation of the Ras/Akt/NF-B
signaling pathway is responsible for the inhibitory effects of PCA.
The inhibition of this pathway is considered a promising approach
in cancer treatment, because it is involved in cell migration and
invasiveness, most likely by the up-regulation of MMP-2, which
degrades the ECM. According to this hypothesis, the same study
shown that PCA inhibits MMP-2 secretion in AGS cells.
Furthermore, PCA decreases the migration of the cells in a dose-
and time-dependent manner probably by inhibiting NF-B nuclear
translocation.
ANTI-MICROBIAL ACTIVITY OF PCA
Polyphenols, including PCA, exert antimicrobial activity, which
represents an interesting field of application both in health
protection and in food preservation in order to avoid food-borne
illnesses [225-227]. Furthermore, the in vitro antimicrobial activity
of natural compounds could provide useful information for
developing novel antibiotics. Medicinal plants could be used as a
possible way to treat diseases caused by multidrug-resistan t bacteria
[228,229]. The antimicrobial activity of purified flavonoids may
result in susceptibility differences against species with different
origins and background [225]. PCA has been shown to be active
against both Gram-negative and Gram-positive strains, including
E.Coli, Ps. Aeruginosa, S. Aureus, B. cereus, and Enterobacteria
[230-232] Notably, PCA, as well as other phytochemicals, interacts
synergistically with selected antibiotics against Ps. aeruginosa, that
is a major nosocomial pathogen resistant to many antibacterial
drugs and particularly dangerous to immune-depressed population.
PCA used in combination with antibiotics increases th e
susceptibility of the pathogen to the drugs and reduces drug toxicity
in vitro [230,232,233].
Finally, PCA, as well as diallyl disulphide and diallyl
trisulphide, exhibited in vitro inhibitory effects against the growth
of susceptible, drug-resistant, H. pylori in broth and mouse stomach
homogenate. This finding indicates that PCA might be useful in the
prevention or therapy of H. pylori that is implicated in peptic ulcer,
duodenal ulcer and gastric cancer [234].
CONCLUSION
A growing body of evidence provides new insight in the
comprehension of the cellular and molecular mechanisms
responsible for the potential preventive/therapeutic activity of PCA
against a number of human diseases. The experimental data suggest
a multifaceted action of PCA through the modulation of cell redox
environment and signals and biochemical pathways involved in the
control of inflammation and oxidative stress. Consequently, PCA
modulates the balance between cell survival and cell death.
Notably, PCA can exert opposite effects depending on the
concentration, the cell system, and the type or stage of th e
degenerative process. It is worth mentioning that PCA seems to
specifically behavior as a chemotherapeutic agent, without harmful
effects on normal cells. However, much evidence of such properties
has been collected from cellular and animal studies, while clinical
studies are still lacking. Although the in vitro systems represent a
useful tool for unraveling the mechanisms of action that allow the
identification of the specific molecular targets, data obtained in
vitro may not be physiologically relevant. This means that further
studies and clinical trials are needed to fully establish the
preventive and therapeutic effectiveness of PCA, and to prove its
safety in humans.
CONFLICT OF INTERESTS
None of the authors have conflict of interest.
ACKNOWLEDGEMENTS
This work was partially supported by Italy/USA cooperation
program (Targeted Research Project no. 11US/23)
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Received: November 28, 20 11 R evised: Mar ch 26, 2012 Accepted: March 26, 20 12
