PHARMACEUTICALS 39 NOVEMBER/DECEMBER 2010

MARZENA MISIAK *
ELZBIETA LODYGA-CHRUSCINSKA
Institute of General Food Chemistry
ul. Stefanowskiego 4/10
90-924 Lodz, Poland
* Corresponding author
Tel (+4842) 631 34 17
Fax (+4842) 636 28 60
marzena.misiak1@wp.p
PHARMACEUTICALS
INTRODUCTION
Flavonoids are naturally occurring
compounds which are widely distributed
in vegetables, fruits and beverages such
as tea and red wine. They have an
important influence on the colour and
flavour of these foods and have a broad
pharmacological activity (1,2). Their
beneficial effects have been described for
allergy, cancer, viral infections, headache,
stomach and duodenal ulcer, and
inflammations (1,36). It is known that
they can bind to biologic polymers such
as enzymes, hormone carriers and DNA,
chelate transition metal ions, catalyse
electron transport and scavenge free
radicals. The ability to chelate Fe
2+
and
Cu
2+
and scavenge free radicals renders
the flavonoids very good antioxidants,
and many of the pharmacological actions
are explained by their action as
antioxidants (1,2).
CHARACTERIZATION OF
TRANSITION METAL IONS SUCH
AS Cu2+ AND Fe
2+
Copper is a microelement essential to
life. It is one of the most prevalent
biological transition metals, second only
to iron, being an essential micronutrient
required for survival by all organisms,
from bacteria to humans (7).
Iron ions exist in two forms, ferrous
Fe
2+
or ferric Fe
3+
. In organisms iron ions
are tightly bound by enzymes and
storage transporter proteins. As with
copper(II), unregulated iron ions
concentration can lead to undesired
oxidative reactions (8). Furthermore, iron
ions are necessary to hemoglobin
synthesis, DNA synthesis, and electron
transport. Iron ions are also involved in
the peroxidation of the mitochondrial
membrane lipids (9).
CLASSIFICATION AND
CHARACTERIZATION OF
FLAVONOIDS
Flavonoids are a group of polyphenolic
compounds, diverse in chemical structure
and characteristics, mainly found in fruits,
vegetables and cereals. Therefore,
flavonoids are part of the human diet.
Over 4000 different flavonoids have
been identified within the major flavonoid
classes which include flavonols, flavones,
flavanones, catechins, anthocyanidins,
isoflavones, dihydroflavonols, and
chalcones. Flavonoids are potent
antioxidants, metal chelators and inhibit
lipid peroxidation (10).
Flavonoids behave as antioxidants
primarily because of their polyphenolic
nature and the greater the number of
hydroxyl groups present on the flavonoid
structure, the greater the antioxidant
capacity of the flavonoid (11).
Because of structural differences
flavonoids are divided into eight different
groups: flavonols, (quercetin, myricetin,
kaempferol and rutin), flavanones
(taxifolin), flavones (luteolin and
apigenin), isoflavones (daidzein and
genistein), catechins, anthocyanidins,
dihydroflavonols and chalcones (12,13).
The carbon atoms in flavonoid
molecules are assembled in two aromatic
rings, commonly denoted as A and B,
which are connected by a three-carbon
bridge: C6-C3-C6. Flavonoids are
classified as flavones, flavanonols,
flavonols, flavanones or isoflavones
(Figure 1) (20).
Flavonoids are weak polybasic acids
that are polyphenolic in nature and have
a number of hydroxyl groups that can be
subjected to protonation and
deprotonation depending on their pK.
Flavonoids have the ability to help
mediate ailments such as iron induced
lipid peroxidation (10) and can act as
Interactions of
flavonoids with
transition metal ions
The medical properties of naturally
occurring compounds such as flavonoids
have been well known for many years.
However, the discovery that their
complexes with metal ions are more
effective than flavonoids alone changed
the course of chemistry research.
Flavonoids can be effective drugs for
neuronal diseases because of their potent
iron-chelating, radical scavenging, anti-
inflammatory and neuroprotective
properties. This paper is a literature
review of flavonoids and complexes
which are generally used in medicine and
pharmacy.
A
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PHARMACEUTICALS 40 NOVEMBER/DECEMBER 2010
anti-cancer agents (14).
Flavonoids are important natural
antioxidants and have been extensively
studied because of their numerous
biological activities (15-19). Many
pharmaceutical preparations containing
flavonoids as active substance are
commercially available today. For example
quercetin, the most biologically active and
popular dietary flavonoid, is generally used
as a dietary supplement (20).
FLAVONOIDMETAL COMPLEXES
In 1962 the first complex of flavonoids
with aluminum as a central ion was
obtained. Since the early 1980s scientists
have investigated about
40 metalflavonoid complexes. The
complexation causes a bathochromic shift
in both I and II absorption bands. Similar
shift has been noticed in all classes of
flavonoids which have 5-hydroxy-4-keto,
3-hydroxy-4-keto and/or o-dihydroxy
groups, suggesting that these kinds of
substituents are important for
chelation (20).
Flavonoids are effective metal ion
chelators and play a key role in the
initiation of free radical processes (via the
Fenton reaction). Metal chelation is
thought to be another mechanism of
flavonoids antioxidant activity, which, along
with some biological effects of flavonoids,
can be altered by flavonoidmetal
interaction (21).
Most of flavonoids are good metal
chelators which can chelate many metal
ions to form different complexes due to
their high superdelocalizability and
conjugated system. The interaction of
metals with flavonoids contributes to the
anti-oxidant activity (10,21-28).
Flavonoids easily chelate metal ions
and create complex compounds. Their
additional antioxidant activity results from
binding of metal ions like Fe
2+
, Fe
3+
and
Cu
+
, which participate in free radical-
generating reactions. Therefore, they act
on two antioxidant pathways: i) direct
reactions with free radicals; ii) chelating of
metal ions involved in production of
reactive oxygen species (20,29-31). Their
chelation depends on the presence of
certain chelation sites in the structure of
the flavonoid as well as the solvent type
and pH conditions (32).
Flavonoids act as weak polybasic
acids, so pH plays an important role in
complex formation. The optimal pH for
complex formation is around 6, although
it strongly depends on the metal ion. At
pH below 3.0, flavonoids remain
undissociated, which is unfavourable for
complex formation. At high pH values
flavonoids are
deprotonated and form
more complex species.
Furthermore, at higher
pH values metal ions
cause side reactions
(hydrolysis) and hydroxo-
complexes are
formed (20).
CHEN et al have
found successful
chelation with transition
metal species such as
Cu
2+
and Ni
2+
to
biochanin A (4'-methoxy-
5,7-dihydroxy-
isoflavone) (33).
Isoflavones are a
subclass of flavonoids, an
isomerised form
of flavones,
with key
structural
features such as a 4-keto group and a C-
C bridge between the 1' carbon on the
B ring and the 3 carbon on the C ring
(Figure 1) (20).
Isoflavone metal chelates are of
scientific interest as flavonoids have been
shown, in previous studies of flavonoid
metal chelates, to exhibit modulation of
biological activity such as enhancing
anti-viral, anti-cancer and, of interest to
this group, antioxidant activity (34).
It was demonstrated that some
interactions with metal ions showed
increases in antioxidant activity while
others showed a prooxidant effect on
flavonoids (35,36). JURD et al. found that
in the case of the isoflavones, the most
likely metal chelation site would be from
the 4-keto and 5-hydroxy groups (37).
The first attempts to chelation of
isoflavones such as daidzein and
genistein, with Cu
2+
and Fe
3+
were
reported by MIRA et al. (32).
The first direct evidence for isoflavone
ligand species that can chelate with
transition metals ions, was presented by
DOWLING et al. (38). This study shows the
partial structural characterisation and
assessment of the antioxidant
characteristics of isoflavone metal
chelates. Isoflavones were found to bind
with Cu
2+
and Fe
3+
. Specifically, the
isoflavones biochanin A and genistein
bind with Cu
2+
and Fe
3+
whereas
daidzein was found not to chelate with
any of the metals. The metals were
binding at the 4-keto and 5-OH site of
biochanin A and genistein and indicated
the presence of coordinated water.
The antioxidant ability of the
isoflavones is affected by metal type, with
copper having an antioxidant effect and
iron having a prooxidant effect (38).
Isoflavones also show antioxidant qualities
but have weaker antioxidant capacity
relative to flavonols like quercetin or
kaempferol which possess more hydroxyl
groups (11).
Quercetin (Figure 2) is a strong
hydroxyantioxidant and a major dietary
flavonoid (flavonols) most commonly
present in nature. The biological activities
of quercetin are influenced by the
presence of metal ions. Hydroxy- and oxo-
groups present in the quercetin structure
have the ability to form
complexes with various
metal ions. It is commonly
found in many common
foods including apple, tea,
onion, nuts, berries,
cauliflower and
cabbage (13). Quercetin
is able to chelate metal
ions via 3- or 4-
phenolic groups
(Figure 3). Upon
complexation, the metal
ion removes phenolic
hydrogen from the flavonoid (39).
S.B. BUKHARI et al. found that the
antioxidant activity of flavonoids depends
on the number and positions of OH
groups in the flavonoid structure. The
Cuquercetin complex shows higher
antioxidant activity as compared to the
pure quercetin. This suggests that the
metal ions for example Cu
2+

significantly change the chemical

Figure 1
Figure 2 Structure of the quercetin
3,3,4,5,7-pentahydroxyflavone (13)
PHARMACEUTICALS 41 NOVEMBER/DECEMBER 2010
properties of the quercetin (13). The
same results were received for
Co-quercetin complex (22). and
Al
3+
-quercetin complex (40).
As well complex formation between
copper (II) sulfate and morin
(3,5,7,2,4-pentahydroxyflavone) showed
that the antioxidant activity of complexed
morin has higher value as compared to
the free morin (Figure 4). In this complex,
morin is chelated with the Cu
2+
ion via
four C=O and three C-O to form a fully
planar five-membered ring coplanar with
the C-ring.
Free naringin (Figure 5) binds Cu
2+
ion through the interaction of the Cu
2+
ion with the condensed ring via the
carbonyl group in position 4 and by the
oxygen of the hydroxyl group in
position 5 (42). The complex
demonstrates higher anti-inflammatory
and antioxidant activity than free naringin.
The increase in biological activity of
complex could be associated with the
coordination of Cu
2+
in positions 4 and 5
of the condensed naringin ring (44).
Generally, it is assumed that the ability
of flavonoids to chelate metals is very
important for their antioxidant activity.
Several authors have reported the
chelation of Cu
2+
and Fe
3+
by various
polyhydroxy-compounds, proposing that
the 3,4-dihydroxy moiety of the catechol
rings, the 3-hydroxy-4-keto or the
5-hydroxy-4-keto groups function as
possible binding sites (45,46).
BROWN et al. found that it is evident
that quercetin and rutin have a greater
capacity to delay oxidation in the Cu
2+
ion
system, whereas luteolin is the most
effective in the haem protein oxidation
system. Kaempferol, in contrast, responds
poorly in both systems. This emphasizes
the importance of the catechol
arrangement in the B ring structure, which
kaempferol lacks, in the radical scavenging
process The comparative effects of the
flavonoids under the infuence of the two
pro-oxidants also highlights the role of
partitioning and the interplay between
radical scavenging and metal chelation. In
considering the Cu
2+
ion system, rutin is
more soluble in aqueous media and thus
has a greater tendency to form Cu
2+
complexes than does luteolin, which is
more lipophilic (47).
M.T. FERNANDEZ et al. provided direct
evidence for chelation between flavonoids
and copper
and iron ions.
Therefore, it is
expected that
flavonoids
can be very
effective
antioxidants
since they
can be
operative by
both antiradical and chelating
mechanisms. The stoichiometries of the
iron complexes and the oxidation states of
iron are more variable than for copper.
This showed that myricetin and quercetin
were the more effective in reducing iron,
followed by a group of intermediate
activity comprising cathechin, luteolin and
kaempferol, with naringenin being the
least effective (48).
The researchers MIRA et al. show that
only flavones myricetin and quercetin
reduced Fe
3+
effectively. Rutin, catechin
and taxifolin were moderately active and
kaempferol and luteolin were relatively
poor reductants. All other flavonoids
studied showed no activity.
While reduction study about Cu
2+
shows that myricetin, with six hydroxyl
groups, was the better reductant followed
by the flavonoids with five hydroxyl groups
quercetin, taxifolin and catechin. The
flavones luteolin (5,7,3,4-OH) and
kaempferol (3,5,7,4-OH) show the same
Cu
2+
reducing activity and apigenin
(5,7,4-OH) has about half of their
activities. The flavone apigenin and the
isoflavone genistein possessing the same
hydroxyl groups (5,7,4-OH) were less
active than the flavonoids with four
hydroxyl groups; the flavanone naringenin
(5,7,4-OH), however, was twice more
effective on reducing the copper ions. This
results clearly demonstrated that from the
interaction of flavonoids with iron and
copper ions can result the formation of
chelates and the reduction of the metal
ions, both depending on flavonoid
structures (32).
Complexes of flavonoids play an
important role in limiting metal
bioavailability and suppressing metal
toxicity. For example, aluminum has
been implicated in neurological and
bone disorder. The complexation of Al
3+
by quercetin reduces aluminum
overload in the diet. By forming
complexes, flavonoids appear to be a
suitable antidote for heavy metal
poisoning in vivo. Quercetin, as an
active biological ligand, might be an
appropriate Mo
6+
chelator in the case of
molybdenum deficiency caused by
irradiation, since the use of
molybdenum salts is undesirable
because of their high toxicity (20).
CONCLUSION
This paper is a literature review of the
interactions between flavonoids with
some metals ions. The complexed
flavonoids were much more effective
free radical scavengers than the free
flavonoids.
Cu
2+
and Fe
2+
as transition metal
ions play a vital role in the initiation of
free radical processes, metal chelation is
widely considered as another
mechanism of the antioxidant activity of
flavonoids. The interaction of flavonoids
with metal ions may change the
antioxidant properties and some
biological effects of the flavonoids.
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42 NOVEMBER/DECEMBER 2010
IRISH PHARMA COMPANY BEGINS MOVE
INTO NEW PENNSYLVANIA HEADQUARTERS
Almac, an Irish pharmaceutical-services
company has completed and begun moving
into a new North American headquarters
on a 40-acre site in Souderton.
Philadelphia Inquirer
CEPHALON ABANDONS JET LAG INDICATION
FOR NUVIGIL AFTER SECOND FDA REJECTION
Cephalon is abandoning an indication for
Nuvigil as a treatment for excessive
sleepiness associated with jet lag after
receiving a second complete response letter
for its sNDA. The FDA restated its previous
concerns with the robustness of patient
assessment findings for Nuvigil
(armodafinil), although Cephalon believes it
met the agreed-upon safety and efficacy
endpoints under its special protocol
assessment. Following several
conversations with the agency and given
this second complete response letter, the
company believes that further
communications with the FDA will not result
in an approval of this application, LESLEY
RUSSELL, Cephalons chief medical officer,
said.
Drug Industry Daily
MANNKINDS INHALED INSULIN VERDICT
DELAYED
MannKind Corp said US regulators need an
additional four weeks to complete their
review of its experimental diabetes
treatment, lifting the hopes of some
investors who had expected the drug to be
rejected outright and sending the
companys stock up 6.4% in premarket
trading.
KUAR
FACTORY BLAST KILLS FIVE IN CHINA
A blast at a Chinese pharmaceutical factory
has killed at least five people and injured
eight others.
Seattle Times
ROCHE ACQUIRES MARCADIA BIOTECH
Marcadia Biotech Inc., a
biopharmaceutical company founded by
former Eli Lilly and Co. executives in 2006,
has been acquired by Swiss life sciences
giant Roche.
Indianapolis Business Journal
ASTRAZENECAS BRENNAN POACHES PFIZER
TO REBUILD RESEARCH
While competitors such as
GlaxoSmithKline Plc and Sanofi-Aventis
SA expand into flea sprays and energy
drinks to offset slowing drug revenue,
AstraZeneca is sticking to one business
developing pharmaceuticals innovative
enough to command premium prices.
Bloomberg
AKORN SELLS AKORN-STRIDES JOINT
VENTURE PRODUCT PORTFOLIO TO PFIZER
Akorn, Inc. reported that its Akorn-Strides
LLC joint venture has entered into a
purchase agreement with Pfizer Inc. to sell
16 ANDAs and 6 filed ANDAs.
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