Pharmacology & Pharmacy, 2012, 3, 474-480
http://dx.doi.org/10.4236/pp.2012.34065 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1
Effects of Long-Term Use of Flavonoids on the Absorption
and Tissue Distribution of Orally Administered Doses of
Trace Elements in Rats
Ausama Ayoob Jaccob1, Saad Abdulrahman Hussain2*
1Department of Pharmacology and Toxicology, College of Pharmacy, Al-Basra University, Basra, Iraq; 2Department of Pharma-
cology and Toxicology, College of Pharmacy, University of Baghdad, Baghdad, Iraq.
Received July 26th, 2012; revised August 28th, 2012; accepted September 12th, 2012
ABSTRACT
The risk of pharmacokinetic polyphenols-trace elements interaction may undesirable therapeutic outcomes. We evaluate
the long-term use of silibinin, epigallo catechin (ECGC), quercetin and rutin on the absorption and tissue distribution of
zinc, copper and iron after single oral doses in rats. Five groups of rats received either with olive oil as control or one of
the polypheno ls silibinin, EPGC, quercetin o r rutin, administered orally as oily so lutions for 30 days. At day 30, a so lu-
tion contains sulphate salt of zinc, copper and iron was administered orally; 3 hrs later blood samples, tissues of brain,
kidney and liver were obtained for evaluation of the elements levels. The results showed that the polyphenols increased
both serum and tissue levels of these elements compared with controls. This effect was relatively varied according to
the structural differences among flavonoids. In conclusion, long-term use of supraphysiological doses of flavonoids
increase absorptio n of Zn, Cu and Fe and their tissue availability in brain, kidney and liver; this effect seems to be dif-
ferent with variations in structural features.
Keywords: Flavonoids; Trace Elements; Absorption; Tissue Distribution
1. Introduction
Flavonoids (polyphenolic compounds) are one of the bio-
active compounds widely availa ble i n fruit s and veget a bl es
[1]. Flavonoids have long been associated with a variety
of biochemical and pharmacological properties, including
antioxidant, antiviral, anticarcinogenic, and anti-inflam-
matory activities [2], and believed to be beneficial to
human health. Many peoples are motivated by scientific
research that is widely carried in the news media, which
indicated these flavonoids and polyphenols could prevent
cancer, ageing, and cardiovascular diseases [3,4]. How-
ever, these researches are often carried out in animals
and their effects in humans remain uncertain [5]. A large
body of evidence, mainly derived from preclinical studies
in animals, has concluded that dietary polyphenols, when
given in large qu antities, can ha ve desirab le o utco mes [6].
There is currently an extensive range of flavonoid sup-
plements on the market [7]. Su ppliers of su ch s uppl emen ts
recommend daily flavonoid intakes in amounts that are
many times higher than those doses which can normally
be achieved from a flavonoid-rich diet. The question
arises whether supplements containing such supra-physio-
logical flavonoid levels may exhibit adverse effects. In
addition, it is likely th at a large proportion of individuals
taking dietary flavonoid supplements are also taking con-
ventional drugs or trace elements [8]. The concomitant
intake of “supra-nutritional” flavonoid doses together with
conventional drugs may lead to flavonoid-drug inter-
actions [8]. Approximately 38 million adults in the US
(18.9% of the population) use herbal p roducts that contain
flavonoids or other natural supplements, but only one
third tell their physician about this use [9]. This lack of
information, combined with the fact th at natural products
are usually a mixture of many active ingredients, in-
creases the likelihood of harm. Moreover, this additionally
raises concerns about the safe use of dietary flavonoids.
The risk of pharmacokinetic polyphenols-trace elements
interaction poses two major extremity challenges, phar-
macotoxicity and treatment failure. The former can result
from the inhibition of the homeostatic mechanisms
responsible for the absorption, tissue distribution and
clearance of the trace elements, while the latter may be
the consequence of inducing processes the lead to faster
clearance. This is in addition to the intrinsic pharma-
codynamic actions of the polyphenols themselves which
may include potentiating, additive, antagonism, or neu-
*Corresponding a uthor.

Effects of Long-Term Use of Flavonoids on the Absorption and Tissue Distribution of
Orally Administered Doses of Trace Elements in Rats 475
tralization effects. The present study was designed to
evaluate the effect of long-term use of supraphysiological
doses of silibinin, epigallocatechin gallate, quercetin and
rutin on the absorption and tissue distribution of orally
administered doses of the trace elements zinc copper and
iron in rats.
2. Materials and Methods
2.1. Chemicals and Reagents
Silibinin dihemisuccinate (SDH) (98% purity) was ob-
tained from Tolbiac SRL, Argentina; Quercetin d ihydrate
(98% pure standardized extract) was purchased from
Xian Co, China; Epigallocatechin gallate (EGCG) was a
gift from Al-Razi Pharm Ind, Syria; Rutin was obtained
from Merck Laboratories, Germany; Ferrous sulphate,
Copper sulphate and Zinc sulphate were obtained from
SD Fine Chemicals, India.
2.2. Animals and Study Design
Thirty male adult Sprague Dawly rats of body weight
200 - 250 g were obtained from the Animal House,
Department of Pharmacology and Toxicology, College of
Pharmacy, Baghdad University, and the experiments
were carried out in Department of Pharmacology, College
of Pharmacy, Al-Basra University, Iraq. The rats were
housed under controlled conditions (22˚C - 25˚C) on a 1 2
h light/12 h dark cycle, and received the standard pellet
diet (National Center for Drug Research and Quality
Control, Baghdad) and water ad libitum. The study
protocol was approved by the Institutional Animal Ethi cal
Committee (IAEC), College of Pharmacy, University of
Baghdad. After acclimatization for a period of one week,
the animals were allocated into five groups consisting of
6 rats each; first group was treated with vehicle (olive oil)
as control group; the other four groups are treated with
one of the flavonoids: SDH (100 mg/kg), EGCG (25
mg/kg); Quercetin (50 mg/kg) and Rutin (500 mg/kg).
All flavonoids are prepared as o ily solutions dissolved in
olive oil and introduced as single daily doses admi-
nistered orally using gavage tube for 30 consecutive days;
the control group receives 0.2 mL/day of olive oil in the
same way. At day 30, all groups of rats received orally
single doses of Zinc sulphate (60 mg/kg), Copper sulphate
(60 mg/kg) and Fe sulphate (60 mg/kg), all these ele men ts
were administered 2 hrs after administration of the last
doses of the flavonoids and the vehicle.
2.3. Samples Preparation
After 3.0 hrs of administration of trace metals, all ani-
mals are sacrificed after short duration anesthesia with
anesthetic ether; blood samples were dra w n and collected
in polyethylene tube, centrifuged at 10000 rpm for 20
min and the resulted serum was kept frozen at –20˚C
until trace elements analysis. The liver and both kidneys
were quickly removed and perfused with ice-cooled sa-
line; the brain was carefully excised, rinsed with ice-
cooled saline and the arachnoid membrane was carefully
removed. One gram tissue of the obtained organs and 1.0
ml of serum were digested utilizing the wet digestion
method [10,11]; the digested samples were stored in re-
frigerator and used later for analysis of tissue and serum
levels of zinc, copper and iron [12].
2.4. Analysis of Trace Elements
The contents of Zn, Cu and Fe in serum and tissue
samples were first released from the protein matrix by
wet digestion method as mentioned previously, and their
concentrations were determined using atomic absorption
spectrophotometer (Buck Scientific, Model 211-VGI,
USA) at wavelength of 214 nm for zinc, 247 nm for Fe
and 324 for Cu [13]. Standard solutions of these elements
were used to prepare calibration carve for quantitative
analysis.
2.5. Statistical Analysis
Values were expressed as mean ± S.D; the values were
statistically evaluated using unpaired Student's t-test and
one way analysis of variance (ANOVA), supported by
Bonferroni’s post hoc analysis. Values with P < 0.05
were considered significantly different. Analysis was
performed using GraphPad Prism software for Windows
(version 5.0, Grap hPad Software, Inc., San Die g o, CA) .
3. Results
Figure 1 showed that all the flavonoids used in the pre-
sent study significantly increased (P < 0.05) the GI ab-
sorption of Zn, when administered as single oral dose
compared to control group; meanwhile, no significant
differences in serum Zn levels were reported among the
effect of the four flavonoids (P > 0.05) in this respect.
Concerning long-term effects of the tested flavonoids on
the absorption of Cu, all of them produced significant
increase (P < 0.05) in serum Cu levels compared to the
values reported in controls; only SDH and quercetin
demonstrated significantly different effects in this respect
(lower effect for quercetin), when the effects of the four
flavonoids compared with each others (Figure 2). In
Figure 3, treatment of rats with one of the four flavon-
oids used in the study for 30 days resulted in significant
increase (P < 0.05) in the oral absorption of Fe when
administered as single dose of ferrous sulphate compared
to control group. When the effects of the studied flavon-

Effects of Long-Term Use of Flavonoids on the Absorption and Tissue Distribution of
Orally Administered Doses of Trace Elements in Rats
476
oids were compared, non-significant differences were re-
ported between the effects of SDH and quercetin, while
the others showed significant differences in the order:
EGCG > SDH = quercetin > rutin (Figure 3).
The effects of flavonoids on the organ availability and
percent amounts of Zn, Cu and Fe distributed to the brain,
kidneys and liver relative to serum levels were evalu ated
after administration of single oral doses of those elements.
In Table 1, the results showed that all administered
flavonoids significantly increased the tissue availability
Serum Zinc Level
Figure 1. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on serum levels of Zn in rats after single
oral dose of this element.
Serum Copper Level
Figure 2. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on serum levels of Cu in rats after single
oral doses of this element.
of the essential metals (Zn, Cu and Fe) compared to the
value reported in control group. The most prominent
effect for the studied flavonoids in this respect was
reported on iron tissue availability, where EGCG pro-
duced consistent increase in the three targeted organs
while rutin showed the lowest effect in this respect com-
pared to others. Concerning the effects on the tissue
availability of Zn and Cu, the influence is relatively
comparable for all flavonoids in all organs (especially for
Cu) and EGCG demonstrates the lowest effect on Zn
availability in the three organs. In Figure 4, the influence
of the flavonoids on percent Zn availability in the brain
tissue relative to serum levels indi cated comparable effects,
Serum Iron Level
Figure 3. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on serum levels of Fe in rats after single
oral dose of this element.
Figure 4. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on tissue availability of Zn in Brain, Kid-
neys and Liver relative to serum levels in rats after single
oral dose of this metal.

Effects of Long-Term Use of Flavonoids on the Absorption and Tissue Distribution of
Orally Administered Doses of Trace Elements in Rats
477
Table 1. Effects of long-term administration of silibinin (100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and rutin (500
mg/kg) on tissue availability of Zn, Cu and Fe in Brain, Kidneys and Liver of rats after single oral doses of these metals.
Tissue Levels of Trace Elements μg/g Tissue
Zn Cu Fe
Treatment
groups
Brain Kidney Liver Brain Kidney Liver Brain Kidney Liver
Control 32.9 ± 2.1 35.6 ± 3.1 44.8 ± 5.7 34.2 ± 3.739.4 ± 5.547.6 ± 4.223.2 ± 5.7 20 5.2 ± 27.8 318.3 ± 34. 2
Silibinin 70.8 ± 4.9*a 75.3 ± 6.0*a 94.0 ± 12.2*a 63.5 ± 4.6*a 72.0 ± 2.6*a 85.6 ± 3.7*a 64.8 ± 12.1*a 456.8 ± 53.0*a 738.9 ± 90.3*a
EGCG 58.4 ± 6.3*b 79.3 ± 6.6*a 95.4 ± 9.7*a 60.3 ± 4. 3*b75.6 ± 7.6*a 84.3 ± 9.6*a 71.2 ± 9.1*a 727.3 ± 81.4*b 954.6 ± 125.4*b
Quercetin 61.0 ± 7 .5*b 95.7 ± 9.0*b 114.6 ± 10.7*b 67.3 ± 7.0*b 70.0 ± 4.7*a 80.0 ± 5.7*a 67.3 ± 7.0*a 579.3 ± 111.0*c 634.8 ± 67.4*a
Rutin 63.7 ± 6.4*b 81.7 ± 2.8*a 94.2 ± 5.4*a 68.3 ± 9.1*b 74.6 ± 11.1*a80.6 ± 12.5*a42.5 ± 3 .2*b 352.6 ± 46.7*d 461 ± 34.4*c
Values are presented as mean ± S .D; n = 6 animals in each gro up, *significantly different compared to control group (P < 0.05); values with non-identical su-
perscripts (a, b, c, d) for the same metal in the same organ are considered significantly different (P < 0.05).
with exception of a significant difference between the
effect of EGCG and rutin (rutin > EGCG); the same
pattern of activity was reported reg arding the distribu tion
of Zn in kidney tissues. Moreover, Figure 4 indicated
that all the tested flavono ids produced comparable effects
(P > 0.05) on the tissue availability of Zn in the liver
relative to serum level after single oral dose of Zinc
sulphate. Figure 5 sowed that the effects of the flavo-
noids on percent Cu distribution to the brain tissue rela-
tive to serum levels were comparable, with exception of
a significant difference between the effect of silibinin
and quercetin (Quercetin > SDH); the same pattern of
activity was reported regarding the distribution of Cu in
kidney tissues.
Additionally, Figure 5 indicated that all the tested
flavonoids produced comparable effects (P > 0.05) on the
liver tissue availability of Cu relative to serum level after
single oral dose of Copper sulphate. Concerning the in-
fluence of the tested flavonoids on tissue availability of
Fe after single oral dose of ferrous sulphate, Figure 6
indicated that no significant differences were reported
regarding their effects on brain and liver tissues avail-
ability of Iron. Meanwhile, significant differences were
reported between the effects of silibinin compared with
those produced by quercetin and rutin respectively (Quer-
cetin > SDH and rutin > SDH); the effect of EGCG in
this respect was found non-significantly different com-
pared with the othe r th ree fl avon oi d s used in the study .
Figure 5. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on tissue availability of Cu in Brain,
Kidneys and Liver relative to serum levels in rats after sin-
gle oral dose of this metal.
protection from many diseases, the conditions and the
levels of flavonoid intake that may pose a potential
hazard remains to be determined. Globally, dietary intake
o f m ix e d fl a vo n oi d s i s es t im a te d to b e i n the range of 500
- 1000 mg, but it can be as high as several gr ams i n t hose
persons supplementing their diets with flavonoids or
flavonoid-containing herbal preparations such as ginko
biloba or grape seed extract [14]. These high doses may
lead to pharmacological concentrations in body fluids
and tissues. In the present study, orally administered
doses of SDH, EGCG, quercetin and rutin to rats for 30
days, significantly improved both serum levels and
tissue availability of orally administered doses of the
essential elements, Zn, Cu and Fe compared control
animals. The explanation of such finding seems to be a
little bit difficult, since conflicting reports are available
4. Discussion
High consumption of flavonoids rich diet may potentiate
other deleterious effects of drugs or trace elements
because of their diverse pharmacological properties; more-
over, it may modulate drugs activity and the activities of
environmental toxins and metalloenzymes. Thus,
although there is evidence that a flavonoid-rich diet or
supplements may promote good health and provide

Effects of Long-Term Use of Flavonoids on the Absorption and Tissue Distribution of
Orally Administered Doses of Trace Elements in Rats
478
Figure 6. Effects of long-term administration of silibinin
(100 mg/kg), EGCG (25 mg/kg), quercetin (50 mg/kg) and
rutin (500 mg/kg) on tissue availability of Fe in Brain, Kid-
neys and Liver relative to serum levels in rats after single
oral dose of this metal.
regarding the influence of polyphenols intake on trace
elements homeostasis. Flavonoids can act as transition
metal ion chelators [15,16]. This feature plays an
important role in their antioxidant activity because the
free radical generation is mainly catalyzed by transition
metals in vivo and in vitro. However, excessive intake of
flavonoids may cause a decrease in essential trace
elements (Cu and Zn) and their related enzyme activities.
According to many previously reported data, flavonoids
as transition metal chelators, when used in excessive
amounts may cause a decrease of trace minerals, such as
iron, copper, and zinc [17]. In contrast to this idea, we
have shown in this study that with oral administration of
supraphysiological doses of SDH, EGCG, quercetin and
rutin, the availability of iron, copper, and zinc levels in
the serum and tissues after single oral doses of these
elements was increased compared to controls. In the
present study, the higher serum and tissue availability of
the essential elements can be explained according to the
fact that absorption and membrane transport of some
metals ions were enhanced when they form complexs
and chelates with organic ligands. In tune with this
finding, many data were reported on metal binding to
proteins in the cells [18,19] an d the higher availability of
chelated elements may be linked to the shielding of the
minerals positive charge during chelation. This allows
the mineral to withstand the binding activity of the
negatively charged mucin layer and results in lower
competition between minerals of similar charge in their
resorption from the gut and transfer to the enterocyte.
These phenomena, combined with lower complex for-
mation in the intestinal lumen with compounds such as
phytate, may contribute to the higher absorption of
minerals from the gut. Moreover, feeding trials in mam-
malian species have shown that complexes of organic
compounds with trace minerals have higher relative bio-
availability than inorganic ones and provide alternative
pathways for absorption, thus leading to a reduction in
the excretion of minerals [20-22]. Another possible ex-
planation for this behavior is based on the metal chelat-
ing ability of polyphenols, which is related to the pr es en c e
of ortho-dihydroxy polyphenol, i.e., molecules bearing
catechol or galloyl groups and condensed tannins; the
possibility of occurren ce of chelation in physiolog ical pH
also supports the physiological significance of this phe-
nomenon [23]. In tune with our finding, in an in vitro
study, polyphenol-rich beverages such as red wine, red
grape juice, and green tea or certain specific polyphenols
(tannic acid and quercetin) have the ability to enhance the
uptake of zinc in Caco-2 cells [24]. Luminal interactions
with ligands have drastic consequences for the bioavaila-
bility of metals. Some metal complexes are very stable.
Depending on the lipophilicity, such a complex may be
absorbed, distributed and possibly excreted without re-
leasing its metal moiety. Thus, in spite of sufficient ab-
sorption, the metal may not be metabolically available.
Although the available information suggests that poly-
phenolic compounds can chelate many essential elements
(Zn, Cu and Fe) and may affect their availability for
absorption [25], Coudray et al. (2000) reported that shor t-
or long-term consumption of polyphenols present in wine
did not have a negative effect on intestinal absorption or
tissue levels of zinc and Cu in rats [26]; the results of the
present study are found relatively comparable with this
finding, even when supraphysiological doses of poly-
phenols were introduced in pure form, and for the first
time showed some differences between certain flavo-
noids in this respect. The differences between flavonoids
reported in the present study could be related to the
differences in certain structural properties, including the
number and distribution of hydroxyl groups at specific
parts of the structural formula; this will consequently
affect the physicochemical properties of these flavonoids,
especially lipid solubility and interactions with b iological
targets. Meanwhile, the interaction between dietary fla-
vonoids and trace minerals may affect metal homeostasis
in a structure-specific fashion. Since fluid properties of
biological membranes were essential for numerous cell
functions including solute transport and membrane-
associated enzymatic activities [27], it is possible that
even mild alterations produced by the lipophilic struc-
tures of flavonoids on membrane fluidity could cause
aberrant function and changes in membrane permeabi-
lity [28]. The D-ring hydroxyl groups of the flavonoid
structure occupy the first coordination sphere around
metal io n to form a diolate combina tion rin g, wh ile B - ri n g

Effects of Long-Term Use of Flavonoids on the Absorption and Tissue Distribution of
Orally Administered Doses of Trace Elements in Rats 479
hydroxyl groups have weak interactions with metal ions
[29]. As suggested by previous reports [30,31], the
number of hydroxyl groups on the B-ring in many
flavonoids structures, the presence of a galloyl moiety,
and the steric characters on the D-ring could affect its
affinity for the lipid bilayers. As a result of this, incor-
poration of the flavonoids into the lipid bilayers was
enhanced, which could lead to the formation of another
ion channels after complexing of flavonoids with metal
ions, which is expected to induce structural variation and
influence the effects of structurally different flavonoids
in this respect. In conclusion, long-term use of supra-
physiological doses of flavonoids increase gastr oin test inal
absorption of essential elements (Zn, Cu and Fe) and
their tissue availability in brain, kidney and liver; this
effect seems to be different with variations in structural
features of the flavonoids.
5. Acknowledgements
The presented data was abstracted from PhD thesis sub-
mitted to the Department of Pharmacology and Toxico-
logy, College of Pharmacy, University of Baghdad. The
authors thank University of Baghdad for supporting the
project.
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