Full text of DOI:10.1385/BTER:73:3:251

©Copyright 2000 by Humana Press Inc.
All rights of any nature, whatsoever, reserved.
0163–4984/00/7303–0251 $12.00
Natural Polyphenols–Iron Interaction
Its Biological Importance
LEOPOLD J. ANGHILERI* AND PIERRE THOUVENOT
Biophysics Laboratory, Medicine Faculty,
University of Nancy, B.P. 184, Avenue de La Forêt de Haye,
54505 Vandoeuvre lès Nancy, France; and Nuclear
Medicine Service, University of Nancy, Medical Center,
Nancy, France
Received April 12, 1999; Accepted May 10, 1999
ABSTRACT
The iron-binding capacity of different fractions of natural
polyphenols extracts was determined by chromatographic and elec-
trophoretic methods. Their effects on iron-induced calcium homeo-
stasis changes in liver tissue suspension showed that mate tea and
green tea extracts provoke a very significant inhibition of the iron
effects, whereas it is much less significant with red wine extract. The
biological importance of this phenomenon is discussed.
Index Entries: Natural polyphenols; iron-induced cell calcium
changes.
INTRODUCTION
Iron binds to polyphenols and catechins, forming colored anionic com-
plexes (1). Plant polyphenols have been suggested as cancer-preventive
agents and an abundant bibliography supports this assumption (2). The
role of intracellular calcium ion concentration variations in cellular repro-
duction and proliferation seems to be an important factor in neoplastic
transformation and spreading of the transformed cell (3). The aim of this
experimental work has been to study the iron interaction with several nat-
ural extracts rich in polyphenols and to assay their effects on liver tissular
calcium homeostasis.
*Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
251
Vol. 73, 2000

252
Anghileri and Thouvenot
MATERIALS AND METHODS
Polyphenol Extraction
Polyphenols were extracted from leaves of green tea (GT) (Thea sinen-
sis) and mate tea (MT) (Ilex paraguariensis) by aqueous decoction at 80°C,
followed by evaporation under vacuum at 60°C to reduce the original vol-
ume to 1/30, and from red wine (WI) (Bordeaux type) by reducing its
original volume to 1/15 under the same conditions of evaporation. The
polyphenols content of the final solution was determined using the Folin–
Ciocalteaus reagent (4) and is referred to as catechin molarity by compar-
ison with a catechin (Sigma, St. Louis, MO) solution.
The ferric ATP complex was prepared as described elsewhere (5);
ferric citrate and ferric chloride were from Merck (Darmstadt, Germany).
When 59Fe-labeled iron compounds were used, they were obtained with
the general preparation technique already described (6).
Chromatographic and Electrophoretic Studies
of the Extracted Polyphenols
A separation of the different polyphenols in each extract was per-
formed by ascending chromatography on 3MM Whatman (Clifton, NJ)
paper using 0.02M sodium bicarbonate as solvent. With this solvent, fer-
ric citrate and ferric ATP complex migrate with the solvent front,
whereas ferric chloride remains at the origin. This analytical determina-
tion was done with aliquots of the original extracts and with aliquots of
the same extracts incubated with ferric citrate, the ferric ATP complex, or
ferric chloride (Fig. 1). This technique produces different migration zones
of a characteristic color, without any reagent spray, that were cut off,
eluted with water, and the elution liquid used for polyphenols quan-
titation with Folin–Ciocalteous reagent, and for iron estimation with
o-phenanthroline (7).
Electrophoresis of aliquots were performed using 0.02M sodium
bicarbonate as buffer, and a 1-h run with a 20-V/cm gradient.
In Vitro Study of Polyphenol Effects on Liver
Calcium Homeostasis
Liver tissue suspension of Swiss mouse liver was prepared according
the technique published elsewhere (8). To assess the changes in calcium
uptake by liver tissue suspensions induced by the polyphenols, aliquots
of the extracts were incubated with ferric ATP complex and 45Ca-chloride.
The radiocalcium uptake and the lipid peroxidation (absorption at 532 nm)
were determined as described in a recent publication (9).
The significance of change from control liver suspension was deter-
mined by statistic analyses of data using Student’s t-test; p < 0.05 was
considered significant.
Biological Trace Element Research
Vol. 73, 2000

Natural Polyphenols, Iron, and Cell Calcium
253
Fig. 1. Polyphenol chromatographic separation of different fractions cor-
responding to A = red wine, B = mate tea, C = green tea, and D = catechin. Sam-
ple volume = 10 µL (1 µmol) and 0.02M sodium bicarbonate as solvent.
RESULTS
The chromatography of the polyphenol solutions has shown spots of
characteristic colors (Fig. 1): For WI, spot 1 was black, spot 2 was blue,
spot 3 was yellow, and spot 4 was pink. For MT, spot 3 was light yellow
and spot 4 was dark green on a light pink-yellow background. For GT,
the diffuse spot 3 showed a superposition of dark green and pink-yellow.
Standard catechin (CA) migrates as a yellow spot coincident with frac-
tion 3 of the polyphenol solutions. The iron load produced by incubation
with ferric citrate, the ferric ATP complex, and ferric chloride in all of the
cases strongly increases the color intensity of fraction 4.
The chromatographic analyses of the original polyphenols extracts
show that there is a direct relationship between the polyphenols and iron
contents of each migrating fraction (Figs. 1 and 2) and that most of the
iron (over 60%) is present in the upper migrating fractions (fractions 3
and 4 in Fig. 2). On the other hand, after incubation with ferric citrate
and the ferric ATP complex, the higher iron incorporation is in those frac-
tions (Fig. 3). When the incubation was done with ferric chloride, this
Biological Trace Element Research
Vol. 73, 2000

254
Anghileri and Thouvenot
Fig. 2. Polyphenols and iron distribution (as percentage of the total) in the
different chromatographic fractions of original extracts. Number 1 to 4 corre-
spond to spots in Fig. 1.
form of inorganic iron remains on the origin and makes the evaluation
of its combination with the fraction 1 uncertain; however, in all the cases,
the iron present in the other migrating fractions reflects a very significant
incorporation in these fractions, particularly in fraction 4 of MT (over
50% of the total iron). When incubated with ferric citrate, WI polyphe-
nols show an iron distribution that is similar to one of the other two
extracts, but with ferric ATP complex, a decrease in the upward migrat-
ing iron, that is concomitant with an increase in nonmigrating iron, is
observed. GT shows a similar behavior, and the black deposit on the ori-
gin is similar to that observed with WI and GT extracts alone (without
incubation with iron compounds). This black deposit is missing with the
MT extract.
The electrophoretic study shows that the migrating fractions are sim-
ilar to that obtained by chromatography, but the use of 59Fe-labeled iron
compounds that permits the use of much lower amounts of solution
shows a slightly higher iron in the forward migrating fraction. The
migration in the electric field is an indication of the anionic character of
the fraction, where a high polyphenol content is reflected by a high
migration.
The electrophoresis of the CA–59Fe–iron chloride interaction product
has shown that with a stoichiometric ratio (µmol CA : µmol FeCl3) of
1 : 0.5, only 22% of the iron migrates with CA; for a 2 : 0.5 ratio, the amount
increases to 79%, and for a 4 : 0.5 ratio, it is 91%. On the other hand,
Biological Trace Element Research
Vol. 73, 2000

Natural Polyphenols, Iron, and Cell Calcium
255
Fig. 3. Iron distribution (as percentage of the total iron) in the chro-
matographic fractions obtained from original extracts (2 mol) incubated with
A = 1 µmol ferric citrate, B = 1 µmol ferric ATP complex, and C = 1 µmol fer-
ric chloride. Incubation for 30 min at 37°C. Numbers 1 to 4 correspond to spots
in Fig. 1.
increased combined iron deceases the migration distance from 5 to 2 cm.
Similar electrophoretic analyses of the interaction product of each natural
polyphenols extract with the 59Fe–ferric ATP complex (ratio = 1 : 0.5) have
shown that for WI about 73% of the iron remains on the origin line,
whereas in the case of MT, GT, and CA, more than 80% migrates with the
polyphenols as an anionic moiety.
Table 1 shows that compared with liver suspension alone, the pres-
ence of 1 µmol catechin equivalent of the natural polyphenol extracts
produce a decrease in calcium uptake: (A) for WI, 54%; for MT, 60%; for
GT, 38%; for CA, 57%. When 0.5 µmol of ferric ATP complex was present
in the incubation medium, the decrease in calcium uptake compared to
the uptake of the ferric ATP complex control (without polyphenols) was
as follows: (B) for WI, 29%; for MT, 84%; for GT, 68%; for CA, 42%. On
the other hand, the ferric ATP complex control increased 3.3-fold the cal-
cium uptake of liver tissue suspension alone.
The observed lipid peroxidation values, as a percentage of the liver
control value were as follows: (A) for WI, 115; for MT, 106; GT, 87; for
CA, 71. In case B, as a percentage of the ferric ATP complex control value,
they were as follows: for WI, 102; for MT, 74; for GT, 53; for CA, 49. The
presence of only the ferric ATP complex (ferric ATP control) increases
1.6-fold the value of liver tissue suspension alone (liver control).
Biological Trace Element Research
Vol. 73, 2000

256
Anghileri and Thouvenot
Table 1
Calcium Uptake and Lipid Peroxidation Induced by 0.5 µmol of Ferric ATP
Complex in the Presence of 1 µmol (as Catechin Equivalent)
of Natural Polyphenols Extracts
Note: All data after 1 h incubation at 37°C of liver tissue suspension (4 mg pro-
tein). The values are the mean SD of four incubations for calcium uptake and three
incubations for lipid peroxidation. * = p < 0.05.
DISCUSSION
The experimental data indicate that there is a clear correlation be-
tween the polyphenols and iron content in the original extracts and that
most of both are present in the fast ascending fraction (Fig. 2). On the
other hand, the incubation with iron compounds shows a chromato-
graphic iron distribution that is characteristic of each polyphenols extract
and of the used iron compound (Fig. 3). The differences between the dif-
ferent iron compounds are related to the different stability constant and
degree of dissociation.
The choice of liver as an assay tissue for the polyphenols–iron inter-
action was done considering the reported in vitro and in vivo suscepti-
bility of liver to the action of low-molecular-weight iron complexes (8)
and the fact that liver is the body’s largest pool of enzymatic activity
where metabolic elimination of a part of polyphenols and catechins may
Biological Trace Element Research
Vol. 73, 2000

Natural Polyphenols, Iron, and Cell Calcium
257
take place. It is interesting to mention that an iron–catechin complex for-
mation is known to be implicated in the action of phenolic oxigenases
that catalyze the cleavage of the aromatic ring of polyphenolic com-
pounds in the presence of molecular oxygen (10).
An explanation of the different effects of WI, MT, GT, and CA on the
ferric ATP complex-induced calcium uptake by liver can be inferred from
their chromatographic and electrophoretic behavior. In a previous work
(11), it has been shown that the iron responsible for the modificationn of
the cell calcium homeostasis is bound to molecular structures behind the
cell plasma membrane barrier and that there is an iron threshold con-
centration that must be exceeded to trigger calcium-ion influx to the cell.
On the other hand, the iron effects depend on the availability of ionic
iron at physiological pH that is determined by the stability constant of
the iron complex (12). When ionic iron [Fe(H2O)6]3+ reacts with the
polyphenols, it can be linked by coordination to give nondissociable
complexes, in which it is incorporated into the molecular structure, as in
the case of Na3[Fe(catechin)3] (1), or less stable complexes with a resid-
ual coordination capacity that can lead, at physiological pH, to hydroly-
sis and insolubilization. This last possibility seems to be the WI and GT
cases, which show a decreased iron in fraction 4 of Fig. 3 at the same
time that there is an increase in the nonmigrant fraction 1. This forma-
tion of an hydrolyzed insoluble fraction has been corroborated by the
electrophoretic analyses: In the case of WI, 73% of iron does not migrate,
whereas more than 80% does, as an anion in the cases of MT, GT, and
CA. The fact that ferric ATP complex-induced calcium uptake by liver is
decreased by only 29% in the presence of WI, whereas the values of 60%
and 38% for MT and GT, respectively (Table 1), can be related to a highly
dissociated iron in the presence of WI and a much lower dissociation
with MT and GT, that at physiological pH determines a lower availabil-
ity of ionic iron with WI.
Iron is required for the growth of all living cells. It is quite obvious
that the chelating capacity of polyphenols is of importance in the bio-
logical systems. Enzyme iron is the smallest iron compartment, but it is
very important to living organisms. There are enzymes that contain
iron in their molecules such as the cytochromes, lipoxidase, catalase,
peroxidases, iron–flavoproteins–cytochrome C reductase, succinate oxi-
dase, acyl-CoA dehydrogenase, NADH dehydrogenase, and xanthine
oxidase, and enzymes requiring iron as cofactor such as aconitase and
succinate dehydrogenase.
Considering the importance of iron in the biochemical functions of the
cell, these experimental results emphasize the possibilities of polyphenols
to control certain biological processes, as they do in the polyphenol con-
centration and ionic iron-dependent inhibition of iron-induced liver cal-
cium homeostasis modification. Work is in progress to evaluate the effects
of MT and GT polyphenols extracts on the experimental carcinogenesis
induced by ferric ATP complex.
Biological Trace Element Research
Vol. 73, 2000

258
Anghileri and Thouvenot
ACKNOWLEDGMENTS
The authors thank the Association pour la Promotion des Recherches
Appliquées en Biologie, Nancy, France for the support to this work, and
to P. Gerard, Biochemistry Laboratory, Medicine Faculty, Nancy Univer-
sity, for his technical assistance.
REFERENCES
1. D. Nicholls, Iron, in Comprehensive Inorganic Chemistry, J. C. Bailar, H. J. Emeleus, and
A. F. Trotman-Dickenson, eds., Pergamon, Oxford, Vol. 3, p. 1048 (1973).
2. H. L. Newmark, Plant polyphenols as potential cancer preventive agents, Adv. Exp.
Med. Biol. 401, 25–34 (1996).
3. J. F. Whitfield, Calcium, Cyclic AMP and Cancer, CRC, Boca Raton, FL, pp. 175–212 (1990).
4. R. W. Keith, D. Le Tourneau, and D. Mahlum, Quantitative paper chromatography
determination of phenols, J. Chromatogr. 1, 534–536 (1958).
5. L. J. Anghileri and J. Robert, In vivo distribution of ferric ATP complex, Eur. J. Drug
Metab. Pharmacokinet. 19, 1–3 (1994).
6. L. J. Anghileri, Effects of Fe3+–tumor cell interaction on Ca2+ uptake by Ehrlich ascites
tumor cells, Cell Calcium 12, 371–374 (1991).
7. L. K. Reitz, A. S. O’Brien, and T. L. Davis, Evaluation of three iron methods using a
factorial experiment, Anal. Chem. 22, 1470–1474 (1950).
8. L. J. Anghileri, P. Malecki, and J. Robert, Changes in calcium uptake by liver induced
by ferric lactate, Arch. Biochem. Biophys. 292, 529–533 (1992).
9. L.J. Anghileri and P. Thouvenot, ATP in cellular calcium-overload by trivalent metal
ions, Biol. Trace Element Res. 62, 115–122 (1998).
10. A. H. Mehler, Phenolytic oxygenases, in Oxygenases, O. Hayaishi, ed., Academic,
New York, p. 87 (1962).
11. L. J. Anghileri, P. Maincent, and A. Cordova-Martinez, On the mechanisms of soft tis-
sue calcification induced by complexed iron, Exp. Toxicol. Pathol. 45, 365–368 (1993).
12. L. J. Anghileri and P. Thouvenot, ATP in iron overload-induced intracellular calcium
changes, Int. J. Mol. Med. 1, 869–873 (1998).
Biological Trace Element Research
Vol. 73, 2000