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Archives of Biochemistry and Biophysics

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Review

Antioxidant actions of avonoids: Thermodynamic and kinetic analysis

Mnica Galleano a, Sandra V. Verstraeten b, Patricia I. Oteiza c, Cesar G. Fraga a,c,*
a

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina
Department of Biological Chemistry, IIMHNO (UBA) and IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Buenos Aires, Argentina
c
Department of Nutrition, University of California, Davis, CA, USA
b

a r t i c l e

i n f o

Article history:
Received 6 February 2010
and in revised form 5 April 2010
Available online xxxx
Keywords:
Flavonoids
Polyphenols
Antioxidants
Free radicals
Chelation
Adsorption
Wine
Tea
Chocolate

a b s t r a c t
The benets of avonoids on human health are very often ascribed to their potential ability to act diminishing free radical steady state concentration in biological systems providing antioxidant protection. This
is an assumption based on the chemical structures of avonoids that support their capacity to scavenge
free radicals and chelate redox-active metals. In this paper we will use thermodynamic and kinetic
approaches to analyze the interactions of avonoids with biological material and from there, extrapolate
the physiological relevance of their antioxidant actions. Thermodynamic analysis predicts that both,
scavenging of oxygen-derived radicals and the sequestration of redox-active metals are energetically
favored. Nevertheless, the actual concentrations reached by avonoids in most animal and human tissues
following dietary ingestion are incompatible with the kinetic requirements necessary to reach reaction
rates of physiological relevance. This incompatibility becomes evident when compared to other antioxidant compounds, e.g. a-tocopherol (vitamin E), ascorbate (vitamin C), and glutathione. Alternatively,
lipidavonoid and proteinavonoid interactions can indirectly mediate a decrease in oxidant (free radical) production and/or oxidative damage to both cell and extracellular components. The nal mechanisms mediating the antioxidant actions of avonoid will be determined by their actual concentration
in the tissue under consideration.
2010 Elsevier Inc. All rights reserved.

Introduction
Polyphenols are secondary metabolites of plants and include a
myriad of chemical structures, from simple molecules such as phenolic acids, to highly polymerized compounds, such as condensed
tannins. We have focused the present analysis on one of the most
abundant family of polyphenols present in human diets, the avonoids. Flavonoids are a chemically dened family of polyphenols
that have a basic structure of two aromatic rings (ring A and ring
B) linked through three carbons that frequently form an oxygenated heterocycle (ring C) (Fig. 1). Different arrangements of hydroxyl and/or carbonyl groups, and carboncarbon double bonds in
the basic structure dene the different subgroups of avonoids as
shown in Fig. 1 [1].
The benets of avonoids on human health are often ascribed to
their potential ability to act diminishing free radical steady state
concentrations in biological systems. Such ability could be possible
considering that polyphenols have chemical structures supporting
the scavenging of free radicals and the chelation of redox-active
metals. In parallel, it has been reported that certain avonoids

* Corresponding author at: Department of Nutrition, University of California,

Davis, CA, USA. Fax: +1 530 752 8966.
E-mail address: cgfraga@ffyb.uba.ar (C.G. Fraga).

can provide benets in pathological situations associated with high

free radical production, e.g. hypertension and cardiovascular disease [29]. However, the physiological mechanisms linking the
antioxidant chemical characteristics of polyphenols with their
health effects are still subject of discussion [3,10,11]. Based on
chemical, thermodynamic and kinetic data, we will analyze the
mechanisms proposed for polyphenol biological actions, especially
antioxidant reactions. Although we will focus the analysis on avonoids, and sometimes particularly on avanols (also known as avan-3-ols), most of the points addressed in this paper are valid for
many other polyphenols, as well as their metabolites able to retain
the antioxidant capacity. This is extremely important given that
most of the ingested avonoids are metabolized by hepatic and
intestinal enzymes, and are present as metabolites in blood and
other tissues.

Thermodynamics versus kinetics

Thermodynamic analysis is used to predict the possibility that a
physical interaction or a chemical reaction occur, that is, to proceed spontaneously as dictated by the distribution of the energy
present in reactants and products. The spontaneity of a chemical
reaction is dened by a decrease in the Gibbs energy (DG < 0). In
biological systems, DG is normally equalized to the change in

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Fig. 1. Basic structures of avonoids and different avonoid subfamilies.

apparent standard Gibbs energy (DG0 ) which is calculated under

biological standard conditions (P = 1 atm, and concentrations =
1 M, except for [H+] that is assumed 107 M).
The DG can be calculated from DG = DH  TDS. From this equation, spontaneous reactions can be dened as enthalpy- or entropy-driven reactions depending on the relationship between the
values of DH and TDS.
To complete a chemical analysis and to assess if the proposed
reactions can occur, it is necessary to know the rate of the chemical
reactions that requires a kinetic analysis. To be physiologically relevant a reaction has to occur in a biologically appropriate time-scale
which will be dictated by both the thermodynamic parameters and
the actual concentrations of reactants and products.

Flavonoids as antioxidants by breaking free radical chain

reactions
Lipid oxidation, which is a major event in the oxidation of biological systems, occurs as a chain reaction involving lipids (LH) and
molecular oxygen as substrates, and metals as catalysts (Fig. 2A).
The inhibition of the lipid oxidation chain reaction (Fig. 2B), may
be considered as one of the most important mechanisms explaining the antioxidant actions of avonoids. Generally, chain-breaking
antioxidants (AH)1 inhibit or retard lipid oxidation by interfering
with chain propagating reactions. As shown in reactions 2 and 3
(Fig. 2B), avonoids (FOH) must donate hydrogen atoms to lipid
(L) or lipid peroxyl (LOO) radicals to hamper lipid oxidation propagation. The products of these reactions are non radical species, i.e.
the original lipid (LH), or the corresponding lipid hydroperoxide
(LOOH), and the radical derived from the antioxidant or avonoid
(A or FO). The effectiveness of FOH as a chain-breaking antioxidant
is basically associated with two thermodynamic conditions [12]: (i)
the occurrence of the reaction between L or LOO and FOH that must

Abbreviations used: AH, antioxidant; A, antioxidant radical; EC, ()-epicatechin;

ECG, ()-epicatechin gallate; EGC, ()-epigallocatechin; EGCG, ()-epigallocatechingallate; FOH, avonoid; FO, avonoid radical; LH, lipid, L, lipid radical; LOOH, lipid
hydroperoxide, LOO, lipid peroxyl radical.

be energetically favored; and (ii) the appropriate stability of the

resultant radical (FO), which must be relatively non-reactive.
Reaction between a radical and a avonoid
To predict the spontaneity of a free radical (redox) reaction, that
is to calculate the DG of the reaction, it can be used DG0 = n F
DE0 . For free radical reactions n (the number of electrons transferred from a reduced to an oxidized compound) is one. F represents the Faraday constant (96,485 coulombs/mol of electrons);
and DE0 is the difference between the biological standard reduction potentials (E0 ). This difference is calculated as the E0 of the
species that are being reduced minus the E0 of the species that
are being oxidized. The antioxidant (redox) reactions involve two
couples (E0 for the reduction half-reaction: L (or LOO) + e +
H+ ? LH (or LOOH); and E0 for the oxidation half-reaction:
FOH ? FO + e + H+).
Adding those two equations, the values of DE0 and then DG0
for the reactions between a radical (for example LOO) and different antioxidants can be calculated. As shown in Table 1 the DG0
values obtained are negative, pointing out the energetically favorable free radical scavenger action of the analyzed avonoids.
The possibility that some avonoids can be prooxidant has
also been suggested [13]. Such prooxidant activity is allegedly
based on a sum of complex reactions involving the reaction between the FOH and O2 rendering O2, and the subsequent reaction between a second FOH and O2 yielding H2O2. The rst
reaction, i.e. FOH autoxidation, can occur under certain conditions
[14,15] although it is thermodynamically unfavorable (E0 O2/
O2 = 330 mV [16], or 160 mV [17]). In contrast, the second
reaction is energetically favored (E0 O2/H2O2 = 940 mV [18]),
but since it is controlled by the occurrence of FOH autoxidation,
it has low possibilities to occur at a signicant extent under physiological conditions.
Stability of the avonoid radical formed
Pioneer studies demonstrated that FOHs having similar reduction potentials can originate radicals with very different reactivity

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Initiation
LH L

Initiation
LH L

(reaction 1)

Propagation

Propagation

L + O2 LOO

O2

LOO

(reaction 2)

FOH
LH +FO
LOO + LH LOOH + L

LOO

LH

(reaction 2)

LOOH + L

(reaction 3)

LOOH + FO

(reaction 3)

FOH

Termination
L + LOO

Termination
L + LOO non radical products
L + L non radical products
LOO + LOO non radical products

non radical products

L + L non radical products

LOO + LOO non radical products
L + FO non radical products
LOO + FO non radical products
2 FO non radical products

Fig. 2. Flavonoids interfering in the lipid oxidation chain reaction. Sequence of reactions involved in the lipid oxidation chain process in the absence (A) or the presence of a
avonoid (FOH) acting as antioxidant. LH, lipid molecule; L, lipid radical; LOO, lipid peroxyl radical; LOOH, lipid hydroperoxyl; AH, antioxidant; FOH, avonoid; A,
antioxidant derived free radical; FO, avonoid derived free radical.

Table 1
Reduction potentials (E0 ) for select compounds; and free energy change (DG0 ) for the
reactions between these compounds and lipid peroxyl radical (LOO).
Compound (redox couple)

E0 (mV)

DG0 (kJ)

Lipid hydroperoxide (LOO/LOOH) [18]

Glutathionecysteine (RS/RS) [79,80]
a-Tocopherol (TP/TP) [81]
Trolox C (TO/TOH) [79,80]
Ascorbate (Asc/Asc) [82]
Catechol (CatO/CatOH) [83]
()-Epigallocatechin (EGC/EGC) [71]
()-Epigallocatechin gallate (EGCG/EGCG) [71]
()-Epicatechin (EC/EC) [71]
()-Epicatechin gallate (ECG/ECG) [71]
Quercetin (Q/QH) [84]
Kaempferol (K/KH) [84]
Rutin (R/RH) [84]

1000
920
500
480
282
530
430
430
570
550
330
750
600

8
48
50
69
45
55
55
41
43
65
24
39

Biological standard reduction potentials (E0 ) are expressed in relation to the

standard hydrogen electrode; biological standard conditions are 1 M concentration
for the chemical species with the exception of [H+], that is 107 M (pH 7) [85].
DG0 for each reaction was calculated as DG0 = n F DE.

towards other molecules present in biological systems [19]. A stable and relatively non-reactive FO will inhibit or decrease the rate
of lipid oxidation decreasing the rate of propagation. On the contrary, a highly reactive FO would propagate rather than interrupt
the reaction chain. Some structural requirements for FO stabilization were hypothesized based on both disproportionation reaction
constants (2FO ? FOH + F = O), and the spectral features of transient aroxyl radicals. These requirements are: (i) the presence of
a catechol moiety in the B-ring; (ii) a 2,3-double bond in conjugation with a 4-oxo function in the C-ring; and (iii) the additional
presence of 3- and 5-hydroxyl groups in the A-ring [19] (see structures in Fig. 1).

Polyphenols as antioxidants by metal sequestration

The spontaneous decomposition rates of hydrogen peroxide and
lipid hydroperoxides to generate hydroxyl radical (OH), peroxyl
radicals (LOO), or alcoxyl radicals (LO), are very slow. However,
in the presence of certain metals that can act as catalysts, these
rates are increased and can become relevant in biological systems.
Iron and copper are examples of metals having more than one redox state that can catalyze radical formation. Consequently,
sequestration of Fe and/or Cu to prevent metal-catalyzed free radical formation is considered an antioxidant strategy [2022].
In general terms, catechol moieties in avonoid molecules possess a high afnity for metal ions. In addition, the electronic properties of the avonoid ring system make them good ligands for the
d electrons of the elements from the fourth period, e.g. iron [23]. A
basic parameter to analyze the capacity of avonoids to chelate a
metal is the stability constant (Kst) for the metalavonoid complex. Table 2 shows the Kst corresponding to complexes of several
avonoids with Fe2+, Fe3+, and with Cu2+. The Kst values of querceTable 2
Stability constants (Kst) of complexes formed by catechol, select avonoids, deferoxamine, and bathocuproine with Fe2+, Fe3+, and Cu2+.
Compound

Deferoxamine
Bathocuproine
Catechol
Quercetin
(+)-Catechin
Baicalein

Log Kst
Fe2+

Fe3+

Cu2+

7.2 [86]

12.1 [86]
13.3 [30]

12 [90]

30.6 [86]
0 [87]
43.8 [30,26]
44.2 [30,88]
47.4 [30]
6 [90]

14.1
19.1

19.3
14.5

Stability constants are dened

[compound].
Dashes indicate absence of data.

as

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[86]
[87]
[88]
[89]

Kst = {[metal]  [compound]}/[metal]

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tin and catechin for both metals are similar or even higher than the
values of widely used iron and copper chelators. Thus, many avonoids sharing the chemical characteristics of catechin and quercetin could be used as chelating agents.
However, the Kst is a thermodynamic parameter that does not
consider other characteristics dening the metalavonoid interaction, as avonoid protonation, denticity, metalligand stoichiometry, etc. The concentration of the free metal, dened under
certain experimental conditions, can be used as an indicator of chelation capacity [24,25]. For example, pFe3+ is the negative logarithm of the concentration of free Fe3+ that remains in solution
in the presence of 10 lM total ligand concentration and 1 lM
Fe3+ at pH 7.4 [24]. Generally, a pFe3+ P 20 is required for efcient
iron scavenging from biological matrices [26]. Using these values,
it can be observed that although catechin (pFe3+ = 17.2) [26] is less
efcient than deferoxamine (pFe3+ = 26.0) as iron-chelator, it still
can provide a good chelating capacity.
Regardless the large differences observed in the metal-chelating
capacity of different avonoids [2730] some molecular aspects
are common: (i) the presence of 30 40 hydroxyl groups in the Bring; (ii) the presence of hydroxyl groups at positions 3 and 5;
and the presence of the 4-oxo function [29] (see structures in
Fig. 1). However, it is important to note that being an efcient metal chelator is not equivalent to have a signicant antioxidant action. Flavonoidmetal complexes could favor the removal of the
metal from the reaction milieu depriving the reaction from a catalyst, but could also remain in the reaction site. In the latter case, to
operate as an antioxidant the avonoidmetal complex has to be
less efcient compared with the free metal as catalyst of free radical formation. However, avonoidmetal complexes can catalyze
free radical formation acting in such cases as prooxidants. The possibility that the avonoidmetal complex could prevent metal redox recycling has also to be considered. For iron, the metal
complexes with E0 higher than 390 mV or lower than 320 mV
would fulll the required conditions for making iron inactive for
redox reactions and then, being an efcient antioxidant through
iron sequestration [31,32].
Interestingly, it has been reported that metals bound to avonoids can have superoxide dismutase-like activity [33]. The
authors consider that this activity can explain that certain avonoids have showed antioxidant activity higher than those expected
by the solely action of sequestering metals.
Interactions of avonoids with membrane lipids
The interactions with membrane lipids and proteins are events
that can mediate certain biological and antioxidant effects of avonoids. Consistent observations show that certain avonoids exert
biological actions despite the fact that they are not internalized
by cells [3436].
Flavonoids are generally amphiphilic molecules, but the different substituents in their chemical structure give each avonoid
particular physical characteristics. In general, at the cellular level
avonoids can: (i) interact with lipids at the surface the bilayer
(adsorption); and/or (ii) insert into the bilayer and interact with
the hydrophobic chains of lipids.

Direct interactions of avonoids with lipids could alter

membrane physical properties, and/or modulate different membrane-associated biological events including the activity of membrane-associated enzymes, ligandreceptor interactions, ion and/
or metabolite uxes, and the modulation of signal transduction.
In addition, the insertion of avonoids into lipid bilayers could
bring these molecules in close proximity to L, LOO, and other
lipid-soluble radicals. Flavonoids could then react with these radicals and prevent further propagation of free radical reactions,
which could potentially cause membrane damage and/or alter
membrane functions.
The experimental data on the thermodynamic features that
govern avonoidlipid interactions is limited. As shown in Table 3
phloretin interacts with phosphatidylcholine [37] and morin interacts with sodium dodecylsulfate (SDS) micelles spontaneously in
enthalpy-driven reactions [38]. The fact of being enthalpy the driving force, is probably reecting hydrogen bonding and/or dipolar
interactions, as it was reported for other amphiphilic molecules
[3941]. By contrast, on cationic micelles of hexadecyltrimethylammonium bromide morin interacts spontaneously, but in an entropy-driven process. The transfer of hydrophobic molecules into
lipid membranes is classically associated with large DS values
and negligible DH values [42].
Regarding the relative contribution of adsorption/absorption
processes, available data are very heterogeneous. In dipalmitoylphosphatidylcholine vesicles, (+)-catechin interactions consists
essentially in adsorption to the vesicle surface, although those
interactions generate effects deep inside the membrane [43].
Molecular dynamics simulations based on experiments with bilayers of a mixture of fatty acids suggested that (+)-catechin insert
into the bilayer [44]. Thus, additional information is required, specially at concentrations with biological relevance.
The adsorption to membranes surfaces could provide a plausible mechanism for the prevention of lipid oxidation by avonoids.
Working with liposomes composed of phosphatidylcholine and
phosphatidylserine, we showed that ()-epicatechin (EC) oligomers adsorb onto liposome surface and inhibit lipid oxidation induced by a hydrophobic oxidant [45]. The strength of the
interactions established between EC oligomers and membrane surface was strongly dependent on the chemical nature of the phospholipids polar headgroup, following the order galactolipids 
phosphatidylcholine > phosphatidylserine [46]. This nding is consistent with the potential formation of hydrogen-bonds, making
galactolipids (glycosphingolipids containing ve hydroxylgroups
in their polar headgroup) excellent candidates to provide a surface
for avonoid binding. Through this kind of interactions, avonoids
could be sequestered onto the cell surface and exert local protective effects. In this regard, hexameric procyanidins protect membranes from external stressors that induce lipid oxidation and
mechanical damage.
Structural features are also relevant factors in the behavior of
the avonoidlipid interactions. EC and ()-epigallocatechin
(EGC) interact with 1,2-dimyristoyl-sn-glycero-3-phosphocholine
multilayers with afnity constants (Ka) of 1.86  103 M1 and
1.33  103 M1, respectively [47]. This is indicating that an extra
hydroxyl group in the B-ring does not affect the interaction. On

Table 3
Thermodynamic parameters for the interaction between select avonoids and lipid systems.
Compound

Lipid system

DG (kJ mol1)

DH (kJ mol1)

DS (J mol1 K1)

TDS (kJ mol1)

Phloretin[37]
Morin[38]
Morin[91]

Phosphatidylcholine bilayers
SDS micelles
CTAB micelles

25.6
17.9
26.5

20.5
15.0
+6.8

+16.7
+9.9
+108

5.1
3.1
33.3

Afnity constants are dened as Ka = {[lipid system]  [compound]}/[lipid system] [compound].

Calculations of TDS were done using T = 308 K; CTAB, hexadecyltrimethylammonium bromide.

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the other hand, a gallate group in the position 3 of the EC (()-epicatechin gallate, ECG), or of EGC (()-epigallocatechin gallate,
EGCG) causes a 1000-fold enhancement of the afnity with those
lipid multilayers (Ka 106 M1) [47]. Accordingly, the calculated
average time involved in the interaction of these avanols with
1-palmitoyl-2-oleoylphosphatidylcholine vesicles followed the order EC  EGCG > EGC = ECG [44]. From these ndings can be concluded that the interaction of avanols with membranes is
dependent on the number and positions of the hydroxyl groups
present in the avonoids molecule, and that is probably due to
their facility to form H-bonds.

The interactions of avonoids with proteins

The interactions between avonoids and proteins are complex
as can be expected from the variety of avonoids and proteins in
nature. Similarly to published evidences for avonoidlipid interactions, most of the thermodynamic studies were performed using
single proteins. The study of these interactions is usually based on
the characterization of direct chemical interactions avonoidprotein, or in the functional consequences of such interactions, e.g. enzyme activity, ligand/receptor function (physiological approach).
These approaches should be complementary but only in very few
cases they have been simultaneously studied, and even less in biological systems or conditions.
Albumin is a protein extensively used in the study of protein
avonoid interactions. Albumin has two major binding sites (sites
I and II). These sites are hydrophobic cavities, and most compounds
bind to them with Ka of 104 to 106 M1 [48,49]. An important number of avonoids has been studied for their interaction with albumin and have showed that the binding is thermodynamically
favored, with DG < 0, and Ka similar to those of select non-phenolic compounds (Table 4). Accordingly, the Ka is not signicantly
inuenced by the chemical structure of avonoids. However, glycosylation of certain avonoids does lower the afnity for albumin
in a magnitude that depends on the conjugation site and on the
type of sugar moiety. For example, the glucopyranosylation of
daidzein and genistein lowers the Ka in about one order of magnitude, and rhamnosylation of quercetin lowers the Ka in about four
orders of magnitude [50].
DG values for the binding of several avonoids to albumin are
around 28 kJ mol1, with the exception of the DG for albumin
EC interaction that is 14 kJ mol1 (Table 4). In that work, authors
hypothesized that the process was less spontaneous because of a

non specic adsorption of EC to the albumin surface rather than

to a binding to specic sites [51]. This interpretation is based on
the number of molecules of EC (more than 60) required for saturating one molecule of albumin as compared to that expected (about
1) for an interaction with a dened site. For other avonoids, i.e. 7hydroxyavone [52] or quercetin [53], it has been proposed that
the binding is targeted to particular interdomain areas of albumin.
Furthermore, it has been also suggested that different avonoids
specically interact with the site I of albumin [5458].
In the examples compiled in Table 4 the enthalpy was predominant over the entropy component. Enthalpy-driven binding processes suggest the presence of one or more non covalent bonding
(hydrogen bonding, electrostatic interactions, or van der Waals
forces), beyond potential hydrophobic forces.
a-Amylase has been also been extensively studied for its interaction with avonoids. Gao et al. [59] reported that a-amylase can
spontaneously bind quercetin, isoquercetin, and rutin with DG
values of 34, 35, and 31 kJ mol1, respectively [59]. The inhibitory effect of these avonols can be explained by their binding to
the A-domain of the protein where the catalytic site is located. In
an extensive study assessing 19 avonoids for their capacity to inhibit salivary a-amylase, authors found that only 7 of them inhibited a-amylase activity [60]. The inhibition of a-amylase activity
was strong (IC50 lM), and could be caused by the interaction of
the Asp197 side chain carboxyl oxygen atom and the hydroxyl
group of avonoids that mimicked the glycosidic bond cleaved
by the enzyme [60].
The interactions between tannins (avanols polymers) with salivary proline-rich proteins (PRPs) lead to the precipitation of the
proteins in the oral cavity producing an astringent sensation [61].
The process has been described as a sequence of successive stages:
(i) ligand binding and saturation of the PRP binding sites; (ii) formation of small sizes avanolsprotein aggregates; and (iii) precipitation upon further avanol addition [62]. Factors that affect
protein precipitation by tannins, include tannin characteristics,
protein features, and the experimental conditions of the reaction,
for example, the polyphenol/protein ratio. The thermodynamic
parameters associated with the interaction of different avanols
and a procyanidin-rich fraction (DP4) with poly-(L-proline) were
determined [62]. Under their experimental conditions, the process
was enthalpy-driven for monomers (ECG and EGCG), whereas was
entropy-driven for the procyanidin-rich fraction. These results
show that EC oligomerization modies the interaction avonoid
proline. Similarly, procyanidin size-related actions were observed
by our group for the inhibition of angiotensin converting enzyme

Table 4
Afnity constants (Ka) and thermodynamic parameters for the interaction between salycilate, warfarin and select avonoids with albumin.
Family/compound

Log Ka (M1)

DG (kJ mol1)

DH (kJ mol1)

DS (J mol1 K1)

TDS (kJ mol1)

Salicylate [92]
Warfarin [93]
Quercetin [57]
Quercetin [94]
Quercetin [53]
Wogonin [58]
7-hydroxyavone [52]
()-Epicatechin [51]
Morin [95]
Puerarin [54]
Genistein [56]
Genistein [96]
Genistein [97]
Kaempferol [98]

5.3
5.3
7.5
4.2
5.3
5.3
5.0
2.5
4.4
4.6
4.7
5.0
5.2
5.5

29.9

14

26.3

28.0
28.7

16.1

37

28.0

22.2
55.1

+46.2

78

5.63

+19.6
87.8

13.8

+23.2

+1.7

5.8
+26.2

Afnity constants are dened as Ka = {[albumin]  [compound]}/[albuminl] [compound].

Dashes indicate that thermodynamic parameters are not available.
Studies were carried out with human serum albumin (HAS).
Calculations of TDS were done using T = 298 K.

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I [6365]. IC50 decreased from 1.8 mM for EC to 4.7 lM for EC hexamer [64]. From the linear relationship observed between DG and
procyanidin size (16 EC units), and the value of 2.6 kJ mol1 per
EC unit for the decrease of G for each EC unit in the procyanidin,
can be inferred the formation of hydrogen-bonds stabilizing the
binding to the protein [64].
EC and dimeric procyanidins can interact with transcription factor NF-jB protein components i.e. p50 and Rel A. As a consequence,
EC and dimer B2 inhibit NF-jBDNA binding and transcriptional
activity (interleukin-2 and NF-jB-driven reporter gene expression)
[6668]. Conformational analysis showed that B2 mimics the
guanine pairs present in the DNA binding site for NF-jB proteins.
Although with similar chemical composition, A-type dimers (A1
and A2) do not inhibit NF-jB activation as could be inferred by
their tridimensional conformation [68].
Kinetic analysis and physiological relevance
The thermodynamic analysis only predicts whether or not a
particular reaction has a spontaneous tendency to form products.
The actual concentrations of reactants (oxidants and antioxidants)
and catalysts are the nal determinants for the occurrence of the
product formation in an appropriate time-scale.
Kinetic studies can be applied to any of the physical and chemical actions of avonoids, i.e. free radical scavenging, metal chelation, and avonoidlipid and avonoidprotein interactions. We
will subsequently focus on the kinetic analysis of the reactions of
free radicals with avonoids, but design and results could be applied to the other interactions.
In a biological system under a constant free radical generation,
the chemical reactions for antioxidants can be thought as a competence among parallel reactions (Fig. 2B, reactions 2 and 20 ; 3 and
30 ). For example, LOO is capable to react with a cellular component, e.g. with a lipid (LH), as well as with an antioxidant. It is also
possible, and more realistic, the occurrence of multiple parallel
reactions, one for each cellular component and one for each antioxidant present. As a consequence, the reaction with a specic antioxidant (FOH) is only one of the multiple potential targets for
LOO. Reducing those reactions to a minimal situation of one lipid
and several antioxidants, and assuming those parallel reactions
as second-order reactions, it is possible to have an idea of the antioxidant potentiality of avonoids (Fig. 3). The rate of each reaction
will depend on the rate constant (k, particular for each reaction and
mostly dened by thermodynamic aspects), and on the concentration of the reactants. The reaction with the higher k and/or with the
highest reactant concentration will prevail.
Fig. 3 shows the relative reaction rates between a lipid peroxyl
radical (LOO) and ascorbate (vitamin C), a-tocopherol (vitamin E),
and EC in human blood plasma. The low plasma (and tissue) con-

+ ascorbate

centrations of avonoids, even after the consumption of foods rich

in these compounds [69], leads to a kinetically unfavorable situation respect to the other compounds regardless similar thermodynamic free radical scavenging capabilities (E0 s). Reaction rates for
ascorbate and a-tocopherol are 25- and 14-times higher, respectively, than that of EC. These relative rates severely constrain the
free radical scavenging hypothesis for EC in plasma and most tissues, and also can be extrapolated for other avonoids present in
similar amounts.
This disadvantaging situation for avonoids as antioxidants in
humans is due to their very low bioavailability. Although, no denitive assessment about tissue and cellular concentrations of avonoids can be done at the moment, based on plasma bioavailability
and cell culture experiments; it is expected to be relatively low.
Flavonoids have been measured low lM or upper nM ranges after
a realistic consumption in human plasma (Table 5). Furthermore
these concentrations are transient, peaking at 26 h after their
consumption in human and rodents [69,70,6]. By contrast, atocopherol, ascorbate, and glutathione are present in blood and/
or in the intracellular milieu in lM and even mM concentrations,
and their concentrations are maintained in time, inclusive for
ascorbate, regardless its hydrosolubility.
Even assuming the highest avonoid concentration found in
plasma (low lM range), the relative reaction rate with the more
common oxygen radicals does not support the in vivo free radical
scavenging effects in blood and in the vasculature. These kinetic
limitations imply the existence of other mechanisms, compatible
with the levels reached by avonoids in those tissues that may explain the observed changes in cell or tissue redox status and oxidative damage [10,7173]. On the other hand, a direct free radical
scavenging action of avonoids can be relevant in tissues directly
exposed to avonoids as the gastrointestinal tract where it has
been estimated that avonoids can reach concentrations higher
than 300 lM [74].
In cells, avonoids also compete with other relevant antioxidants, such as glutathione. It can be estimated that cells have
around 107 to 1010 molecules of glutathione, and less than 103 avonoid molecules, making the scavenging of radicals by avonoids
of very limited relevance. In addition, glutathione is able to function as both, a direct antioxidant by scavenging free radicals, and
indirectly as the reducing cofactor of glutathione peroxidase, a major component of the enzymatic antioxidant defenses. Additionally,
most of biological systems are able to synthesize reduced glutathione and to reduce oxidized glutathione, allowing the maintenance
of an adequate steady state concentration of reduced glutathione.
By contrast, avonoids are not synthesized in animals, and it is unknown if they can be recycled. Furthermore, avonoid levels in the
organism are transient, requiring a permanent consumption of avonoid-rich foods to sustain a steady state concentration.

[LOOH]+ [Asc]

vASC = kASC [LOO] [Asc]

vASC/ [LOO] = 50 s-1

[LOOH]+ [TP]

vTP = kTP [LOO] [TP]

vTP/ [LOO] = 28 s-1

[LOOH]+ [EC]

vEC = kEC [LOO] [EC]

vEC / [LOO] = 2 s-1

kASC

[LOO]

+ -tocopherol

kTP

+ EC

kEC

Fig. 3. Estimation of relative rate reactions of different antioxidants with peroxyl radical (LOO) in plasma. Rate constants (v) were estimated by using kASC = 1  106 M1 s1
[99]; kTP = 1  106 M1 s1 [100], and kEC = 7.3  106 M1 s1 [101] as second-order reactions constants, and [Asc] = 50 lM [71], [TP] = 28 lM [102], and [EC] = 0.3 lM [70] as
plasma concentrations. Asc, ascorbate; TP, a-tocopherol; EC = ()-epicatechin; Asc, ascorbyl radical; TP = a-tocopheryl radical; EC = ()-epicatechin radical.

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M. Galleano et al. / Archives of Biochemistry and Biophysics xxx (2010) xxxxxx

Table 5
Estimated avonoid presence in different body compartments.

Total avonoid content

Flavonoid oligomers or polymers
Glycosylated/methylated
avonoids
Metabolites from avonoids
rupture
a

Gastrointestinal tract

Extracellular/vascular compartments

Intracellular compartment

lM
High concentration
High amount

6lM
Low concentration
??

<lM
Very low concentration or absence
??

Principally from colonic

microora

Principally from enterocytes and extracellular

metabolism

Principally from intracellular

metabolism

Considered as the sum of the different forms (original compound and metabolites) in which each individual avonoid can be measured in human tissues and uids.

Similar kinetic analysis can be done for metal chelation by

avonoids. The conclusions are similar: it is unlikely that avonoids can exert a relevant antioxidant action through metal chelation. This can be concluded considering the panoply of metal
chelators present in tissues at concentrations signicantly higher
than those of avonoids, and similar chelation capacity.
Interactions with lipid and proteins should be considered under
a different perspective. Alterations in membrane and protein functions can happen at very low avonoid concentrations, and have
major effects on biological events. Then, lipidavonoid or proteinavonoid interactions can be relevant at concentration much
lower than those necessary to cope with a constant free radical
production. In terms of inhibition of free radical formation, the
mentioned SOD-like activity [33], the inhibition of NADPH oxidase activity [7577], and the modulation of nitric oxide synthase
[35,78] are examples of indirect antioxidant effects derived from
avonoidprotein interactions.
We have observed a degree of specicity lipidavonoid (procyanidin) interactions. It could explain the differential inhibition by
hexameric procyanidins of NF-jB activation initiated by various
stimuli in intestinal cells [34,35]. We hypothesize that such differential effects can be related to specic molecular association between select lipids present in certain areas of the membrane, and
large procyanidins.
Finally it has to be noted, that the discussed kinetic limitations
are not always considered in many in vitro studies, in which avonoid concentrations exceed by far those attainable through human
and animal dietary consumption, and in consequence have limited
physiological value.
Concluding remarks
Thermodynamic and kinetic conditions dictate the occurrence
of a chemical reaction. For the study and analysis of the biological
and antioxidant actions of avonoids it should be taken into account the actual concentration of avonoid and avonoid metabolites in different biological compartments.
An important body of research is currently based on the possibility for ascribing the documented benecial antioxidant effects of
avonoids to other mechanisms beyond their free radical scavenger
and metal chelation activities. These mechanisms include avonoidlipid and avonoidprotein interactions. These interactions
are ascribed to a higher molecular specicity than that for trapping
radicals, and can have physiological relevance at avonoid concentrations attainable through normal dietary habits.
Acknowledgments
This work was supported by Grants of the University of Buenos
Aires, UBACyT B081 and B082; and CHNR-State of California Vitamin Price Fixing Consumer Settlement Fund. M.G., S.V.V., P.O.,
and C.G.F. are members of the Scientic Investigator Career, CONICET, Argentina.

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