Does hydrophobicity always enhance antioxidant drugs? A cut-off effect of the chain length of functionalized chlorogenate esters on ROS-overexpressing fibroblasts

Article abstract of DOI:10.1111/j.2042-7158.2010.01216.x

Objectives: Phenolic antioxidants are currently attracting a growing interest as potential therapeutic agents to counteract diseases associated with oxidative stress. However, their high hydrophilicity results in a poor bioavailability hindering the devel

Full text of DOI:10.1111/j.2042-7158.2010.01216.x

Research Paper
JPP 2011, 63: 531–540
© 2011 The Authors
JPP © 2011 Royal
Pharmaceutical Society
Received January 30, 2010
Accepted October 19, 2010
DOI
Does hydrophobicity always enhance antioxidant drugs?
A cut-off effect of the chain length of functionalized
chlorogenate esters on ROS-overexpressing fibroblastsjphp_1216 531..540
10.1111/j.2042-7158.2010.01216.x
ISSN 0022-3573
Mickaël Laguerrea, Chantal Wrutniak-Cabellob, Béatrice Chabib,
Luis J. López Giraldoa, Jérôme Lecomtea, Pierre Villeneuvea and
Gérard Cabellob
aCIRAD, UMR Ingénierie des Agropolymères et Technologies Émergentes, and bINRA, UMR
Différenciation Cellulaire et Croissance, Montpellier, France
Abstract
Objectives Phenolic antioxidants are currently attracting a growing interest as potential
therapeutic agents to counteract diseases associated with oxidative stress. However, their
high hydrophilicity results in a poor bioavailability hindering the development of efficient
antioxidant strategies. A promising way to overcome this is to increase their hydrophobicity
by lipophilic moiety grafting to form the newly coined ‘phenolipids’. Although hydropho-
bicity is generally considered as advantageous regarding antioxidant properties, it is never-
theless worth investigating whether increasing hydrophobicity necessarily leads to a more
efficient antioxidant drug.
Methods To answer this question, the antioxidant capacity of a homologous series of
phenolics (chlorogenic acid and its methyl, butyl, octyl, dodecyl and hexadecyl esters)
toward mitochondrial reactive oxygen species (ROS) generated in a ROS-overexpressing
fibroblast cell line was investigated using 2′,7′-dichlorodihydrofluorescein.
Key findings Overall, the long chain esters (dodecyl and hexadecyl esters) were more
active than the short ones (methyl, butyl, and octyl esters), with an optimal activity for
dodecyl chlorogenate. Moreover, dodecyl chlorogenate exerted a strong antioxidant capac-
ity, for concentration and incubation time below the cytotoxicity threshold, making it a
promising candidate for further in-vivo studies. More importantly, we found that the elon-
gation of the chain length from 12 to 16 carbons led unexpectedly to a 45% decrease of
antioxidant capacity.
Conclusion The understanding of this sudden collapse of the antioxidant capacity through
the cut-off theory will be discussed in this article, and may contribute towards development
of a rational approach to design novel amphiphilic antioxidant drugs, especially phenolipids
with medium fatty chain.
Keywords chlorogenic acid; cut-off effect; fibroblasts; lipophilization; phenolipids
Introduction
More than 50 years ago it was hypothesized that aging could be the result of the accumu-
lation of oxidative damage from free radicals generated endogenously in cells during normal
metabolism.[1] Growing interest in these oxidative phenomena has progressively emerged as
the implication of oxidative stress in multifactorial diseases such as cancers[2] and cardio-
vascular diseases[3] has been demonstrated. It is well established that the leakage of electrons
from the respiratory chain located within the inner mitochondrial membrane is the major
biological process leading to oxidant species generation, especially reactive oxygen species
(ROS). Because a common feature in such diseases is an increased level of mitochondrial
ROS, the use of antioxidant molecules, especially phenolic compounds, has been considered
as a promising way to counteract ROS-mediated damage. However, the high hydrophilicity
of these antioxidant compounds results in a poor bioavailability hindering the development
of efficient antioxidant strategies. A promising way to enhance their cellular uptake is
to increase their hydrophobicity by grafting a lipid onto a phenolic moiety.[4,5] These
functionalized molecules can be coined as ‘phenolipids’ and seem to be very promising as
therapeutic agents to counteract oxidative stress. In Caco-2 cells, Tammela et al.[6] showed
Correspondence: Pierre
Villeneuve, Bât. 33, 2 Place
Pierre Viala, 34 060 Montpellier
Cedex 02, France.
E-mail: pierre.villeneuve@cirad.fr
531

532
Journal of Pharmacy and Pharmacology 2011; 63: 531–540
Methyl moiety (C1)
Chlorogenic Acyl
Butyl moiety (C4)
O
Octyl moiety (C8)
Dodecyl moiety (C12)
Hexadecyl moiety (C16)
O
O
HO
HO
O
OH
HO
OH
Phenol
Lipid
Phenolipid
Figure 1 Chemical structure of investigated phenolic compounds.
that octyl gallate was extremely rapidly and almost totally is only determined by the drug incubation time sufficient to
reduced from the apical solution whereas the uptake of methyl observe a significant antioxidant effect.
and propyl gallate was slower. Similarly, it has been suggested
In this study, this strong endogenous production of oxidant
in mouse B cell lymphoma Wehi 231 that dodecyl gallate species from mitochondria was advantageous to evaluate the
exhibited a better membrane crossing than its propyl homo- ability of a homologous series of phenolipids to decrease
logue.[7] Recently, Trujillo et al.[8] demonstrated in brain the level of ROS. To monitor this level, a fluorogenic probe
homogenates that hydroxytyrosyl linoleate is more efficient (2′,7-dichlorodihydrofluorescein diacetate (H2DCFDA)),
than hydroxytyrosol in protecting both lipids and proteins exhibiting fluorescence in visible spectrum (lex: 492 nm /lem:
from oxidation induced by cumene hydroperoxides. In their 525 nm) when oxidized, was employed. The modulation of
review paper, Rice-Evans et al.[9] highlighted that the partition ROS level by phenolic compounds was thus estimated by
coefficient (or hydrophobicity) is an important parameter in means of fluorescence modulation. In this paper, we evaluated
predicting antioxidant activity in lipophilic media, such as the antioxidant capacity of these novel phenolipids and gave
liposomes and low-density lipoproteins (LDL).
new insight on the relationship between hydrophobicity and
Although hydrophobicity is generally considered as antioxidant properties.
advantageous regarding antioxidant properties, it is worth
asking whether increasing hydrophobicity necessarily leads to
Materials and Methods
a more efficient antioxidant. This point is of particular interest
since it may contribute to the development of a more rational
approach to designing novel amphiphilic antioxidant drugs
such as phenolipids.
Chemicals
Hoechst 33258, DNA salmon sperm, chlorogenic acid
(5-caffeoylquinic acid), methanol, Amberlite IR 120 H,
chloroform, Candida antarctica lipase B, hexane, acetoni-
trile, silica gel, toluene and ethyl acetate were purchased
Recently, we have shown that chlorogenic acid (5-
caffeoylquinic acid, Figure 1), exhibiting antioxidant[10] and
antiradical[11] properties in test tube-based assays, can be
lipophilized by various fatty alcohols in a two-step lipase-
catalysed transesterification strategy.[12] This reaction leads to
a homologous series of alkyl chlorogenate esters (from methyl
to hexadecyl, Figure 1), which constitutes an interesting phe-
nolic model to study the impact of the hydrophobicity on the
antioxidant capacity in biological samples. Nevertheless, the
major challenge remains to develop analytical tools to assess
from
Sigma
(Saint
Quentin,
France).
2′,7′-
Dichlorodihydrofluorescein diacetate (H2DCFDA), Dulbec-
co’s modified Eagle’s medium (DMEM, 4.5 g/l glucose),
D-PBS, gentamicin and amphotericin B were from Invitrogen
(Cergy Pontoise, France).
Synthesis of chlorogenate esters
these properties in a relevant and reliable manner. To date, the The chemo-enzymatic esterification of chlorogenic acid to
experimental models involve chemical assays, cultured cells, obtain chlorogenate esters was carried out following the pro-
animal models of oxidative stress and clinical test in humans. cedure described by López Giraldo et al.[12] Briefly, 10 mmol
Oxidative stress studies generally use exogenic inductor(s) of chlorogenic acid was dissolved in 240 ml methanol. Amber-
oxidative stress such as antimycin A (inhibition of complex lite IR 120 H (10 g), previously dried at 110°C for 48 h, was
III), oxidized LDLs, transient metals, diazo compounds or added to the reaction mixture which was then stirred in an
hydroperoxides. However, in such a system, it is difficult to orbital shaker (250 rpm) for 9 h at 55°C. After cooling to
distinguish the intrinsic antioxidant effect from more complex room temperature, the reaction medium was filtered on a
effects induced by interaction between antioxidant and exo- 1.6 mm glass microfiber filter (Whatman International Ltd,
genic inductor(s). To partially overcome these difficulties, we Maidstone, UK) and the methanol was removed under
have developed a new cellular line of fibroblasts characterized vacuum. Chloroform (150 ml) was then added, and the solu-
by a strong production of oxidant species of mitochondrial tion was dried over sodium sulfate, filtered on a 1.6 mm glass
origin.[13] This feature allows evaluation of antioxidant capac- microfiber filter, and evaporated under vacuum at 50°C. The
ity without any exogenic inductor(s) of oxidation, with the resulting methyl chlorogenate (5 mmol) was then added to
equivalent analysis duration generally encountered in cellular 375 ml of desired fatty alcohol, and the mixture was then
assays. Indeed, with these cellular lines, the analysis duration placed in sealed flasks and stirred on an orbital shaker

Cut-off effect of phenolipid antioxidants
Mickaël Laguerre et al.
533
(250 rpm, 55°C) until complete dissolution of methyl chloro- (covered by aluminium foil) was then incubated for 10 min
genate. Candida antarctica lipase B (5% wt/wt; calculated at 37°C before fluorescence measurement (lex: 485/lem:
from the total weight of both substrates) was then added to 530 nm).
start the transesterification reaction. The suspensions were
Raw fluorescence was calculated for each well, subtracting
then heated at 55°C for 96 h under a nitrogen flow in order to the fluorescence background (without probe) from that mea-
eliminate continuously the formed methanol and favor the sured in presence of probe (Equation 1).
displacement of the reaction equilibrium towards the synthe-
sis. The final lipophilized esters were then purified in a two
step procedure. First, a liquid-liquid extraction using 250 ml
of hexane and 1000 ml of a solution of acetonitrile/water
(3 : 1, v/v) was realized to remove the fatty alcohol in excess.
In a second step, the alcohol traces were eliminated using
silica gel column chromatography (length 25 cm, i.d. 1.6 cm)
using toluene/ethyl acetate (90 : 10, v/v) as eluant. All recov-
ered esters were then characterized by mass spectrometry as
described by López Giraldo et al. [12]
Raw Fluo = Fluo (cells with probe) −
(1)
Fluo (cells without probe)
Given that the tested substances are likely to exert an influence
on the cell growth, a correction of the probe fluorescence by
the DNA content (mg DNA/well, see section on DNA Quan-
tification, below) was achieved using Equation 2.
Corrected FluoH2DCFDA
raw FluoH2DCFDA / DNA quantity (in μg) per well
=
(2)
Finally, to consider the potential effect of carrier solvent
(ethanol) which can act as antioxidant in some case,[14] the
corrected fluorescence was expressed as the percentage of
the control (with ethanol) (Equation 3).
Cell culture preparation
Dark 96-well microplates with flat and transparent bottoms
(Greiner, Frickenhausen, Allemagne) were seeded at a cellular
density of 10 000 cells/well. Cell cultures were allowed to
grow in DMEM (4.5 g/l glucose) supplemented with genta-
micin (50 mg/ml), amphotericin B (50 mg/ml) and fetal calf
serum (10%) at 37°C, 5% CO2 and 100% humidity.
FluoH2DCFDA (% of control) =
(3)
(Corrected FluoH2DCFDA ×100)/ FluoH2DCFDA of control
The antioxidant capacity was thus calculated as the per-
centage of fluorescence inhibition in presence of the tested
phenolic compared with the control (phenolic free, but con-
taining ethanol), this value being normalized by cell DNA
content.
Cell treatment with phenolic compounds
Phenolic compounds (chlorogenic acid and its methyl, butyl,
octyl, dodecyl and hexadecyl esters), being either hydrophilic,
amphiphilic or hydrophobic according to their alkyl chain
length, were dissolved in ethanol at the desired concentration.
A 1000-fold dilution was performed in culture medium to
obtain final concentrations of 0, 10, 25, 50 and 100 mm. After-
wards, 100 ml of this culture medium containing phenolic
compounds was dispensed into each well of a 96-well micro-
plate 24 h after cell seeding. The experiment involving each
concentration was repeated four times (n = 4) in four diff-
erent microplates seeded from different cell passages (each
microplate comprising already triplicate wells for each
concentration).
DNA quantification by the Hoechst
33258 method
Cell DNA content was measured using the Hoechst 33258
method previously described by De Arcangelis et al.[15] with
some modifications. Cells of each well (previously measured
for the antioxidant capacity determination) were lysed with
100 ml of TE5N buffer solution (10 mm Tris-HCl pH 8,
0.1 mm EDTA and 0.5 m NaCl) containing 0.05% Triton
X-100. The microplate was incubated for 10 min under stir-
ring at room temperature. Afterwards, 100 ml/well Hoechst
33258 solution at 0.4 mg/ml in TE5N were added, then incu-
bated for 30 min at room temperature protected from light.
The fluorescence (lex: 340/lem: 440 nm) was then read in the
same spectrofluorimeter as used in the section on Measure-
ment of Reactive Oxygen Species, above.
To calibrate the fibroblasts’ DNA content, the fluorescence
of a standard of salmon sperm DNA solutions (0, 0.5, 1, 2 and
4 mg/ml TE5N) incubated with 100 ml Hoechst 33258 solu-
tion in TE5N, was analysed on a separate microplate. It
is worth mentioning that the DNA concentration range was
prepared in the same TE5N solution as samples.
Measurement of reactive oxygen species using
2Ј,7Ј-dichlorodihydrofluorescein diacetate probe
After 24, 48 or 72 h incubation with phenolic compounds,
the medium was removed. Adherent cells were washed
twice with 50 ml/well Locke’s buffer (140 mm NaCl, 5 mm
KCl, 1.2 mm MgCl2, 1.8 mm CaCl2, 10 mm d-glucose and
10 mm hydroxyethylpyperazinethane acid (HEPES)), then
each well was filled with 100 ml Locke’s buffer. A measure-
ment (lex: 485 nm/lem: 530 nm) of the fluorescent back-
ground was achieved with a microplate spectrofluorimeter
(Synergy 2 BioTek, Bio-tek Instruments, Inc., Winooski,
USA).
Cells were incubated with 100 ml of a 10 mm solution of
H2DCFDA prepared in Locke’s buffer, for 20 min at 37°C in
the dark. After the removal of the H2DCFDA solution, each
well was washed twice using 50 ml Locke’s buffer and finally
was filled with 100 ml Locke’s buffer. The microplate
First, all fluorescence values were subtracted from
the fluorescence of a 0 mg/ml sperm salmon DNA solution
(Equation 4).
Corrected FluoHoechst = RawHoechst
−
(4)
FluoHoechst (0 μg /ml DNA)

534
Journal of Pharmacy and Pharmacology 2011; 63: 531–540
Then, a linear regression was established between DNA stan- Finally, to take into account the potential influence of the
dard concentrations and observed fluorescence (expressed carrier solvent (ethanol) on this measurement, the cytotoxicity
as arbitrary units); the same calculation being achieved for was expressed as a percent of control (phenolic free, but
samples. Thus, the sample DNAconcentration was determined containing ethanol) (Equation 9).
from the calibration curve slope as follows (Equation 5):
Cytotoxicity (% of control) =
Cytotoxicity (%)/Cytotoxicity of control
(9)
DNA concentration (μg/ ml) =
Corrected FluoHoechst /calibration curve slope
(5)
Light microscopy observations
The DNA quantity expressed in mg/well was then calculated
taking into account the total volume of the solution per well
(200 ml) (Equation 6).
The microstructure of the self-assembled aggregates formed
by dodecyl chlorogenate in water was assessed using phase
contrast microscopy with a 60¥ oil immersion objective lens
(Olympus optical BX60F5, Japan). Before observation, the
sample was gently mixed, and a droplet was placed on a
microscope slide and covered with a cover slide. The images
of the sample were captured using a digital camera directly
connected to the computer equipped with image processing
software (Image Pro Plus, version 6; Media Cybernetics, Inc.
Bethesda, USA).
DNA quantity (μg/ well) =
(DNA concentration (μg/ ml) × 200)/1000
(6)
This DNA quantity was used in Equation 2 for antioxidant
capacity calculation.
Cytotoxicity measurement using lactate
dehydrogenase assay
Bond dissociation energy calculation
Molecular modelling was performed with Hyperchem
(Autodesk, Sausalito, USA). Parameters and hydrogen
bonding patterns are detailed in Table 1.
The cytotoxicity was estimated using the enzymatic detection
kit CytoTox-ONE G7891 (Promega, Madison, USA). After
24, 48 or 72 h of incubation with phenolic compounds, micro-
plates were incubated at room temperature for 30 min. After
dispensing 100 ml CytoTox-ONE reagent into each well, the
microplate was incubated for 10 min at room temperature
in the dark. Then, 50 ml/well stop solution was added and
the fluorescence was immediately measured (lex: 560/lem:
590 nm).
Because the serum used in culture medium may have con-
tained lactate dehydrogenase (LDH), a fluorescence measure-
ment of DMEM supplemented with 10% fetal calf serum was
achieved in the absence of cells on the same microplate, thus
constituting the culture medium background.
Statistical methods
Statistical analysis of the effects of increasing concentration
of phenolics (10, 25, 50 and 100 mm), exposure time (24, 48
and 72 h), and chain length of phenolipids (0, 1, 4, 8, 12 and
16 carbon atoms) on antioxidant capacity and cytotoxicity
using ROS-overexpressing fibroblasts was performed using
the Kruskall–Wallis test. In all cases, post-hoc comparisons of
the means of individual groups were performed using Dunn’s
test. P < 0.001 denoted significance in all cases.
Curve-fitting
The corrected fluorescence was then calculated by sub-
tracting the background fluorescence from the fluorescence
measured in the presence of cells (Equation 7).
The CurveExpert 1.3 shareware (Copyright © 1995–2001
Daniel Hyams) was used in the curve-fitting procedures using
a quadratic formula for the calculation of the area under the
concentration curves.
Corrected FluoLDH = Raw FluoLDH (with cells) −
(7)
Background FluoLDH (without cells)
Results and Discussion
Taking into account that phenolics are liable to exert an
influence on the cell growth, and consequently to decrease
the releasable quantity of LDH, values were corrected con-
sidering the total amount of intracellular and extracellular
LDH. Therefore, some wells containing cells were exposed
to 2 ml total lysis solution (9% Triton X-100 in phosphate
buffer) to release all LDH in the extracellular medium and
finally treated identically as other wells. The absolute cyto-
toxicity level (percent of control) for phenolic compound
was calculated by dividing the corrected fluorescence
obtained without lysis solution by that obtained after total
lysis (Equation 8).
Antioxidant capacity measurement
Since hydrophobicity is generally considered as advantageous
regarding antioxidant properties in cellular systems, a
growing number of lipophilized phenolics (known as pheno-
lipids) has been designed and synthesized this last decade.[16]
In this context, the present study was implemented to answer
the question of whether increasing hydrophobicity necessarily
leads to a more efficient antioxidant. To focus the study on the
influence of the hydrophobicity on the antioxidant capacity of
phenolic compounds, a homologous series of alkyl esters of
chlorogenic acid was used. These phenolipids were obtained
by lipase-catalysed esterification of the carboxylic function of
chlorogenic acid by aliphatic alcohols of various chain lengths
(8) (1, 4, 8, 12 and 16 carbons, Figure 1). Their antioxidant
capacity was evaluated at various concentrations (0, 10, 25, 50
Cytotoxicity (%) = 100 ×[Corrected FluoLDH (without lysis
solution)/Corrected FluoLDH
(with lysis solution)]

Cut-off effect of phenolipid antioxidants
Mickaël Laguerre et al.
535
Table 1 Bond dissociation energy (BDE) calculation
Compound
Hydrogen bond
Energy (kcal-1·mol-1)
BDEa (kcal-1·mol-1)
Chlorogenic acid
Chlorogenic acid radical
Methyl chlorogenate
Methyl chlorogenate radical
Butyl chlorogenate
Butyl chlorogenate radical
Octyl chlorogenate
Octyl chlorogenate radical
Dodecyl chlorogenate
Dodecyl chlorogenate radical
Hexadecyl chlorogenate
Hexadecyl chlorogenate radical
O4′-H · · · O3′-H
O4′-H · · · O3′•
O4′-H · · · O3′-H
O4′-H · · · O3′•
O4′-H · · · O3′-H
O4′-H · · · O3′•
O4′-H · · · O3′-H
O4′-H · · · O3′•
O4′-H · · · O3′-H
O4′-H · · · O3′•
O4′-H · · · O3′-H
O4′-H · · · O3′•
-4559.84
-4489.59
-4826.79
-4756.61
-5667.62
-5595.73
-6789.78
-6719.53
-7911.92
-7841.67
-9034.09
-8961.93
70.25
70.18
71.89
70.25
70.25
72.16
aBDE = E (aryloxyl radical) – E (parent phenol). Energies of parent phenols and aryloxyl radicals calculated by semi-empirical quantum mechanic
(parametric method 3 (PM3), unrestricted Hartree–Fock (UHF) mode, in vacuo, 0 K at constant temperature)
and 100 mm) and incubation durations (24, 48 and 72 h). incubation time tested (24, 48, and 72 h), then arbitrarily
Because phenolics were introduced in the culture medium as multiplied by 10 000) (Figure 3).
an ethanolic solution, which can act in some cases as an
On one hand, it appeared that the short chain lengths (1, 4
antioxidant,[14] the evaluation of ethanol influence (0.1% v/v) and 8 carbons) did not affect antioxidant capacity, whereas a
on DCF fluorescence was achieved, and no significant effect maximal activity was reached for the dodecyl chain. On the
was observed (data not shown). Regarding the influence of the other hand, an elongation from 12 to 16 carbons led to a 45%
alkyl chain length on antioxidant capacity, Figure 2 shows decrease in the efficiency of the corresponding chlorogenate
that free chlorogenic acid along with methyl, butyl and octyl ester. This particular behaviour may explain why some
esters does not exert a significant inhibition of the probe lipophilization strategies lead sometimes to an unexpected
fluorescence during 24, 48 and 72 h of incubation. In contrast, decrease in the antioxidant effect, especially those using long
a significant inhibition (P < 0.001) was noticed for dodecyl aliphatic chains. To the best of our knowledge, this is the first
and hexadecyl esters with a dose-dependent effect (Figure 2e time such a non-linear relationship between hydrophobicity
and 2f). The dodecyl ester, which exhibited a maximal pro- and antioxidant capacity has been demonstrated in cultured
tective effect against H2DCF oxidation, was the only one to cells. This finding may contribute to the development of a
lead to a complete inhibition of the probe fluorescence at rational approach in the design of new phenolic-based anti-
50 mm. Unexpectedly, increasing the alkyl chain length from oxidants through lipophilization.
12 to 16 carbon atoms resulted in a decrease in the antioxidant
capacity. The same trend was observed when microplates
were seeded with 3000 cells/well (data not shown). Finally, no
time-dependent relationship was found suggesting that chlo-
H-donation ability, self-assembly properties and
cut-off theory
rogenate esters such as dodecyl and hexadecyl exhibited a First, it is worth trying to elucidate the mechanism of action
complete antioxidant action within 24 h.
that could explain the non-linear influence of the alkyl chain,
To express antioxidant capacity in a quantitative way, the with an optimal antioxidant effect for the dodecyl chloroge-
area under the concentration curves has been calculated: the nate. Interestingly, we have observed similar antioxidant
smaller the area under the curve, the higher is the antioxidant behaviour for chlorogenic acid and its alkyl esters in a non-
capacity. This calculation method supposes a good fitting living system consisting of a stripped tung oil-in-water
of experimental data by a suitable mathematic model. A emulsion (without any protein).[17] Figure 4 shows the good
quadratic model (y = a - [(a-d) x ∧ c]/(b ∧ c + x ∧ c)) was relationship (R2 = 0.872) between antioxidant capacity values
*
empirically developed for each curve displayed in Figure 2. measured in the present study (fibroblasts) and those previ-
The first advantage of this method of calculation is that the ously obtained in emulsified medium. This suggests that the
antioxidant capacity of all substances can be quantified what- non-linear effect observed in fibroblasts does not necessarily
ever its amplitude. Secondly, all the concentrations used are result from a biological mechanism, but rather from a simple
taken into account with the same weight, whatever the kinetic physicochemical process, which incited us to search for an
profile. The integration of the area under the concentration explanation in the physicochemical realm.
curve allowed the classification of phenolic compounds
In this context, the simplest physicochemical mechanism
in terms of antioxidant effectiveness as follows: dodecyl of action for antioxidant phenolics is the reduction of free
chlorogenate (4.53) > hexadecyl chlorogenate (2.52) > butyl radicals by hydrogen transfer from their phenolic hydroxyl
chlorogenate (1.63) ~ methyl chlorogenate (1.26) ~ octyl groups, which can be theoretically estimated by their bond
chlorogenate (1.14) ~ chlorogenic acid (1.12) (antioxidant dissociation energy (BDE): the smaller the BDE, the higher
capacities were expressed as 1/area calculated from all the the ability to give up a hydrogen atom.[18] To investigate the

536
Journal of Pharmacy and Pharmacology 2011; 63: 531–540
Chlorogenic acid (a)
Methyl chlorogenate (b)
120
120
80
40
0
80
40
24 h
24 h
48 h
72 h
48 h
72 h
0
0
25
50
75
100
0
25
50
75
100
Octyl chlorogenate (d)
Butyl chlorogenate (c)
120
80
40
0
120
80
40
0
24 h
48 h
72 h
24 h
48 h
72 h
0
25
50
75
100
0
25
50
75
100
Dodecyl chlorogenate (e)
Hexadecyl chlorogenate (f)
120
80
40
0
120
80
40
0
24 h
48 h
72 h
24 h
48 h
72 h
*
*
*
*
*
0
25
50
75
100
0
25
50
75
100
Medium phenolic concentration (μM)
Figure 2 Influence of chlorogenic acid and its alkyl esters on 2′,7′-dichlorofluorescein formation (lex: 485 nm/lem: 530 nm), which is an estimation
of the level of reactive oxygen species. Fluorescence values on the y-axis were calculated according to Equation 3. Cells were cultivated with culture
medium containing 10% (v/v) fetal calf serum, with 0, 10, 25, 50 and 100 mm phenolic compound and 0.1% (v/v) ethanol and then stained with 10 mm
of H2DCFDA. Results were expressed as mean Ϯ SD of four independent measurements carried out on four separate microplates seeded with four
different cell suspensions (n = 4). *P < 0.001 compared with the control without phenolic (Kruskall–Wallis followed by Dunn’s multiple pairwise
comparison).

Cut-off effect of phenolipid antioxidants
Mickaël Laguerre et al.
537
In contrast, it is well documented that hydrophobicity
can strongly favour the cell membrane uptake or penetration
of amphiphilic molecules by giving them the so-called
‘membrane-like character’.[19,20] In this way, a passive or
assisted membrane transfer enabling an intracellular location
of phenolics (presumably close to the oxidizing species)
could strongly favour an antioxidant action and therefore
could explain the strong activity of dodecyl and hexadecyl
chlorogenates (Figure 2e and 2f). Besides, Serrano et al.[7]
reported that the dodecyl ester of gallic acid presents a better
membrane-crossing action than its propyl homologue.
Interestingly, we have observed by optical microscopy
that dodecyl and hexadecyl chlorogenates were present (both
at 50 and 100 mm) in the culture medium (DMEM + foetal
calf serum + antibiotics + cells) under a self-assembled colloi-
dal structure, which was not observed for short chain esters (1, 4
and 8 carbons) and free chlorogenic acid (data not shown).
Further observations by light microscopy (60¥ oil immersion
objective lens) of 100 mm dodecyl chlorogenate in water have
shown a wide range of lipid polymorphs, most of which are
characterized by a crystalline structure of an average size of
10–50 mm (Figure 5). It has also been visually observed that
a temperature increase from 25 to 37°C leads to an irreversible
phase transition from a gel-like structure to an aggregate. This
interesting observation could explain the size and the shape
of the microstructures depicted in Figure 5, which are much
larger than simple micelles. Indeed, it is admitted that amphi-
philic molecules self assemble spontaneously in aqueous solu-
tion to form a variety of thermodynamically stable structures
known as association colloids such as micelles. It is also well
known that micelles can aggregate in larger structures when
the temperature is increased above the cloud point. This is due
to the progressive dehydration of the hydrophilic head groups of
amphiphilic molecules, which alters their molecular geometry
and decreases the repulsion forces between them.[21,22]
*
*
5
24h
4
3
2
1
0
48h
72h
*
*
0
1
4
8
12
16
Alkyl chain length (carbon number)
Figure 3 Influence of the alkyl chain length on the antioxidant capacity
of chlorogenic acid and its alkyl esters. The antioxidant capacity was
expressed in arbitrary units taking into account the reverse of the area
under the concentration curves presented on Figure 2. *P < 0.001 com-
pared with the control without phenolic (Kruskall–Wallis followed by
Dunn’s multiple pairwise comparison).
3
C16
C12
2.5
C0
y = 0.3145x + 1.5486
R2 = 0.872
According to McClements,[23] the properties of amphi-
philic molecules dispersed as monomers are very different
from those of aggregates. Indeed, ester monomers are
amphiphilic and have a high surface activity whereas associa-
tion colloids can have low surface activity because their
surface is covered by a hydrophilic head group. Therefore, in
our case, a colloidal drug delivery system could favour the
transport of amphiphilic chlorogenate esters (dodecyl and
hexadecyl esters) across biological membranes, compared
with others tested esters, for which such a structure was not
observed by optical microscopy.
2
C1
C4
C8
1.5
1
2
3
4
5
Antioxidant capacity (fibroblasts)
Figure 4 Correlation between antioxidant capacity values observed
in this study (fibroblasts) and those obtained in emulsion for the same
Finally, if the higher efficiency could be tentatively explained
for long chain esters by self-assembly properties, it is far more
homologous series[17]
.
possible influence of the hydrophobicity on the free radical difficult to speculate about the possible mechanism by which
scavenging properties of chlorogenic acid and its alkyl esters, an elongation from 12 to 16 carbons leads to a decrease in
the BDE of phenolic O-H was deduced from semi-empirical the antioxidant ability. Interestingly, this finding is consistent
quantum mechanic calculations after optimization of geom- with numerous published studies on a diverse range of biological
etry and hydrogen bonding in both the parent phenol and the properties, such as anaesthetic,[24,25] antimicrobial,[26,27] cyto-
corresponding aryloxyl radical (Table 1). However, it can be toxic[28] and spermicidal[29] activities. In these studies the depen-
observed that the alkyl chain length does not significantly dence is quasi-parabolic, which means that the efficiency of
influence the BDE of the corresponding O-H groups since the interaction of such compounds with biological membranes
BDE values are between 70.18 and 72.16 kcal/mol. Such a grows with an increase in their hydrophobic parts up to a certain
result can be explained since there is no conjugation between length and then begins to diminish. Ferguson[30] in 1939 was
the carboxylic function and the aromatic ring that could affect one of the first to document this type of effect when compiling
the BDE through electron delocalisation by mesomeric effect. a combination of studies related to a homologous series of

538
Journal of Pharmacy and Pharmacology 2011; 63: 531–540
20mm
20mm
20mm
Figure 5 Representative light microscopy observations of self-assembled structures formed by dodecyl chlorogenate in water.
compounds. Sometimes named the ‘parabolic case’, this effect molecules (especially the dodecyl ester) to counteract oxida-
is now known under the term of ‘cut-off effect’. Balgavy and tive stress in vivo supposes a lack of toxicity in the concen-
Devinsky[31] reported that this effect is a general phenomenon tration and incubation time ranges used. To assess this factor,
observed in various biological and toxic actions with practi- cytotoxic experiments using various concentrations (0, 10, 25,
cally every amphiphilic homologous series tested so far. This 50 and 100 mm) of chlorogenic acid and its alkyl esters were
study thus constitutes a first attempt to connect the cut-off carried out during 24, 48 and 72 h of incubation using the
theory to the non-linear dependency observed here between lactate dehydrogenase (LDH) kit assay (see experimental pro-
hydrophobicity and antioxidant capacity, in an effort to bridge cedure above). LDH is a cytosolic enzyme present within all
the gap between these different fields.
mammalian cells. Briefly, the normal plasma membrane is
In addition, as already mentioned, we have observed in impermeable to LDH, but damage to the membrane results in
emulsion on the same homologous series, a cut-off influence a change in its permeability and a subsequent leakage of LDH
of the alkyl chain length, with an optimal antioxidant effect into the extracellular fluid. Release of LDH from cells pro-
for the dodecyl chlorogenate.[17] The fact that a similar cut-off vides an accurate measure of cell membrane integrity and,
effect was observed in a non-living system (i.e. oil-in-water consequently, gives access to the cytotoxicity. The extra-
emulsion) without any protein, could suggest that the cut- cellular LDH level has been expressed as the percentage of the
off effect observed in the ROS-overexpressing fibroblasts total releasable LDH after total lysis. Similarly to antioxidant
results from a ‘simple’ physicochemical interaction between capacity measurement, cytotoxicity values were expressed
phenolics, water and lipids. This interaction, which remains as the percentage of the control (phenolics free, but with
unclear, could possibly take place at the membrane level. ethanol).
Unexpectedly, we demonstrated in an emulsified system that
First of all, Figure 6 shows that the LDH release is
dodecyl chlorogenate exhibited a higher partition in the oily largely dependent on the nature and concentration of com-
phase than longer esters (16, 18 and 20 carbons).[17] This pounds, along with their incubation time. Indeed, only four
suggests that in a system containing an interfacial membrane tested compounds (chlorogenic acid and its methyl, dodecyl
(emulsion) or a biological membrane (fibroblasts), the and hexadecyl esters) exerted a significant cytotoxicity
relationship between hydrophobicity and partitioning in the (P < 0.001) in the concentration range used. In terms of
lipidic phase is not as linear as expected, and that the mem- concentration, it is worth noting that up to 25 mm, the
brane acts as a selective barrier for some of these esters. dodecyl ester was the only one able to induce cytotoxicity
Interestingly, Walters et al.[32] reported that in many biological after 72 h of incubation with cells. Thus, the concentration
systems, molecules show maximum membrane activity of the dodecyl ester necessary to exert significant cytotox-
if they possess a medium chain length (~10–12 carbons). icity is less important than that of the hexadecyl ester by a
Taken together, these arguments suggest that the dodecyl factor of two. Moreover, these two compounds induce dose-
chain confers to its corresponding ester the highest affinity dependent cytotoxicity, whereas the cytotoxicity appeared
for lipid membrane. Finally, the fact that aggregates were only for the highest concentration (100 mm) in the cases of
observed with dodecyl chlorogenate but not with short chain free chlorogenic acid and its methyl ester. In addition, con-
esters suggests that the better antioxidant capacity for dodecyl cerning the effect of incubation time, one can notice that
ester (which is conveyed by the phenolic hydroxyls) is largely when a strong cytotoxicity was detected (highest concentra-
due to its self-assembly properties.
tions), the maximal effect was generally reached for an incu-
bation time of 48 h, and even 72 h in some cases. In other
words, while the antioxidant action needed less than 24 h to
Cytotoxicity measurement
Regardless of the mechanism of action of the chlorogenic acid become apparent, the cytotoxicity needed at least 48 h of
and its alkyl esters, the putative application of some of these incubation.

Cut-off effect of phenolipid antioxidants
Mickaël Laguerre et al.
539
Chlorogenic acid (a)
Methyl chlorogenate (b)
*
125
125
100
75
50
25
0
*
24h
100
24h
48h
72h
*
*
48h
75
72h
*
*
50
25
0
0
50
Butyl chlorogenate (c)
10
25
100
0
10
25
50
100
Octyl chlorogenate (d)
125
100
75
50
25
0
125
100
75
50
25
0
24h
48h
72h
24h
48h
72h
0
10
25
50
100
*
0
10
25
50
100
*
Dodecyl chlorogenate (e)
Hexadecyl chlorogenate (f)
125
100
75
125
100
75
50
25
0
*
*
24h
24h
48h
72h
*
48h
72h
*
50
25
0
*
*
*
0
10
25
50
100
0
10
25
50
100
Medium phenolic concentration (μM)
Figure 6 Cytotoxicity of chlorogenic acid and its alkyl esters estimated by lactate dehydrogenase release (lex: 560 nm/lem: 590 nm). Cytotoxicity
values on the y-axis were calculated according to Equation 9. Cells were cultivated with culture medium containing 10% (v/v) fetal calf serum, with
0, 10, 25, 50 and 100 mm phenolic compound and 0.1% (v/v) ethanol and lactate dehydrogenase activity was measured. Results were expressed as
mean Ϯ SD of four independent measurements carried out on four separate microplates seeded with four different cells suspensions (n = 4). *P < 0.001
compared with the control without phenolic (Kruskall–Wallis followed by Dunn’s multiple pairwise comparison).
of modulating the hydrophobicity of phenolic antioxidants
Conclusion
through lipophilization reactions using medium chain length.
It can be concluded from this study that the antioxidant capac- This study paves the way for a systematic investigation of
ity of chlorogenate derivatives on a ROS-producing fibro- the effect of chain length. A hypothesis according to which
blasts cell line increases as the alkyl chain is lengthened, with medium and long alkyl chains exhibit a good affinity for lipid
a threshold for the dodecyl chain, after which further chain phase and favoured internalisation of phenolic drugs via their
extension leads to a drastic decrease in antioxidant capacity. self-assembly properties has been put forward to explain the
This non-linear (or cut-off) trend is underlined here for the better antioxidant activity of dodecyl and hexadecyl esters
first time in a cellular system and demonstrates the interest over short chain esters. In contrast, the origin of the cut-off

540
Journal of Pharmacy and Pharmacology 2011; 63: 531–540
13. Grandemange S et al. Stimulation of mitochondrial activity by
p43 overexpression induces human dermal fibroblast transforma-
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and other oxidants. Influence of organic solvents. Exp Gerontol
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15. De Arcangelis V et al. Hypertrophy and transcriptional regula-
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phenomenon was not elucidated and requires further investi-
gation. In addition, the self generation of ROS from the cell
instead of the questionable induction of oxidation by artificial
agents (diazo-compounds, transient metals) may be seen as
an interesting achievement. Finally, it was shown that the
dodecyl ester demonstrates a potent antioxidant capacity,
for concentration and exposure time below the cytotoxicity
threshold, making it a good candidate for further in-vivo
study.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
to disclose.
17. Laguerre M et al. Chain length affects antioxidant properties
of chlorogenate esters in emulsion: the cutoff theory behind the
polar paradox. J Agric Food Chem 2009; 57: 11335–11342.
18. Laguerre M et al. Evaluation of the ability of antioxidants to
counteract lipid oxidation: existing methods, new trends and
challenges. Prog Lipid Res 2007; 46: 244–282.
Funding
Luis Javier López Giraldo acknowledges the support from
Programme Alban, the European Union Programme of
High Level Scholarships for Latin America, scholarship N°
E05D055786CO.
19. Puelo A et al. Chemical and enzymatic stability evaluation
of lipoamino acid esters of idebenone. Eur J Pharm Biopharm
2004; 57: 343–346.
20. Pignatello R et al. A calorimetric evaluation of the interaction of
amphiphilic prodrugs of idebenone with a biomembrane model.
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22. Evans DF, Wennerstrom H. The Colloidal Domain: Where
Physics, Chemistry, Biology and Technology Meet. New York:
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