Effect of resveratrol-related stilbenoids on biomembrane models

Article abstract of DOI:10.1021/np400188m

The interactions of the two resveratrol analogues 2-hydroxy-3,5,3′, 5′-tetramethoxystilbene (4) and 2-hydroxy-3,5,3′,4′- tetramethoxystilbene (5) with model biomembranes were studied. The aim of this investigation was to highlight possible differences in the interactions with such biomembranes related to the minimal structural differences between these isomeric stilbenoids. In particular, different experiments on stilbenoid/biomembrane model systems using both differential scanning calorimetry (DSC) and Langmuir-Blodgett techniques were carried out to evaluate stilbenoid/multilamellar vesicle and stilbenoid/phospholipid monolayer interactions, respectively. Dimyristoylphosphatidylcholine was used as constituent of the biomembrane models and permitted the experiments to be carried out at 37 C, close to body temperature. Kinetic studies were also run by DSC to evaluate the uptake of the resveratrol derivatives by the biomembrane model in an aqueous medium and when transported by a lipophilic carrier. The results indicated that both of the resveratrol analogues influenced the behavior of multilamellar vesicles and monolayers, biomembrane models, with 4 producing a larger effect than 5. These results are useful for better understanding the mechanism of action of these compounds. Moreover, the kinetic results could be of importance for future design of lipophilic delivery systems for these stilbenoids.

Full text of DOI:10.1021/np400188m

Article
pubs.acs.org/jnp
Effect of Resveratrol-Related Stilbenoids on Biomembrane Models
Maria Grazia Sarpietro,*,† Carmela Spatafora,‡ Maria Lorena Accolla,§ Orazio Cascio,⊥ Corrado Tringali,‡
and Francesco Castelli†
†Dipartimento di Scienze del Farmaco, Universita
̀
degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy
degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy
“Magna Græcia” di Catanzaro, Campus Universitario “S. Venuta’”, Viale S. Venuta,
‡Dipartimento di Scienze Chimiche, Universita
̀
§Dipartimento di Scienze della Salute, Universita
88100 Germaneto (CZ), Italy
̀
⊥Dipartimento di Anatomia, Biologia e Genetica, Medicina Legale, Neuroscienze, Patologia Diagnostica, Igiene e Sanita
̀
Pubblica (G.
̀
F. Ingrassia), Universita degli Studi di Catania, Via S. Sofia 87, 95123 Catania, Italy
S
* Supporting Information
ABSTRACT: The interactions of the two resveratrol analogues 2-hydroxy-3,5,3′,5′-
tetramethoxystilbene (4) and 2-hydroxy-3,5,3′,4′-tetramethoxystilbene (5) with model
biomembranes were studied. The aim of this investigation was to highlight possible
differences in the interactions with such biomembranes related to the minimal structural
differences between these isomeric stilbenoids. In particular, different experiments on
stilbenoid/biomembrane model systems using both differential scanning calorimetry
(DSC) and Langmuir−Blodgett techniques were carried out to evaluate stilbenoid/
multilamellar vesicle and stilbenoid/phospholipid monolayer interactions, respectively.
Dimyristoylphosphatidylcholine was used as constituent of the biomembrane models and
permitted the experiments to be carried out at 37 °C, close to body temperature. Kinetic
studies were also run by DSC to evaluate the uptake of the resveratrol derivatives by the
biomembrane model in an aqueous medium and when transported by a lipophilic carrier.
The results indicated that both of the resveratrol analogues influenced the behavior of multilamellar vesicles and monolayers,
biomembrane models, with 4 producing a larger effect than 5. These results are useful for better understanding the mechanism of
action of these compounds. Moreover, the kinetic results could be of importance for future design of lipophilic delivery systems
for these stilbenoids.
resveratrol analogues with minimal differences in their
esveratrol (E-3,5,4′-trihydroxystilbene, 1, Figure 1) is a
Rnatural stilbenoid found in some components of the structures show significantly different antiproliferative activities
human diet, especially in grapes and red wine.1 It has gained
against SW480 human colorectal cells. In particular, (E)-2-
hydroxy-3,5,3′,5′-tetramethoxystilbene (4) resulted in greater
activity than (E)-2-hydroxy-3,5,3′,4′-tetramethoxystilbene
(5).17 However, binding affinity calculations based on docking
to tubulin, a biological target of resveratrol analogues,18 are not
always in agreement with observed antiproliferative activity,
probably because of crossing biological membranes and, more
generally, of ADME (absorption, distribution, metabolism, and
excretion) implications.19 In this context, the study of the
interactions of the two resveratrol analogues 4 and 5 with
model biomembranes was carried out with the aim of
highlighting possible differences in the interaction with model
membranes related to the minor structural differences between
these isomeric stilbenoids. In particular, different experiments
on stilbenoid/biomembrane model systems were carried out
using both differential scanning calorimetry (DSC) and
Langmuir−Blodgett (LB) techniques. DSC was used to
evaluate the stilbenoid/multilamellar vesicles (MLVs) inter-
action20 and the Langmuir−Blodgett technique to afford
popularity as a cardioprotective agent since the formulation of
the so-called French paradox.2 Compound 1 exerts various
biological activities that may be beneficial to human health,3
including anti-inflammatory,4 antioxidant,5 antiproliferative,6
proapoptotic,7 and anticancer effects.8 In view of the potential
of 1 against a variety of diseases, and in particular as an
anticarcinogenic agent,9 many resveratrol analogues have been
synthesized not only to improve its chemopreventive properties
but also to enhance its metabolic stability. Thus, a number of in
vivo studies indicate that 1 has a short half-life, rapid clearance,
and low bioavailability, being rapidly metabolized especially via
phase II glucuronide or sulfate conjugation.10,11 Among the
several synthetic analogues of 1 produced,12 polymethoxys-
tilbenes are promising in displaying both a higher antiprolifer-
ative activity13 and greater metabolic stability14,15 than 1. Of
some importance is the number and position of methoxy and
hydroxy groups of the stilbene core: as an example, (E)-
3,5,3′,4′-tetramethoxystilbene (2) was found to be more potent
than (E)-3,5,3′,5′-tetramethoxystilbene (3) as an inhibitor of
ABCG2, an efflux pump protein involved in multidrug
resistance.16 In a further study,17 it was observed that
Received: March 5, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Chemical structures of compounds 1−5.
information on the interaction of test compounds and a
phospholipid monolayer.21 In both cases, dimyristoylphospha-
tidylcholine (DMPC) was used as constituent of the
biomembrane models, as it permits experiments to be carried
out at 37 °C. In addition, kinetic studies were run by DSC to
evaluate the uptake of the resveratrol derivatives by the
biomembrane model in an aqueous medium and when
transported by a lipophilic carrier. Finally, the results were
compared with earlier published studies on 1.22,23
pretransition peak and the main transition peak and, in
particular, can affect the thermotropic parameters of the peaks:
pretransition and transition temperature and enthalpy
change.31−33 The calorimetric curves of MLVs prepared with
4 or 5, together with that of MLVs prepared without either
compound, are shown in Figure 2A and B. Compound 4 caused
the disappearance of the pretransition peak at all molar
fractions used, whereas the transition peak broadened and
moved toward lower temperature, as the compound molar
fraction increased. In addition, at 0.09 molar fraction, a hint of
phase separation was observed, represented by the splitting of
the peak into two components at different temperatures, and
this became more evident at a 0.12 molar fraction. It is likely
that the low-temperature component is caused by the melting
of phospholipid domains containing a high amount of 4,
whereas the higher temperature component is due to the
melting of phospholipid domains containing a low amount of 4.
When MLVs were prepared with increasing amounts of 5, the
disappearance of the pretransition peak was observed, whereas
the transition peak widened and shifted toward lower
temperature. The values of the transition temperature (Tm),
enthalpy change (ΔH), and T1/2 (width at half-height of the
main phase transition peak) are plotted in Figure 3A−C as a
function of compound molar fraction. Transition temperature
decreases as a result of an increase of compound molar fraction.
Up to 0.06 molar fraction, the results highlight an ideal
behavior described by the van’t Hoff equation, which predicts
an almost linear relationship between the lowering of the
solvent melting temperature and concentration of the solute34
and has been applied to several compounds such as
anesthetics35 and perfluoroalkyl acids.36 With a molar fraction
greater than 0.06, deviations from the ideal behavior become
evident that can be ascribed to nonideal solute−solvent
mixing.37,38 Even the enthalpy change decreases when the
molar fraction of compounds increases; however, the decrease
was stronger for 4 when compared to 5. On considering the
T1/2 values, it can be noted that they increase with an increase
in the molar fraction. With regard to T1/2, 4 showed a larger
effect than 5. Taking these results together, both compounds
interacted with the biomembrane model. It is known that
RESULTS AND DISCUSSION
■
The importance of an interaction between a bioactive molecule
and a biological membrane in obtaining a better understanding
of the mechanism of action is widely recognized. Each molecule
can be characterized by its own special fingerprint, when it
interacts with membrane bilayers,24 and several studies have
been carried out to relate drug activity with its interaction with
a membrane.25 Different resveratrol derivatives show various
potencies in antiproliferative activity toward SW480 human
colorectal cells that cannot be justified only by the tubulin
binding affinity based on docking data;17 thus, it was attempted
to establish if this behavior could be related to a different
interaction with biomembrane models. The analogues 4 and 5
(Figure 1), which differ only in the position of one methoxy
group, were evaluated in order to highlight the possible role of
the substituent position on the stilbene core. The interaction
was studied by analyzing the effect exerted on the thermotropic
behavior of MLVs in the presence of a compound of
interest.26,27 MLVs based on DMPC, when submitted to
heating, pass from an ordered gel phase (in which the chains
are in an all-trans configuration) to an undulated gel phase (in
which the bilayer surface displays a periodic ripple structure)
and then to an ordered liquid crystalline phase (in which the
chains become more disordered due to the introduction of
gauche conformers).28 These two events produce two well-
defined calorimetric peaks, respectively. The first is less
energetic and occurs at about 17 °C, and is called the
pretransition peak, whereas the second, at about 25 °C, is
termed the main transition peak.29,30 Any compound
interacting with MLVs can cause variations of both the
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Figure 3. (A) Temperature variations. (B) enthalpy variations. and
(C) T1/2 (width at half-height of the main phase transition peak) of
MLVs prepared with 4 or 5, as a function of compound molar fraction.
pound 4 (more active than 5 against SW480 tumor cells)
interacted with the biomembrane model more than 5,
indicating that the minimal modification of the methoxy
group position produces significant changes in the interaction.
In an earlier paper reporting the interaction of 1 with DMPC-
based MLVs,22 it was found that 1 produces a decrease of the
transition temperature. It was conjectured that the hydroxy
groups of compound 1, forming hydrogen bonds, localize the
molecule between the phospholipid headgroups. A similar
hypothesis was made by Wesołowska and co-workers,23 who
studied the interactions of resveratrol and piceatannol (the
latter compound bears one additional hydroxy group when
compared to 1) with DMPC liposomes. These stilbenoids
produced a concentration-dependent decrease of the main
phase transition temperature of DMPC, with piceatannol
reducing the melting temperature to a greater extent than 1.
The influence of these stilbenoids on transition enthalpy was
smaller than in the case of transition temperature. Both
compounds influenced the half-height width of calorimetric
peaks to a similar extent. It was concluded that both
Figure 2. Calorimetric curves, in heating mode, of MLVs prepared
with increasing molar fractions of (A) 4 and (B) 5.
phospholipids do not melt independently of each other, but
rather the transition has a cooperative domain. Pure
phospholipids exhibit a sharp transition at a critical temper-
ature, showing no coexisting region between ordered and
disordered domains.39,40 On the other hand, mixtures with
foreign molecules give rise to a coexisting region, of which the
width increases with the concentration of foreign molecules
within the bilayer. The widening of the coexistence region is
41,42
reflected in the parameter T1/2
.
Broadening of the
transition peak caused by 4 and 5 suggests that these
compounds intercalate within the DMPC molecules. Com-
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compounds interact preferentially with the polar headgroup
region of the bilayer and that small amounts of these molecules
also penetrate into the deeper membrane regions.23 The results
obtained in the present work are similar to those obtained with
1 and piceatannol and support the hypothesis that resveratrol-
type stilbenoids can preferentially localize next to the DMPC
headgroup through the formation of hydroxyl bonds.
Another approach that gives information on the interaction
of a test compound with phospholipids uses the Langmuir−
Blodgett technique. This measures the surface pressure as a
function of the mean molecular area occupied by a molecule in
a monolayer adjacent to an aqueous subphase affording surface
pressure (mN/m)/molecular area (Å2) isotherms. In Figures
S1 and S2 (Supporting Information), the isotherms of DMPC
monolayers as well as of the compound/DMPC mixed
monolayers, registered at 10 and 37 °C, respectively, are
shown. No isotherm for either 4 and 5 was observed. This
could indicate that the compounds leave the interface and
transfer to the aqueous subphase. This behavior is similar to
that observed with naproxen43 and some coumarins.44 At 10
°C, the isotherm of DMPC, going from 120 to about 42 Å2, is
characterized by a gaseous state (120−100 Å2), a liquid
expanded state (100−65 Å2), a liquid expanded−liquid
compressed transition (65−50 Å2), and a liquid compressed
state (50−42 Å2).45 Compound 4 did not produce significant
variation of the isotherm up to a 0.12 molar fraction; from 0.25
molar fraction, the gradual shift of the isotherm toward lower
values of molecular area was observed, and this occurred always
with a decrease of the liquid expanded−liquid compressed
transition. A similar behavior was gained for DMPC/5 mixed
monolayers. At 37 °C, DMPC exhibits only a gaseous state
(120−105 Å2) and a liquid expanded state (105−52 Å2).46 The
presence of either 4 and 5 did not cause strong variations of the
isotherm up to a 0.12 molar fraction; then the isotherms moved
toward lower values of molecular area. Information on the type
of interaction between the test compound and DMPC in the
mixed monolayers can be obtained by reporting in graph form
the mean molecular area as a function of the compound molar
fraction at defined surface pressures (10, 20, and 30 mN/m)
(Figures 4 and 5). The mean molecular area of a monolayer
with two components can be calculated by A = A1X1 + (1 − X1)
A2, where A is the mean molecular area, X1 is the molar fraction
of component 1, and A1 and A2 are the areas per molecule of
the pure component monolayers at the same surface pressure.
The experimental results (continuous line) may be compared
with the ideal graphs (dotted line) referred to two different
cases: (i) the compound and DMPC are ideally mixed and the
interactions DMPC/compound are like those of DMPC/
DMPC and compound/compound, and (ii) the compound and
DMPC are completely immiscible at the air−liquid inter-
face.47,48 Instead, any deviation from the ideal line indicates that
the compound and DMPC interact; in particular, positive
deviations suggest that repulsive interactions occur, whereas
negative deviations suggest the existence of attractive
interactions. In Figure 4A and B (experiments carried out at
10 °C), it can be noted that both 4 and 5 produce a positive
deviation from the dotted line with a bigger effect for 4 with
respect to 5. Even at 37 °C (Figure 5A and B), positive
deviations are present. However, at this temperature, the larger
effect for 4 is still more evident than at 10 °C. The results
indicate that the compounds cause an area expansion in the
monolayers, with 4 being more effective than 5. The results
obtained in this investigation are similar to those obtained with
Figure 4. Molecular area of (A) DMPC/4 and (B) DMPC/5 mixed
monolayers at the air−water interface, plotted as a function of the
molar fraction of compound at various values of surface pressures at 10
°C.
resveratrol by Micieli,46 who found that 1 produced the
expansion of the DMPC monolayer. The results are also
consistent with those of Olas and Holmsen,49 who showed that
1 increased the phospholipid mean molecular area during
compression, suggesting that this stilbenoid is interdigitated
between the phospholipid molecules in the monolayer.
The ability of 4 and 5 to be absorbed by the biomembrane
model was tested. Thus, each compound was placed in contact
with the MLVs in a calorimetric pan that was subjected to
cooling and heating at 1 h intervals, during which the sample
was kept at 37 °C. The calorimetric curves, together with those
of MLVs and of MLVs prepared with a 0.09 molar fraction of
each compound (curve r), are displayed in Figure 6A and B.
Curve r can be considered as a reference, as it belongs to MLVs
prepared with a 0.09 molar fraction of each test compound and
represents the maximum interaction that can occur between
MLVs and the compound. As expected, both 4 and 5 caused
only decreases of the pretransition peak and a slight shift of the
transition peak toward lower temperature, and the curve r was
not reached. This is a clear indication of the inability of each
compound, in an aqueous medium, to be absorbed by the
biomembrane model. A way to circumvent this problem
consists in loading the compound into water-soluble carriers.
Liposomes are biocompatible carriers that can be prepared
from lipids and loaded with compounds of a different
lipophilic−hydrophilic nature. Recently, this strategy was used
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Figure 5. Molecular area of (A) DMPC/4 and (B) DMPC/5 mixed
monolayers at the air−water interface, plotted as a function of the
molar fraction of compound at various values of surface pressures at 37
°C.
successfully to enhance the bioavailability of 1, using liposomes
made mainly by DPPC.50 To enhance the absorption of the test
compounds by the biomembrane model, MLVs were prepared
in the presence of each compound at a 0.12 molar fraction as a
lipophilic carrier (loaded MLVs). To evaluate the delivery of
the test compound to the biomembrane model (unloaded
MLVs), equimolar amounts of loaded MLVs and unloaded
MLVs were left in contact, and each sample was immediately
submitted to DSC analysis. The calorimetric curves are shown
in Figure 7A and B. For comparison, also the calorimetric
curves of unloaded and loaded MLVs and the calorimetric
curve of MLVs prepared with the compound at 0.045 molar
fraction (curve r) are provided. Curve r is considered as a
reference curve, since it contains a molar fraction average of
those contained in MLVs placed in contact, and, if loaded
MLVs deliver half of their content to unloaded MLVs, a curve
similar to the reference curve is obtained. Compound 4 will be
considered first. In the first calorimetric curve a small
pretransition peak, a large shoulder at about 22 °C (belonging
to loaded MLVs), and a main peak at about 22.5 °C (belonging
to unloaded MLVs) were present. From the second scan
onward, the pretransition peak disappeared, whereas the
shoulder and main peak merged into a peak that moves
toward lower temperature closer to the reference curve. With
regard to 5, the pretransition disappeared in the first scan, while
the main peak moved toward lower temperature approaching
Figure 6. Calorimetric curves, in heating mode, of MLVs left in
contact with (A) 4 and (B) 5, at a 0.09 molar fraction, with increasing
incubation time. Curve r belongs to MLVs prepared with the
compound at 0.09 molar fraction.
the reference curve. A comparison is provided in Figure 8,
where the transition temperature is reported as a function of
the calorimetric scans. Both compounds caused a rapid
decrease of the transition temperature, with 4 exerting a larger
effect than 5. Next, 4 and 5 can transfer from loaded to
unloaded MLVs with the amount of 4 transferred being greater
than that of 5.
In conclusion, DSC experiments associated with Langmuir−
Blodgett measurements indicated clearly that the resveratrol
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Figure 8. Transition temperature variation of MLVs left in contact
with MLVs prepared in the presence of test compound at 0.09 molar
fraction, as a function of calorimetric scans. Values r are the transition
temperature of MLVs prepared with 0.045 molar fraction.
importance for future design of lipophilic delivery systems of
these compounds.
EXPERIMENTAL SECTION
■
Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC) (purity = 99%) was supplied by Genzyme Pharmaceuticals
(Liestal, Switzerland). Methanol and chloroform were of LC grade and
were bought from VWR (Darmstadt, Germany). All other reagents
were of analytical grade and were used as supplied.
Calorimetric analyses were performed using a Mettler Toledo
STARe system (Greifensee, Switzerland) equipped with a DSC-822e
calorimetric cell and employing Mettler TA-STARe software (8.1
version). The sensitivity was chosen automatically as the maximum
possible by the calorimetric system, and the reference pan was filled
with 50 mM Tris buffer (pH 7.4). The calorimetric system was
calibrated, in terms of temperature and enthalpy changes, following the
procedure of the DSC 822 Mettler TA STARe instrument, using
indium and palmitic acid (purity ≥99.95% and ≥99.5%, respectively;
Fluka, Buchs, Switzerland).
Monolayer measurements were performed using a KSV mini trough
apparatus provided with a computer interface unit, operating software,
a trough (24 225 mm2 available area) made of Teflon, and two mobile
barriers made of Delrin. As subphase, 5 mM Tris buffer (pH 7.4) in
ultrapure Millipore water with a resistivity of 18.2 MΩ cm was used.
The test compounds 2-hydroxy-3,5,3′,5′-tetramethoxystilbene (4)
and 2-hydroxy-3,5,3′,4′-tetramethoxystilbene (5) were synthesized
through a mild aromatic hydroxylation with m-CPBA of 3,5,3,5′-
tetramethoxystilbene (3) and 3,5,4′,5′-tetramethoxystilbene (2),
respectively, as previously reported.51 The purity of the compounds
4 and 5 was greater than 97%.
MLV Preparation. DMPC and compounds 4 and 5 were
separately dissolved in chloroform−methanol (1:1). Aliquots of
DMPC solution corresponding to 7 mg were distributed in glass
tubes, where aliquots of 4 or 5 were added in order to produce exact
molar fractions (0.00, 0.015, 0.03, 0.045, 0.06, 0.09, and 0.12) with
respect to DMPC. The solvents were evaporated under a nitrogen
flow, and the resulting films were freeze-dried to remove solvent traces
(FreeZone 2.5 Liter Freeze-Dry Systems, Labconco). The resulting
films were hydrated with 168 μL of Tris, maintained at 37 °C in a
water bath for 1 min, vigorously agitated for 1 min, three times, and
finally left in a water bath at 37 °C for 60 min.
Compound/MLV Interaction. An aliquot of 120 μL of an
aqueous dispersion of MLVs was placed in an aluminum calorimetric
pan (volume = 160 μL), which was sealed and immediately submitted
to DSC analysis. The sample was heated from 5 °C to 37 °C, at 2 °C/
min, and cooled from 37 °C to 5 °C, at 4 °C/min, at least three times.
Each experiment was carried out in triplicate. Calorimetric data were
analyzed using the software Mettler TA-STARe version 8.1.
Figure 7. Calorimetric curves, in heating mode, of MLVs left in
contact with MLVs prepared in the presence of a 0.09 molar fraction
of test compound. Curve r belongs to MLVs prepared with the
compound at 0.045 molar fraction.
analogues 4 and 5 influenced the thermotropic behavior of both
MLVs and monolayers, biomembrane models, with 4
producing a larger effect than 5. These results may be useful
for understanding the mechanism of action of these
compounds. In particular, these data are in agreement with
previous observations suggesting that other factors, in addition
to the target affinity, are responsible for the different biological
activities observed for these closely structurally related
stilbenoids.17 In addition, the kinetic results could be of
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Absorption of Compounds by MLVs. An exact amount of 4 or 5
(corresponding to 0.09 molar fraction with respect to DMPC) was
weighed in a calorimetric pan; afterward 120 μL of MLVs was added
and the pan was sealed. Immediately after this, the sample was
subjected to DSC analysis consisting of three consecutive steps: (1) a
heating scan from 5 to 37 °C, at 2 °C/min, (2) an isotherm scan at 37
°C of 60 min, and (3) a cooling scan from 37 to 5 °C, at 4 °C/min.
The three consecutive steps were repeated at least eight times. Each
experiment was carried out in triplicate. Calorimetric data were
analyzed using the software Mettler TA-STARe version 8.1.
Compound Transmembrane Transfer. An aliquot of 60 μL of
MLVs prepared without compound (unloaded MLVs) was placed in a
calorimetric pan, and 60 μL of MLVs loaded with the compound
(prepared in the presence of 0.09 molar fraction; loaded MLVs) was
added. The pan was sealed and submitted to DSC analysis as described
above. Each experiment was carried out in triplicate. Calorimetric data
were analyzed using Mettler TA-STARe version 8.1.
Monolayer Measurements. Equimolar solutions (0.001 mmol/
mL) of DMPC, 4, and 5 in chloroform were prepared. Immediately
before being used, mixed DMPC/compound solutions were prepared
containing, for each compound, the following molar fractions: 0.015,
0.03, 0.045, 0.06, 0.09, 0.12, 0.25, 0.50, and 0.75. Aliquots of 30 μL of
solutions of the pure components or of the mixed solutions (to have
each time the same amount of the molecules spread on the subphase)
were spread drop by drop on the subphase surface by a Hamilton
microsyringe; the solvent was allowed to evaporate for 15 min (at 10
or 37 °C), and then the monolayers were compressed by the two
mobile barriers at a constant speed of 10 mm/min. Surface pressure
versus molecular area isotherms was recorded by the Wilhelmy plate
arrangement attached to a microbalance. Before sample spreading,
blank experiments were run to check that no impurities were present
on the subphase. The experiments were performed at subphase
temperatures of 10 and 37 °C, which were kept constant by a
cryothermostat connected to the trough. Each experiment was
repeated at least three times.
(7) Liao, H. F.; Cheng, A. J.; Huang, H. T.; Shen, M. L.; Hei, T. K.;
Chen, Y.-J. Food Chem. 2012, 132, 2094−2101.
(8) Signorelli, P.; Ghidoni, R. J. Nutr. Biochem. 2005, 16, 449−466.
(9) Pezzuto, J. M. Pharm. Biol. 2008, 46, 443−573.
(10) Baur, J. A.; Sinclair, D. A. Nat. Rev. Drug Discovery 2006, 5, 493−
506.
(11) Walle, T. Ann. N.Y. Acad. Sci. 2011, 1215, 9−15.
(12) Chillemi, R.; Sciuto, S.; Spatafora, C.; Tringali, C. Nat. Prod.
Commun. 2007, 2, 499−513.
(13) Lin, H.-S.; Tringali, C.; Spatafora, C.; Wu, C.; Ho, P. C. J.
Pharm. Biomed. Anal. 2010, 51, 679−684.
(14) Lin, H.-S.; Zhang, W.; Go, M. L.; Tringali, C.; Spatafora, C.; Ho,
P. C. J. Agric. Food Chem. 2011, 59, 1072−1077.
(15) Ma, Z.; Molavi, O.; Haddadi, A.; Lai, R.; Gossage, R. A.;
Lavasanifar, A. Cancer Chemother. Pharmacol. 2008, 63, 27−35.
(16) Valdameri, G.; Pereira Langer, R.; Spatafora, C.; Guitton, J.;
Gauthier, C.; Arnaud, O.; Pereira-Ferreira, A.; Falson, P.;
Winnischofer, S. M. B.; Rocha, M. E. M.; Tringali, C.; Di Pietro, A.
ACS Chem. Biol. 2012, 7, 322−330.
́
(17) Mazue, F.; Colin, D.; Gobbo, J.; Wegner, M.; Rescifina, A.;
Spatafora, C.; Fasseur, D.; Delmas, D.; Meunier, P.; Tringali, C.;
Latruffe, N. Eur. J. Med. Chem. 2010, 45, 2972−2980.
(18) Androutsopoulos, V. P.; Ruparelia, K. C.; Papakyriakou, A.;
Filippakis, H.; Tsatsakis, A. M.; Spandidos, D. A. Eur. J. Med. Chem.
2011, 46, 2586−2595.
(19) Fotakis, C.; Gega, S.; Siapi, E.; Potamitisb, C.; Viras, K.;
Moutevelis-Minakakis, P.; Kokotos, C. G.; Durdagi, S.; Golic
Grdadolnik, S.; Sartori, B.; Rappolt, M.; Mavromoustakos, T. Biochim.
Biophys. Acta 2010, 1798, 422−432.
(20) Castelli, F.; Sarpietro, M. G.; Ceruti, M.; Rocco, F.; Cattel, L.
Mol. Pharmaceutics 2006, 3, 737−744.
(21) Castelli, F.; Sarpietro, M. G.; Micieli, D.; Stella, B.; Rocco, F.;
Cattel, L. J. Colloid Interface Sci. 2007, 316, 43−52.
(22) Sarpietro, M. G.; Spatafora, C.; Tringali, C.; Micieli, D.; Castelli,
F. J. Agric. Food Chem. 2007, 55, 3720−3728.
̌
(23) Wesołowska, O.; Kuzdzał, M.; Strancar, J.; Michalak, K. Biochim.
̇
̇
ASSOCIATED CONTENT
* Supporting Information
■
Biophys. Acta 2009, 1788, 1851−1860.
S
(24) Fotakis, C.; Megariotis, G.; Christodouleas, D.; Kritsi, E.;
Zoumpoulakis, P.; Ntountaniotis, D.; Zervou, M.; Potamitis, C.;
Hodzic, A.; Pabst, G.; Rappolt, M.; Mali, G.; Baldus, J.; Glaubitz, C.;
Papadopoulos, M. G.; Afantitis, A.; Melagraki, G.; Mavromoustakos, T.
Biochim. Biophys. Acta 2012, 1818, 3107−3120.
Surface pressure/molecular area isotherms of DMPC/4 and
DMPC/5 mixed monolayers at the air/water interface, at 10
and 37 °C. This material is available free of charge via the
Internet at http://pubs.acs.org.
(25) Koukoulitsa, C.; Durdagi, S.; Siapi, E.; Villalonga-Barber, C.;
Alexi, X.; Steele, B. R.; Micha-Screttas, M.; Alexis, M. N.; Tsantili-
Kakoulidou, A.; Mavromoustakos, T. Eur. Biophys. J. 2011, 40, 865−
875.
AUTHOR INFORMATION
Corresponding Author
*Tel: +39 095 7385099. Fax: +39 095 580138. E-mail: mg.
sarpietro@unict.it.
■
(26) Castelli, F.; Sarpietro, M. G.; Micieli, D.; Ottimo, S.; Pitarresi,
G.; Tripodo, G.; Carlisi, B.; Giammona, G. Eur. J. Pharm. Sci. 2008, 35,
76−85.
Notes
(27) Librando, V.; Minniti, Z.; Accolla, M. L.; Cascioc, O.; Castelli,
F.; Sarpietro, M. G. Chemosphere 2013, 91, 791−796.
The authors declare no competing financial interest.
(28) Brandenburg, K. O.; Garidel, P.; Howe, J.; Andra, J.; Hawkins,
̈
ACKNOWLEDGMENTS
■
L.; Koch, M. H. J.; Seydel, U. Thermochim. Acta 2006, 445, 133−143.
(29) Walde, P. In ASP Encyclopedia of Nanoscience and Nano-
technology; Nalwa, H. S., Ed.; American Scientific Publishers:
Stevenson Ranch, CA, 2004; pp 43−79.
Financial support for this work was obtained from the
University of Catania (PRA, Catania, Italy) and from MIUR
(PRIN 2009, Rome, Italy).
(30) Yi, Z.; Nagao, M.; Bossev, D. P. Biophys. Chem. 2012, 160, 20−
27.
(31) Marsh, D. In Nonmedical Applications of Liposomes; Barenholz,
Y.; Lasic, D. D., Eds.; CRC Press: Boca Raton, FL, 1996; Vol. II, pp 1−
16.
REFERENCES
■
(1) Fulda, S. Drug Discovery Today 2010, 15, 757−765.
(2) Sun, A. Y.; Simonyi, A.; Sun, G. Y. Free Radical Biol. Med. 2002,
32, 314−318.
(32) Sarpietro, M. G.; Micieli, D.; Rocco, F.; Ceruti, M.; Castelli, F.
(3) Aggarwal, B. B.; Shishodia, S. Resveratrol in Health and Disease;
CRC Taylor & Francis: Boca Raton, FL, 2006.
(4) Wang, K.-T.; Chen, L.-G.; Tseng, S.-H.; Huang, J.-S.; Hsieh, M.-
S.; Wang, C.-C. J. Agric. Food Chem. 2011, 59, 3649−3656.
(5) Rodrigo, R.; Miranda, A.; Vergara, L. Clin. Chim. Acta 2011, 12,
410−424.
Int. J. Pharm. 2009, 382, 73−79.
(33) Sarpietro, M. G.; Ottimo, S.; Paolino, D.; Ferrero, A.; Dosio, F.;
Castelli, F. Int. J. Pharm. 2012, 436, 135−140.
(34) Hill, M. W. Biochim. Biophys. Acta 1974, 357, 117−124.
(35) Heimburg, T.; Jackson, A. D. Biophys. J. 2007, 9, 3159−3165.
(36) Oldham, E. D.; Xie, W.; Farnoud, A. M.; Fiegel, J.; Lehmler, H.-
J. J. Phys. Chem. B 2012, 116, 9999−10007.
(6) Schneider, Y.; Vincent, F.; Duranton, B.; Badolo, L.; Gosse,
́
F.;
Bergmann, C.; Seiler, N.; Raul, F. Cancer Lett. 2000, 158, 85−91.
G
dx.doi.org/10.1021/np400188m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(37) Lee, A. G. Biochim. Biophys. Acta 1977, 472, 285−344.
(38) O’Leary, T. J.; Ross, P. D.; Levin, I. W. Biophys. J. 1986, 50,
1053−1059.
(39) Halling, K. K.; Slotte, J. P. Biochim. Biophys. Acta 2004, 1664,
161−171.
(40) Jemiola-Rzeminska, M.; Rivera, C.; Suwalsky, M.; Strzalka, K.
Thermochim. Acta 2007, 458, 132−137.
(41) Jorgensen, K.; Ipsen, J. H.; Mouritsen, O. G.; Bennett, D.;
Zuckermann, M. J. Biochim. Biophys. Acta 1991, 1062, 227−238.
(42) Basso, L. G. M.; Rodrigues, R. Z.; Naal, R. M. Z. G.; Costa-
Filho, A. J. Biochim. Biophys. Acta 2011, 1808, 55−64.
(43) Micieli, D.; Giuffrida, M. C.; Pignatello, R.; Castelli, F.;
Sarpietro, M. G. J. Colloid Interface Sci. 2011, 360, 359−369.
(44) Sarpietro, M. G.; Giuffrida, M. C.; Ottimo, S.; Micieli, D.;
Castelli, F. J. Nat. Prod. 2011, 74, 790−795.
(45) Sarpietro, M. G.; Rocco, F.; Micieli, D.; Ottimo, S.; Ceruti, M.;
Castelli, F. Int. J. Pharm. 2010, 395, 167−173.
(46) Micieli, D. Tecniche Termoanalitiche Applicate allo Studio di
Composti e Sistemi Colloidali Biologicamente Attivi. Ph.D. Thesis.
̀
Universita degli Studi di Palermo, Palermo, Italy, 2009, p 221.
(47) Gaines, G. L., Jr. In Insoluble Monolayers at Liquid−Gas
Interfaces; Wiley-Interscience: New York, 1966.
(48) Shahgaldian, P.; Coleman, A. W. Langmuir 2003, 19, 5261−
5265.
(49) Olas, B.; Holmsen, H. Food Chem. Toxicol. 2012, 50, 4028−
4034.
(50) Bonechi, C.; Martini, S.; Ciani, L.; Lamponi, S.; Rebmann, H.;
Rossi, C.; Ristori, S. PLoS One 2012, 7 (8), e41438.
(51) Spatafora, C.; Basini, G.; Baioni, L.; Grasselli, F.; Sofia, A.;
Tringali, C. Nat. Prod. Commun. 2009, 4, 239−246.
H
dx.doi.org/10.1021/np400188m | J. Nat. Prod. XXXX, XXX, XXX−XXX