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Introduction
Silybin (or silibinin) is the main biologically active
flavonolignan extracted from the seeds and fruits of milk
thistle (Sylibum
marianum)[1]. Although the beneficial
properties of this plant have been known and used since antiquity,
silybin appeared in official medicine as a hepatoprotective
substance in the 1970s[2]. Similar to other flavonoids, the
range of effects exerted by silybin is very
wide[3]. It was found to play the role of anticancer agent against
lung[4], prostate[5],
ovarian[6] and other cancer types. Silybin
derivative dehydrosilybin was found to exert antimalaric
effects[7]. Three main mechanisms are supposed to lie in the
background of the silybin biological activity: scavenging of
free radicals and the chelation of active metal ions,
membrane stabilizing function and the influence on RNA
synthesis[8]. Recently, silybin was also found to act as a modulator
of transporter proteins involved in multidrug resistance of
cancer cells: P-glycoprotein[9], multidrug resistance-associated protein 1
(MRP1)[10,11] and breast cancer resistance protein
(BCRP)[12,13]. Strong inhibitory effects of silybin on transport carried by
MRP1 protein in human erythrocytes was also recorded in
our laboratory[14]. Trompier et
al[15] have shown that prenylation of dehydrosilybin increases the binding affinity
of flavonoid molecule to the MRP1's nucleotide binding site.
Silybin formulations are commercially often available as
complexes of the flavonoid with phospholipids. As shown
experimentally, such complexes are characterized by better
bioavailability[16] and are more effective free radical
scavengers[17,18]. In the murine model, the silybin-containing
liposomes composed of phospholipids and cholesterol
appeared to be much more effective hepatoprotectors than pure
silybin[19].
Despite the fact that the membrane-stabilizing function
was suggested as one of the basic molecular mechanisms of
silybin activity[8,20] and that silybin-phospholipid complexes
are more effective than plain flavonoid, present knowledge
about the interactions of silybin with lipid bilayers is very
poor. In the study on the influence of silybin on liver
microsomal membranes, Parasassi et
al[21] suggested that flavonoid molecules were incorporated into the
hydrophobic-hydrophilic interface of membranes. Taken into account the
antioxidant properties of this compound, it is obvious that it
has to interact at least with the polar region of the lipid
bilayers. Such interactions seem to be responsible for the
protective role of flavonoids against free radicals or active
metal ions[22_24]. To elucidate the problem of
silybin-phospholipid interactions, we performed this study in which
microcalorimetry, fluorescence spectroscopy and electron
spin resonance techniques were used. The results of the
experiments suggest that silybin interacts with the surface
of lipid bilayers and induces small changes in the
biophysical properties of lipid membranes. Such an interaction may
explain why silybin exerts such beneficial effects without
causing any important side effects.
Materials and methods
Silybin (its chemical structure is presented in Figure 1A)
was purchased from Sigma (St Louis, MO, USA). Laurdan
(6-dodecanoyl-2-dimethylaminonaphthalene), Prodan
(6-propionyl-2-dimethylaminonaphthalene) and calcein were
purchased from Molecular Probes (Eugene, OR, USA).
1,6-diphenyl-1,3,5-hexatriene (DPH) and the lipids used in the
experiments, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC), egg yolk phosphatidylcholine (EYPC), were from
Sigma (St Louis, MO, USA). The spin probes, 5-doxylstearic
acid (5-DSA) and 16-doxylstearic acid (16-DSA), were also
purchased from Sigma (St. Louis, MO, USA).
Tempo-palmitate (TP) was synthesised at the University
of £ódŸ (£ódŸ,
Poland). All other chemicals were of analytical grade.
Small unilamellar liposomes (used in all experiments,
except calcein leakage studies) were prepared by sonication of
2 mmol/L phospholipid suspensions in 20 mmol/L Tris-HCl
buffer, 0.1 mmol/L EDTA, 50 mmol/L NaCl (pH 7.4) using a
UP 200 s sonicator (Dr Hilscher GmBH, Berlin, Germany).
Steady-state fluorescence measurements were performed
using a LS 50B spectrofluorimeter (Perkin-Elmer Ltd,
Beaconsfield, UK) equipped with a xenon lamp.
Fluorescence spectra were collected and processed using FLDM
Perkin-Elmer 2000 software (UK).
Theoretical calculations were made using Titan 1.0.8
software (Wavefunction Inc, Irvine, CA, USA and Schrodinger
Inc, Portland, OR, USA). Silybin properties were modeled
using the AM1 semi-empirical molecular orbital method.
Laurdan and Prodan generalized
polarization Laurdan and Prodan generalized polarization (GP) was determined in
small unilamellar liposomes made of DPPC, DMPC, and EYPC.
Stock solutions of Laurdan (1 mmol/L), Prodan (1 mmol/L)
and silybin (5 mmol/L) were prepared in DMSO. Liposomes
were incubated with fluorescent dye for 15 min, and
afterwards with modulator for 30 min (in dark conditions at room
temperature). The final concentrations of substances in the
examined samples were: 5 µmol/L Laurdan or Prodan, 200
µmol/L lipid, and 100 µmol/L silybin. The excitation
wavelengths for Laurdan ranged from 320 nm to 400 nm (every 10
nm). Prodan fluorescence was excited at 360 nm. Both
excitation and emission slit width was 5 nm. The temperature
was controlled by a water-circulating bath and measured
directly in the cuvette using a platinum thermometer.
Generalized polarization was calculated according to
equation 1 given by Parasassi et
al[25]. Fluorescence emission intensity of a blue edge of the spectrum
(IB) was measured at 440 nm for both fluorescence probes; fluorescence intensity
of a red spectrum edge (IR) was recorded at 490 nm for Laurdan
and 480 nm for Prodan.
(1)
DPH fluorescence Small unilamellar liposomes were
prepared from DPPC, DMPC or EYPC as described earlier. Stock
solution of DPH (1 mmol/L) was prepared in tetrahydrofuran.
Liposomes were incubated with DPH for 30 min, and
afterwards with silybin for the next 20 min (in dark conditions at
room temperature). The final concentrations of the
compounds in the samples were: 5 µmol/L DPH, 200 µmol/L lipid
and 25_150 µmol/L silybin. The experiments were performed
at room temperature. DPH fluorescence was excited at 350
nm and its emission was recorded at 428 nm. Fluorescence
polarization degree was calculated according to the
following equation:
 (2)
Where IVV is emission intensity measured with polarizer
parallel to the direction of polarization of the excitation light,
IVH is the same for the perpendicular polarizer, and G is the
instrumental correction factor calculated by FLDM
Perkin-Elmer 2000 software (Perkin-Elmer Ltd, Beaconsfield, UK).
DPH fluorescence lifetime measurements were performed
with a SLM Aminco 48000S frequency-domain instrument
(SLM Instruments Inc, Urbana, Illinois, USA) using a 450 W
xenon lamp as a light source, and frequency range of 1_250
MHz. The lifetime of DPH fluorescence decay was
calculated by multi-exponential analysis based on phase shifts
and demodulation parameters, using fluorimeter software.
As DPH fluorescence decay was not a single exponential
function, the average lifetime <t> was calculated according
to the following equation:
(3)
Where ti is fluorescence lifetime,
and fi is fractional intensity contribution.
Differential scanning calorimetry
(DSC) For each sample, 2 mg of lipid was dissolved in an appropriate amount
of silybin stock solution (5 mmol/L, in ethanol) to obtain the
desired drug:lipid molar ratio. We studied silybin:lipid
mixtures at 0.03, 0.045 and 0.06 molar ratios because in these
concentrations in the experiments with other flavonoids, we
observed pronounced changes in thermal behavior of lipids,
but bilayers were not perturbed by flavonoids as
much[26]. The samples were dried under the stream of nitrogen and
placed under vacuum for at least 2 h. Then 15 µL of 20
mmol/L Tris-HCl buffer (150 mmol/L NaCl, 0.5 mmol/L EDTA, pH
7.4) was added to each sample. Hydrated mixtures were
heated to the temperature circa about 10 °C higher than
main phase transition temperature of a given lipid, and
vortexed until a homogeneous dispersion was obtained.
Samples were sealed in aluminum pans and scanned at the
rate of 1.25 °C/min. Measurements were performed using
a Rigaku calorimeter (Rigaku, Tokyo, Japan), which was
partially rebuilt in our laboratory. Samples were scanned
immediately after preparation. For each drug:lipid molar
ratio, at least 2 samples were prepared; each sample was
scanned at least 4 times. Calorimetric data were collected
and processed offline using software developed in our
laboratory.
Electron spin resonance spectroscopy
(ESR) Samples for the ESR experiments were prepared from ethanol
solutions similar as those for DSC. Dry lipid films formed from
DPPC, DMPC or EYPC (5 mg per sample) and silybin were
hydrated with the addition of 250 µL of Tris-HCl buffer
(20 mmol/L, 150 mmol/L NaCl, 0.5 mmol/L EDTA, pH
7.4). Then the samples were shaken at room temperature for 5 min to obtain
multilamellar liposomes. All samples were incubated for at
least 12 h at 4 °C before the experiment.
All spin probes (TP, 5-DSA and 16-DSA) were dissolved
in ethanol (10 mmol/L stock solutions). The lipid:spin probe
molar ratio was 0.015 for DMPC and EYPC, and 0.05 for DPPC.
An appropriate amount of spin probe stock solution was
dried on the glass tube walls. Afterwards, the suspension of
liposomes was added to the tube and the mixture was
mechanically shaken. After 20 min, the spin probes were
incorporated into lipid bilayers and the liposomes were sufficiently
labeled for the ESR experiment.
The labeled liposomes were then transferred into a glass
capillary (1 mm inner diameter). All spectra were recorded at
25 °C using a standard SE/X-28 electron spin resonance
spectrometer (Wroclaw Technical University, Wroclaw, Poland)
operating in the X-band. In order to estimate the mobility of
the spin probes TP and 16-DSA during their isotropic
weakly-restricted rotational motion, the tumbling
correlation time (tc) was calculated using Kivelson's
method[27]:
tC=6.5×10-10w
0
[(h0/h-1)
0.5-1]
(4)
where w0,
h0 and h-1, are parameters taken from the ESR
spectrum, w0 is the midfield line width, and
h0, h-1 are mid-
and high-field line amplitudes.
The 5-DSA spin probe under experimental conditions
had strongly restricted motion in the membrane system. In
this case, the degree of restriction of its motion was
expressed by the order parameter (S), which was a measure
of the relative fluidity in the membranes. The order
parameter was calculated from the following equation:
S=[(A||
-A^)/(Azz_A
xx)](a/a') (5)
Where A|| and A^ are the maximal and minimal hyperfine
splitting constants measured, respectively,
Azz and Axx are
the hyperfine splitting tensors measured for probes in a
crystal matrix, and a and a' are the isotropic hyperfine splitting
constants for nitroxides in the crystal matrix:
a=1/3 (Azz+2Axx) (6)
And in the membranes:
a' = 1/3 (A|| +2A^)
(7)
Calcein leakage studies Unilamellar EYPC liposomes
were prepared by the extrusion method. First, the samples
containing chloroform solution of lipid were evaporated
under a stream of nitrogen. The remnants of organic solvent
were removed under vacuum for at least 2 h. Then the lipid
film was hydrated by vortexing with 1 mL of 36 mmol/L
solution of calcein in 10 mmol/L HEPES buffer (pH 7.4).
Liposomal suspensions were extruded through
polycarbonate filters, 400 nm and subsequently 100 nm, using a high pressure
extruder (PPH Marker, Wroclaw, Poland). The
calcein-containing vesicles were separated from the free calcein by
molecular filtration on a Sepharose 4B column eluted with 10
mmol/L HEPES buffer,150 mmol/L NaCl; 1 mmol/L EDTA (pH
7.4). The final lipid concentration in the liposome
suspension was 200 µmol/L. The liposomes containing calcein were
incubated with silybin (4_84 µmol/L) for 5 min (in dark
conditions at room temperature). Longer incubation times did not
change the results. The degree of calcein release was
determined spectrofluorimetrically. The fluorescence was excited
at 490 nm and emission was recorded at 520 nm. Both the
excitation and emission slit widths were set at 4 nm. The
results of the experiments are presented as the percentage of
released calcein (Frelease), calculated according to the
following equation:
 (8)
Where Ft is the fluorescence intensity of liposomes after
the addition of silybin, F0 is the fluorescence intensity of
liposomes, and F¥ is the total (100%) fluorescence intensity
measured after lysis of liposomes with Triton X-100
(detergent final concentration 0.5%).
Results
Molecular modeling of silybin properties Molecular
modeling showed that the silybin molecule was not planar
and had 2 bends between the policyclic structures (Figure
1B). These bends are likely to be responsible for the
flexibility of the silybin molecule. The octanol:water partition
coefficient of silybin was also calculated (lg P=1.53).
Laurdan and Prodan generalized
polarization The fluorescent probe Laurdan was employed to study the influence
of silybin on the thermal behavior of liposome membranes
formed from various lipids. For DPPC and DMPC liposomes,
an abrupt change of the GP value corresponded to the
phospholipid main phase transition. As shown in Figure 2, silybin
did not influence any of the studied phosphatidylcholine
bilayers significantly in the studied temperature range.
Transition temperatures of DPPC and DMPC were almost
unchanged in the presence of the flavonoid. Only in case of
DMPC did silybin cause a slight increase of GP values in the
gel state of the lipid (Figure 2B). The addition of 100
μmol/L silybin to the EYPC model membranes resulted in the slight
decrease of Laurdan GP values in temperatures higher than
30 °C (Figure 2C).
The shapes of the Laurdan fluorescence spectra
depended on the lipid phase state, showing an emission
maximum near 440 nm in the gel state and near 490 nm in the
liquid-crystalline state. Measuring Laurdan generalized
polarization as a function of excitation wavelength allows
not only characterization of the phase state of the lipid bilayer,
but also detects the phase separation, that is, the existence
of lipid microdomains[25]. The dependence of Laurdan GP on
the excitation wavelength recorded for DPPC, DMPC and
EYPC is shown in Figure 3. For DPPC (Figure 3A) and DMPC
(Figure 3B), the GP (lex) functions were plotted for the
temperatures below, above and during the main phase transition.
For pure lipids, the GP was not dependent on excitation
wavelength in temperatures below the phase transition. The curve
representing GP versus the lex relationship ascended during
the phase transition and descended in temperatures above
the lipid melting temperature. The addition of silybin did not
change either the DPPC or DMPC bilayer properties significantly. EYPC bilayers were in a liquid-crystalline state
in all the temperatures studied; that is why the negative slope
of GP on lex dependence was observed (Figure 3C); the
addition of silybin did not influence the EYPC bilayers.
Prodan was additionally used to investigate the effect of
silybin on DPPC membranes as this probe allowed the
monitoring of not only the main phase transition, but also the
phospholipid pretransition. As shown in Figure 4, the
addition of 100 μmol/L silybin to the DPPC liposomes caused the
decrease of Prodan GP values. This decrease was more
pronounced in temperatures in which DPPC was in gel state.
The biggest difference between the curve obtained for pure
lipid and for the DPPC:silybin mixture could be observed in
the region between 30 and 35°C, which suggested that silybin
strongly influenced DPPC pretransition.
DPH fluorescence DPH, a fluorescent probe localizing
deeply in the bilayer core[28], was employed to study the
influence of silybin on the hydrophobic region of model
membranes. The recorded values of DPH fluorescence
polarization were dependent on the type of phospholipid. The
values were near 0.4 in the DPPC bilayers that were in the
gel phase at the temperature of the experiment, 0.2 in
DMPC that was near the phase transition, and 0.1 in the
EYPC liquid-crystalline model systems. The addition of
silybin had almost no effect on DPH fluorescence
polarization in the DPPC membranes, whereas it caused
considerable increase in DPH polarization values in DMPC (about
17% at 100 μmol/L of silybin) and in the EYPC bilayers (near
30% at 100 μmol/L). No significant influence of the solvent
used (DMSO) on DPH fluorescence polarization was recorded.
At the same time, quenching of DPH fluorescence by
silybin was observed. Stern-Volmer plots of DPH quenching
were linear (Figure 5). The extent of quenching was similar in
all lipids studied.
As silybin-induced DPH fluorescence polarization
increase is accompanied by the decrease of fluorescence
intensity, we decided to study the DPH fluorescence
lifetimes too. The values of the DPH fluorescence lifetime in 3
investigated lipids and in their mixtures with silybin are shown
in Figure 6. In pure lipids, DPH had the longest lifetime in
membranes composed of DPPC, and the shortest in the
EYPC bilayers. The addition of silybin into DPPC liposomes caused
no significant changes in the DPH lifetime. Contrary to DPPC,
in the liquid-crystalline DMPC and EYPC membranes, the
shortening of the DPH fluorescence lifetime was observed
when the concentration of silybin was raised.
Differential scanning calorimetry To study the
influence of silybin on the thermotropic properties of lipid bilayers,
microcalorimetry was employed. The changes of transition
parameters of DMPC and DPPC caused by the addition of
the drug are presented in Table 1. In pure lipids, both
pre-transition and gel-liquid crystalline transition were recorded.
Pretransition vanished in both the DMPC and DPPC model
membranes, even at the lowest silybin:lipid molar ratio used
(0.03). Incorporation of silybin into the DMPC model
systems produced the lowering of lipid main phase transition
temperatures accompanied by calorimetric peak broadening.
Transition enthalpy of DMPC decreased in the presence of
silybin, however, no clear dependence of this parameter on
drug concentration was observed. DPPC membranes were
influenced by silybin to a lesser extent than those of DMPC.
First, we observed that the first thermogram recorded
immediately after the sample preparation always looked different
than the following ones. Different calorimetric peak shape
and a stronger lowering of the transition temperature were
observed during the first scan. The transition parameters
for DPPC presented in Table 1 were calculated only for
thermograms recorded after the samples reached equilibrium. The
transition temperature of the DPPC bilayers were slightly
lowered in the presence of silybin, whereas no significant
changes of transition enthalpy were observed. Both
parameters did not show clear dependence on silybin
concentra-tion.
Electron spin resonance spectroscopy Three types of
spin probes used in the ESR experiments (TP, 5-DSA and
16-DSA) enabled us to follow the mobility changes induced
by silybin in different regions of phosphatidylcholine
bilayers. Tempo-palmitate was used to monitor the lipid
polar heads region of the model membrane, 5-DSA probed
polar/apolar interface, while 16-DSA delivered information from
the hydrophobic region close to the bilayer centre. The
spectral parameters obtained for the spin probes are
presented in Figure 7. The results showed that the addition of
silybin to liposomes caused TP spin probe immobilization in
all the studied lipids (Figure 7A). On the other hand, silybin
exerted almost no effect on order parameter of the 5-DSA
spin probe (Figure 7B), only in case of DMPC bilayers slight
immobilization of 5-DSA was visible. The addition of silybin
had no effect also on spin probe 16-DSA located deeply in
the membrane core (Figure 7C). It was also noticed that the
effect of silybin on model membranes was not dependent on
the bilayer phase state. Although all the experiments were
performed at 25 °C, the temperature in which DPPC was in
the gel state, DMPC was slightly above main phase
transi-tion, and EYPC was in the liquid-crystalline state, the
influence of silybin on the model membranes composed of
different lipids was similar, no matter which spin probe was
employed.
Calcein leakage studies The influence of silybin on the
EYPC bilayer structure integrity and stability was
investigated using calcein leakage assay. Calcein was entrapped in
liposomes in such a concentration that its fluorescence was
self-quenched. Disturbance of lipid bilayer by silybin caused
the increase of membrane permeability and
concentration-dependent release of the fluorescence probe from liposomes
(Figure 8). The release of the fluorescence probe grew when
the concentrations of silybin were raised to 25 μmol/L,
whereas for higher flavonoid concentrations, only a slight
further increase of calcein leakage was observed. Maximal
release of calcein caused by the flavonoid was about 29%. It
was observed that DMSO alone was able to cause only slight
calcein leakage (up to 5%).
Discussion
In the present work, we have systematically studied the
influence of silybin on biophysical properties of
phospholipid bilayers. In our experiments, we used model membranes
composed of zwitterionic lipid, phosphatidylcholine. Two
phosphatidylcholine species differing in length of the acyl
chains were used (DPPC and DMPC), as well as a mixture of
natural phosphatidylcholines (EYPC).
The value of the octanol/water partition coefficient
calculated for silybin indicated relatively low hydrophobicity
of this compound. Based on this feature, one should not
expect strong silybin_membrane interactions. On the other
hand, molecular modeling showed that the silybin molecule
was not planar, and high flexibility of its molecule could be
expected. The ability of the silybin molecule to adopt
different conformations is likely to influence its interaction with
phospholipid bilayers. The importance of spatial
conformation of flavonoid molecules for their intercalation into model
membranes has been previously
suggested[29,30].
To further characterize the interactions of silybin with
phospholipid bilayers, fluorescence spectroscopy was
em-ployed. Laurdan is a fluorescent probe possessing lauric
acid tail that anchors in the hydrophobic core of the
membrane, and in which fluorophore resides at the level of
the phospholipid glycerol backbone. The spectral
properties of this probe are extremely dependent on the amount
of the water molecules penetrating into the
hydrophobic-hydrophilic interface of the membrane and on their dynamics,
especially on the dipolar relaxation process in Laurdan's
environment[31]. Since gel and liquid-crystalline phases of
phospholipid bilayers differ in hydration and water dynamics,
Laurdan constitutes a useful tool to study lipid thermotropic
behavior. As observed by Laurdan fluore-scence, the
addition of silybin into the phosphatidylcholine bilayer did not
cause any pronounced effects. GP values for pure lipids and
the lipid:silybin mixtures were virtually the same, only DMPC
in gel state, and EYPC in high temperatures could small
influences of silybin addition be observed. The influence of this
compound on the temperature of the main phase transition
of DMPC and DPPC was also negligible.
Laurdan can also be used to detect the phase separation,
that is, the existence of lipid
microdomains[25]. Measuring Laurdan GP as a function of excitation wavelength allowed
us to check whether the addition of silybin to the model
bilayers affected lateral membrane organization. In no case
such an influence of silybin was detected. In general, the
presence of silybin in phospholipid systems seemed not to
alter significantly the packing of the lipid membrane in the
vicinity of Laurdan molecules.
Prodan possesses the same fluorophore as Laurdan, but
connected to a shorter propionyl tail. That is why it is more
loosely anchored to the lipid bilayer and located closer to
the membrane surface than Laurdan[31]. By following
Prodan's GP as a function of temperature, the pretransition
of the lipid bilayer can be detected. The biggest difference
of the course of GP on temperature dependence recorded in
the DPPC:silybin mixture as compared to pure lipid was
visible in temperatures typical for DPPC pretransition. This
indicates that silybin interacts with the polar head-group
region of the DPPC membrane strongly enough to affect
pretransition. In contrast to Laurdan, Prodan exhibits quite
significant fluorescence in water, which influenced the
values of GP calculated for this probe. The possibility of
interaction between Prodan and silybin present in the buffer was
excluded by the experiment in which we observed no
apparent influence of flavonoid on the emission spectrum of the
fluorescent probe. It was shown that the Prodan partition
coefficient between the phosphatidylcholine bilayer and
water strongly depended on the lipid's phase state, being
higher in liquid-crystalline phase[32]. Lower GP values were
observed for the silybin:DPPC mixture than for pure lipid,
which could be at least partially caused by the higher
number of Prodan molecules residing outside the membrane in
the presence of the flavonoid. Fluorescence originating from
water-immersed Prodan molecules raised the total emission
intensity of a red spectrum edge thus reducing the GP value.
We hypothesized that the presence of silybin decreased
Prodan partitioning into DPPC bilayers, especially in the gel
phase. Such an effect is likely to be caused by silybin
interaction with the same membrane region where Prodan is
located.
Fluorescence of DPH, the probe localizing in the
hydrophobic core of the membrane, was also affected by the
addition of silybin to the liposomes. The increase of
fluorescence polarization was observed in DMPC and EYPC
bilayers, but not in those of DPPC. However, this increase
was likely to be apparent as the presence of silybin resulted
in DPH fluorescence lifetime shortening in these model
systems where the polarization increase was recorded.
Previous researchers[21] reported only a slight influence of
silybin on DPH fluorescence anisotropy when incorporated
into rabbit liver microsomes. We have also observed that
the addition of silybin into liposomes caused a reduction in
DPH fluorescence intensity. Other flavonoids have been
also reported to be able to quench DPH
fluorescence[33]. As Stern-Volmer plots of fluorescence quenching were linear,
and lifetime shortening was seen in EYPC and DMPC model
membranes, we concluded that silybin-induced changes of
DPH fluorescence were the manifestation of collisional
quenching of this probe by the flavonoid. Where such a
quenching mechanism seemed probable in EYPC and DMPC
bilayers, static quenching seemed to prevail in more
compactly-packed DPPC bilayers in which no lifetime
shortening was observed. The difference in quenching mechanisms
between different phosphatidylcholine species was likely to
result from loosely-packed structures of liquid-crystalline
bilayers of EYPC or DMPC, and more compact structures of
DPPC being in gel state in the temperature of the experiment.
DPH fluorescence lifetime shortening observed in EYPC and
DMPC in the presence of silybin could accompany the
quenching, but the DPH lifetime was also reported to be
shortened by increased water penetration into the membrane
interior[34]. In our opinion, this was not the case since
fluorescence of Laurdan was not altered by the flavonoid. It is
doubtful that silybin would have caused distinct hydration
increase deep in the membrane core without even slightly
changing the amount of water associated with the
hydrophobic_hydrophilic interface of membranes.
We assumed the quenching of DPH fluorescence by
silybin was caused by the interaction of the 2 molecules
inside the membrane, requiring direct contact between the 2
entities. Quenching by FRET (fluorescence resonance
energy transfer) was excluded as the absorption spectrum of
the flavonoid did not overlap with the emission spectrum of
DPH. Silybin's molecule is quite long, and when
incorporated into the lipid bilayer, it could penetrate deeply into the
membrane. It has been shown previously that flavonoids
are able to influence the fluorescence of anthroyloxy-fatty
acids with fluorophores located at different depths from the
membrane surface[35,23]. It has also been demonstrated that
the membrane distribution of flavonoids is quite
broad[36] which suggests that at least a portion of silybin molecules
could interact with DPH when incorporated into the lipid
bilayer.
Microcalorimetry was employed to investigate the
influence of silybin on the thermotropic properties of the DMPC
and DPPC bilayers. It has been demonstrated that the
flavonoid altered the structure of the phospholipid bilayer in
the gel state as the addition of the drug resulted in the
vanishing of pretransition in both lipids used. The
disappearance of DPPC pretransition recorded by DSC was in
accordance with silybin-induced changes in Prodan-generalized
polarization observed by fluorescence spectroscopy.
Studying the influence of silybin on main lipid phase transition, we
noticed that the flavonoid affected mainly the transition
temperature, while its impact on the transition enthalpy was
weaker. Such behavior is recognized to be characteristic for
modifiers that interact mainly with the polar head group
region of the bilayer and only partially penetrate into the
hydrocarbon region[37]. Similar changes on the DPPC
thermotropic properties as exerted by silybin have been previously
observed for other flavonoids[29]. Silybin perturbed the
DMPC bilayer structure stronger than the DPPC model
membranes. In our opinion, this could be explained by the
fact that DMPC, possessing shorter acyl chains than DPPC,
is also characterized by weaker interactions between
hydrocarbon chains and more loosely-packed structures which
could facilitate silybin incorporation into DMPC membranes.
It has been also observed that the shapes of calorimetric
peaks of the DPPC:silybin mixtures varied as a function of
time as if the mixture reached equilibrium only after some
time after the sample preparation. Quercetin and hesperetin
have been also demonstrated to exhibit time-dependent
changes in thermograms recorded in DPPC
membranes[29].
ESR experiments showed that silybin did not change the
mobility of spin probes embedded either in polar/apolar
interface or in hydrophobic regions of the membrane. On the
other hand, the tumbling correlation time of
Tempo-palmitate was increased in the presence of silybin which indicated
the immobilization of the spin probe. Based on the results of
the ESR experiments, we concluded that silybin interacted
with the polar head group region of the lipid bilayers while
exerting almost no effect on deeper regions of the membrane.
Similar conclusions were drawn previously in the ESR study
on quercetin influence on human erythrocyte
membranes[38].
We have also shown that silybin was able to increase the
permeability of liquid crystalline EYPC membranes to calcein.
The overall increase of EYPC membrane permeability in the
presence of silybin was not very large. The increase was
most rapid in low silybin concentrations, whereas higher
flavonoid concentrations induced only slight further
permeability increase, as if the effect exerted by this compound
on the membrane became saturated. It seemed likely that
the introduction of low amounts of silybin molecules into
the membrane resulted in the transient changes of bilayer
packing and the appearance of membrane defects.
Flavonoids possessing several hydroxyl groups in the molecule
were postulated to form multiple hydrogen bonds with
polar heads of phospholipids. Such a mechanism was
proposed to explain the protective properties of flavonoids against
membrane oxidation and solubilization by
detergents[23,24]. Therefore, it seemed possible that increasing amounts of
silybin molecules formed a network stabilizing the surface
of liposome membrane. At concentrations high enough,
the silybin-induced processes of membrane destabilisation
and sealing balanced, thus leading to the saturation of
calcein leakage. It should be also noted that EYPC
membranes are more loosely packed and more vulnerable to
perturbation than natural membranes. We observed that
silybin in concentrations up to 150 μmol/L was not able to
induce erythrocyte hemolysis (data not presented). In our
opinion, silybin is not likely to induce strong alterations of
natural membrane integrity and stability.
The results presented show that silybin interacts mainly
with the polar head group region of the lipid bilayers. The
ability of silybin to quench the fluorescence of DPH
molecules in the membranes indicate that at least some of the
flavonoid molecules can partition into the hydrophobic
region of the membrane. This partitioning, however, does not
change the biophysical properties of deeper membrane
regions in a significant way. In our opinion, such a behavior
of silybin in membranes is in accordance with its postulated
biological functions, such as membrane stabilization and cell
protection. Moreover, the absence of serious perturbations
of membrane biophysical properties induced by silybin may
correspond to the observed lack of serious side-effects of
this drug, in spite of its use in therapy in relatively high
doses. Further research is needed, however, to determine
the precise membrane localization of silybin molecules.
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