Biochimica et Biophysica Acta 1858 (2016) 363–373

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Biochimica et Biophysica Acta

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Complexation of phospholipids and cholesterol by triterpenic saponins in

bulk and in monolayers
Kamil Wojciechowski a,⁎, Marta Orczyk a, Thomas Gutberlet b, Thomas Geue c
a
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
b
Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany
c
Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institute, WHGA/110, 5232 Villigen — PSI, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: The interactions between three triterpene saponins: α-hederin, hederacoside C and ammonium glycyrrhizate
Received 16 October 2015 with model lipids: cholesterol and dipalmitoylphosphatidylcholine (DPPC) are described. The oleanolic acid-
Accepted 1 December 2015 type saponins (α-hederin and hederacoside C) were shown to form 1:1 complexes with lipids in bulk, character-
Available online 2 December 2015
ized by stability constants in the range (4.0 ± 0.2)·103–(5.0 ± 0.4)·104 M−1. The complexes with cholesterol are
generally stronger than those with DPPC. On the contrary, ammonium glycyrrhizate does not form complexes
Keywords:
Biosurfactant
with any of the lipids in solution. The saponin–lipid interactions were also studied in a confined environment
Saponin of Langmuir monolayers of DPPC and DPPC/cholesterol with the saponins present in the subphase. A combined
α-Hederin monolayer relaxation, surface dilational rheology, fluorescence microscopy and neutron reflectivity (NR) study
Hederacoside C showed that all three saponins are able to penetrate pure DPPC and mixed DPPC/cholesterol monolayers. Overall,
Ammonium glycyrrhizate the effect of the saponins on the model lipid monolayers does not fully correlate with the lipid–saponin complex
Surface pressure formation in the homogeneous solution. The best correlation was found for α-hederin, for which even the pref-
Dilatational surface rheology erence for cholesterol over DPPC observed in bulk is well reflected in the monolayer studies and the literature
data on its membranolytic activity. Similarly, the lack of interaction of ammonium glycyrrhizate with both lipids
is evident equally in bulk and monolayer experiments, as well as in its weak membranolytic activity. The com-
bined bulk and monolayer results are discussed in view of the role of confinement in modulating the saponin–
lipid interactions and possible mechanism of membranolytic activity of saponins.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction microscopy observations of saponin-treated biological cell membranes

Saponins are glycosidic biosurfactants produced by numerous or-
ganisms, mainly plants [1,2]. Despite a great variety of chemical struc-
tures, the common feature of all saponins is the presence of at least
one glycoside bond linking an aglycone part (steroid or triterpenoid)
with a glycone (mono- or oligosugar) part. The biological role of sapo-
nins is still largely unknown. However, the fact that they are stored in
parts of a plant most susceptible to attack by predators and are often
produced in response to threats [3,4] suggests that they play active
role in plant's self-defense [1,3]. Many saponins display membranolytic
activities towards bacteria, viruses and fungi, and some are even capable
of lysing the red blood cell (erythrocyte) membranes [5]. The mem-
brane activity of both triterpenoid and steroid saponins has been tradi-
tionally associated with their ability to interact with membrane-bound
cholesterol [6]. Following the observations of Ransom [7] and Windaus
[8], saponins were initially believed to simply remove cholesterol from
biological membranes [9], leaving the cytosol-permeable pores. The
works of Schulman and Rideal [10,11], as well as subsequent electron
⁎ Corresponding author.
E-mail address: kamil.wojciechowski@ch.pw.edu.pl (K. Wojciechowski).
[12,13], were however more consistent with the hypothesis of penetra-
tion of saponins into the lipid layers and subsequent disturbance of
cholesterol's interactions with other lipids (phospho- and
sphingolipids) [14]. On the other hand, some reports suggest that cho-
lesterol is not crucial for the saponins' membranolytic activity [15] or
that their membrane activity is related to interactions with specific re-
ceptors [16].
Despite some claims [17], usually no clear correlation can be found
between the surface activity of saponins and their membranolytic activity.
Moreover, the ability to lower surface tension (hence the detergent activ-
ity) of saponins is rather weak at biologically-relevant concentrations. For
example, typical concentrations for 50% cytotoxic activity, IC50, for most
hemolytic saponins are in the range of 10 μM [18]. Our recent studies
with Quillaja bark saponins (QBS) clearly show that interaction of sapo-
nins with lipid membranes is much more complex than one would expect
from a simple surfactant activity [19–21].
To the best of our knowledge no successful attempts have been
described so far to systematically compare the strength of interac-
tions between different saponins and membrane lipids, especially
phospholipids and cholesterol. Some preliminary information can
be deduced from molecular dynamics (MD) investigation which

http://dx.doi.org/10.1016/j.bbamem.2015.12.001
0005-2736/© 2015 Elsevier B.V. All rights reserved.
364 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
showed that the binding free energy for dioscin–cholesterol in dec-
ane is significantly more favorable (− 19.10 kcal/mol) than for cho-
lesterol–cholesterol complexes (− 0.77 kcal/mol) [14].
Three triterpenoid saponins were selected for this study (Fig. 1): α-
hederin, hederacoside C and glycyrrhizic acid (ammonium salt). The
first two originate from the same plant (Hedera helix) and share the
same aglycone (hederagenin, belonging to the oleanolic acid-type
triterpenes). They differ in the extent of sugar substitution: α-hederin
is a monodesmoside with one disaccharide (α-L-rhamnose-(1 → 2)-
α-L-arabinose) group attached at the C3 position of the aglycone,
while hederacoside C is a bidesmoside, additionally substituted at C28
with a trisaccharide group. Both saponins are active components of
cough syrups, where they act as mucolytic and secretolytic agents.
The aglycone part of glycyrrhizic acid (from Glycyrrhiza glabra L) is of
glycyrrhetinic acid type and is substituted only at C3 position
(monodesmoside) with a disaccharide moiety. Because of its low toxic-
ity, it finds many applications in healthcare and food industries, e.g., as a
low calorie sweetener [22].
The three saponins differ significantly in hemolytic activity. α-
Hederin is one of the most hemolytic triterpenoid saponins, with HD50
and HD100 (referring to the concentrations causing 50% and 100% hemo-
lysis, respectively) of 11.3 and 28.8 μM. For comparison, hederacoside C
shows HD50 N 100 μM [23]. Glycyrrhizic acid shows no hemolytic activ-
ity at least up to 48 μM (the highest concentration tested in [24]). In
membrane toxicity study by Böttger et al. [17] using human urinary
bladder epithelial carcinoma cells (ECV-304), the monodesmoside
α-hederin was found by far the most cytotoxic (IC50 = 29 μM). Its
bidesmoside analog, hederacoside C, was much less toxic to the same
cells (IC50 N 238 μM). Glycyrrhizic acid was not toxic either, at least up
to the maximum studied concentration (IC50 N 164 μM). The membrane
toxicity of the three saponins in ECV-304 cell is closely related to their
effect on membrane cholesterol: glycyrrhizic acid and hederacoside C
did not affect its content in the cells, while α-hederin significantly
reduced it [9]. These observations suggest that both the glycone and
aglycone parts of a saponin molecule play important roles in its
membranolytic activity.
The limited knowledge about the mechanism of saponin–lipid inter-
actions prompted us to investigate in more detail the stoichiometry and
stability of the three triterpenoid saponin–lipid complexes using choles-
terol and 1,2-dipalmitoylphosphatidylcholine (DPPC) as model mem-
brane lipids. The common aglycone moiety was chosen in order to
allow for general conclusions concerning the role of nature of glycones
in interactions between saponins and lipids. To the best of our knowl-
edge chemical interactions have not been considered so far as an impor-
tant contribution to the membranolytic activity of saponins. In this
contribution we seek for a possible correlation between the lipid–
Fig. 1. Chemical structures of saponins (upper row, from left to right): α-hederin, hederacoside C and ammonium glycyrrhizate, as well as lipids (bottom row, from left to right): choles-
terol and 1,2-dipalmitoylphosphatidylcholine (DPPC).
saponin complex stability and the extent of lipid monolayer penetration
by the given saponin.
2. Experimental
For all experiments, α-hederin (Cat. No. 9970.1), hederacoside C
(Cat. No. 6775.1) and ammonium glycyrrhizate (ammonium salt of
glycyrrhizic acid, Cat. No. 6247.1) were purchased from Carl Roth
and were used as received. Chloroform (CHROMASOLV for HPLC),
ethanol (pure p.a.) and dimethylsulphoxide (pure p.a.) were pur-
chased from Sigma-Aldrich and POCh. The 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC) with purity of 99% and cholester-
ol with purity of 99.5% were purchased from Sigma-Aldrich. Milli-Q
water (Millipore) was used to prepare all aqueous solutions. All
glassware for Langmuir trough experiments was cleaned with ace-
tone and Hellmanex II solution (Hellma Worldwide) and subse-
quently rinsed with copious amounts of Millipore water.
2.1. Surface pressure
All measurements were carried out with a KSV NIMA small Lang-
muir–Blodgett trough (77.5 cm2) equipped with two movable barriers.
Each measurement was performed at least in duplicate; in case of any
significant discrepancies it was repeated further. The surface pressure
was measured using a Wilhelmy plate made of filter paper (Whatman
Chr1) connected to an electro balance. The speed of barrier movement
was set to 10 mm/min, as suggested by the manufacturer. For the sur-
face pressure vs. time measurements, the trough was filled with an ap-
propriate saponin solution (10−4 M) in Milli-Q water (with addition of
4% DMSO in the case of α-hederin for poor solubility reasons) and the
surface pressure was recorded for 1 h. Temperature was controlled at
21 °C by water circulation from a thermostat. The Wilhelmy balance
was zeroed on pure Milli-Q water just before pouring the saponin
solution.
2.2. Subphase exchange
The subphase exchange setup consisted of a Gilson's MINPULS 3
peristaltic pump and Teflon tubings. The in and out tubings were im-
mersed in the subphase on opposite sides of the trough to introduce the
concentrated saponin solution and to recirculate the subphase, in order
to achieve the final saponin concentration of 10−4 M. Hederacoside C
and ammonium glycyrrhizate were dissolved in Milli-Q water, and for
α-hederin 4% of DMSO was added to improve its solubility. Accordingly,
the subphase consisted of either pure Milli-Q water or water with
addition of DMSO (4%). The lipid (DPPC and DPPC/cholesterol 10:9)
 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
monolayers were spread first on pure water as chloroform solutions and
after the solvent evaporated, the monolayers were compressed to the ini-
tial surface pressure of 32.5 mN/m. Then the concentrated saponin solu-
tion was introduced at the flow rate of 2 ml/min. The flow and the
barrier position were maintained for 1 h and the changes in surface pres-
sure, Π(t), induced by the presence of saponins were monitored.
2.3. Dilatational rheology
Mechanical properties of the Langmuir monolayers and adsorbed
layers were probed using the Langmuir trough. At the end of each Π
vs. time measurement harmonic oscillations of the area per molecule
were applied by moving both barrier positions around the initial loca-
tion with the frequency of 0.1 Hz and amplitude of 2%, which is within
the viscoelastic linear regime for the system under study. Each measure-
ment consisted of several harmonic compression/decompression cycles
for 300 s. The two moduli were obtained from the Fourier transform of
harmonic oscillations of the surface pressure in response to the applied
strain (area oscillations) using the KSV NIMA software.
The dilatational visco-elasticity modulus, | E(ω)|, is defined as
follows:
dπ
jEðωÞj ¼ − A
dA
where A is the molecular area at a given surface pressure π.
The visco-elasticity modulus E(ω) is a complex quantity and consists
of two frequency-dependent contributions: storage (E′) and loss (E″)
moduli:
EðωÞ ¼ E0 ðωÞ þ iE″ ðωÞ:
2.4. Fluorescence microscopy
In order to observe the topography of the monolayers exposed to the
action of triterpenic saponins an OLYMPUS BX51WI inverted fluores-
cence microscope was used. The microphotographs were taken during
the subphase exchange for the appropriate saponin solution, as
described for Π(t) measurements. As a fluorescent dye TopFluor PC (1-
palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-
3-phosphocholine) was used (1 mol% vs the lipid). The dye was of
N99% purity and was supplied by Avanti Lipids. Fluorescence micrographs
from the samples were collected using a MPLFN10X objective and the im-
ages were processed using cellSens software.
2.5. Neutron reflectivity
In a reflectivity measurement the beam intensity, R, reflected
from a surface, normalized to the incoming intensity at a certain
angle of incidence, θ is recorded. In case of a neutron reflectivity
(NR) experiment, R defined as above is measured as a function of
scattering wave vector, q, expressed as the incidence angle θ normal-
ized to the corresponding wavelength, λ, as qz = 4π sin(θ)/λ. NR
measurements were performed at the time-of-flight neutron
reflectometer AMOR (Paul Scherrer Institute (PSI), Villigen,
Switzerland) [25]. The time-of-flight method allows the recording
of a wide range of q values in a single angular setting.
The NR experiments at air/water interface were performed at three
angles of incidence (0.25°, 0.65° and 1.4°) using a wavelength band
from 3 to 12 Å, covering the necessary q range for the experiments of
qzmin = 0.01 Å−1 to qzmax = 0.15 Å−1. The resolution was set by a slit
system on the incident side and the time-of-flight parameters to
Δqz = 0.006 Å− 1. A beam of rectangular cross-section 1 × 35 mm2
(for low angles) impinged on the samples at the air/water Langmuir
365
trough. The scattered neutrons were recorded with a 3He-single detec-
tor tube in time-of-flight mode requiring typically 8 h of beam time.
The experimentally obtained reflectivity curves were analyzed by
applying the standard fitting routine, using the Parratt 32 software
[26]. It determines the optical reflectivity of neutrons from planar sur-
faces using a calculation based on Parratt's recursion scheme for strati-
fied media [27]. The reflecting interface is modeled as consisting of
layers of specific thickness, scattering length density and roughness,
which are the fitting parameters. The neutron scattering length density
(SLD) is defined by the sum of the bound coherent scattering length bc of
the reflecting material normalized by the volume, v, as SLD = ∑bc/v.
The model reflectivity profile is calculated and compared to the mea-
sured data, then the model is recursively adjusted by a change in the
fitting parameters to best fit the data.
3. Results
3.1. Stoichiometry and stability constants of binary complexes of triterpene
saponins with cholesterol and DPPC
The actual location of the saponin–lipid complex formation in real
biological systems is not a priori known. As described in the
Introduction, the complexes could form within the membrane (pene-
tration of a saponin), in the surrounding aqueous phase (solubilization
of cholesterol) or at the interface between the polar aqueous phase sur-
rounding the lipid membrane and the non-polar lipid layer. Attempts
were first made to predict the most probable complex location by
using extraction procedures. Unfortunately, liquid–liquid extraction
using an aqueous solution of saponin and a tetradecane solution of
lipids could not be used for this purpose, because of technical problems
with separating the phases after extraction (emulsion formation).
Instead, a solid–liquid extraction was employed: solid cholesterol
was extracted with an aqueous solution of QBS (10−3 M), and a solid
QBS — with a tetradecane solution of cholesterol (10−3 M). After 24 h
extraction with the respective solids, the liquid phases (aqueous or
tetradecane) were assessed by UV/Vis. No noticeable changes in their
spectra could be found, suggesting that under these conditions the sa-
ponin–cholesterol complex is formed neither within the membrane,
nor in the surrounding aqueous phase. Therefore, to mimic an interme-
diate polarity of the membrane-aqueous interface, ethanol/water mix-
tures were used (95:5 for α-hederin and hederacoside C, and 90:10
for ammonium glycyrrhizate).
The complex stoichiometry was assessed by employing a continuous
variation method (“Job's method”). For this purpose the UV/Vis spectra
of saponin–lipid mixtures with continuously varying molar ratio but
constant total concentration were acquired, as described in the
Experimental part. A representative curve (so called “Job plot”) for α-
hederin/cholesterol mixtures is shown as an inset in Fig. 2, with a char-
acteristic maximum at the molar ratio = 0.5, indicative of 1:1 complex
stoichiometry. Similar curves were obtained for α-hederin and
hederacoside C mixtures with both cholesterol and DPPC.
In order to determine the stability constants of the respective sapo-
nin–lipid complexes, UV/Vis titrations were performed with constant
concentration of the lipid and increasing concentrations of saponins
(see Fig. 2 for a representative set of spectra). For both hederagenin sa-
ponins, clear inflection points around the saponin-to-lipid ratio of one
were observed, consistent with the Job method results. The correspond-
ing titration curves could be fitted using a 1:1 complexation model. The
global fitting procedure (simultaneously at typically two wavelengths)
provided the stability constants of α-hederin and hederacoside com-
plexes as collected in Table 1. On the other hand, for ammonium
glycyrrhizate, the Job plots for both DPPC and cholesterol were almost
flat, suggesting weak interaction of both lipids with this saponin in the
mixed ethanol:water solvent. This was further confirmed by UV/Vis ti-
tration, where no inflection point could be observed on an absorbance
vs saponin-to-lipid ratio.
366 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
Fig. 2. UV/Vis spectra for 2·10−4 M cholesterol solution titrated with 4·10−3 M α-hederin solution (the red arrow shows a direction of changes with increasing saponin concentration).
Inset shows a Job's plot for α-hederin/cholesterol system at λ = 212 nm. All spectra recorded in 95% ethanol.
3.2. Saponin–lipid interactions at the water–air interface
3.2.1. Surface pressure
Spontaneous adsorption of the triterpene saponins on a bare water–
air interface (formation of Gibbs layers) was studied using a Langmuir
trough. The ability to lower surface tension of aqueous solutions
(i.e., to increase surface pressure) for pure saponins at the same concen-
tration (c = 10−4 M) is compared in Fig. 3. The monodesmosidic α-
hederin increases surface pressure by nearly 20 mN/m more than its
bidesmosidic analog, hederacoside C. This suggests that the presence
of the second glycone group located at the opposite end of the
triterpene backbone with respect to C-3 (at C-28) disturbs the
lipophilic-hydrophilic balance of the molecule and renders it more
water-soluble, but also less amphiphilic. Neutralization of the negative
ionic charge of a free carboxylic group (in α-hederin) by its esterifica-
tion (in hederacoside C) might also play a role in lowering the surface
activity. In another monodesmosidic saponin used in this study, ammo-
nium glycyrrhizate, the presence of a ketone group and the location of
the free carboxylic acid group (C-30 vs C-28 in α-hederin) apparently
disfavor adsorption at the water–air interface. Overall, the trend of
lowering ability to increase the surface pressure (decrease surface ten-
sion) follows the order: α-hederin N ammonium glycyrrhizate N
hederacoside C.
A similar trend can be observed for surface dilational storage and
loss moduli of the Gibbs layers after 1 h adsorption (Table 2). Because
of different surface pressures achieved after 1 h adsorption for each sa-
ponin, the values cannot be directly compared. Nevertheless, it is clear
that the layers formed by α-hederin are characterized by exceptionally
Table 1
Stability constants for saponin–lipid binary complexes determined from UV/Vis titration
in 90% (for α-hederin and hederacoside C) or 95% (for ammonium glycyrrhizate) ethanol.
Saponin Lipid Ka, M−1
α-Hederin Cholesterol (5.0 ± 0.4)·104
DPPC (5.0 ± 0.2)·103
Hederacoside C Cholesterol (9.0 ± 0.3)·103
DPPC (4.0 ± 0.2)·103
Ammonium glycyrrhizate Cholesterol No complex formation
DPPC No complex formation
high values of both moduli. This feature is typical for monodesmosidic
saponins capable of interacting by means of both hydrogen bonding
and hydrophobic interactions [28]. The values for ammonium
glycyrrhizate are also high, but significantly lower than for α-hederin.
In agreement with the surface pressure results, the bidesmosidic analog
of a-hederin (hederacoside C) shows negligible values of both moduli.
In the next step, the interaction of the triterpene saponins with
DPPC and cholesterol at the model membrane–aqueous interface
was studied using Langmuir monolayers. Pure DPPC and DPPC/cho-
lesterol (10:9 mol/mol) mixed monolayers were first spread on
Milli-Q water and compressed to 32.5 mN/m. Next, the subphase
was exchanged with the appropriate triterpene saponin solution
to give the final concentration of 10 − 4 M, as described in the
Experimental part. The surface pressure vs time curves are shown
in Fig. 3. Note that the time evolution of surface pressure starts at
the same time as the subphase exchange and that the complete sub-
phase exchange in the employed setup takes about 15 min [19]. For
DPPC monolayers the effect of the saponins follows the same order
as for the respective Gibbs layers on bare water/air interface. This
suggests that it is not the interaction of saponins with DPPC but rath-
er their intrinsic amphiphilicity that drives their penetration into
DPPC monolayers. The weakly adsorbing hederacoside C has practi-
cally no effect on surface pressure of the DPPC monolayer (the relax-
ation curve is very similar to that on pure water, without subphase
exchange, not shown). In contrast, the presence of α-hederin sharply
increases the surface pressure to about 45 mN/m, i.e., 12 mN/m
above the surface pressure value prior to the subphase exchange.
For ammonium glycyrrhizate the increase of surface pressure is
much slower than for the Gibbs layer on bare water/air interface.
The kinetics of glycyrrhizate adsorption is thus slowed down when
the lipid layer is present, or that the latter undergoes slow rearrange-
ment in the presence of ammonium glycyrrhizate.
Similar relaxation behavior can be observed for this saponin also
when cholesterol is present in the mixed lipid monolayer. This might
suggest that ammonium glycyrrhizate does not differentiate between
DPPC and cholesterol. Despite distinct differences between ammonium
glycyrrhizate and hederacoside C on a free water surface and on DPPC
monolayers, in the presence of cholesterol their effect on surface pres-
sure is rather similar. Among the three investigated saponins, α-
 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373 367

Fig. 3. Surface pressure vs time curves for Gibbs layers formed from aqueous solutions (c = 10−4 M) of pure saponins (□) and for Langmuir monolayers during the subphase exchange
following compression to 32.5 mN/m: DPPC ( ) and DPPC/cholesterol 10:9 ( ). Left panel: ammonium glycyrrhizate, middle panel: hederacoside C, and right panel: α-hederin (inset
shows expansion of the curve for DPPC/cholesterol on α-hederin). The green dashed line corresponds to the initial value of surface pressure, Π0 = 32.5 mN/m.

hederin is clearly the most sensitive to the presence of cholesterol. responsible for the high visco-elasticity of its layers on water, are there-
During the first minutes of subphase exchange (even before it is fore weakened by the saponin molecules penetrating the monolayer,
completed) the surface pressure rises very steeply and few sudden especially in the case of hederacoside C. The latter observation is sur-
jumps can be noticed (see the inset in Fig. 3, right panel). These pres- prising in view of the weak effect of this saponin on relaxation behavior
sure jumps might suggest a partial collapse of the lipid monolayer as well as on the neutron reflectivity curve (see below) of the DPPC
penetrated by α-hederin. However, if the lipid layer were replaced monolayer.
by a detergent (as it was observed e.g., for ionic synthetic surfac- On the other hand, the effect of α-hederin on dilatational surface rhe-
tants: SDS or CTAB), the surface pressure would decrease to the ological response of DPPC monolayers is markedly different (note that
levels comparable with those for the respective Gibbs layers of the these experiments were performed on water containing 4% DMSO for sol-
pure surfactant [19]. In the case of α-hederin and DPPC/cholesterol, ubility reasons). The storage modulus, E′ = 188.6 mN/m, seems not to be
the monolayer replacement by the saponin can be excluded given significantly affected by the saponin penetration (for DPPC on water/
the consecutive increase of surface pressure and its stabilization at DMSO 4%, E′ = 181.8 mN/m, close to the value for DPPC on pure water,
about 50 mN/m, i.e., over 17 mN/m higher than the initial value be- E′ = 176.5 mN/m). However, the viscous component (loss modulus) is
fore the subphase exchange, and 25 mN/m higher than α-hederin drastically reduced as compared to the starting DPPC monolayer on
on bare water/air interface. water/DMSO 4% (E″ = 8.5 mN/m vs E″ = 111.2 mN/m). Interestingly, E
″ for the penetrated layer is also smaller than that for the α-hederin's
3.2.2. Surface dilational rheology Gibbs layer. Thus, in contrast to the bare DPPC monolayers and α-
The observations regarding the effect of the three saponins on relax- hederin's Gibbs layers, the α-hederin-penetrated DPPC monolayer
ation of DPPC and DPPC/cholesterol mixed monolayers are also con- behaves as almost purely elastic body. The fact that the loss modulus
firmed by the results of the surface dilational rheology measurements decreases upon penetration by all three saponins despite an increase of
(Table 2). For DPPC layers exposed to both ammonium glycyrrhizate surface pressure indicates that packing of the molecules is different than
and hederacoside C, the storage and loss moduli take values significant- it would be in an analogous monolayer compressed by mechanical
ly higher than those for the bare saponins, yet smaller than for the means. The molecules in the mixed DPPC–saponin monolayers have
starting DPPC layers (E′ = 176.5 mN/m, E″ = 105.0 mN/m [19]). The at- less possibilities to relax the mechanical stress by molecular reorientation
tractive interactions between the closely-packed DPPC molecules, and rearrangements, than in pure DPPC.
When cholesterol is present in addition to DPPC, the effect of
Table 2 α-hederin is again different: both E′ and E″ of the penetrated mixed
Dilational surface visco-elasticity moduli for Gibbs layers of the triterpene saponins on wa- lipid layer are close to the values for the Gibbs layer of α-hederin on
ter–air interface (10−4 M) and for the Langmuir monolayers of DPPC and DPPC/cholester-
free water surface, despite markedly different monolayer relaxation be-
ol (10:9 mol/mol) penetrated with these saponins (10−4 M). “n.d.” refers to the cases
where surface rheological response could not be determined because of a brittle nature havior. Unfortunately, the surface dilational visco-elasticity of the pene-
of cholesterol-rich monolayers. trated DPPC/cholesterol monolayers values cannot be compared
quantitatively with those for the corresponding monolayer on pure
Subphase Gibbs layer DPPC DPPC/cholesterol
water, because of a loss of material during surface area oscillations for
E′ E″ E′ E″ E′ E″
the latter (see Supporting Information, Fig. S1.). This phenomenon orig-
Ammonium glycyrrhizate 21.1 4.3 134.6 62.0 n.d. n.d. inates probably from a very high rigidity of the cholesterol-rich mono-
Hederacoside C 2.6 0.8 84.1 42.1 n.d. n.d. layers on water. For the same reasons, the surface dilational
α-Hederin 242.3 147.0 188.6 8.5 303.2 146.0
rheological response could not be retrieved for the mixed DPPC/
368 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
cholesterol monolayers in contact with the two remaining saponins,
which interact only weakly with the monolayers. As far as the me-
chanical response to the monolayer area perturbation is concerned,
the DPPC/cholesterol mixed layers penetrated by the weakly-
interacting saponins (hederacoside C and ammonium glycyrrhizate)
can be considered similar to those not penetrated.
3.2.3. Fluorescence microscopy
Fluorescence microscopy is a well-established tool to investigate the
phase behavior of amphiphilic systems at the air–water interface [29–
32]. Here it was employed to highlight possible effects of the triterpene
saponins penetration on lipid monolayers initially compressed to Π0 =
32.5 mN/m (Fig. 4).
During compression, bare DPPC monolayers showed typical bean-
shaped LC domains growing within the LE phase [33,34] (see
Supporting Info, Fig. S2). Above surface pressure of 10 mN/m the LC do-
mains filled almost an entire space and the fluorescence microscopy im-
ages did not change significantly upon further compression (Fig. 4).
Introduction of the saponins at Π0 = 32.5 mN/m did not change the sit-
uation (not shown), despite a clear increase of surface pressure, even up
to 45 mN/m (for α-hederin, see Fig. 3). This suggests that all three sapo-
nins are miscible with DPPC, independently of the strength of their in-
teraction with the lipid.
In binary lipid monolayers, the phase transitions are additionally com-
plicated by mutual solubility and complex formation between the lipids
[35,36]. Keller et al. [37] in a systematic study of mixtures of cholesterol
with variable-length phosphatidylcholines (PC) nicely showed that phos-
pholipids with chain-melting temperatures above 23 °C exhibit two
upper miscibility critical points, depending on the amount of cholesterol.
At lower surface pressures, the PC:cholesterol complexes phase separate
from the pure PC and cholesterol, forming the cholesterol-poor (α) and
cholesterol-rich (β) regions in the phase diagram [38]. Even though the
existence of a critical point in the β region of DPPC/cholesterol mixtures
has been initially neglected [39], recent works clearly confirm remixing
at higher surface pressures [40,41]. Similar features have been observed
in other cholesterol/phospholipid mixtures [42–44]. Also our present
fluorescence microscopy results confirm remixing of the DPPC/cholester-
ol (10:9 mol/mol) monolayer above Π0 = 3 mN/m (see Supporting Info,
Fig. S3). Therefore, the DPPC/cholesterol monolayers prior to saponin in-
troduction (Π0 = 32.5 mN/m) likely contain a liquid-ordered (lo)
mixed phase (Fig. 4). The alternate bright and dark stripes (not to be
confused with the stripe phase often observed near critical points [45])
suggest formation of cholesterol-rich/cholesterol-poor, few micrometer-
thick linear domains of very low line tension [30,42]. The increase of the
number of dark stripes with increasing cholesterol concentration (data
not shown) suggests that they may correspond to the condensed phase
and/or cholesterol-rich regions. An alternative explanation for the origin
Fig. 4. Fluorescence microphotographs of DPPC (left) and mixed 10:9 (mol/mol) DPPC/cholesterol (right) Langmuir monolayers compressed to Π0 = 32.5 mN/m.
of the stripe pattern might be formation of folds during compression,
but at the present stage we cannot distinguish between the two possibil-
ities. After introduction of the saponins, the contrast between dark and
bright stripes first increases, and with time the inhomogeneity gets less
visible (Fig. 5). This suggests eventual mixing of the phases, possibly
due to the flow induced by introduction of saponins.
Surprisingly, the two saponins for which the least changes were
noticed in surface pressure showed also the highest incompatibili-
ties with the penetrated mixed monolayer. The circular bright do-
mains seen in microphotographs for ammonium glycyrrhizate and
especially hederacoside C (Fig. 5) correspond probably to a
cholesterol-poor phase [36], separated as a consequence of the
saponin-DPPC and/or saponin-cholesterol complex formation. Inter-
estingly, α-hederin–lipid complexes are rather well miscible with
the lipid matrix of the DPPC/cholesterol monolayer, which is consis-
tent with the fact that the α-hederin-penetrated mixed monolayer
spontaneously approaches a surface pressure of about 50 mN/m
(Fig. 3). The α-hederin-penetrated monolayers were also distinct
in their sharply increasing viscosity, which practically blocked any
movement due to the subphase exchange after 20 min. No fluores-
cence changes were noticed during the sudden surface pressure
drops observed in the first 3–5 min of penetration, confirming that
the pressure jumps are not related to any processes within the
monolayer (e.g., a collapse), but are rather artifacts due to partial
wetting of the trough's Teflon rim.
3.2.4. Neutron reflectivity
The effect of the three triterpene saponins on the structure of DPPC
and DPPC/cholesterol monolayers was investigated using neutron re-
flectivity (NR). The lipid chain-perdeuterated DPPC (DPPC-d62) and
protonated cholesterol were used for this purpose. In order to highlight
the changes in the lipid layer induced by the saponin, the scattering
length density (SLD) of the subphase was matched to that of air (so-
called null reflecting water, NRW). The reflectivity curves for bare
DPPC-d62 and DPPC-d62/cholesterol (10:9 mol/mol) monolayers on
pure NRW and NRW containing dissolved α-hederin, hederacoside
and ammonium glycyrrhizate at a concentration of 10−4 M are shown
in Fig. 6. The saponins were introduced to the subphase in the same
way as for the surface pressure measurements, by the subphase ex-
change following compression to 32.5 mN/m. For comparison, also the
corresponding reflectivity curves for the Gibbs layers of each saponin
at the same concentration at the NRW/air interface are shown.
All three saponins spontaneously form Gibbs layers on free NRW
surface, as evidenced by an increased reflectivity of neutrons (NRW
without saponins was not reflecting neutrons at all). The NR curves
for the saponins' Gibbs layers could be fitted assuming a 51.0 Å-
thick (for ammonium glycyrrhizate and hederacoside C) or a 19.4
 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373 369

Fig. 5. Fluorescence microphotographs of mixed DPPC/cholesterol (10:9) Langmuir monolayers penetrated by ammonium glycyrrhizate (left), hederacoside C (middle) and α-hederin
(right) after 2 min (top), 10 min (middle) and 30 min (bottom). The monolayers were initially compressed to 32.5 mN/m.

Fig. 6. NR curves for DPPC-d62 ( ) and DPPC-d62/cholesterol (10:9 mol/mol, ) monolayers on NRW containing 10−4 M of: (A) ammonium glycyrrhizate, (B) hederacoside and α-hederin
(C). For comparison, the NR curves for Gibbs layers of each saponin on the free surface of NRW (■), as well as for DPPC-d62 ( ) and DPPC-d62/cholesterol (10:9 mol/mol, ) monolayers on
pure NRW are shown. Solid lines correspond to the best fits from Tables 3 and 4. All experiments were performed at 21 °C.
370 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373

Å-thick (α-hederin) layers (see Table 3). The two former saponins Table 4
seem to favor formation of thicker Gibbs layers, with adsorbed mol- The best-fit parameters (for neutron reflectivity of bare DPPC-d62 and DPPC-d62/cho-
lesterol (10:9 mol/mol) monolayers on pure NRW and NRW containing α-hederin,
ecules oriented presumably perpendicular to the surface. For hederacoside C and ammonium glycyrrhizate (10−4 M). Surface roughness was fixed
bidesmosidic hederacoside C, this orientation is probably forced by at 2 Å in all fittings.
the presence of two glycone groups attached to the opposite sides
Subphase DPPC-d62 DPPC-d62/chol
of the triterpenoid aglycone (C3 and C28 atoms), while for ammoni-
um glycyrrhizate, a hydrophilic free carboxylic group (see Fig. 1) Layer thickness Scattering Layer thickness Scattering
(fitted), length (fitted), length
probably plays the decisive role in forcing the perpendicular orienta-
τ (Å) density τ (Å) density
tion. In contrast, much smaller thickness of the monodesmosidic α- (fitted), (fitted),
hederin suggests an orientation parallel to the surface, which is SLDlayer SLDlayer
probably favored by the presence of only one glycone group and (10−6/Å2) (10−6/Å2)
the free carboxylic group attached to C-28. The corresponding best- α-Hederin 26.2 5.80 23.2 2.70
fit values of SLD for the Gibbs layers vary among the saponins, – – 15.0 0.90
reflecting different volume fractions in the layer (φsaponin) and differ- – – 33.9 0.54
Hederacoside C 26.2 5.56 23.8 3.02
ent adsorbed amounts (Γsaponin ). The two parameters calculated
38.0 0.15 15.0 0.49
using Eqs. 1 and 2 are collected in Table 3. Ammonium 26.2 5.56 23.8 3.02
glycyrrhizate 36.3 0.24 10.2 0.76
SLDlayer ¼ SLDsaponin  φsaponin ð1Þ NRW 26.2 5.56 23.8 3.02
NRW/DMSO 28.0 5.56 27.1 3.02
τ
Γ saponin ¼ φ ð2Þ
NAv V saponin saponin
hydrated glycone parts extending beyond the lipid layer towards the
where SLD layer is the scattering length density obtained from the aqueous phase, hence its low thickness and SLD.
best-fit of the NR curve, SLDsaponin and Vsaponin are theoretical values For α-hederin the situation is different. Despite a clear increase of
of the scattering length density, and molar volume of the given sapo- surface pressure, the NR curve for DPPC-d62 penetrated with this saponin
nin. NAv is the Avogadro's number. lays slightly below that for the bare monolayer on pure NRW. This is
The adsorbed amount calculated from the NR data is the highest for α- probably caused by the presence of only one glycone chain, which can
hederin (3.0·10−6 mol/m2) and the lowest for hederacoside C be accommodated almost entirely within the DPPC layer, only slightly
(0.8·10−6 mol/m2), in good agreement with the order of their ability to changing its thickness and SLD. Unfortunately, the resolution of NR
increase surface pressure in the Langmuir trough measurements measurements is not sufficient to quantify these small changes of SLD
(Fig. 3). The distinctly different Γα-hederin is a consequence of much lower for α-hederin penetrated DPPC layers. It should be noted here that all
extent of the α-hederin layer hydration (φ = 1), as compared to the the measurements for α-hederin were performed in the presence of
other two saponins (φ = 0.2 and 0.3). DMSO in the subphase (4% v/v). As shown by Dabkowska et al., at such
The NR curve for bare DPPC monolayer is consistent with the literature low DMSO content, the structure of the lipid layer is not significantly
results [46,47]. The NR results confirm that the DPPC monolayers com- affected [48]. Nevertheless, the effect of DMSO was taken into account
pressed to 32.5 mN/m can resist the detergent activity of all three sapo- by using the reference measurements for lipid layers spread also on
nins. Thanks to perdeuteration of the alkyl chains in DPPC used for our NRW containing DMSO. The NR curves for DPPC-d62 in the absence of
NR experiments, any removal of the highly neutron-reflecting DPPC-d62 α-hederin are shifted slightly upwards with respect to those on pure
would immediately result in a decrease of reflectivity. With the progres- NRW (see Supporting Information, Fig. S3). Our results show that in the
sive loss of deuterated material from the monolayer to the subphase, presence of DMSO (4% v/v), thickness of the lipid layers slightly increased
the resulting reflectivity curve would then tend towards that for the re- (by 3.3 Å, see Table 4).
spective Gibbs layer on bare NRW surface. No such trends were observed Similarly to the pure phospholipid monolayers, the mixed DPPC-d62/
even on the eight-hour timescale of the NR experiment. On the contrary, cholesterol (10:9 mol/mol) ones are not solubilized by hederacoside C
for hederacoside C and ammonium glycyrrhizate the reflectivity curves or ammonium glycyrrhizate. The reflectivity curves are shifted upwards,
were even slightly shifted upwards, suggesting deposition of additional as for the pure DPPC-d62 layers, suggesting accumulation of additional
neutron-reflecting material (with SLD slightly higher than that of NRW). adsorbed material. Alternatively, because the lipid layers contain now a
For both saponins, an additional low-SLD layer had to be included to fit protonated cholesterol (SLDcholesterol = 0.21·10−6 Å2), the upwards shift
the NR curves for DPPC-d62 after the subphase exchange (Table 4). In could be explained by the replacement of cholesterol with other mole-
both cases, the additional layer is thinner and has lower SLD than the cor- cules of higher SLD (e.g., with saponins, see Table 3). However, the NR
responding freely adsorbing Gibbs layer. This effect could be explained by curve cannot be fitted with a single layer, and the presence of an addition-
partial inclusion of the hydrophobic triterpenoid part of the adsorbing sa- al thin layer of 10.2 and 15.0 Å (for ammonium glycyrrhizate and
ponin molecules into the topmost DPPC-d62 monolayer. Such a partial in- hederacoside C, respectively) had to be assumed. In analogy to the results
clusion could also explain the increase of surface pressure observed in the for pure DPPC, they might correspond to the glycone parts of both sapo-
Langmuir trough measurements during the subphase exchange. The ad- nins extending towards the aqueous phase, while the less hydrated agly-
ditional layer seen in NR is probably composed mostly of the NRW- cone parts would be intermixed within the lipid layer. The reduced depth

Table 3
The best-fit parameters for neutron reflectivity of Gibbs layers of free saponins on pure NRW and NRW containing α-hederin, hederacoside C and ammonium glycyrrhizate (10−4 M).
Surface roughness was fixed at 2 Å in all fittings.

Subphase Gibbs layers

Molar volume Scattering length density Layer thickness Scattering length density Volume Adsorbed amount,
(theoretical), Vsaponin (Å3) (theoretical), SLDsaponin (10−6/Å2) (fitted), τ (Å) (fitted), SLD (10−6/Å2) fraction, φ Γ (mol/m2)

α-Hederin 1250 0.77 22.6 0.77 1 3.0·10−6

Hederacoside C 2030 0.91 51.0 0.20 0.2 8.4·10−7
Ammonium glycyrrhizate 1395 0.99 51.0 0.28 0.3 1.8·10−6
 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
of the second layer (containing mostly the glycone groups) for the
cholesterol-containing lipid layers indicates that the presence of choles-
terol promotes penetration of these two saponins into the lipid layer.
This is also in agreement with the more pronounced surface pressure in-
crease observed for cholesterol-containing lipid layer, as compared to
those composed solely of DPPC (Fig. 2).
The most pronounced changes in NR curves can be noticed for α-
hederin penetrating the mixed DPPC-d62/cholesterol monolayer on
NRW containing DMSO (4%). It should be noted that the NR curves for
DPPC-d62/cholesterol in the absence of α-hederin are shifted slightly
upwards with respect to those on pure NRW, analogously to DPPC-d62
(see Supporting Information, Fig. S4). Thickness of the lipid layers in-
creased slightly by 1.8 Å in the presence of DMSO (4% v/v) (Table 4).
To obtain a reasonable fit of the reflectivity for the DPPC-d62/
cholesterol/α-hederin system, two additional layers had to be taken
into account (Table 4). The first, 15 Å-thick layer with SLD of
0.9·10− 6 Å− 2 (higher than that for pure cholesterol, SLDcholesterol =
0.21·10−6 Å2, or α-hederin, SLDα-hederin = 0.77·10−6 Å2) must contain
some higher-SLD material, e.g. DPPC-d62. The second layer of lower SLD
(0.54·10−6 Å− 2) and a thickness of about 34 Å probably comprise
mostly cholesterol and the saponin (although the presence of DPPC-
d62 cannot be excluded). Thus, α-hederin seems to pull part of choles-
terol and DPPC out of the lipid layer. The latter, being less strongly
bound by α-hederin remains partially immersed in the monolayer,
while the stronger α-hederin–cholesterol complex is shifted more to-
wards the aqueous phase. A similar picture of a digitonin-penetrated
mixed lipid bilayer was recently provided by Frenkel et al. using X-ray
reflectivity (XR) and QCM study [49]. The XR approach, however, does
not allow for contrast variation, hence the authors could not resolve
the composition of the observed 45 Å-thick additional layer.
4. Discussion
Even though the saponin–membrane interactions are generally be-
lieved to be dominated by cholesterol, our study shows that phospho-
lipids may also actively participate in attracting saponins to biological
membranes. The saponin–lipid complexes formed in solution are only
slightly less stable for DPPC than for cholesterol (Table 1). These obser-
vations are in line with suggestions of Demana et al., who used diffuse
reflectance IR (DRIFT) spectroscopy for studying complexes formed be-
tween a saponin mixture from Quillaja saponaria Molina (Quil A) and
phosphatidylcholine (PC) [50]. They showed that the PC's CO, PO groups
may form hydrogen bonds with the\\OH groups of the saponin's sugar
moieties. In parallel, the free carboxylic acid group of the latter may
electrostatically bind with the PC's (CH3)3N+ group. The differences in
stability constants for the saponin–lipid complexes observed in our
studies point to the crucial role of proper spatial arrangement of the
complimentary groups in these complexes. Despite several structural
similarities between the three saponins, clear differences in lipid-
binding properties can be noticed even in solution, where the effects
of spatial arrangement of the host and guest molecules are less
constrained than in the mono- or bilayer. The most striking example is
provided by comparison of ammonium glycyrrhizate and α-hederin.
Both possess a two-sugar glycone attached to C-3, but only α-hederin
is capable of complexing DPPC or cholesterol. A single carboxylic
group of α-hederin (at C-28 position) is more effective than the three
present in ammonium glycyrrhizate (one attached to the glycone at
C-30 position, and two in the glucuronic acids of the glycone part). Ap-
parently, their spatial arrangements in the latter molecule do not allow
for complementarity of hydrogen and ionic bonding as effectively as in
α-hederin or Quil A.
Some authors noted that the presence of a free\\COOH group is im-
portant for lipid binding [23], but the small difference in stability con-
stants for DPPC complexation between α-hederin and hederacoside C
observed in the present study suggests that this effect is more likely
linked with spatial confinement in the membrane. Somehow
371
surprisingly, a previous electrospray ionization mass spectroscopy
(ESI-MS) study of Lekar et al. showed that in the gas phase cholesterol
is complexed by α-hederin, but not by hederacoside C [51,52]. However,
as far as the bulk solution interaction is concerned, the location of the
carboxylic group seems to be the most important, as proven by the
lack of interaction of DPPC and cholesterol with ammonium
glycyrrhizate. Moreover, the comparison of chemical structures of the
three saponins points to an important role of van der Waals interactions
between the steroid backbone of cholesterol with the triterpene agly-
cones of the saponins in bulk. In view of this, the presence of a ketone
group in ammonium glycyrrhizate may act as a steric hindrance
preventing a close approach and consequently efficient van der Waals
interaction with cholesterol and DPPC.
The ability to form lipid–saponin complexes in the homogeneous
bulk solution does not fully correlate with the strength of interactions
in a confined environment of lipid monolayers. The best correlation be-
tween the bulk complexation and the monolayer activity can be found
for α-hederin, for which the highest affinity to the lipids is evident
from both the bulk and monolayer studies. Even the preference for cho-
lesterol over DPPC (Table 1) is well reflected in the monolayer studies in
this case (Fig. 2). The highest value of the complex stability constant as
well as the most pronounced monolayer response correspond well also
to the highest membranolytic activity of α-hederin. Similarly, the lack of
interaction of ammonium glycyrrhizate with both lipids is evident
equally in the bulk and monolayer experiments, and corresponds well
to its weak membranolytic activity. However, the relatively high affinity
to both cholesterol and DPPC for hederacoside C observed in bulk is nei-
ther reflected in monolayer studies, nor in its membranolytic activity.
This again highlights the important role of spatial organization of the
lipids in their interactions with saponins. One possible explanation has
been recently provided by Lorent et al. who extensively discussed the
differences between hederagenin, δ-hederin and α-hederin in view of
different curvature of the molecules [53,54]. The failure of our solid–liq-
uid extraction experiments could also be explained by the crucial role of
the lipid state in its interaction with saponins.
Hederacoside C and ammonium glycyrrhizate easily penetrate
monolayers of both DPPC and DPPC/cholesterol without significantly
disturbing them. This correlates well with their mild activity against
both red blood cells (hemolysis) and cancer cells [55]. For α-hederin
penetrating the mixed DPPC/cholesterol monolayers, the situation is
clearly more complex, in line with its pronounced hemolytic and cyto-
toxic activity. Not only the surface pressure response is much more pro-
nounced than for other saponins, but also the structure and mechanical
properties of the penetrated ternary α-hederin/DPPC/cholesterol layer
are distinct. Bare DPPC/cholesterol monolayers are too rigid to reliably
determine their surface dilational rheological parameters. Penetration
with α-hederin softens them to the extent allowing for quantitative
measurements without breaking the monolayer. The visco-elasticity
modulus for the penetrated layers is in fact similar to that for the α-
hederin Gibbs layers. On the other hand, both the surface pressure and
neutron reflectivity results assure us that the lipid layer is still present
after penetration. This prompts us to propose that α-hederin/DPPC/
cholesterol mixtures could form surface-confined structures analogous
to those suggested by Frenkel et al. [49] for digitonin, or to the open
cage-like colloidal immunostimulatory complex matrices (ISCOM)
[56]. The latter structures are often found in cholesterol/phosphatidyl-
choline layers hydrated with saponin solutions, especially with purified
QBS extracts (Quil A) [57]. They were shown to exist in mixtures con-
taining between 10 and 30% of cholesterol after 1 day of hydration.
However, if hydration was prolonged to 2 months, they could even be
observed in mixtures containing up to 70% of cholesterol. Whether the
α-hederin complexes with cholesterol and DPPC in the monolayer are
of an adduct-type, as suggested by Frenkel et al. [49], or cage-like, re-
mains to be determined in the future. In both cases, an additional
layer built-up from the saponin and parts of the original lipid layer
would strongly affect mechanical and physiological properties of the
372 K. Wojciechowski et al. / Biochimica et Biophysica Acta 1858 (2016) 363–373
biological membrane (e.g. in erythrocytes), and can be responsible for
the enhanced membranolytic activity of α-hederin. Despite better
packing of molecules suggested by the increased surface pressure, the
overall structure might be more fluid and permeable than the original
non-penetrated structure.
5. Conclusions
Adsorption of three triterpene saponins (α-hederin, hederacoside C
and ammonium glycyrrhizate) onto a free water surface and onto Lang-
muir monolayers of DPPC and DPPC/cholesterol (10:9 mol/mol) was de-
scribed. A combined monolayer relaxation, surface dilational rheology,
fluorescence microscopy and neutron reflectivity (NR) study showed
that all three saponins are able to interact and partially penetrate the
pure DPPC and mixed DPPC/cholesterol monolayers. At the surface of
pure water, α-hederin (10−4 M) forms densely packed layers with the
thickness of 22.6 Å and decreased surface tension by 25 mN/m. In con-
trast, hederacoside C and ammonium glycyrrhizate decrease surface
tension only slightly and form highly hydrated (70–80%), rather loose
layers of 51.0 Å thickness. The surface activity of the saponins correlates
well with their effect on lipid monolayers: α-hederin was shown to af-
fect the layers to the highest extent, especially when cholesterol is
present. The mixed DPPC/cholesterol monolayers in contact with
α-hederin develop an additional thick layer extending towards the sub-
phase and consisting probably of α-hederin-DPPC and α-hederin-
cholesterol complexes. We believe that similar structures might be re-
sponsible for the pore formation during α-hederin's membranolytic ac-
tivity (e.g., during hemolysis). The other two saponins partially
penetrate the lipid layers, probably only with their hydrophilic glycones
extending towards the aqueous subphase.
For comparison, the saponin–lipid complex formation was also stud-
ied in ethanol-aqueous homogeneous solutions using UV/Vis spectros-
copy. The oleanolic acid-type saponins (α-hederin and hederacoside
C) were shown to form complexes with lipids characterized by stability
constants in the range (4.0 ± 0.2)·103–(5.0 ± 0.4)·104 M−1. The com-
plexes with cholesterol are stronger than those with DPPC. On the con-
trary, ammonium glycyrrhizate does not form complexes with any of
the lipids in solution. Among the studied saponins, only α-hederin in-
teracts strongly with the lipid monolayers and displays membranolytic
activity towards red blood cells and cancer cells. Ammonium
glycyrrhizate does not interact strongly with the lipids neither in bulk,
nor in monolayers. It also shows negligible membrane activity. These re-
sults suggest that the chemical interactions of the aglycone part are not
decisive in the strength of interactions with cholesterol, and/or that the
interactions with glycone parts are important. The ability to form a
lipid–saponin complex in bulk solution seems to be a necessary but
not sufficient condition for efficient interaction with the same lipids in
a confined environment of the monolayer or real biological membranes.
The fact that hederacoside C, despite similar cholesterol-binding ability
as α-hederin, is less active in membrane permeabilization might reflect
its hindered access to the cholesterol molecules embedded in the
bilayer.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
This work was financially supported by the Polish National Science
Centre, grant no. DEC-2011/03/B/ST4/00780 and COST CM1101 Action.
Ms. Katarzyna Krzeminska is acknowledged for assistance with UV/Vis
measurements. The work is based on experiments performed at the
neutron reflectometer AMOR at Swiss spallation neutron source SINQ,
Paul Scherrer Institute, Villigen, Switzerland.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbamem.2015.12.001.
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