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Biochimica et Biophysica Acta 1848 (2015) 1963–1973

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Biochimica et Biophysica Acta

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Impact of two different saponins on the organization of model lipid membranes

Beata Korchowiec a,⁎,MarcelinaGorczycab, Kamila Wojszko a,c, Maria Janikowska b,d, Max Henry c, Ewa Rogalska c,⁎

a Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland b Department of Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland c Structure et Réactivité des Systèmes Moléculaires Complexes, BP 239, CNRS/Université de Lorraine, 54506 Vandoeuvre-lès-Nancy cedex, France d Faculty of Physics, Astronomy, and Applied Computer Science, Jagiellonian University, ul. S. Lojasiewicza 11, 30-348 Krakow, Poland

article info abstract

Article history: Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This Received 23 February 2015 activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous so- Received in revised form 2 June 2015 lution and interact with membrane components. In this work, Langmuir monolayer techniques combined with Accepted 4 June 2015 polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy Available online 6 June 2015 were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, Keywords: Digitonin cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1- Saponin–membrane interactions glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin–lipid interaction was Langmuir films characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both Lipid monolayers saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis. © 2015 Elsevier B.V. All rights reserved.

1. Introduction have been detected in oats, tomato seeds, alliums, asparagus, yam, yucca, fenugreek, ginseng, capsicum peppers and aubergine, whereas Saponins are naturally occurring surface active glycosides, which triterpenoid saponins have been found in many legumes and also in al- possess amphiphilic character originating from lipophilic aglycone (sa- liums, tea, spinach, sugar beet, liquorice, sunflower, ginseng, horse pogenin) and hydrophilic sugar moieties [1,2]. The hydrophobic part chestnut and quinoa. Their main role is protection of organisms from may be steroid (27C) or triterpenoid (30C) [3]. Most known saponins harmful effects of pathogens, thanks to their anti-fungal, anti-virial are monodesmosidic, which means that only one position of the agly- and anti-bacterial characteristics [1,7]. cone is glycosylated. The sugar moiety is attached by an ether linkage The name ‘saponin’,reflects a wide-spread ability of saponins to to the C3 hydroxyl group occurring in the majority of sapogenins [4,5]. form soap-like foams in aqueous solutions. An important property of sa- Saponins can possess from one to three straight or branched sugar ponins is their lytic action on erythrocyte membranes as a result of affin- moieties. The most often occurring sugar moieties are D-glucose, L- ity of the aglycone moiety to membrane cholesterol [2]. An interesting rhamnose, D-galactose, D-glucuronic acid, L-arabinose, D-xylose or D- property of saponins is cytotoxity. Most studies concerning the latter fucose. The sugar chain contains from one to several monosaccharide effect focus on the impact of saponins on cancer cell lines. The effect of residues [3]. saponins on different human cancers such as leukemia, melanoma, Saponins are widely distributed in plants, but also in lower marine CNS, non-small cell lung, colon, ovarian, breast, renal and prostate is animals, such as starfish or sea cucumber [1,4,6]. Steroidal saponins described in the literature [3,7]. Saponins can act extracellularly or in- tracellularly on tumor cells. The first way of interaction is direct inhibi- tion of membrane proteins or changing membrane permeability. ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Korchowiec), Induction of apoptosis and cell cycle arrest are the most frequent intra- [email protected] (E. Rogalska). cellular pathways. Nowadays, saponins are used in cancer therapy to

http://dx.doi.org/10.1016/j.bbamem.2015.06.007 0005-2736/© 2015 Elsevier B.V. All rights reserved. 1964 B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 inhibit angiogenesis and drug efflux. They also possess anti-metastatic spontaneously incorporate into the membrane. Subsequently, the ste- properties [7]. roidal glycoalkaloid-cholesterol 1:1 complexes would form and accu- In this work, a commercial Merck Saponin and a digitonin, two types mulate in the membrane. The mechanism of this process is not of saponins with different structures, were studied for their ability to in- completely understood. However, Armah et al. [14] proposed that steric teract with membrane lipids. The chemical structures of saponins are properties of saponin–cholesterol complexes may cause membrane cur- shown in Fig. 1. Digitonin is a steroidal, monodesmosidic saponin vature and, in consequence, may provoke vesiculation or pore forma- (Fig. 1A, B). It contains a pentasaccharide moiety consisting of two ga- tion in the cell membrane. lactose, two glucose and one xylose monosaccharide residues. Digitonin As a second compound, the commercial Merck Saponin was used naturally occurs in a ubiquitous plant, Digitalis purpurea [8].Likemostof (Fig. 1C, D). Merck Saponin is a saponin fraction isolated from the saponins, digitonin increases permeability of biological membrane and roots of Gypsophila paniculata L. and Gypsophila arrostii Guss. The chem- disperses membrane-bound proteins [9]. Digitonin may also act as a ical structure of two major components of Merck Saponin was competitive inhibitor for P-glycoprotein (P-gp), which is an important established using NMR techniques [15,16]. The aglycone moiety was protein in cancer therapy; P-gp is responsible for removing many for- identified as gypsogenin, while seven or eight monosaccharide units eign substances from the cell [10]. However, the most common proper- were identified in the sugar moiety (Fig. 1). Due to its high hemolytic ty of digitonin is its affinity for cholesterol. One possible application of properties, Merck Saponin was widely used as a standard for hemolytic digitonin is detection of cholesterol in the cell. Raj and coworkers [11] tests [17]. Its toxic effect is much lower when given orally, as it cannot developed a method of detection of cholesterol with digitonin- enter the blood stream by crossing the intestine, and the presence of conjugated gold nanoparticles. Vermeer et al. [12] and Fornas et al. blood plasma reduces the hemolytic effect [18].TheeffectofGypsophila [13] used digitonin as a protective agent for preparing liver tissue for saponins on plasma cholesterol concentration was studied by different electron microscopy to avoid loss of cholesterol. Augustin and co- groups [19,20]. In all cases, the authors showed that in rats, ingestion workers [4] studied complexation of cholesterol with digitonin. Due to of saponins resulted in a significant reduction of blood cholesterol the fact that aglycone part of digitonin is lipophilic, digitonin can concentration.

Fig. 1. Structures of digitonin (A, B) and two major components of Merck Saponin; R = H or β-D-xylopyranosyl (C, D). (A, C): chemical structures, and (B, D): 3-D structures obtained at HF/ 6-31G(d) level of theory; in Merck Saponin panel D: R = H. Molecular volumes of Merck Saponin and digitonin (B, D) are, 6241 Å3 and 4435 Å3, respectively. Molecular volume was defined by 0.001 a.u. isosurface of electron density of molecule. This calculations were performed at HF/6-31G(d) level of theory using Gaussian 0.9 software [52]. Color code: carbons in black, oxygens in red, and hydrogens in gray. B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 1965

Despite a number of studies dealing with saponin–membrane inter- of 2.5 Å2 molecule−1 min−1. A PC computer and KSV software were actions, very few involved Langmuir monolayers [14,21–23]. In the last used to control the experiments. Each compression isotherm was per- years, Langmuir lipid monolayers were extensively used to investigate formed at least three times. The Π–A and ΔV–A isotherms were record- interactions with different compounds co-spread at the interface or dis- ed at 20 ± 0.1 °C. The standard deviation obtained from compression solved in the subphase [24–27]. These model membranes are useful for isotherms was ±0.5 Å2 on molecular area (A), ±0.2 mN m−1 on surface obtaining information on the mechanism of saponin–membrane inter- pressure and ±0.01 V on surface potential. actions. Here, to better understand the effect of Merck Saponin or digi- The morphology of the films was imaged with a computer-interfaced tonin on the lipid model membranes, the Langmuir film technique KSV 2000 Langmuir balance combined with a Brewster angle microscope was used to simultaneously measure the surface pressure and surface (KSV Optrel BAM 300, Helsinki). The Teflon trough dimensions were potential. Langmuir monolayers formed with cholesterol (Chol), 1,2- 58 × 6.5 × 1 cm; other experimental conditions were as described above. dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn- glycero-3-phospho-rac-(1-glycerol) (DPPG), as well as DPPC/Chol or 2.3. Adsorption kinetics measurements DPPG/Chol mixtures were formed on subphases containing Merck Sa- ponin or digitonin. Brewster angle microscopy was used to study the Adsorption experiments were carried out in a Teflon dish with a morphological properties of pure lipid and mixed lipid/saponin mono- subphase volume of 20 mL. The pure Chol, DPPC, DPPG, and mixed layers. To examine the molecular organization and molecular interac- DPPC/Chol and DPPG/Chol solutions were spread at the air–water tion in model membranes, polarization modulation infrared reflection interface using a Hamilton syringe to obtain an initial surface pressure, absorption spectroscopy was used. Π =30mNm−1. This value corresponds to the lateral pressure in the biological bilayers [28]. After 20 min of equilibration, a small volume 2. Materials and methods of the concentrated saponin solution was injected into the subphase be- neath the lipid film. The subphase was gently stirred with a magnetic 2.1. Materials and reagents stirrer for 30 s. The final saponin concentration in the subphase was 78.5 μgmL−1. The variation of the surface pressure was recorded as a Digitonin (66% pure) and cholesterol (Chol, ~99% pure) were function of time using a KSV 2000 Langmuir balance (KSV Instruments, from Sigma-Aldrich. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Helsinki). The registration of adsorption kinetics started immediately (DPPC; 99% pure) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac- after the injection of saponin into the subphase. All measurements (1-glycerol) sodium salt (DPPG; 99% pure) were from Avanti were performed at 20 °C. Polar Lipids. The structures of two major saponins present in the commercial Merck Saponin (Merck No. 7695; supply interrupted), 2.4. Polarization–modulation infrared reflection–absorption spectroscopy were determined by high-field gradient-enhanced and homo- (PM-IRRAS) and heteronuclear 2D NMR methods [15,16]. The two major compo- nents were identified as: 3-O-β-D-xylopyranosyl-(1 → 3)-[β-D- The PM-IRRAS spectra of pure Chol, DPPC, DPPG and mixed galactopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl gypsogenin DPPC/Chol or DPPG/Chol monolayers formed on pure water and sapo- 28-O-(6-deoxy-β-D-glucopyranosyl)-(1 → 4)-[β-D-xylopyranosyl- nin solution subphases were registered at 20 °C. The Teflon trough di- (1 → 3)-β-D-xylopyranosyl-(1 → 4)]-α-L-rhamnopyranosyl- mensions were 36.5 × 7.5 × 0.5 cm; other experimental conditions (1 → 2)-β-D-fucopyranoside, and 3-O-β-D-xylopyranosyl-(1 → 3)- were as described in the preceding paragraph. The PM-IRRAS measure- [β-D-galactopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl gypsogenin ments were performed using a KSV PMI 550 instrument (KSV Instru- 28-O-β-D-glucopyranosyl-(1 → 3)-[β-D-xylopyranosyl-(1 → 4)]-α-L- ments, Helsinki). The PMI 550 instrument contains a compact Fourier rhamnopyranosyl-(1 → 2)-β-D-fucopyranoside. The chemical struc- Transform IR-spectrometer equipped with a polarization–modulation tures of the saponins studied in this work are shown in Fig. 1. (PM) unit on one arm of a goniometer, and a MCT detector on the The aqueous solutions of Merck Saponin and digitonin were pre- other arm. The incident angle of the light beam can be freely chosen be- pared using Milli-Q water, which had resistivity of 18 MΩ cm and sur- tween 40° and 90°; here the incident angle was 79°. The spectrometer face tension of 72.8 mN m−1 at 20 °C. Spectrophotometric grade and the PM unit operate at different frequencies, allowing separation chloroform (Sigma-Aldrich) was used for preparing lipid solutions. of the two signals at the detector. The PM unit consists of a photoelastic modulator (PEM), which is an IR-transparent ZnSe piezoelectric lens. 2.2. Compression isotherms and Brewster angle microscopy The incoming light is continuously modulated between s-andp- polarization at a frequency of 74 kHz. This allows simultaneous mea- The surface pressure (Π) and electric surface potential (ΔV) mea- surement of spectra for the two polarizations, the difference providing surements were carried out with a KSV 2000 Langmuir balance (KSV In- surface specific information and the sum providing the reference spec- struments, Helsinki). A Teflon trough (58 × 15 × 1 cm) with two trum. As the spectra are measured simultaneously, the effect of water symmetrically moving, hydrophilic Delrin barriers was used in all ex- vapor is largely removed. The PM-IRRAS spectra of the film-covered sur- periments. The surface pressure was measured by the Wilhelmy meth- face, S(f), as well as that of the subphase, S(w), were measured and the od using a platinum plate. Surface potential was monitored by means of normalized difference ΔS/S =[S(f) − S(w)]/S(w) is reported. 6000 in- KSV Spot 1 with a vibrating electrode and a stainless steel as a counter terferogram scans (10 scans per second) have been acquired for each electrode immersed below the water surface. Before each measure- spectrum. In the mid-IR region, the wavenumber at which the half- ment, the subphase surface was cleaned by sweeping and suction. All wave retardation takes place can be freely selected. Here, the maximum solvents used for cleaning the trough and the barriers were of analytical of PEM efficiency was set to either 1500 or 2900 cm−1 for analyzing the grade. The stability of the surface potential signal was checked before different regions of the spectra. The spectral range of the device is each experiment, after cleaning the water surface. After the ΔV signal 800–4000 cm−1 and the resolution is 8 cm−1. had reached the constant value it was zeroed, and the film was spread on the subphase. The pure Chol, DPPC and DPPG as well as DPPC/Chol 3. Results and discussion and DPPG/Chol mixtures of 0.3 molar fraction of Chol, were spread with a Hamilton syringe on water subphase or aqueous saponin solu- 3.1. Compression isotherms and Brewster angle microscopy tion and left for 15 min to allow solvent evaporation and to reach an equilibrium state of the monolayer. All isotherms were recorded upon Merck Saponin and digitonin are water soluble saponins. They lower symmetric compression of the monolayer at a constant barrier speed the surface tension of aqueous solutions and form micelles when 1966 B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 reaching the critical micelle concentration (CMC). Since the CMC of Chol/digitonin surface pressure isotherm and lack of the collapse point Merck Saponin was unknown, the surface tension (γ)–bulk concentra- on the isotherm. This effect correlates well with ΔV–A results, which tion (c) dependency was established (Fig. 2A). This was done by show relatively small changes of the surface potential values, indicating collecting the equilibrium surface pressure (П) data for a number of almost unchanged orientation of molecules upon compression of the Merck Saponin solutions with different bulk concentrations. П was film. In the case of the other lipid/saponin systems, the ΔV–A dependen- monitored as a function of time (Fig. 2B), to ensure that the calculated cies are modified in the presence of the saponin in the subphase com- values of γ used to plot the γ-log c dependencies correspond to the ther- pared to pure water. Indeed, at high molecular areas the ΔV values modynamic equilibria of the solutions. These results show, that the ad- show lower fluctuations and in the most cases are higher compared to sorption time increases with the decreasing concentration of Merck the pure lipid films; this indicates the monolayer stabilization and order- Saponin. For the high bulk concentrations reaching the equilibrium is ing. On the other hand, the lower ΔV values observed in the most con- of the order of minutes (Fig. 2B, black line), whereas for the low bulk densed phase regions with the Chol/Merck Saponin, Chol/digitonin, concentrations the time to attain the equilibrium is above 20 h (yellow DPPC/Merck Saponin, DPPC/digitonin and DPPG/digitonin compared to line). The CMC value determined from the inflection point on the γ-log c thepurelipidfilms may indicate interactions between the saponin pres- plot is 2.3 mg mL−1. In the case of digitonin, the CMC value provided by ent in the subphase and the lipid polar heads leading to some conforma- Sigma-Aldrich [29] is approximately 0.6 mg mL−1. tional disordering of the lipid molecules. The increase of the surface The interaction of Merck Saponin or digitonin with lipids was stud- potential observed in the case of the DPPG/Merck Saponin film com- ied using monolayers spread on low concentration solutions below pared to the pure water subphase indicates that the DPPG hydrocarbon the CMC, to avoid saponin aggregation; concentration of 78.5 μgmL−1 chains reorient and acquire a more vertical orientation in the presence wasusedwithbothsaponinsinallexperiments. of Merck Saponin. −1 In order to investigate the affinity of Merck Saponin and digitonin to It can be also observed that the compressibility modulus (CS ; −1 lipid Langmuir monolayers, compression isotherms of the monolayers CS = −A(∂Π/∂A)T [30])valuescalculatedfromtheΠ–A dependency formed with Chol, DPPC or DPPG were registered. The lipid monolayers (Fig. 3B, D and F) are lower for the monolayers spread on the Merck Sa- were spread on pure water, as well as on Merck Saponin or digitonin ponin or digitonin solutions compared to pure water, indicating a aqueous solutions. The Π–A and ΔV–A isotherms obtained upon com- higher fluidity of the films containing either molecule. This result to- pression of the Chol, DPPC or DPPG monolayers at 20 °C are shown in gether with the surface potential results suggest that the saponins Fig. 3A, C and E, respectively. In Fig. 3B, D and F, the compressibility inserted in the film modify the orientation of the lipids spread at the −1 modulus–surface pressure (CS –Π) plots are presented. air–water interface. The presence of Merck Saponin in the subphase in- The Π–A isotherms of all three pure lipids spread on the aqueous so- duces a more liquid-like character of the films compared with digitonin. −1 lutions of Merck Saponin and digitonin differ considerably compared to The CS values obtained from the isotherms at the most condensed the pure water subphase. Indeed, the isotherms registered in the states of the monolayers are much lower for saponin solutions than presence of Merck Saponin are strongly shifted to high surface pressures; for pure water subphase. The latter is in accordance with the other re- the shift is lower in the case of digitonin. This indicates that the saponins sults showing the presence of the saponin in the highly condensed −1 penetrate from the subphase into the phospholipid film. It has to be no- Chol, DPPC and DPPG monolayers. Interestingly, the CS value obtained ticed, both in the case of Π–A and ΔV–A dependencies, that saponins in- from the Chol/digitonin isotherm at the most condensed state of the sert in the low and high surface pressures lipid film. The only exception monolayer is very low (89 mN m−1) and supports the conjecture that is Chol/digitonin system for which Π–A isotherm illustrates the insertion cholesterol and digitonin were squeezed out from the more packed film. of digitonin at low surface pressures and a loss of the film forming mate- The impact of saponins on the film structure can be observed using rial at higher surface pressures. It suggests, that at low surface pressures BAM. The BAM snapshots of the Chol, DPPC and DPPG monolayers on digitonin adsorbed to the Chol film and was maintained in the film by in- the pure water subphase and aqueous solutions of Merck Saponin and teractionwithChol,whileathighsurfacepressuresbothCholanddigito- digitonin are presented in Fig. 4. The images of the lipid film spread on nin were gradually expelled from the film, shown by the low slope of three different subphases were recorded at 45 Å2 for Chol and 55 Å2 for DPPC or DPPG. At the chosen molecular area the Chol film formed on water and Merck Saponin solution is isotropic, as shown in Fig. 4A 90 and B respectively. However, it has to be noticed, that the image obtain- 40 A B ed on Merck Saponin subphase is brighter compared to that of the pure ) 80 -1 30 water subphase. This effect, which shows as well in the analysis of the isotherms, indicates a higher density of the film due to the incorporation 20

(mN m of Merck Saponin. On the contrary, some anisotropy can be observed at Π ) 70

-1 10 this molecular area in the Chol film spread on the digitonin solution (Fig. 4C); the bright pattern observed with BAM indicates that digitonin 0 60 0510152025 is present in the monolayer and forms mixed Chol/digitonin structures.

mN m Time (h) ( Formation of the digitonin-cholesterol structures may favor removing γ 50 of the latter to the aqueous subphase from the pure, condensed Chol film, while at low surface pressures, the adducts can be accumulated fi 40 in the lm. Overall, the results obtained in our work are in accordance with the literature. Indeed, while cholesterol alone is insoluble in water, the cholesterol/digitonin complexes solubility is 0.6 mg L−1 [12]. 30 The images corresponding to the LE–LC phase transition in the pure -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 DPPC and in DPPC/Merck Saponin and DPPC/digitonin films are present- log c ed in Fig. 4D, E and F, respectively. It can be observed that the condensed phase domains formed in the DPPC film spread on the digitonin solution Fig. 2. Surface activity of Merck Saponin. (A) the adsorption isotherm of Merck Saponin at (Fig. 4F) are smaller and less condensed compared to the pure water the air/water interface. (B) Π (t) adsorption curves obtained for different concentrations subphase. The latter indicates that digitonin is present in the monolayer of saponin in aqueous solutions: 17.7 μgmL−1 (yellow), 78.5 μgmL−1 (navy), − − − − and alters the density of the monolayer. In the case of the DPPC/Merck 157.0 μgmL 1 (gray), 392.5 μgmL 1 (orange), 785.0 μgmL 1 (magenta), 1.3 mg mL 1 −1 −1 −1 fi (cyan), 2.7 mg mL (blue), 9.2 mg mL (green), 13.1 mg mL (red) and Saponin lm, the condensed phase domains are not observed. Instead, 30.5 mg mL−1 (black). irregular bright spots can be seen (Fig. 4E), indicating formation of a B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 1967

A 0.5 B 1000 70 0.4 60 0.3 800 ) -1

) 50 0.2 Δ -1 600 40 0.1 V (V) 0.0 30 (mN m 400 -1 S (mN m -0.1 C Π 20 -0.2 200 10 -0.3 0 -0.4 0 30 40 50 60 70 80 90 0 1020304050 2 -1 A (Å ) Π (mN m ) C D 400 90 0.6 350 80 0.5 0.4 70 300 )

) 0.3 -1 -1 250 60 0.2 Δ V (V) 50 0.1 200 40 0.0 (mN m (mN m 150 -1 S

Π -0.1

30 C -0.2 100 20 -0.3 50 10 -0.4 0 -0.5 0 30 40 50 60 70 80 90 100 110 0 10203040506070 2 -1 A (Å ) Π (mN m ) E F 1000 90 0.4 80 0.3 800 70 0.2 ) -1 ) 60 0.1 -1 Δ 600

0.0 V (V) 50 -0.1

40 (mN m 400 S -0.2 -1 (mN m C Π 30 -0.3 20 -0.4 200 10 -0.5 0 -0.6 0 30 40 50 60 70 80 90 100 110 010203040506070 A (Å2) Π (mN m-1)

−1 Fig. 3. Compression isotherms of Chol (A), DPPC (C) and DPPG (E) films. Solid lines, Π–A isotherms; dashed lines, ΔV–A isotherms. (B, D and F) CS –Π dependency for Chol (B), DPPC (D) and DPPG (F) films. Subphases: pure water (black); Merck Saponin (blue); digitonin (red).

−1 mixed, phospholipid/Merck Saponin film. Indeed, the LE–LC phase tran- [31,32], are shown in Fig. 5A and C. The plots of CS –Π dependency sition was not observed in the corresponding Π–A isotherm, which is are presented in Fig. 5BandD. strongly shifted towards the higher surface pressures compared to In the case of the DPPC/Chol and DPPG/Chol mixed monolayers those formed on water subphase. (Fig. 5A and C, respectively), the shift of the Π–A isotherm to higher mo- BAM images of pure DPPG, DPPG/Merck Saponin and DPPG/digitonin lecular areas in the presence of digitonin in the subphase is more signif- films (Fig. 4G, H and I, respectively) show that DPPG in the presence of icant than that observed with pure DPPC and DPPG monolayers. For digitonin forms a homogenous, isotropic film, while in the presence of example, at the surface area of 55 Å2 the DPPC isotherm shifts to Merck Saponin reveals an anisotropic morphology similar to the pure 14 mN m−1, while that of mixed DPPC/Chol film to 45 mN m−1.Thelat- DPPG monolayer. A brighter shade of the DPPG/Merck Saponin and ter suggests that a higher number of the digitonin molecules is incorpo- DPPG/digitonin film snapshots compared to those of the pure DPPG rated into the mixed DPPC/Chol film compared to the pure DPPC. As film suggests adsorption or penetration of saponins to the DPPG mono- previously, the isotherms of the pure DPPG and mixed DPPG/Chol layers and, consequently, a higher interfacial molecule concentration. monolayers shift respectively to 15 and 16 mN m−1. The smaller shift To better understand the interaction between saponins and lipid of mixed DPPG/Chol isotherm compared to the DPPC/Chol indicates model membranes, the properties of mixed DPPC/Chol and DPPG/Chol that the affinity of digitonin for the mixed DPPG/Chol film is much monolayers upon interaction with Merck Saponin and digitonin were lower compared to DPPC/Chol film. A strong penetration and interac- investigated. The compression isotherms obtained with mixed films tion of digitonin with the lipid molecules was also confirmed by the containing 30 mol% of cholesterol, the biologically relevant content collapse area values. As shown in Fig. 5A and C, the isotherms 1968 B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973

Fig. 4. Brewster angle micrographs of Chol (A–C), DPPC (D–F) and DPPG (G–I) monolayers spread on pure water (A, D, G) and on the subphases containing Merck Saponin (B, E, H) and digitonin (C, F, I). The micrographs (A–C) and (D–I) were taken at 45 Å2 and 55 Å2, respectively. Scale: the width of the snapshots corresponds to 400 μm. corresponding to the mixed films formed on digitonin solution (red The latter suggests that the digitonin molecules incorporated into the lines) show higher molecular area values at the collapse point, com- mixed lipid films were not squeezed out from the highly compressed pared to the isotherm obtained on pure water subphase (black lines). monolayers. This effect is more important for the zwitterionic DPPC

A B 90 0.6 500 80 70 0.4 400 ) ) 60 -1 -1 0.2 300 50 Δ 0.0 V (V) 40 (mN m (mN m

-1 S 200 Π 30 -0.2 C 20 100 -0.4 10 0 -0.6 0 30 40 50 60 70 80 90 100 0102030405060 A (Å2) Π (mN m-1) C D 600 80 0.4 70 500 0.2

60 ) )

-1 400 -1 0.0

50 Δ V (V) 300 40 -0.2 mN m (mN m

30 -1 S

Π ( 200 -0.4 C 20 100 10 -0.6 0 -0.8 0 30 40 50 60 70 80 90 100 0102030405060 A (Å2) Π (mN m-1)

−1 Fig. 5. Compression isotherms of DPPC/Chol (A) and DPPG/Chol (B) mixed films. Solid lines, Π–A isotherms; dashed lines, ΔV–A isotherms. (C, D) CS –Π dependency for DPPC/Chol (C) and DPPG/Chol (D) films. Subphases: pure water (black); Merck Saponin (blue); digitonin (red). T = 20 °C. B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 1969 compared to the negatively charged DPPG. Moreover, the results variations (at a constant film area) as a function of time. The corre- obtained for DPPG film spread on a Merck Saponin solution are in accor- sponding adsorption isotherm profiles are shown in Fig. 7. dance with those obtained for digitonin. At the surface area of 55 Å2 the The results show that both saponins penetrate from the subphase shift of the pure and mixed DPPG isotherms is 31 and 34 mN m−1,re- into the condensed lipid monolayers and lower their surface tension. spectively, showing a stronger penetration of Merck Saponin to the However, the ΔΠ effect observed upon the penetration of Merck mixed system compared to the pure one. On the contrary, the penetra- Saponin to the lipid layers (Fig. 7A) is significantly lower compared to tion of Merck Saponin to the DPPC/Chol film (22 mN m−1 shift) is lower digitonin (Fig. 7B). For example, the maximum ΔΠ effect for Chol/ compared to DPPC film (32 mN m−1 shift). Merck Saponin and Chol/digitonin systems is observed at 14 and The properties of the mixed phospholipid/Chol films were evaluated 31 mN m−1, respectively, showing a higher affinity of digitonin based on their compressibility (Fig. 5B and D). It can be seen that intro- for Chol, compared to Merck Saponin. A higher ΔΠ effect was also ob- ducing of cholesterol into the zwitterionic DPPC film results in film con- served for DPPC/Chol/digitonin or DPPG/Chol/digitonin compared to densation (Fig. 5B). The compressibility modulus, calculated at the most DPPC/Chol/Merck Saponin or DPPC/Chol/Merck Saponin, respectively. condensed state of DPPC/Chol mixed film spread on a digitonin solution It should be also noted that the ΔΠ–t dependencies of pure phospho- −1 −1 is CS = 429 mN m , which is higher than that for the DPPC film lipids and their mixtures with Chol (red, green, magenta and blue −1 −1 (CS = 256 mN m ). Similarly to DPPC, the increase of condensation lines) registered for digitonin reach the equilibrium surface pressure was also observed in the case of DPPG/Chol/digitonin system (Fig. 5D). value within few seconds after injecting the digitonin into the water −1 −1 The maximum value for DPPG/Chol mixed film CS = 387 mN m , subphase. This process is significantly lower in the case of Merck Sapo- −1 −1 while for the pure DPPG CS = 354 mN m . Interestingly, the opposite nin. These results indicate that adsorption/penetration of saponin into effect was observed in the case of mixed films formed on Merck Saponin the lipid film is correlated with the molecular structure of the saponins. −1 solution; an important decrease in the CS values, namely 94 and 173 mN m−1, respectively, for DPPC and DPPG, indicate a more fluid- 3.3. PM-IRRAS like character of the mixed films. These results may suggest modifica- tion of the conformation of both lipids upon the interaction with The PM-IRRAS technique [33–35] was used to analyze changes in the Merck Saponin. orientation of lipid chains induced by saponins, as well as the molecular The run of ΔV–A isotherms of the mixed systems corresponds to that interactions between saponins and lipid membranes. The PM-IRRAS described earlier for the pure lipid films. The higher ΔV values, observed spectra recorded at surface pressure of 30 mN m−1 are shown in in the presence of Merck Saponin, can be explained by a more upright Fig. 8; the values of the characteristic vibrations of the spectra are orientation of the phospholipid hydrocarbon chains. given in Table 1. The BAM snapshots of the DPPC/Chol and DPPG/Chol mixed films Changes in the lipid acyl chains packing and conformation can be de- presented in Fig. 6 complete the compression isotherm results. The tected based on lipid CH2 antisymmetric and symmetric stretching vi- brightness of the images in Fig. 6CandFreflects a higher condensation brations located in the spectral region from 3000 to 2800 cm−1.Inthe of monolayers formed on digitonin solution compared to the respective absence of saponins, two dominant bands were observed in the spec- monolayers on Merck Saponin solution (Fig. 6BandE). trum of Chol, namely, at 2912 and 2847 cm−1 (Table 1), which were assigned to the all-trans conformation of acyl chains [36–38]. The shift 3.2. Adsorption kinetics of these bands to higher frequencies in the presence of saponins reflects the increased number of gauche conformers of the acyl chains. The shift

In order to compare the effect of Merck Saponin and digitonin on the of νas (CH2) is much more pronounced for digitonin than for Merck Sa- lipid membranes, adsorption kinetics were determined. First, a pure ponin, showing that the incorporation of digitonin to the Chol layer lipid or mixed solution was spread at the water–air interface to obtain leads to a marked decrease in conformational order of the acyl chains. an initial surface pressure of approximately 30 mN m−1. Then a small This is not surprising, since digitonin easily penetrates to the Chol volume of the concentrated saponin solution was injected into the monolayers which results in the formation of less ordered films. On water subphase, beneath the lipid film, to reach a final concentration the other hand, the bands due to ν (C–O) stretching appear in the fin- of 78.5 μgmL−1. The adsorption of Merck Saponin and digitonin to gerprint region of the PM-IRRAS spectrum of Chol. This band is sensitive the pure Chol, DPPC and DPPG films, as well as to the mixed DPPC/Chol to hydrogen bonding and shifts to lower frequency upon group hydra- and DPPG/Chol films was followed by recording the surface pressure tion [39,40]. According to our results, the ν (C–O) band is observed at

Fig. 6. Brewster angle micrographs of DPPC/Chol (A–C) and DPPG/Chol (D–F) monolayers spread on pure water (A, D) and on the subphase containing Merck Saponin (B, E) and digitonin (C, F). The micrographs were taken at 55 Å2. Scale: the width of the snapshots corresponds to 400 μm. 1970 B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 A 35 B 35 30 30 ) ) -1

25 -1 25 m 20 20 mN mN m (

15 ( 15

ΔΠ 10 10 ΔΠ 5 5 0 0 0 1200 2400 3600 4800 6000 7200 0 1200 2400 3600 4800 6000 7200 Time (s) Time (s)

Fig. 7. Adsorption kinetics of Merck Saponin (A) and digitonin (B) to different lipid monolayers at initial surface pressure Π =30mNm−1. ΔΠ is the difference between the final and initial surface pressures. Monolayer lipids: Chol (black); DPPC (red), DPPG (green), DPPC/Chol (magenta); DPPG/Chol (blue). Final saponin concentration in the subphase was 78.5 μgmL−1. Temperature: 20 °C.

−1 1061, 1052 and 1047 cm , respectively for Chol, Chol/Merck Saponin effects were observed in the case of DPPG. The values of the ν (CH2) and Chol/digitonin system. These results can be explained by an are generally similar for pure and mixed DPPG indicating that the addi- increased hydrogen bonding of the cholesterol –OH groups in the pres- tion of Chol does not affect the conformation of the phospholipid acyl ence of both saponins; the effect is more important in the case of digito- chains. The blue-shift of the ν (C_O) band visible upon incorporation nin. Indeed, H-bond formation between the bulky sugar headgroups of of Chol (from 1732 to 1738 cm−1) indicates a dehydration of carbonyl digitonin and hydroxyl groups of cholesterol as well as the hydrophobic ester groups in the negatively charged phospholipid. The impact of sa- interaction may result in formation of more soluble and less stable Chol/ ponins on the mixed model membranes is also noticeable. The νas digitonin complex, which can be squeezed out into the aqueous (CH2) signals are blue-shifted in the PM-IRRAS spectra registered on sa- −1 subphase at higher surface pressures. This effect of cholesterol–sugar ponin subphases. The small shift of 1 and 2 cm of the νas (CH2) band headgroup interaction was reported in our previous paper. Indeed, observed with DPPG/Chol in the presence of Merck Saponin and digito- cholesterol and glycolipids can establish a hydrogen bond network nin, respectively, indicates a limited penetration of saponin into the hy- linking the cholesterol hydroxyl group and the maltooligosaccharide drophobic part of the negatively charged phosphoglyceride, while the headgroup of the glycolipid [41]. important shift for DPPC/Chol mixture (8 and 18 cm−1, respectively The spectra of the pure DPPC and DPPG monolayers exhibit charac- for Merck Saponin and digitonin) shows that both saponins penetrate teristic vibrations of phospholipids, namely methylene and carbonyl into the hydrophobic region of the monolayer. It can be observed that ester stretching vibrations [42–44]. While no significant influence of Chol favors hydration of the phospholipid carbonyl groups in the pres- the saponins on the organization of the hydrocarbon chains in DPPG ence of saponins, as shown by the red shift of the ν (C_O) bands. We can be observed, the shift of the νas (CH2)andνs (CH2) to higher propose that saponin–cholesterol interaction results in a higher expo- wavenumbers in the case of DPPC indicates lower conformational or- sure of the phospholipid carbonyl ester groups to water and, conse- dering and higher freedom of acyl chains in the presence of saponins. quently, a higher hydration of these groups compared to saponin-free This effect, more significant for digitonin, is in accordance with the ΔV systems. and penetration results, showing molecule disordering in the monolay- er and a more fluid-like character of the phospholipid/saponin mono- 4. Conclusions layers. Moreover, the impact of saponins on the polar heads of lipids was monitored via the frequencies of the stretching vibration of the car- Here, two different saponin preparations, namely Merck Saponin bonyl ester group ν (C_O) observed in the PM-IRRAS spectra at ap- and digitonin were used to improve our understanding of the interac- proximately 1730 cm−1.AsseeninFig. 8D and F (respectively for tion between amphiphilic glycosides and cell membranes. The results DPPC and DPPG), the presence of saponins in the subphase induces a obtained show that molecules present in both preparations penetrate blue-shift of ν (C_O) band, indicating dehydration of the ester groups. into the lipid monolayers at a concentration below the CMC, and sig- The latter effect is larger in the presence of digitonin and may result nificantly increase the fluidity of the monolayers. It was found that from an increasing interaction with the digitonin sugar headgroups. the saponins used here induce a conformational disorder of the It is interesting to compare the PM-IRRAS spectra of the pure phos- lipid acyl chains. The fluidizing effect was more significant in the pholipids with their mixtures with cholesterol. According to our results case of Merck Saponin bearing a large glycone moiety. It can be ob- obtained on a pure water subphase, the position of methylene served that molecular volumes of Merck Saponin and digitonin are, stretching bands in the DPPC/Chol spectrum indicates a greater role of 6241 Å3 and 4435 Å3, respectively; this volume difference can play a trans conformers in the arrangement of molecules in the mixed film, role in membrane fluidization. compared to the pure DPPC. Moreover, the analysis of the C_O band Both saponins used in this study show a higher affinity for cholesterol frequencies reveals that the addition of cholesterol leads to a small in- compared to phosphatidylglycerols. It has to be noted that saponins con- crease in the number of hydrated carbonyl ester groups. These results tain structural motifs that are similar to membrane sterols. The structural are consistent with the literature reports [45–47], which show a reduc- likeness between the aglycone part of these glycosides and the sterol tion in the number of gauche conformers and a larger contribution of part of cholesterol may be the main reason for a facilitated interaction. the hydrated ester groups of DPPC when mixed with Chol. No such Moreover, the saponin sugar residues may interact with the cholesterol

Fig. 8. PM-IRRAS spectra of Chol (A, B), DPPC (C, D), DPPG (E, F), DPPC/Chol (G, H) and DPPG/Chol (I, J) monolayer in the CH2 (A,C,E,G,I),C–O(B)andC_O (D, F, H, J) stretching regions. Subphases: pure water (black); Merck Saponin (blue); digitonin (red). Saponins were injected beneath the lipid monolayer compressed to 30 mN m−1. T = 20 °C. Dashed lines: deconvoluted bands. B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973 1971

A 0.008 B 0.008 0.006 0.006

0.004 0.004

0.002 0.002

0.000 0.000 3000 2960 2920 2880 2840 2800 1100 1080 1060 1040 1020 -1 Wavenumber (cm ) Wavenumber (cm-1)

C 0.010 D 0.006

0.008 0.005 0.004 0.006 0.003 0.004 0.002

0.002 0.001

0.000 0.000 3000 2960 2920 2880 2840 2800 1800 1780 1760 1740 1720 1700 -1 Wavenumber (cm ) Wavenumber (cm-1) E 0.008 F 0.006 0.006

0.004 0.004

0.002 0.002

0.000 0.000 3000 2960 2920 2880 2840 2800 1800 1780 1760 1740 1720 1700 Wavenumber (cm-1) Wavenumber (cm-1) G 0.008 H 0.008 0.006 0.006

0.004 0.004

0.002 0.002

0.000 0.000 3000 2960 2920 2880 2840 2800 1800 1780 1760 1740 1720 1700 Wavenumber (cm-1) Wavenumber (cm-1) I J 0.008 0.010

0.008 0.006 0.006 0.004 0.004 0.002 0.002

0.000 0.000 3000 2960 2920 2880 2840 2800 1800 1780 1760 1740 1720 1700 Wavenumber (cm-1) Wawenumber (cm-1) 1972 B. Korchowiec et al. / Biochimica et Biophysica Acta 1848 (2015) 1963–1973

Table 1 significantly affect the interaction of OSW-1 with its putative target in Characteristic vibrational wavenumbers of lipid bands obtained from PM-IRRAS spectra. the cells.

νas (CH2) νs (CH2) ν (C–O) While our results do not allow identifying a precise site in the sapo- (cm−1) (cm−1) (cm−1) nins studied, which are responsible for the interaction with the mem- brane lipids, it may be proposed that the interaction with cholesterol Chol/H2O 2912 2847 1061 Chol/Merck Saponin 2932 2863 1052 results in a higher exposure of the phospholipid carbonyl ester groups Chol/digitonin 2937 2859 1047 to water and, consequently, a higher hydration of these groups com- pared to saponin-free systems. This effect may be the driving force for ν ν ν _ as (CH2) s (CH2) (C O) fi (cm−1) (cm−1) (cm−1) the cholesterol-dependent modi cation of cell membranes by saponins. To determine the role of cholesterol in the mechanism of digitonin action DPPC/H O 2920 2852 1728 2 at an atomic level further systematic studies using chemically pure sapo- DPPC/Merck Saponin 2928 2854 1738 DPPC/digitonin 2930 2855 1742 nins are required.

DPPG/H2O 2919 2843 1732 DPPG/Merck Saponin 2922 2851 1733 Acknowledgments DPPG/digitonin 2920 2851 1737

DPPC/Chol/H2O 2914 2847 1726 DPPC/Chol/Merck Saponin 2922 2865 1735 M. G. acknowledges the financial support from an Interdisciplinary DPPC/Chol/digitonin 2932 2856 1732 PhD Studies project entitled “Molecular Sciences for Medicine” co- DPPG/Chol/H2O 2919 2851 1738 financed by the European Social Fund within the Human Capital Opera- DPPG/Chol/Merck Saponin 2920 2850 1731 tional Programme. M. G. thanks the Université de Lorraine for facilitating DPPG/Chol/digitonin 2921 2850 1730 her stay at the laboratory of “Structure et Réactivité des Systèmes Moléculaires Complexes”. hydroxyl group. Interestingly, a higher affinity for the lipid monolayers was observed with digitonin having a higher surface activity. According References to the results obtained, the number of the digitonin molecules incorpo- rated into the phospholipid films was higher compared to Merck Sapo- [1] G. Francis, Z. Kerem, H.P.S. Makkar, K. Becker, The biological action of saponins in an- – nin. 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