Vibrational Spectroscopy 62 (2012) 64–69
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Interaction of quercetin, genistein and its derivatives with lipid bilayers – An ATR IR-spectroscopic study Katarzyna Cie´slik-Boczula a,∗ , Jadwiga Maniewska b , Grzegorz Grynkiewicz c , Wiesław Szeja d , Aleksander Koll a , Andrzej B. Hendrich e a
Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland Department of Biophysics, Wrocław Medical University, ul. T. Chałubi´ nskiego 10, 50-368 Wrocław, Poland c Pharmaceutical Institute, ul. L. Rydygiera 8, 01-793 Warszawa, Poland d Department of Organic, Bioorganic Chemistry and Biotechnology, Silesian Technical University, ul. B. Krzywoustego 4, 44-100 Gliwice, Poland e Department of Medical Biology and Parasitology, Wrocław Medical University, ul. J. Mikulicza-Radeckiego 9, 50-367 Wrocław, Poland b
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 10 May 2012 Accepted 14 May 2012 Available online 24 May 2012 Keywords: Flavonoids Genistein IR spectroscopy Lipid bilayers Antioxidant
a b s t r a c t Genistein, a main soy isoﬂavone, is well known as phytoestrogen and antioxidant but details of its interactions with lipid membranes are poorly understood. The aim of this work was to elucidate the interaction of genistein, its derivatives and quercetin with lipid bilayers using attenuated total reﬂection infrared spectroscopy technique. Measurements performed mainly on liposomes and assistantly on dehydrated lipid ﬁlms enabled us to ﬁnd that studied ﬂavonoids intercalate into phospholipid bilayers. The temperature of chain-melting phase transition for isoﬂavone-mixed DPPC liposomes was determined. The changes of population of the trans/gauche conformers of lipid chains were studied. They exert rigidifying effect in hydrophobic core of bilayer and due to the formation of hydrogen bonds induce a new spatial arrangement of lipid molecules. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Genistein is a main soy isoﬂavone and therefore in humans consuming soy-rich diet its concentration in plasma reaches up to 2.4 M . Due to its diverse biological activities and beneﬁcial health effects genistein substantially attracts the interest of many laboratories and becomes the subject of the numerous studies . Most of the published papers concentrate on the effects exerted by genistein on the intracellular targets and only few deal with the problems related to interaction of this isoﬂavone with biological membranes. This aspect of genistein activity, however, seems to be also important for at least three independent reasons: (i) to reach its intracellular targets isoﬂavone must cross plasma and/or nuclear membranes, (ii) alteration of the membrane biophysical properties contributes to the modulation of integral proteins and ﬁnally (iii) lipid peroxidation is inhibited by genistein molecules already incorporated into the membrane.
Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ATR-IR, attenuated total reﬂection infrared spectroscopy. ∗ Corresponding author. Tel.: +48 71 7357209; fax: +48 71 3282348. E-mail address: kasia [email protected]
(K. Cie´slik-Boczula). 0924-2031/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vibspec.2012.05.010
Antioxidant properties of genistein in model liposomal systems were studied by Arora et al. . Comparison with other isoﬂavones revealed that genistein is more potent antioxidant than daidzein, formononetin and biochanin A. The inhibition of lipid peroxidation by genistein, and also other isoﬂavones, was attributed by some research groups to the preferential partition of these compounds into the hydrophobic core of lipid bilayers [4,5] but also the interactions with polar head groups were considered . The results of calorimetric and ﬂuorescence spectroscopic experiments performed by our group  pointed also to more superﬁcial interactions of genistein with lipid bilayers. According to these data genistein after its incorporation into membrane is located close to the polar-heads region of the bilayer. Almost the same location of daidzein, an isoﬂavone structurally similar to genistein, was proposed on the basis of calorimetric measurements by Lehtonen et al. . Apart from the studies on the location of genistein in lipid bilayers also positions of other ﬂavonoids within membranes were determined. Since those ﬂavonoids differ signiﬁcantly in their structure (i.e. number and positions of hydroxyl groups, presence of additional methyl groups or sugar substitutions) from genistein the results of these studies could not be directly used in the discussion on the genistein–lipid interactions. It is worth to mention only one study in which 1 H magic angle spinning NMR spectroscopy was used to demonstrate that structurally different ﬂavonoids (ﬂavone,
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Fig. 1. Chemical structures of studied ﬂavonoids.
chrysin, luteolin, myricetin, and luteolin-7-glucoside) do not adopt a single position within the membrane but are distributed along the bilayer normal . Since the calorimetric measurements, which were used in our previous study , provide only rough information about the isoﬂavone–lipid bilayer interactions in present work we used ATR-IR spectroscopy to follow the inﬂuence of genistein and its derivatives on the different parts of lipid molecules organized in a bilayer structure. Infrared spectroscopy offers a possibility to observe the alterations of the vibration frequencies characteristic for different parts of lipid molecules, and therefore the different regions of lipid bilayer could be monitored simultaneously in one experiment. This paper will focus mainly on the effect of isoﬂavones on the hydrophobic part of the DPPC membrane (e.g. chain-melting phase transition) and point out that studied compounds also have an inﬂuence on the hydrophilic region of the lipid bilayer. The study of the dehydrated lipid ﬁlm provides the information about the direct interactions between the lipid ﬂavonoid molecules. Apart of genistein itself we also studied few 7-O-derivatives possessing additional benzene ring modiﬁed by different substitutions (see Fig. 1 for details). According to Arora et al. , a replacement of the hydroxyl group in position 7 of the ﬂavonoid molecule by other substitutions does not signiﬁcantly affect the antioxidant activity of molecules modiﬁed in this way. Such a modiﬁcation alters, however, the dipolar moment and hydrophobicity of molecules and changes the liposome membrane integrity . Presence (or not) of the 7-OH group might also inﬂuence several membrane-related processes like the uncoupling efﬁciency of the membrane potential in cytochrome c oxidase vesicles . 2. Materials and methods 2.1. Chemicals Genistein and its derivatives were synthesized in the Department of Organic, Bioorganic Chemistry and Biotechnology of
Silesian Technical University; their chemical structures are shown ´ Poland). in Fig. 1. Quercetin was purchased from Sigma (Poznan, Since those ﬂavonoids were almost not soluble in water, their ethanol or chloroform solutions were used in the experiments. 1,2-Dipalmitoyl-n-glycero-3-phosphatidylcholine (DPPC) was ´ Poland). The lipid was used as purchased from Sigma (Poznan, delivered, without further puriﬁcation. All other chemicals used in experiments were of analytical grade. 2.2. Preparation of liposomes For each sample 12.5 mg DPPC was dissolved in the appropriate amount of ethanol or chloroform stock solution (5 mM) of the studied compound. The ﬂavonoid/lipid molar ratio in the samples was 0.05. Then the organic solvent was evaporated by a stream of nitrogen and the residual solvent was removed under vacuum for 2 h. Samples were hydrated by 1 ml of Tris–EDTA–NaCl (20 mM Tris, 0.5 mM EDTA, 150 mM NaCl) buffer (pH = 7.4) and sonicated to obtain unilamelar DPPC liposomes, using an UP 200 s sonicator (Dr Hilscher GmbH, Berlin, Germany). 2.3. ATR-IR measurements The ATR-IR measurements were performed using a Nicolet Avatar 360 FT-IR spectrometer equipped with a ZnSe-ATR crystal (face angle: 45◦ , 6 reﬂections, Specac). For each measurement 256 scans were collected at a resolution of 2 cm−1 . Interferograms were processed with Happ-Genzel apodization. The correction of the data for the wavelength dependent penetration depth was done by applying a ramp function to the uncorrected ATR spectra. The liposome suspensions were prepared according to procedure described above and spread on the surface of the ZnSe-ATR crystal. The dehydrated ﬂavonoid-doped DPPC ﬁlms were prepared by spreading 200 l of chloroform solution of the ﬂavonoid-doped DPPC mixture on the surface of the ZnSe-ATR crystal and evaporating the solvent under a stream of nitrogen. The concentration of DPPC in
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1.0 νas,sCN 0.5 ν OH 00 0.0
wavenumber / cm-1 Fig. 2. ATR IR-spectra of dehydrated DPPC ﬁlm (solid line) and dehydrated DPPC/genistein (90/10 mol%) ﬁlm (dashed line) at room temperature with the assignment of the main absorption bands of DPPC.
chloroform solution was 10 mg/ml. The spectra of dehydrated ﬁlm and liposome suspensions were recorded in a heating cycle from 10 ◦ C to 60 ◦ C. The sample temperature was equilibrated for 5 min before acquisition of each spectrum. The sample (lipid ﬁlm and liposome suspension) was heated to 60 ◦ C then cooled to 10 ◦ C and in the second heating cycle the sample was measured. The data were analyzed by Grams software. For monitoring the level of hydration in a lipid ﬁlm, the procedure proposed by Pohle et al. [12,13] was applied. The Awr parameter (relative, calibrated water-absorptivity parameter) was calculated as a ratio of the integral absorbance Aw of the broad 1,3 (OH) band centered near 3400 cm−1 due to the stretching vibration of water and the total integrated absorbance of the overall (C H) bands (from lipids) between ca. 2800 and ca. 3050 cm−1 . The Awr in the lipid ﬁlm and in the doped DPPC ﬁlm was not more than 0.5 and was kept at approximately constant level during the experiment. 3. Results and discussion 3.1. Dehydrated ﬁlm of genistein/DPPC mixtures The study of the dehydrated lipid ﬁlm provides the information about the direct interactions between the lipid and biologically active molecules. The inﬂuence of genistein on the hydrophilic part of the DPPC bilayer in the dehydrated state was characterized using the ATR-IR method. The review spectra of dehydrated DPPC ﬁlm before and after complexation with genistein are shown in Fig. 2. The interaction of genistein with DPPC molecules are manifested in changes of spectral parameters of (OH), (C O), (PO2 − ), as (PO), and (CN) bands. 3.1.1. The OH stretching band The identiﬁcation of hydrogen bonds formed by OH groups originating from drug and lipid molecules is based on a very characteristic (OH) vibrational band. The (OH) band is located in the 3600–2000 cm−1 vibrational region of the IR spectrum. The shape and position of this band strongly depend on the strength of hydrogen bond formed by hydroxyl groups. The wavenumber of stretching vibrations of non-hydrogen bonded OH groups is located in the region between 3600 cm−1 and 3400 cm−1 . The band of the stretching vibration of “free” OH groups is sharp. The shift to lower wavenumber region accompanied by broadening of this (OH) band is characteristic for hydrogen bonded OH groups. This red shift increases proportionally to the increase of the strength of the hydrogen bond [14,15]. The ATR-IR spectra of the dehydrated genistein and the dehydrated genistein/DPPC (10/90 mol%) ﬁlms are shown in Fig. 3. The genistein molecule has both proton-donor
and proton-acceptor groups which provide a lot of different possibilities to form both inter- and intramolecular hydrogen bonds. The crystallographic studies showed the existence of an intramolecular H-bond between C O and the adjacent OH group [16,17]. The network of intermolecular H-bonds formed by C O and all OH groups with different combinations is also present in the crystal of genistein . Since, we were studying the dehydrated ﬁlm of genistein molecules with a limited number of water molecules, the type of intra- and intermolecular interactions should be similar to that observed in the crystal. Both intra- and intermolecular H-bonds are manifested in the infrared spectrum as a broad (OH) band with a maximum centered near 3190 cm−1 (see Fig. 3). Additionally, the presence of a sharp (OH) band at around 3400 cm−1 indicates that some of genistein OH groups are non-hydrogen bonded. Mixing of genistein and DPPC molecules (10/90 mol%) results in the disappearing of the sharp (OH) band at 3400 cm−1 and shifting of the broad (OH) band to lower frequency region. Thus, in genistein/DPPC mixtures new and stronger H-bonds are formed. The weaker hydrogen bonds, which are present in genistein dehydrated ﬁlm, are replaced by stronger interactions between proton-donor OH groups in genistein and proton-acceptor groups like phosphate or ester in DPPC molecules. 3.1.2. The 1800–700 cm−1 vibration region Fig. 4 shows the spectrum of the 1800–700 cm−1 vibration region of genistein molecules in the pure genistein ﬁlm and in the mixture with DPPC lipids. As can be seen, the infrared spectrum of genistein shows several characteristic peaks, the most prominent being at around 1651 cm−1 (the (C O) band), 1613 cm−1 (the (C C) band), 1520–1404 cm−1 (the ı(5OH), (C C) bands), 1320–1150 cm−1 (the (C O C) band), 930–790 cm−1 (the (C H), (C C) bands) [18–20]. The subtraction the spectrum of pure DPPC ﬁlm from the spectrum of genistein/DPPC (10/90 mol%) ﬁlm reveals the spectrum of genistein in lipid environment with the additional bands associated with mutual inﬂuences of lipid and isoﬂavone molecules; see Fig. 4B. The hydrogen bond interaction, mentioned in the previous chapter, change most of the substantial vibrations of genistein, for comparison see Fig. 4A and B. The ı(5-OH) bands centered at 1503 cm−1 and 1426 cm−1 are not present in the spectrum of genistein incorporated into the lipid bilayer, which indicates the involvement of these functional groups in a new type of interaction, e.g., in a hydrogen bond with lipid molecules. The literature data shows that the position of the (C O) band of genistein occupies mostly the 1670–1630 cm−1 region and depends on the type of interactions with that moiety, i.e., if the hydrogen bond is formed or not [18–20]. Thus, the maximum of that band is not present for wavenumbers larger than 1700 cm−1 , the additional two bands centered at around 1748 cm−1 and 1718 cm−1 , at positions characteristic for C O vibrations of DPPC molecules, see the difference spectrum
dehydrated film of genistein
dehydrated fim of i t i /DPPC C genistein/DPP (10/90mol%)
wavenumber wavenum ber / cm-1 Fig. 3. Representative absorption spectra of dehydrated genistein (dashed line) and dehydrated genistein/DPPC (10/90 mol%) ﬁlm (solid line) in the (OH) region at 25 ◦ C.
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0.45 0.30 0.15
genistein in DPPC liposomes
0.15 0.1 0.05 0.0 -0.05 1800
wavenumber / cm-1 Fig. 4. The spectrum of the 1800–700 cm−1 vibration region of the pure genistein dehydrated ﬁlm (A) The difference spectrum of the dehydrated genistein/DPPC (10/90 mol%) spectrum and the dehydrated pure DPPC spectrum at room temperature (B). *The appearance of two additional carbonyl stretching bands and the low-wavenumber shift of the as (PO2 − ) band.
in Fig. 4B, originate from the C O stretching vibrations of DPPC molecules. The ester carbonyl groups are located in the polar–apolar interface of DPPC bilayer. The C O stretching band is present in the 1750–1700 cm−1 region of the IR spectrum. This is one of the most intensive lipid bands, and it is useful for probing the polar/apolar interface region of lipid assemblies [21–31]. The position of the (C O) band maximum is sensitive to the conformation and/or orientation of ester groups and to the hydration level of the carbonyl region in DPPC bilayer. The studies of Lewis et al.  demonstrated that the subcomponents of the (C O) absorption band of diacyl PC bilayers in the fully hydrated and dry state are not due to the non-equivalent location of two ester carbonyl groups (sn-1 and sn-2) on the PC glycerol backbone. The experiments with 13 C O labeled PC lipid in hydrated bilayers showed that the sn-1 and sn-2 ester (C O) bands are themselves each resoluble into two components with similar maxima for both ester conformers [26,31]. Thus, the subcomponent at lower wavenumber reﬂects the contribution of subpopulations of hydrogen bonded carbonyl groups, while the high wavenumber band arises from free (non H-bonded) groups [26,31]. The degree of hydration, phase state of the lipid and the presence of some compounds and drugs in a lipid bilayer change the shape and position of the (C O) band [26,28,29]. The genistein-adduct causes the appearance of two additional components in the DPPC’s carbonyl band centered at around 1748 cm−1 and 1718 cm−1 , see Fig. 4B. The lower wavenumber band can result from the hydrogen bond interactions between proton-donor OH groups of genistein and proton-acceptor C O moieties. Our IR spectroscopic studies were not performed under vacuum condition. Hence, it was not possible to obtain a completely dry lipid ﬁlm. These water molecules can form hydrogen bonds with carbonyl groups of DPPC molecules. The genistein/DPPC ﬁlm was prepared in the same way as the pure DPPC ﬁlm, without vacuum condition. Therefore, the presence of water molecules can also be expected in a mixed DPPC ﬁlm. Water molecules can be replaced by genistein molecules, which lead to H-bonds with ester groups. In a dehydrated ﬁlm formed from pure DPPC we observed two strong bands around 1258 cm−1 and 1095 cm−1 , which appeared
due to the PO2 − antisymmetric and symmetric stretch vibration, respectively . Both these bands shifted gradually to the lower wavenumber region with increasing hydration as a result of the increase of the number of hydrogen bonds between water proton-donor OH groups and PO2 − proton-acceptor groups of DPPC molecules [21,25]. The wavenumber shift of as (PO2 − ) to lower wavenumber region observed in a genistein-doped DPPC ﬁlm is induced by the interactions of hydroxyl groups of genistein with proton acceptor PO2 − groups of DPPC. The maximum of the as (PO2 − ) band shifts gradually from position 1257 cm−1 , through 1254 cm−1 , to 1243 cm−1 and 1239 cm−1 , for 10, 20, 30 and 40 mol% of the concentration of genistein in the DPPC medium, respectively. Additionally, the appearance of H-bonds in doped DPPC bilayer also shifts gradually the maximum of the s (PO2 − ) band from position 1093 cm−1 , through 1091 cm−1 , to 1089 cm−1 and 1086 cm−1 , for 10, 20, 30 and 40 mol% of the concentration of genistein in a DPPC ﬁlm, respectively. Fig. 4B shows the differential spectrum obtained by subtraction of a pure DPPC ﬁlm spectrum from the spectrum of DPPC mixed with 10 mol% genistein with a very distinguished shift of as (PO2 − ) band from high to lower wavenumber region as a consequence of forming H-bonds between lipid PO2 − and genistein OH groups. 3.2. Flavonoid-doped DPPC liposomes 3.2.1. Aliphatic chain region The vibrational 3000–2900 cm−1 region contains several stretching vibrations of C H groups of DPPC hydrocarbon chains . In the spectrum of DPPC liposomes and other phospholipids, the antisymmetric and symmetric CH2 stretching bands are located at around 2920 and 2850 cm−1 , respectively. Since the positions, intensities, and widths of these bands are conformation sensitive, they are a sensitive marker of the lipid order [21,32–37]. The increase of gauche conformers population in the aliphatic lipid chains is accompanied by the shifting to higher wavenumbers, intensity reduction and bandwidth increase of the as,s (CH2 ) bands [33,34]. This conformational ﬂuidization of the lipid bilayer can be caused by the increase of temperature, water content, or by incorporation of some biologically active compounds into the lipid membrane . A dramatic increase of the gauche conformer population occurs during the main thermotropic phase transition of lipid bilayer. Changes of the position of the s (CH2 ) band maximum in DPPC liposomes doped with a 0.05 mol fraction of genistein and its derivatives are shown in Fig. 5A. For DPPC as well as for doped DPPC liposomes, close to the temperature of main phase transition, the sharp shift of the position of s (CH2 ) band maximum was observed. In IFG41-, IFG70-, IFG71-, IFG74-doped DPPC liposomes, this shift was exactly the same as in pure DPPC liposomes. On the other hand, when genistein or quercetin was present in studied systems, the observed chain melting phase transition was broader comparing to that in DPPC liposomes. In the presence of these two ﬂavonoids the ﬂuidization of DPPC membrane started also at lower temperatures; this temperature shift was more pronounced in the quercetin/DPPC liposomes than in genistein/DPPC ones (see Fig. 5A, Table 1). Since for temperatures below the main phase transition, all studied samples showed the same position of s (CH2 ) band maximum, one can conclude that in the gel phase all investigated liposomes the trans/gauche ratio was similar. Similar results were found in the analysis of the position of as (CH2 ) band maxima (see Fig. 5B). 3.2.2. The carbonyl group region In the gel state of hydrated DPPC bilayer the (C O) band is centered at 1737 cm−1 . In the disordered, more hydrated liquid-crystalline phase this band is located at 1733.5 cm−1 . During
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IFG 71 IFG 74
2850.5 2850 2849.5 2849
wavenumber / cm-1
waven number / cm-1
temperature / °C
temperature / °C
Fig. 5. The position of the symmetric (A) and antisymmetric (B) CH2 stretching band s,as (CH2 ) as a function of temperature in pure and ﬂavonoid-doped DPPC liposomes. Table 1 Values of Tm (the chain-melting phase transition) obtained from the analysis of the position of s (CH2 ) band as a function of increase of temperature in pure and genistein- and its derivatives-doped DPPC liposomes. The chemical composition of liposomes
Tm (◦ C)
DPPC IFG41/DPPC IFG70/DPPC IFG71/DPPC IFG74/DPPC Genistein/DPPC Quercetin/DPPC
41.5 41.5 41.7 41.5 41.5 39 32.5
the main phase transition the sharp shift of the (C O) vibrations to the lower frequency region is observed in DPPC liposomes (see Fig. 6). It is evoked by the increase of hydration of the polar–apolar interface in DPPC membrane, when the conformational disorder in hydrophobic part is introduced . The temperature increase causes the rise of the gauche population in hydrocarbon chains, which weakens the force of van der Waals interactions. At the temperature of the chain-melting phase transition, the distances between DPPC molecules markedly increase, and more water molecules are able to penetrate deeper into the DPPC bilayer. The relationship of the maximum position of the (C O) band at different temperatures in pure DPPC and doped DPPC liposomes is shown in Fig. 6. In all cases the change of the stretching vibration of carbonyl groups to the lower frequency region is observed close to the temperature of chain-melting phase transition. This indicates
IFG 70 IFG 71 IFG 74
wavenumber / cm m-1
that the presence of quercetin, genistein and 7-O-derivatives of genistein in DPPC membrane does not vanish the increase of the hydration of interface region of DPPC bilayer, which is characteristic for the main phase transition in pure DPPC liposomes. The position of the maximum of the ester C O band in both gel and liquid-crystal phase in doped DPPC membranes clearly differs from that in pure DPPC one, see Fig. 6. The shift of this band to the lower wavenumber region in the liquidcrystal state of DPPC membranes increases in the following order: IFG71 < quercetin < IFG41 < IFG74. On the contrary, the genistein increases the wavenumber of the maximum to around 1739 cm−1 and 1738.5 cm−1 in the gel and liquid-crystalline phase, respectively. Only one genistein benzyl derivative, IFG70, does not change the ester stretching band position in both phases of the DPPC membrane. In the gel phase the red shift of the (C O) vibrations grows with the following order: IFG41 < IFG74 < quercetin and for IFG71 it is slightly blue shifted compared to the position of this band in pure DPPC liposomes. For all observations mentioned above, we conclude that the presence of quercetin, genistein and its benzyl derivatives (except IFG70) have an inﬂuence on ester carbonyl groups properties. The above presented results clearly show that quercetin, genistein as well as its derivatives interact with lipid bilayers. It seems that due to the formation of hydrogen bonds with several components of lipid polar heads, the studied compounds introduce a new spatial arrangement of these lipid parts and loosen the packing of molecules. This bilayer structure loosening is also visible at the level of hydrocarbon chains – as an increase of the number of gauche conformers induced by the presence of ﬂavonoids. The incorporation of ﬂavonoid molecules into the lipid bilayer structure might also inﬂuence the efﬁcacy of these molecules as antioxidants. Located in the polar region of the lipid bilayer they can play a role of “guarding” molecules, preventing penetration of acyl chain regions by reactive oxygen species (ROS).
This work was supported by a grant N N204 150338 from the Polish Ministry of Science and Higher Education. We thank the OEAD (Proj. PL-05/2011) for additional ﬁnancial support.
1734 1733 1732
1731 1730 10
temperature / °C Fig. 6. Temperature dependence of the wavenumbers of the ester C O stretching group in pure and doped DPPC liposomes.
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