Impact of quercetin and fish oil encapsulation on bilayer membrane and oxidation stability of liposomes

Food Chemistry 185 (2015) 48–57

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Impact of quercetin and fish oil encapsulation on bilayer membrane and oxidation stability of liposomes M. Frenzel ⇑, A. Steffen-Heins Institute of Human Nutrition and Food Science, Kiel University, Kiel, Germany

a r t i c l e

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Article history: Received 4 August 2014 Received in revised form 6 March 2015 Accepted 30 March 2015 Available online 3 April 2015 Keywords: Liposomes Quercetin Fish oil EPR ESR Oxidation Spin probing Spin trapping

a b s t r a c t Unsaturated soy phosphatidyl choline (PC) liposomes were systematically analyzed for chemical and physical stability and for influence on membrane fluidity, when quercetin and fish oil were encapsulated. The physical stability of liposomes was lowered with loading, which is mainly due to fish oil leakage. While fish oil did not induce oxidative acceleration, quercetin did not reduce lipid-derived radical formation but it did inhibit hexanal formation. It also showed no relevant effects on membrane fluidity, polarity or partitioning of the spin probe TEMPOL-benzoate (TB), as proved by EPR measurements and simulation. However, increasing concentration of fish oil in a membrane might increase the acyl chain dynamics and therefore apply a more attractive environment for TB. In contrast to the encapsulates increasing fluidity of saturated membranes by disturbing the lipid packing, membrane properties of unsaturated systems with a Tm below 0 °C were not influenced by encapsulation of quercetin or fish oil. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Quercetin and fish oil are lipophilic compounds, which are of great interest for human nutrition and functional foods (Bigliardi & Galati, 2013; Russo, Spagnuolo, Tedesco, Bilotto, & Russo, 2012). Due to their poor solubility in hydrophilic matrices, their application in hydrophilic food systems is limited (Pool, Mendoza, Xiao, & McClements, 2012). Carrier systems can help to overcome this drawback by encapsulation of these compounds in lipid-based systems such as single and multilayer emulsions, solid-lipid nanoparticles, micelles or liposomes. As liposomes resemble the structure of biomembranes, they are suitable for use as delivery systems in food and to ensure a good cellular uptake (Cadena et al., 2013). The physical instability and high semi-permeability of membranes, below their phase transition temperature Tm, are challenging tasks when incorporating liposomes into foods. For stability

Abbreviations: TB, TEMPOL-benzoate; EPR, electron paramagnetic resonance; EE, encapsulation efficiency; PC, phosphatidyl choline; Tm, phase transition temperature; pdi, polydispersity index. ⇑ Corresponding author at: Kiel University, Institute of Human Nutrition and Food Science, Heinrich-Hecht-Platz 10, D-24118 Kiel, Germany. Tel.: +49 431 8805034; fax: +49 431 8805544. E-mail address: [email protected] (M. Frenzel). http://dx.doi.org/10.1016/j.foodchem.2015.03.121 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

reasons and low leakage of encapsulates, it is now recommended and approved to use phospholipids with high saturation degree (Pawlikowska et al., 2007) and also by optionally adding cholesterol (Ionescu & Ganea, 2012), which increases membrane stiffness. As application of synthesized phospholipids or cholesterol is not appropriate in food applications, the use of mixed lecithins, e.g., soy lecithin, is cost efficient, unproblematic to food legislation and more nutritionally valuable (Laye, McClements, & Weiss, 2008). However, the low Tm of below 0 °C causes liposome instability that needs to be overcome by other strategies, such as application of a biopolymer coating layer, e.g. chitosan (Laye et al., 2008) or whey protein isolate (Frenzel & Steffen-Heins, 2015). For an efficient application of liposomal carriers as food delivery systems, it is necessary to obtain deeper insights into the solubilization site, stability and impact of encapsulated compounds on bilayer membrane properties, such as fluidity, micropolarity and molecule dynamics. A powerful tool for investigating the effect of lipophilic compounds on the liposomal bilayer is electron paramagnetic resonance spectroscopy (EPR) spin probing (Jia, Joly, & Omri, 2008; Pawlikowska-Pawle˛ga, Gruszecki, Misiak, & Gawron, 2003). TEMPOL-benzoate (TB) is a lipophilic and non-site-directed spin probe and, as such, is very sensitive to its microenvironment. It shows strong dynamic properties and coexists in different spectral populations that differ in site, micro polarity and molecular tumbling. To calculate spectral parameters, such as hyperfine

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splitting, proportion, rotational correlation time, and order parameter of each spin probe population, that are sensitive to alterations in membrane fluidity and hydrophobicity, superimposed EPR spectra were simulated by EPRSim-C software (Štrancar et al., 2005). In this work the influence of quercetin and fish oil on soy PC liposomes was investigated. Spin probing was used to characterize the impact of bioactive compounds on membrane fluidity and integrity. The encapsulation efficiency of quercetin-loaded liposomes was tested over a period of more than two months. The impact of fish oil and quercetin on lipid oxidation in liposomes was evaluated, using EPR spin trapping, and compared with established methods for the study of lipid oxidation. 2. Materials and methods 2.1. Materials All used solvents were purchased from Carl Roth (Karlsruhe, Germany). They were of analytical grade and used without further purification. Quercetin was purchased from Dr. Behr (Bonn, Germany) and of 95% purity. Phospholipon 90 G was kindly provided by Lipoid AG (Switzerland). The fish oil, Omevital 1812 TG Gold (with 30% of omega-3 fatty acids), was purchased from BASF Personal Care and Nutrition GmbH (Illertissen, Germany). Crude rapeseed oil was obtained from a local supermarket and used as received. All substances used for liposome production and loading were of food grade. TEMPOL-benzoate (TB), a-phenyl-tert-butyl-nitrone (PBN), crosslinked dextran G-50 medium, ferrous-II-heptahydrate, ammonium thio sulfate, barium chloride and sodium dodecyl sulfate (SDS) were purchased from Sigma– Aldrich (Steinheim, Germany) and used as received. 2.2. Methods 2.2.1. Liposome preparation and encapsulation of quercetin or fish oil Liposomes were produced by a thin-film-hydration method, as previously described (Frenzel & Steffen-Heins, 2015). Phospholipid isolate Phospholipon 90 G from soy was used to form liposomes; it consists of more than 95% of phosphatidyl choline and the main fatty acids C18:2 cis 6 (60.5 ± 0.7%), C16:0 (12.8 ± 0.1%), C18:1 cis 9 (9.9 ± 0.1%) and C20:3 n6 (5.4 ± 0.8%). 100 mM sodium acetate buffer (pH value 3) was used as rehydration medium. For the experiment, different concentrations of quercetin in ethanol or of fish oil in ether were added to the phospholipid-ether solution prior to solvent evaporation. Encapsulation efficiency and total concentration of liposomal quercetin were investigated at 3 g/ 100 ml Phospholipon liposomes, with increasing quercetin concentrations between (750 and 4500 lM) (Fig. 1b and c). Storage experiments were carried out at 1 g/100 ml of Phospholipon liposomes with a concentration of 800 lM quercetin (Fig. 2). For lipid oxidation experiments, liposomes consisted of 3 g/100 ml of Phospholipon and either of quercetin encapsulated at the concentrations of 2 lM or 10 lM (Fig. 3) or of fish oil at concentrations of 0.06 g/100 ml or 0.3 g/100 ml, referred to the entire liposomal solution (Fig. 4). For EPR probing experiments liposomes containing 2 g/100 ml of Phospholipon and 1 mM TEMPOL-benzoate (TB) were used with either 0–0.675 g/100 ml of fish oil and 0– 2000 lM quercetin referred to the entire liposomal solution (Figs. 5 and 6). 2.2.2. Preparation of micellar solutions and emulsions Micellar solutions were prepared by dissolving 3 g of SDS in 100 ml of distilled water and stirring it at 30 °C for 30 min. For preparation of emulsions, SDS was dissolved in a third of the

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continuous phase (distilled water). This emulsifier solution was mixed with crude rapeseed oil and pre-homogenized with an ultrasound probe for 30 s (cycle 9, 40% power, Bandelin sonopuls HD 2200). The remaining continuous phase was added stepwise and emulsion was further sonicated for 150 s. The final emulsion contained 3 g of SDS and 10 g of rapeseed oil per 100 ml of emulsion. 2.2.3. Particle size and zeta potential Particle size and zeta potential were assessed by dynamic light scattering and measurement of electrophoretic mobility on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), using a laser with 173 °C backscatter (refractive index 1.45, 0.0872 cp, 25 °C). The samples were diluted 1:10 in their original solvent. Measurements were conducted in triplicate. Particle size was calculated as scattering intensity based average hydrodynamic diameter (z-average). As indicator for system polydispersity, the particle distribution index, pdi (0 = completely monodisperse, 1 = polydisperse) was assessed in addition. 2.2.4. Determination of quercetin concentration, solubility and encapsulation efficiency The quercetin concentration in liposomes and other systems was determined on an Agilent 1100 HPLC system (Waldbronn, Germany) equipped with quaternary pump, UV–Vis detector and CC 125/4 Nucleodur Sphinx RP, 5 lm column (Macherey Nagel, Germany). The following method was used: 50/50 v/v methanol/ ultrapure water acidified with 0.5% formic acid (isocratic), flow rate 1 ml/min, injection volume 10 ll, column temperature, 30 °C, detection wavelength 365 nm. Analyses were conducted in triplicate. Encapsulation efficiency (EE) was determined by size exclusion chromatography on crosslinked dextran, using the mini column centrifugation method modified from Fry, White, and Goldman (1978). In brief, a dextran slurry was filled into 5 ml syringes. Excess fluid was removed by a 3 min centrifugation at 1000g and the syringes were locked with filters. 250 ll of diluted liposome solution was pipetted onto the columns, followed by a second centrifugation step to collect the liposome fraction freed from non-encapsulated material in a tube. The first eluate was analyzed for solubilized quercetin concentration as described above. The encapsulation efficiency (EE) was calculated by Eq. (1):

EE ½% ¼ 100 

liposomal quercetin total quercetin

ð1Þ

For determination of quercetin solubility, a stock solution of dihydroquercetin was prepared in ethanol. Equal aliquots of this stock solution were pipetted into glass tubes and ethanol was removed by a stream of nitrogen. The dried quercetin residue in the tubes was redissolved in ethanol, bidistilled water, rapeseed oil, 3% SDS-micelles or rapeseed oil emulsion from 3% SDS. The samples were warmed at 40 °C, bath-sonicated for 30 s and filtered. The permeate was analyzed for solubilized quercetin concentration as described above. 2.2.5. Lipid oxidation 2.2.5.1. Hydroperoxides. The ferric thiocyanate method was modified from Stöckmann and Schwarz (1999). In brief, the thiosulfate reagent was prepared by dissolving 30 g of ammonium thiosulfate in 100 ml of distilled water and the ferrous reagent by dissolving 0.5 g of ferrous sulfate heptahydrate in 50 ml of distilled water and 0.4 g of barium chloride dihydrate in 50 ml of distilled water and combining both solutions with simultaneous addition of 3 ml of 25% hypochlorous acid. The phospholipids were extracted, using 50/50 v/v isopropanol/isooctane and vortexing three times for ten seconds, followed by centrifugation at 2000 rpm for 10 min. 500 ll

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Fig. 1. Survey of the solubilized proportion of 1000 lM quercetin in different media such as ethanol (EtOH), water (H2O), rapeseed oil (RO), SDS emulsion (SDS E), SDS micelles (SDS M) and liposomes (Lipo). Non-solubilized proportion of quercetin was separated by SEC for liposomes and by a filtration method for all other media used. The quercetin concentration measured in ethanol was set as 100% (a). Encapsulation efficiency and total concentration of encapsulated quercetin (750–4500 lM) in liposomes (3 g/100 ml) (b), as well as particle size and polydisperity index (pdi) of those liposomes (c).

Fig. 2. Scattering intensity based average liposome hydrodynamic diameter (a), zeta potential (b), encapsulation efficiency (c) and total quercetin concentration (d) of 800 lM quercetin encapsulated in liposomes (1 g/100 ml) and stored for 80 days at 4 °C in the dark.

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Fig. 3. Scattering intensity based average hydrodynamic diameter (a), polydispersity index, pdI (b), zeta potential (c), radical formation, I0 (d), hydroperoxide formation (e) and hexanal formation (f) of liposomes (3 g/100 ml) containing 0 lmol/l (control,d), 2 lmol/l (j) or 10 lmol/l (N) of quercetin. Samples were stored for 21 days at 40 °C in the dark.

of isooctane phase was mixed with 4500 ll of isopropanol, set with reagents and incubated for 30 min at 60 °C. Solutions were cooled to room temperature and absorbance was measured on a Thermo Scientific Genesys 10S UV–Vis spectrophotometer (Braunschweig, Germany) at 484 nm. Analyses were carried out in triplicate. The exact lipid content of extracts was determined by drying another 500 ll of the isooctane phase under a stream of nitrogen and weighing. 2.2.5.2. Hexanal. As the main unsaturated fatty acids of phospholipid isolates were the n6-fatty acids C18:2 cis 6 (60.5 ± 0.7%) and C20:3 n6 (5.4 ± 0.8%), hexanal was used to follow the formation of secondary oxidation products. According to Serfert, Drusch, and Schwarz (2009), one gramme of sample was weighed into 20 ml headspace vials. An Agilent 6890 series GC-system, coupled with an Agilent G1888 Headspace sampler (Waldbronn, Germany), was used for analysis. 1 ml of volatiles was injected onto a J&W DB1701 column (60 m  0.32 m  3 lm, Agilent Technologies,

Waldbronn, Germany) after 15 min of incubation in the oven at 70 °C with the following auto sampler settings: oven temperature 70 °C, loop temperature 120 °C, tray temperature 150 °C and 15 min vial equilibration at 70 °C; GC cycle time of 28 min, loop fill time 0.2 min, loop equilibration time 0.05 min; GC method: helium flow of 12.7 ml/min at 16.15 psi, split ratio 4:1, split flow, 8 ml/ min, helium flow 2 ml/min. Oven ramp: temperature was set to 45 °C, held for two minutes, raised up to 85 °C at 15 °C/min and maintained for 4 min, and further raised to 220 °C at 15 °C/min and held for 3 min; total run time was 20.67 min. A flame ionization detector was used at 250 °C. Hexanal content was assessed by peak integration and calculated as peak area (p.a.). 2.2.5.3. Free radicals assessed by EPR spin trapping. The spin trap PBN was used at a concentration of 45 mM. 190 ll of sample were mixed with 10 ll of PBN solution and incubated at 55 °C for 25 min. 25 ll of the sample were drawn into a glass capillary and placed in the EPR cavity. EPR spectra of PBN-adducts were

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Fig. 4. Scattering intensity based average hydrodynamic diameter (a), polydispersity index, pdI (b), zeta potential (c), radical formation, I0 (d), hydroperoxide formation (e) and hexanal formation (f) of liposomes (3 g/100 ml) containing 0 g/100 ml (control,d), 0.06 g/100 ml (j) or 0.3 g/100 ml of fish oil (N). Samples were stored for 30 days at 40 °C in the dark.

acquired on a Bruker Elexsys E 500 EPR spectrometer (Rheinstetten, Germany), operating with an X-band microwave frequency of 9.85 GHz and modulation frequency of 100 kHz. Microwave power was set at 20 mV, modulation amplitude at 1.1 G, receiver gain at 80 dB, time constant at 81.92 ms and conversion time at 40 ms. The center field was optimized to 3502 G with a field sweep of 48.7 G. Spectra were accumulated in three scans. The radical concentration was assessed by double integration of the central peaks (H0) and expressed as radical formation, I0. 2.2.6. Membrane properties assessed by EPR spin probing EPR spectra of the spin probe TB (1 mM) in liposomes were recorded on a Bruker Elexsys E 500 EPR spectrometer (Rheinstetten, Germany) in the presence and absence of increasing concentrations of either quercetin or fish oil. The modulation amplitude was set at 1.0 G, microwave power at 0.6325 mW and receiver gain at 60 dB. The center field was optimized to 3510 G and the sweep width to 94.3 G. The time constant used was

81.92 ms and the conversion time was 39.16 ms. Spectra were simulated by EPRsim-C software for nitroxide fitting developed by Štrancar et al. (2005) to distinguish between individual probe populations of TB in different chemical microenvironments. To prove the best fit parameters of the spectra, different numbers and kinds of models were evaluated and checked for lowest value of the chi-squared test of goodness of fit, as well as for minimum difference after subtraction of fitted from measured spectra. Biophysical models were used for approximation of experimental spectra based on the following parameters: line shape (Lw), rotational correlation time sc), relative weight (d), order parameter (S), as well as polarity correction factors for the A (pa) and g tensors (pg), and an additional broadening constant (W). A model for an isotropic tumbling spin probe in a fast motional regime (index 1), and a model for a fast tumbling spin probe in an anisotropic environment, e.g., membranes (indices 2 + 3), were used for simulations. As tensors for TB those of TEMPO were used. According to the collection of Berliner (1976), the g tensors were set at

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Fig. 5. Measured and simulated EPR spectra of TEMPOL-benzoate (TB) in unloaded liposomes (2 g/100 ml) (a). Simulated spectra of the three different spectral populations (b). Measured EPR spectra of liposomes loaded with two concentrations of quercetin (c) and of fish oil-loaded liposomes (d). TB was used at a concentration of 1 mM.

Fig. 6. Hyperfine splitting constants (a), proportion (b), order parameter (c) and rotational correlation time (d) of all three populations (population 1 (d), population 2 (j) and population 3 (.) in the presence of increasing concentrations of fish oil (filled symbols) and quercetin (unfilled symbols). Spectral populations were simulated by using a model for an isotropic tumbling spin probe in a fast motional regime (population 1) and a model for a fast tumbling spin probe in an anisotropic environment, such as membranes (populations 2 + 3).

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gxx = 2.0103, gyy = 2.0069, gzz = 2.0030 and the A tensors on Axx = 7.60 G, Ayy = 6.00 G and Azz = 31.80. Spectral parameters for superimposed spectra were calculated from simulated spectra and recorded as the means of three individual samples. For isotopic spectra of TB, the overall micropolarity of the spin probe was calculated by means of the hyperfine splitting parameter, aN, and recorded as the distance between low field peak (H+1) and middle field central peak (H0).

3. Results and discussion 3.1. Quercetin solubility in different media Quercetin is a semi lipophilic flavonol, which is poorly watersoluble. For assessing the solubility of quercetin in media of different polarities, the same concentration of quercetin was solubilized in ethanol, water, crude rapeseed oil, SDS-emulsions and micelles and phospholipid liposomes. As it was not possible to use size exclusion chromatography (SEC) on crosslinked dextran in all media investigated to separate the non-solubilized from the solubilized quercetin, only liposomes were purified by SEC. Although the SEC gave more reliable results, both methods could be compared to get a general idea about quercetin solubility in different media as they separated the free from the solubilized quercetin content whereas the solubilized content was measured. A complete solubility was given in 100% ethanol (Fig. 1a). Low solubility, of approximately 10% of quercetin, was found in water but also in crude rapeseed oil. In SDS emulsion and micelles, solubility of more than 90% was found. For comparison reasons, the same concentrations of quercetin and the emulsifier Phospholipon were used to form liposomes, resulting in an encapsulation efficiency of 80% quercetin in liposomes. The results emphasize that sufficient quercetin solubility is only given in amphiphilic systems, whereas micelles, emulsions and liposomes showed the same high solubilization capacity. This conclusion is also proved by other groups, who investigated the solubility of quercetin in 50% ethanol, in different PEG solutions (Priprem, Watanatorn, Sutthiparinyanont, Phachonpai, & Muchimapura, 2008), in MCT emulsions (Pool et al., 2012), in nano micelles (Tan, Liu, Chang, Lim, & Chiu, 2012) or in solid lipid nano particles (Li et al., 2009). However, the use of liposomal systems exhibits additional advantages amongst simple micelles and emulsions, when those systems are applied as delivery systems in food. As the liposome bilayer membrane bears resemblance to human cell membranes, it can enhance the cellular uptake of encapsulated compounds (Priprem et al., 2008). In this work, a focus was set on quercetin encapsulation in soy PC liposomes and the concentration-dependent solubility of quercetin was investigated and calculated as encapsulation efficiency. Up to 3200 lM quercetin could be encapsulated without any increase in liposome size (z-average) or heterogeneity of liposome sizes (polydispersity index, pdi) and having an EE between 65% and 79% (Fig. 1b). From 3400 lM a strong increase in z-average and polydispersity could be observed, which was probably provoked by excess quercetin that was no longer soluble in the system and may accordingly have formed aggregates (Pohjala & Tammela, 2012). At this concentration, encapsulation efficiency decreased as well (Fig. 1c). However, it is difficult to properly determine non-encapsulated lipophilic compounds, due to their low solubility in the surrounding buffer. Cadena et al. (2013) found an even higher EE of 97.3% when encapsulating quercetin into elastic liposomes from PC and cholesterol. However, Priprem et al. (2008) found an EE of 61–68% for their liposome system comprised of egg PC and cholesterol. The range for EE demonstrates the sensitivity to the phospholipid composition and liposome production method used.

3.2. Storage stability of quercetin-loaded liposomes at 4 °C To assess the long term physical stability of quercetin-loaded liposomes that were stored at 4 °C in the dark, the development of size, polydispersity, zeta potential and encapsulation efficiency was followed over a period of 80 days. When 800 lM quercetin was encapsulated into liposomes, encapsulation efficiency (Fig. 2c) and average size of liposomes (Fig. 2a) remained stable over 53 days. From that day on, a decrease in EE and a slight size increase could be observed. The overall quercetin content remained stable for 80 days (Fig. 2d) and no alteration in zeta potential was observed (Fig. 2b). The size increase may be explained by liposome swelling and increased membrane fluidity, which may have resulted in the observed decreased EE. Liposomes also tend to lose their encapsulated compounds over a longer storage period; in particular non-polar compounds might easily permeate through the bilayer membrane (Marsanasco et al., 2011) The slight increase in liposome size over the storage period of 80 days is in good accordance with results from Thompson, Haisman, and Singh (2006) who tested the stability of unloaded soy PC liposomes at 4 °C and a pH-value of 5. 3.3. Chemical and physical stability of liposomes loaded with quercetin at 40 °C Liposomes containing either none (control), 2 mM or 10 mM quercetin were stored over a period of 3 weeks at 40 °C in the dark. While an immediate increase in liposome size (Fig. 3a) and polydispersity (Fig. 3b) could be observed, the zeta potential remained stable (Fig. 3c). The amount of radicals detected by EPR spin trapping increased equally for all samples until day 11, but the overall radical concentration was lowest for the 10 mM quercetin sample (Fig. 3d). The subsequently decreasing radical concentration was accompanied by a drastic increase in hydroperoxides and hexanal in all samples, whereas the lowest formation of both oxidation products was found in the quercetin samples (Fig. 3e/f). Thus, primary and secondary oxidation products went into propagation phase, when the radical concentration reached its highest level, which is in accordance with general knowledge about lipid oxidation: after radical formation, hydroperoxides are formed as primary oxidation products and are then degraded into volatiles, e.g., hexanal and propanal, by metal ion-induced catalysis (Frankel, 1998). In contrast to our results, Nieto, Huvaere, and Skibsted (2011) found a strong inhibiting effect of quercetin on the lag phase (induction period) when catalyzing oxidation in liposomes by the Fenton reaction. A concentration-dependent elongation of the lag phase in PC liposomes was also found when oxidation was initialized by AAPH radical addition (Becker, Ntouma, & Skibsted, 2007). These contradictory findings may be explained by the mechanism by which lipid oxidation was induced, as in both studies lipid oxidation was catalyzed by radical generators in the aqueous phase. These approaches result in immediate generation of high concentrations of very reactive hydroxyl or peroxyl radicals that properly react with quercetin, leading to a clear elongation of the induction phase. However, heat-induced lipid oxidation is the classic, very slow autoxidation mechanism that results in the formation of low alkoxyl and peroxyl radical concentrations. The influence of quercetin on hydroperoxide and hexanal formation might be less pronounced at such low radical concentrations while, in the propagation phase, hydroperoxides and hexanal are exponentially formed and the antioxidant effect of quercetin is in evidence. It is also conceivable that the solubilization site of quercetin and radicals generated from radical initiators differs from radicals generated by autoxidation, which in turn may result in a reduced antioxidant activity (Heins, McPhail, Sokolowski, Stöckmann, &

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Schwarz, 2007). As quercetin is located in the phospholipid headgroup area at the surface of liposomes (Pawlikowska-Pawle˛ga et al., 2007), it might be in close proximity to the Fenton- or AAPH-derived hydroxyl and peroxyl radicals. In our experiment, lipid oxidation was induced by heat, which will induce oxidation in the deeper fatty acid tail region. Since quercetin is not located in this area, its radical-scavenging activity is limited here. In addition, in the presence of naturally occurring alpha-tocopherol in sunflower oil, quercetin is not a potent radical-scavenger (RoedigPenman & Gordon, 1998). However, in the presence of citric acid or in stripped oil, quercetin acted as a potent radical-scavenger. The same group produced liposomes from isolated egg phosphatidylcholine, probably free from alpha-tocopherol, and did find an induction period elongating effect. The soy-derived lecithin used in this work also contains low amounts of naturally occurring alpha-tocopherol, which may explain the lack of radical-scavenging activity of quercetin in the induction phase. Thirdly, hexanal formation by hydroperoxide degradation is metal ion induced (Frankel, 1998). The metal-chelating ability of quercetin may contribute to the dose-dependent hexanal inhibition found in the propagation phase. 3.4. Chemical and physical stability of liposomes loaded with fish oil at 40 °C Unlike the unloaded control sample, in the presence of 0.06 g/ 100 ml and 0.3 g/100 ml fish oil, particle size immediately increased by more than 200% within the first 24 h. From day 24, all samples strongly increased in size (Fig. 4a) and in polydispersity from below 0.300 to a maximum of 0.750 at the end of the storage period (Fig. 4b). As the zeta potential remained constant for all three samples (Fig. 4c), the size increase found for fish oil-loaded samples might be provoked by leakage of fish oil out of liposomes and systems became accordingly more polydisperse. Obviously, no fish oil adhered to the liposome surface as this would have resulted in a changed zeta potential. EPR spin trapping with PBN showed an increase in radical concentration within the first 48 h, which was most pronounced in the presence of 0.3 g/100 ml of fish oil. During the further storage period, radical concentration remained constant with fluctuations, keeping the highest oxidation level at 0.3 g/100 ml of fish oil (Fig. 4d). The formation of hydroperoxides and hexanal did not differ between the unloaded and fish oil loaded samples (Fig. 4e/f) whereas, in the propagation phase, the final hexanal content was even highest for the control sample. One reason might be the shift in the omega-6 to omega-3 fatty acid ratio in favor of the omega-3 fatty acid content which might result in a higher propanal formation, corresponding to a lower hexanal content. Due to the coelution of propanal with traces of ether, it was not possible to accurately quantify the propanal signal. This unexpected result might also be explained by sticky phospholipid-oil fractions that sedimented after approximately two weeks during storage at 40 °C. This phenomenon was observed, to the greatest extend, in the 0.3 g/100 ml of fish oil sample, but not in the control. For that reason, it could be assumed that the fat content was probably highest in those HS-GC samples that did not contain any fish oil, thus resulting in the highest hexanal concentrations. However, this could not be observed in the hydroperoxide results, as those values were corrected for the exact fat weight. Data thus imply that, unlike for physical stability, liposomal loading with 0.06 and 0.3 g/100 ml of fish oil did not negatively affect chemical stability of liposomes. 3.5. Influence of quercetin and fish oil on TB solubilization in the membrane bilayer The influence on the membrane properties was investigated for increasing concentrations of quercetin and fish oil, when

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encapsulated in the liposomal membrane. For this reason, the non-site-directed, lipophilic and dynamic spin probe (TB) was coencaspulated in the membrane and analyzed by EPR. In multiphase systems, spin probes give rise to a coexistence of different rotational motions and polarities of probe populations that, in turn, result in spin probe spectra composed of several superimposed spectral components (Fig. 5a/b). The hyperfine splitting aN of the probe populations indicates the micropolarity of the spin probe environment. In water, the isotropic aN value of the spin probe TB was found to be aN1 = 16.94 ± 0.000 G (Fig. 6a) and is in line with that reported in the literature, indicating a strong polar environment (Jores, Mehnert, & Mader, 2003). When encapsulated into liposomes, the TB spectra broadened and appeared as the superimposition of different motionally hindered populations (Fig. 5a/ba). TB indicated three different populations (Fig. 5b), which agrees with the findings for solid lipid nanoparticles and nanoemulsions, as reported by Jores et al. (2003). In contrast to our previous observation for liposomes that were WPI-coated and therefore diluted with coating solutions at a pH value of 3 (Frenzel & Steffen-Heins, 2015), in these non-diluted liposomal solutions the proportion of TB was shifted, to a greater extent, to the liposomal surface. Only 4% of TB was still solubilized in the aqueous phase and showed isotropic behavior (aN1 = 17.08 ± 0.05 G, sc1 = 0.03 ± 0.02 ns), resembling the isotropic spectra in pure water. 91% of TB was located in a moderate polar environment (aN2 = 15.86 ± 0.09 G, sc2 = 0.623 ± 0.05 ns) that resembles the aN value of a glycerol-rich environment as reported by Mäder (2007) and may be indicative of an association at the surface in the glycerol/phosphatidyl group moiety. The residual population 3 of about 5% was solubilized in the more lipophilic environment (aN3 = 15.01 ± 0.154 G, sc3 = 0.12 ± 0.04 ns). The order parameter, that is indicative of membrane fluidity, ranging from a completely ordered (S = 1.0) to a completely fluid environment (S = 0) (Chin & Goldstein, 1977), revealed a very fluid membrane, around population 2, of about S = 0.17 ± 0.02. This is in a range of values typical for the liquid crystalline phase (Horasan, Sünnetçiog˘lu, & Sungur, 2006) and is caused by the high amount of unsaturated fatty acids, mainly composed of C18:2 cis 6 (60.5 ± 0.7%), C16:0 (12.8 ± 0.1%), C18:1 cis 9 (9.9 ± 0.1%) and C20:3 n6 (5.4 ± 0.8%) (data not shown). For comparison, saturated DPPC-liposomes are still in the gel phase at 37 °C. Jia et al. (2008) reported a rigid membrane, of S = 0.72 ± 0.01 for the main population of 5-DSA of about 80%, indicating a restricted fast motional domain close to the phospholipid headgroups. In the absence of any quercetin or fish oil, the TB high field peak revealed a small shoulder to higher field (Fig. 5a) as a result of superimposition of the three populations (Fig. 5b). The encapsulation of increasing fish oil concentrations induced a reduction of this shoulder (Fig. 5d) while, with encapsulation of quercetin, this shoulder became slightly more pronounced (Fig. 5c). To deduce the impact of the encapsulates on the membrane properties, the superimposed spectra were simulated to investigate the hyperfine splitting (aN), proportion (d), rotational correlation time (s0), and order parameter (S) for each spectral population (Fig. 6). The aN2 values of the main TB fractions of population 2 were constant with increasing fish oil concentration, indicating unchanged micropolarity of this population (Fig. 6a). In contrast, the aN value of the smaller TB populations, 1 and 2, decreased at a concentration above 0.075 g/100 ml of fish oil, indicating a shift of these populations into a more lipophilic microenvironment. While the small population 3, with high fluctuation in standard deviation, showed only a trend in shifting, population 1 obviously shifted to a more lipophilic environment (aN1 = 15.1 G) than the glycerol/phosphatidyl group moiety. This is accompanied by a small reduction in the amount of population 2, whereas around 10% of TB shifted from

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the glycerol/phosphatidyl group moiety to the more lipophilic environments of population 3 (Fig. 6b). Likewise, in the absence of any encapsulates, the tumbling of population 1 resembles isotropic motion in a polar environment, but in the presence of fish oil, it increased in sc1, which is indicative of a less mobile population than that of control liposomes (Fig. 6d). sc2 of the main TB population was reduced with increasing fish oil concentration, which is indicative of greater dynamics of population 2. Severcan, Bayari, and Karahan (1999) investigated the encapsulation of fish oil into DPPC-liposomes by FTIR and demonstrated interaction of fish oil with the cooperativity region (C2–C8) of the fatty acyl chain and thus increasing the dynamics of the acyl chains and reduction of Tm. For that reason, it might be conceivable that the increase of acyl chain dynamics provides a more attractive environment for the dynamic spin probe TB, resulting in the shift of population 1 into the membrane. Likewise, this would explain the decrease of the shoulder, when fish oil was encapsulated in the liposomal membrane (Fig. 5c). The order parameter tended to increase slightly, which is indicative of less membrane fluidity, as was shown for the main TB population 2 (Fig. 6c). Data thus imply that the encapsulation of fish oil into the fluid membrane of soy PC, with a low phase transition temperature, below 0 °C (O’Neill and Leopold, 1982), results in a small tendency to increase membrane fluidity and a complete solubilization of dynamic spin probe TB in the membrane into more lipophilic regions. Other studies investigating the encapsulation of fish oil (Severcan et al., 1999) or walnut oil in liposomes (Horasan et al., 2006) used more saturated phospholipids for liposome production (DPPC Tm = 41 °C and DMPC = 24 °C). Below the phase transition temperature, in those studies small effects, such as enhanced membrane fluidity and reduced phase transition temperature, were reported and explained by the distortion of lipid packing by the intercalation of the oils. However, above Tm in the liquid crystalline state, minor effects on membrane fluidity were observed, in line with our findings for liposomes with a very fluid membrane. Regardless of increasing quercetin concentrations, the individual TB populations resembled the micropolarity of that in the absence of quercetin (Fig. 6c) and the rotation correlation time of TB populations remained unchanged (Fig. 6d). The main population 2 of TB revealed a slight increasing order tendency, this being indicative of a slight decrease in membrane fluidity (Fig. 6c). This agrees with the observations of Arora, Byrem, Nair, and Strasburg (2000) who reported that flavonoids and isoflavanoids, that are encapsulated in unsaturated 1-stereoyl-2-linoleoyl PC-liposomes, increased the ordering of the acyl chains and therefore reduced membrane fluidity and increased the Tm. However, it cannot be ruled out that our effects would be greater with higher concentrations of quercetin, as here we used concentrations below the maximum concentration of quercetin in soy PC liposomes (Fig. 1a). A slight shift of around 5% of the proportion of population 2 into the environment of population 3 might be implied. This is in line with the study of Pawlikowska-Pawlega and colleagues (2007), who found no effect of 5 mol% quercetin on membrane fluidity of DPPC-liposomes investigated by 5- and 16-DSA, but demonstrated its influence on the partition dynamics of non-site-directed spin probe TEMPO. They postulated that, below the phase transition temperature, quercetin enhanced partitioning of the spin probe into the polar headgroup region, since it lowered the highly ordered structure of DPPC and the spin probe could easier penetrate the liposome easier. Nevertheless, in the fluid phase above Tm, quercetin would prevent further penetration of spin probe due to its own incorporation, which might be seen as a quercetin saturation at the head group region. As we proved that TB’s main population 2 is solubilized in the glycerol/phosphatidyl group moiety, the small proportion of TB shifting to the more lipophilic environment may be provoked by quercetin saturation in the acyl

group moiety beneath the phospholipid headgroups (Košinová, Berka, Wykes, Otyepka, & Trouillas, 2012). In summary, it is known that encapsulates alter physical properties of saturated membranes by disturbing the order and lipid packing, and therefore induce an increase of the membrane fluidity and reduction of the phase transition temperature (Horasan et al., 2006; Pawlikowska-Pawle˛ga et al., 2007; Severcan et al., 1999). However, this could not be found in our study, since we used unsaturated systems with a phase transition temperature below 0 °C. Thus we have found neither a stabilizing nor a destabilizing effect of quercetin or fish oil, when encapsulated in the fluid membrane of soy PC liposomes. 4. Conclusions To systematically investigate the applicability of soy PC liposomes for encapsulating lipophilic substances in foods, the chemical and physical stability, as well as the influence on membrane fluidity and polarity, were analyzed. The physical stability of liposomes is lowered with loading of fish oil relative to unloaded liposomes. In contrast, chemical stability was not affected by either of the encapsulates. Unexpectedly, quercetin did not reduce radical formation but lowered the formation of hexanal. This might be attributed to: (I) a different solubilization site of quercetin and lipid radicals, so that proximity of both reactants was not close enough for reaction, (II) the low radical-scavenging activity of quercetin in the presence of naturally occurring alpha-tocopherol, or (III) the improved metal-chelating ability of quercetin relative to a chain-breaking ability that might result in dose-dependent hexanal inhibition in the propagation phase. EPR measurements revealed no obvious effects on membrane fluidity and polarity after increasing quercetin concentrations. However, increasing concentration of fish oil in membrane might increase the acyl chain dynamics and thereby supply a more attractive environment for the dynamic spin probe TB, in which the free tumbling of the spin probe is enhanced. It has already been proved that encapsulates alter physical properties of saturated membrane by disturbing the order/lipid packing and this results in a higher membrane fluidity and a lower phase transition temperature. However, membrane properties of our unsaturated systems with Tm below 0 °C were not influenced by encapsulation of quercetin or fish oil. Acknowledgements We would like to thank Nadine Schulz for her experimental support. The project was funded by the German Ministry for Education and Research and is part of the network project Food Chain Plus (Grant No. 0315539A). References Arora, A., Byrem, T. M., Nair, M. G., & Strasburg, G. M. (2000). Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids. Archives of Biochemistry and Biophysics, 373, 102–109. Becker, E. M., Ntouma, G., & Skibsted, L. H. (2007). Synergism and antagonism between quercetin and other chain-breaking antioxidants in lipid systems of increasing structural organisation. Food Chemistry, 103, 1288–1296. Berliner, L. J. (1976). Spin labeling: Theory and applications. Molecular Biology, 21. New York: Academic Press. Bigliardi, B., & Galati, F. (2013). Innovation trends in the food industry: The case of functional foods. Trends in Food Science & Technology, 31, 118–129. Cadena, P. G., Pereira, M. A., Cordeiro, R. B., Cavalcanti, I. M., Barros Neto, B., Pimentel, M. d. C. C., et al. (2013). Nanoencapsulation of quercetin and resveratrol into elastic liposomes. Biochimica et Biophysica Acta (BBA) – Biomembranes, 1828, 309–316. Chin, J., & Goldstein, D. (1977). Drug tolerance in biomembranes: A spin label study of the effects of ethanol. Science, 196, 684–685. Frankel, E. N. (1998). Lipid oxidation. Oily Press lipid library. Dundee, Scotland: Oily Press.

M. Frenzel, A. Steffen-Heins / Food Chemistry 185 (2015) 48–57 Frenzel, M., & Steffen-Heins, A. (2015). Whey protein coating increases bilayer rigidity and stability of liposomes in food-like matrices. Food Chemistry, 173, 1090–1099. Fry, D. W., White, J. C., & Goldman, I. D. (1978). Rapid separation of low molecular weight solutes from liposomes without dilution. Federation Proceedings, 37, 1708. Heins, A., McPhail, D. B., Sokolowski, T., Stöckmann, H., & Schwarz, K. (2007). The location of phenolic antioxidants and radicals at interfaces determines their activity. Lipids, 42, 573–582. Horasan, N., Sünnetçiog˘lu, M. M., & Sungur, R. (2006). EPR spin label study of walnut oil effects on phosphatidylcholine membranes. Chemistry and Physics of Lipids, 140, 1–10. Ionescu, D., & Ganea, C. (2012). A study of quercetin effects on phospholipid membranes containing cholesterol using Laurdan fluorescence. European Biophysics Journal, 41, 307–318. Jia, Y., Joly, H., & Omri, A. (2008). Liposomes as a carrier for gentamicin delivery: Development and evaluation of the physicochemical properties. International Journal of Pharmaceutics, 359, 254–263. Jores, K., Mehnert, W., & Mader, K. (2003). Physicochemical investigations on solid lipid nanoparticles and on oil-loaded solid lipid nanoparticles: A nuclear magnetic resonance and electron spin resonance study. Pharmaceutical Research, 20, 1274–1283. Košinová, P., Berka, K., Wykes, M., Otyepka, M., & Trouillas, P. (2012). Positioning of antioxidant quercetin and its metabolites in lipid bilayer membranes: Implication for their lipid-peroxidation inhibition. Journal of Physical Chemistry B, 116, 1309–1318. Laye, C., McClements, D., & Weiss, J. (2008). Formation of biopolymer-coated liposomes by electrostatic deposition of chitosan. Journal of Food Science, 73, N7. Li, H., Zhao, X., Ma, Y., Zhai, G., Li, L., & Lou, H. (2009). Enhancement of gastrointestinal absorption of quercetin by solid lipid nanoparticles. Journal of Controlled Release, 133, 238–244. Mäder, K. (2007). Characterization of nanoscaled drug delivery systems by electron spin resonance (ESR). In S. S. R. Kumar, Challa (Ed.), Nanotechnologies for the life sciences. Germany: Wiley-VCH Verlag GmbH & Co. KGaA. Marsanasco, M., Márquez, A. L., Wagner, J. R., del, V., Alonso, S., & Chiaramoni, N. S. (2011). Liposomes as vehicles for vitamins E and C: An alternative to fortify orange juice and offer vitamin C protection after heat treatment. Food Research International, 44, 3039–3046. Nieto, G., Huvaere, K., & Skibsted, L. H. (2011). Antioxidant activity of rosemary and thyme by-products and synergism with added antioxidant in a liposome system. European Food Research and Technology, 233, 11–18.

57

O’Neill, S. D., & Leopold, A. C. (1982). An assessment of phase transitions in soybean membranes. Plant Physiology, 70, 1405–1409. Pawlikowska-Pawle˛ga, B., Gruszecki, W. I., Misiak, L. E., & Gawron, A. (2003). The study of the quercetin action on human erythrocyte membranes. Biochemical Pharmacology, 66, 605–612. Pawlikowska, B., Ignacy, W., Misiak, L., Paduch, R., Piersiak, T., Zarzyka, B., et al. (2007). Modification of membranes by quercetin a naturally occurring flavonoid via its incorporation in the polar head group. Biochimica et Biophysica Acta (BBA) – Biomembranes, 1768, 2195–2204. Pohjala, L., & Tammela, P. (2012). Aggregating behavior of phenolic compounds – A source of false bioassay results? Molecules, 17, 10774–10790. Pool, H., Mendoza, S., Xiao, H., & McClements, D. J. (2012). Encapsulation and release of hydrophobic bioactive components in nanoemulsion-based delivery systems: Impact of physical form on quercetin bioaccessibility. Food & Function, 4, 162. Priprem, A., Watanatorn, J., Sutthiparinyanont, S., Phachonpai, W., & Muchimapura, S. (2008). Anxiety and cognitive effects of quercetin liposomes in rats. Nanomedicine: Nanotechnology Biology and Medicine, 4, 70–78. Roedig-Penman, A., & Gordon, M. H. (1998). Antioxidant properties of myricetin and quercetin in oil and emulsions. Journal of the American Oil Chemists Society, 75, 169–180. Russo, M., Spagnuolo, C., Tedesco, I., Bilotto, S., & Russo, G. L. (2012). The flavonoid quercetin in disease prevention and therapy: Facts and fancies. Biochemical Pharmacology, 83, 6–15. Serfert, Y., Drusch, S., & Schwarz, K. (2009). Chemical stabilisation of oils rich in long-chain polyunsaturated fatty acids during homogenisation, microencapsulation and storage. Food Chemistry, 113, 1106–1112. Severcan, F., Bayari, S., & Karahan, D. (1999). FTIR and turbidity studies of fish oildipalmitoylphosphatidylcholine model membrane interactions. Journal of Molecular Structure, 480–481, 413–416. Stöckmann, H., & Schwarz, K. (1999). Partitioning of low molecular weight compounds in oil-in-water emulsions. Langmuir, 15, 6142–6149. Štrancar, J., Koklicˇ, T., Arsov, Z., Filipicˇ, B., Stopar, D., & Hemminga, M. A. (2005). Spin label EPR-Based characterization of biosystem complexity. Journal of Chemical Information and Modeling, 45, 394–406. Tan, B.-J., Liu, Y., Chang, K.-L., Lim, B. K. W., & Chiu, G. N. C. (2012). Perorally active nanomicellar formulation of quercetin in the treatment of lung cancer. International Journal of Nanomedicine, 7, 651–661. Thompson, A. K., Haisman, D., & Singh, H. (2006). Physical stability of liposomes prepared from milk fat globule membrane and soya phospholipids. Journal of Agricultural and Food Chemistry, 54, 6390–6397.