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Research ArticleAction of Chicory Fructooligosaccharides onBiomimetic Membranes

A F Barbosa1 R S Henrique1 A S Lucho2 V Paffaro Jr3 and J M Schneedorf1

1 Biochemistry Laboratory Federal University of Alfenas R Gabriel Monteiro da Silva 700 Centro 37130-000 Alfenas MG Brazil2Materials Group Lab Chemistry Institute Federal University of Alfenas 37130-000 Alfenas MG Brazil3 Integrative Animal Biology Laboratory Institute of Biomedical Sciences Federal University of Alfenas 37130-000 Alfenas MG Brazil

Correspondence should be addressed to J M Schneedorf zemasfsgmailcom

Received 23 May 2014 Revised 17 October 2014 Accepted 19 October 2014 Published 16 November 2014

Academic Editor Shen-Ming Chen

Copyright copy 2014 A F Barbosa et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fructooligosaccharides from chicory (FOSC) are functional prebiotic foods recognized to exert several well-being effects in humanhealth and animal production as decreasing blood lipids modulating the gut immune system enhancing mineral bioavailabilityand inhibiting microbial growth among others Mechanisms of actions directly on cell metabolism and structure are however littleknown In this sense this work was targeted to investigate the interaction of FOSC with biomimetic membranes (liposomes andsupported bilayer membrane s-BLM) through cyclic voltammetry impedance spectroscopy spectrofluorimetry and microscopyFOSC was able to disrupt the membrane structure of liposomes and s-BLM from the onset of molecular pores induced on itThe mechanism of interaction of fructans with biomimetic membranes suggests hydrogen bonding between the polyhydroxylatedstructure of the oligosaccharides and the negative polar group of L-120572-phosphatidylcholine (PC) present in both liposomes ands-BLM

1 Introduction

As stated almost two decades ago prebiotics are considereda nondigestible food ingredient that beneficially affects thehost by selectively stimulating the growth andor activityof one or a limited number of bacteria in the colon andthus improving host health [1] A prebiotic group largelystudied is the chicory fructooligosaccharides (FOSC) FOSCare fructans (carbohydrates with a great extent of fructo-sylfructose links) extracted on a commercial basis from thechicory root namely the Compositae family (Cichoriumintybus) [2] FOSC are also present in several fruits andvegetables species and are produced by transfructosylationof sucrose These compounds comprise a functional foodgroup containing mixed 120573-D-fructans with two to four 120573(2-1) linked fructosyl units displaying a terminal 120572-D-glucoseresidue as kestose nystose fructosylnystose and fructofu-ranosylnystose among others [3] FOSC differs from inulina well-known fructan of a high degree of polymerization(DP) as well as oligofructose a small FOS (DP about 5) pro-duced during endoglycolitic hydrolysis of inulin FOSC are

considered to arrive the human gastrointestinal tract almostwithout hydrolysis being a carbon source for short-chainfatty acids by bifidobacteria and lactobacilli living into thelumen [4] Some properties of FOSC as their nondigestibleand fermentable nature as well as their sweetening power andlow caloric value make them attractive to be used in pastryconfectionery and dairy industries [5]Moreover both FOSCand inulin are also known as uniquely 120573(2-1) fructans withrecognized prebiotic activities [3] The reported beneficialeffects of oral intake of FOSC include the improvementof gastrointestinal metabolism together with short-chainfatty acid production promotion of mineral absorption andenhancement of bone calciumbioavailability [6] reduction ofserum lipids acting as ROS scavenger in gut [7] modulationof immune system [5] and antimicrobial activity againsta large broad of pathogenic strains [4] Although FOSC isrecognized to inhibit cellular responses at diverse levels [8 9]little is known about their molecular mechanisms directlyinvolved in cell metabolism or structure [10 11] as theirplausible interaction with cell membranes In this goal thiswork tests the molecular effects of FOSC against biomimetic

Hindawi Publishing CorporationInternational Journal of ElectrochemistryVolume 2014 Article ID 186109 8 pageshttpdxdoiorg1011552014186109

2 International Journal of Electrochemistry

membranes using reconstituted liposomes and supportedbilayer membranes (s-BLM) as target models

2 Material and Methods

21 Reagents L-120572-Phosphatidylcholine (PC) from fresh eggyolk fructooligosaccharides from chicory (FOSC) andcholesterol were of the highest obtainable purity and weresupplied by Sigma-Aldrich (St Louis MO USA) All otherchemicals were of the highest quality as possible and allchemicals were used without further purification The waterused in all experiments was twice distilled

22 Supported Lipid Bilayer Formation The formation of s-BLMs on a Pt electrode has been conducted as reportedbefore [12ndash14] Briefly L-120572-phosphatidylcholine (PC) wasdissolved in chloroform at 20mgmL of final concentrationand cholesterol (CH) at 7mgmL (BLM solution) A workingPt electrode (050mm diameter) was first polished with sandpaper followed by alumina slurry on polishing cloth Then itwas sonicated in pure water for 2min rinsed and immersedin a solution of 10molL H

2SO4 Cyclic voltammetry was

performed after Pt electrode activation in 1molL H2SO4

The Pt electrode was immediately taken out rinsed with purewater again and sonicated with a highly power supersonicwave generator inwater and ethanol bath for 5min Following5 120583L of BLM forming solution was dropped on the electrodesurface The electrode was then rinsed again with redistilledwater to remove chloroform traces and transferred into a01molL KCl solution for 20min in which the supportedlipid layer was formed spontaneously [15] All experimentswere conducted in triplicate

23 Liposome Preparation The procedure was done fol-lowing S Basu and M Basu [16] In short a chloroformsolution containing PC at 10mmolL was evaporated in testtubes to dry with argon following evacuation for 60minfor total remotion of the solvent Subsequently the film washydrated in sodium phosphate buffer 01molL pH 74 or10mmolL dopamine solution after complete dispersion atroom temperature (DA-liposome) Then the multilamellarvesicles produced were ultrasonicated in water bath for 1minand sized by extrusion through Sephadex G-50 This lastprocedure was conducted whenever 10mmolL dopaminewas mixed with PC solution for dopamine incorporation(DA-liposome) The eluted volume was measured by spec-trofluorimetry to avoid dopamine freely available in solutionThe liposome structure was confirmed by phase contrast andfluorescence microscopy with Rhodamin B with a NikonEclipse 80i (Nikon Co Tokyo Japan)

24 Electrochemical Assays Cyclic voltammetry (CV) wasperformed on a potentiostat-galvanostat instrument PG-39MCSV (Omni Metra Instr Cient Ltda RJ Brazil) Theapparatus used for electrochemical impedance spectroscopy(EIS) was composed of Autolab with potentiostatgalvanostatworking station 128N and the Frequency Response Analysis

System Software FRA (Metrohm Autolab BV The Nether-lands) Impedance measurements were performed from100 kHz to 420mHz with a signal amplitude of 10mVThe experiments were carried out with a three-electrodesystem composed of anAgAgCl (KCl-saturated) as referenceelectrode and a platinum wire (05mm diameter) as thecounter electrode The working electrode comprised a Ptdisc of 05mm diameter melted in a glass tube EIS runswere performed in the presence of 1mmolL Fe(CN)

6

3minus4minus

as a redox probe contained in 01molL KCl as supportsolution and at a system potential of E0 of 235mV Theworking electrode was cleaned before use with MaxiClean1400 (Unique Ind Com Ltda SP Brazil) The experimentswere conducted at room temperature

25 Spectrofluorimetry of Liposomes The action of FOSCagainst reconstituted liposomes containing dopamine wasalso monitored by spectrofluorimetry [17] The fluorescencemeasurements were performed with a Cary Eclipse spec-trofluorophotometer (Varian Australia) with a 10mm quartzcuvette at an excitation wavelength of 279 nm The fluores-cence emission spectra were recorded in the 300ndash400 nmwavelength range with a 5 nm bandwidth Spectrofluorimet-ric runs were carried out after 5min of preincubation ofliposomes with 5mmolL SDS (sodium dodecyl sulfate) orFOSC (up to 75mgmL)

26 Data Analysis All of the experiments were conducted intriplicateThe data are expressed as themeansplusmn SE Statisticalanalysis was done with the free computing environment R (RCore Development Team) [18]

3 Results and Discussion

31 Characterization of s-BLM and Study of Interaction ofFructans on the Pt Electrode for EIS Surface-modified elec-trodes can also be assessed by electrochemical approachesas EIS and CV [19] In this sense EIS was carried outaiming to get further information about s-BLM integrityupon the action of tested FOS The complex impedance canbe presented as the sum of the real 119885re and imaginary 119885imcomponents that are originated mainly from the resistanceand capacitance of the cell respectively (Nyquist plot [14])The resulting data can be analyzed by a relatively equivalentRandles circuit consisting of an ideal and nonideal electri-cal analogs to the real physical and chemical processes atheoretical abstraction of an interfacial system [20] Figure 1illustrates the results of impedance spectroscopy measure-ments on the bare electrode whereas Figure 2 presents theresults obtainedwith the electrode coatedwith s-BLM (a) andafter interaction for 10min with 50 gL of fructooligosaccha-rides from chicory (b) in 01molL KCl solution containing1mmolL Fe(CN)

6

4minus3minus measured at the formal potential ofthe system

For the untreated electrode a very small circle probed byhigh frequencies can be visualized near the origin followedby a Warburg-like mass transfer impedance in the low

International Journal of Electrochemistry 3

700

600

500

400

300

200

100

0

7006005004003002001000

Z998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)

Figure 1 Electrochemical impedance spectroscopy of bare Ptelectrode in 1mmolL Fe(CN)

6

4minus3minus solution containing 01molLKCl

0 1000 2000 3000 4000 5000 6000 7000

0

1000

2000

3000

4000

5000

6000

7000

042 kHz480 kHz

W

R

(a)

(b)

195 kHzZ998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)

Rm

Cm Zw

CdlRsol

Rct

Figure 2 Electrochemical impedance spectroscopy of (a) Pt elec-trode modified with s-BLM and (b) after interaction for 20minwith 50 gL of fructooligosaccharides from chicory in 1molLFe(CN)

6

4minus3minus solution containing 01molL KCl Inset modifiedRandles equivalent circuit presenting the fitting curve 119877sol denotesthe electrolyte resistance119877

119898denotes the lipidmembrane resistance

119862119898denotes the lipid membrane capacitance 119862dl denotes the double

layer capacitance 119877 is resistance of the monolayer defects 119877ctdenotes the charge-transfer resistance and119885

119908denotes theWarburg

element

frequency region (Figure 1) This finding suggests a kinetic-driven control for the faradaic current produced from voltageexcitation [15] On the other side a random motion of redoxspecies around the interfacial region seems to have appearedas observed by the unitary slope of Nyquist plot (Figure 1) inthe low frequency region of spectra [21]

Figure 2(b) shows the results of impedance spectroscopyof s-BLM after interacting with 50 gL of fructooligosac-charides from chicory for 10min in equimolar 1mmolLFe(CN)

6

4minus3minus solution with 01molL KCl as supporting

electrolyte A smaller diameter with a decrease in the charge-transfer resistance can be devised in Figure 2(b) as comparedto Figure 2(a) suggesting an effectiveness of s-BLM onprevention of the redox probe from accessing the electrode[20 22] Furthermore this result suggested a Warburg-likemass transfer impedance occurring in the low frequencyportion which indicated that s-BLM after interaction wasdeficient to eliminate electron transfer between Fe(CN)

6

4minus3minus

and the covered electrode [23] This result suggests a s-BLMas having a fractal surface with defects permeating throughmembrane to the electrode after fructans binding

A major difference in the spectra however can beobserved whenever the s-BLM was treated with FOSC (Fig-ure 2(b)) with the presence of one semicircle at higherfrequency and another one in the average frequency of thespectra This finding is expected when a resistive film ispresent on electrode surface and with higher impedancevalues [21] The two capacitive arcs can be hardly separatedas the nearness of two time constants for the capacitors Athigher frequencies the data suggest the presence of anothermembrane behaviour whereas at medium frequencies aninterfacial contact with the electrode solution due to the s-BLM pores A prevention of the electron-ion probe transfercan also be attested from the observed semicircle in themodified electrode A relatively equivalent circuit modelconsisting of ideal and nonideal electrical analogs of thephysical-chemical process has been chosen from severalelectrical models to the impedance data to extract the circuitcomponents [12ndash14 24 25] Hence the modified Randlesequivalent circuit similar to that presented by Huang et al[12] has fitted better the measured data (Figure 2 inset)Through simulation it was confirmed that the s-BLM isformed by the resistance of the solution (119877sol) two capacitorsin series (119877

119898 119862119898 119877ct and 119862dl) and Warburg impedance

(119885119908) In this model 119877

119898is the membrane resistance 119862

119898is

the membrane capacitance per unit area 119877ct is the charge-transfer resistance and 119862dl is the double layer capacitanceThe values for equivalent electrical circuit were 119877sol of 0128mΩsdotcm2 (00 error) 119862

119898of 016 120583Fcm2 (00 error) 119877

119898

of 346 kΩsdotcm2 (00 error) 119877ct of 139 kΩsdotcm2 (28 error)

and 119862dl of 011 120583Fcm2 (33 error) These values are inaccordance with those reported before [12 13 24ndash26]

From these findings aWarburg impedance highlights thepresence of molecular pores in the s-BLM structure [24]Furthermore defects in s-BLM related to monolayers can beassigned to the resistance 119877 values merging the capacitorsFrom the 119862

119898value found in this work close to the ones

published before [24 27] the thickness 119889 of the biomimeticmembrane can be estimated according to Du et al as follows[25]

119862119898=

1205760sdot 120581

119889

(1)

where 1205760is the dielectric permittivity of free space (120576

0= 885times

10minus14 Fsdotcmminus1) and 120581 is the dielectric permittivity of the lipid

(120581 = 205 [13]) From (1) a value of 113 nm for the thicknessof the lipid membranes close to range of 4ndash10 nm was foundand observed for phosphatidylcholine bilayers [27 28]


32 Characterization of s-BLM on the Electrode Surface byCyclic Voltammetry Cyclic voltammetry with Fe(CN)

6

4minus3minus

as ion probe [29 30] was carried out to examine the s-BLM integrity upon challenge with FOSC Figure 3 showsthe voltammograms of (a) the bare Pt electrode and (b) thePt electrode coated with s-BLM in 10mmolL K

3[Fe(CN)

6]

K4[Fe(CN)

6] (1 1) solution containing 01molL KCl The

s-BLM is considered a highly effective barrier for electrontransfer between the bulk and the electrode surface In thissense a pair of well-defined reversiblewaves can be visualizedfor bare Pt [31] with decreasing current signal as s-BLM isformed on the electrode surface up to the disappearance ofboth redox peaks (Figure 3(b)) [27 28 32]

33 Preincubation Effects of Fructans on s-BLM Pt electrodescoated with s-BLM immersed into 50 gL FOSC solutionsfor 0 5 10 15 30 and 50min were transferred into a01molL KCl solution containing 10mmolL K

3[Fe(CN)

6]

K4[Fe(CN)

6] (1 1) The resulted voltammograms are dis-

played in Figure 4 As expected no voltammetric peaks werefound in the absence of FOSC (Figure 4(a)) However whenthe fructanswere preincubatedwith s-BLMa faradaic currentdue to the probe transport to the electrode surface appearedfor different times (Figure 4 (b)ndash(f)) These results evokemembrane defects arising around 5min after preincubationwith the fructans and can be explained by adsorption of FOSCmolecules [12] andor rearrangement of s-BLM structure [13]

Aiming to test the hypothesis of the reappearance offaradaic current as due to pore formation in Pt-s-BLM wehave used the treatment of Amatore et al [33] for partiallyblocked electrodes According to this theoretical approachthe half-wave potential of a limiting current (119894lim) of a bareelectrode can be represented by the standard potential of aredox couple in a pure diffusion-controlled electron transferThe relationship between the fractional coverage of electrode(120579) and 119894lim can then be given by [28]

120579 = 1 minus

(06 sdot 119894lim sdot 119903)

119865119860119862119863

(2)

where 119903 is the average radii of active pinhole sites 119865 is theFaraday constant119860 is the surface area119862 is the concentrationof oxidant or reductant (molsdotdm3) and 119863 is the diffusioncoefficient This approach resulted in pore radii for s-BLMFOSC-induced 33 A [34]

34 Concentration Effects of FOSC on s-BLM After interac-tion of the fructans with s-BLM increasing redox peaks dueto Fe(CN)

6

4minus3minus could be attained in 01molL KCl solutionas depicted in voltammograms at Figure 5 The peak separa-tion decreased and peak current increased with increasingfructans concentration Nevertheless below 25 gL of FOSno faradaic current for themarker ion was observed (data notshown)

To evaluate the electron transfer rate in the presence ofthe fructans the Tafel approach [32] was applied to the risingpart of anodic branch of the voltammograms The electrontransfer rate Ks changed linearly with FOSC concentration(22 times 10minus4 sdot sminus1 at 25 gL to 46 times 10minus4 sdot sminus1 at 125 gL

600

400

200

0

minus200

minus400

minus600

400 5003002001000minus100minus200

I(n

A)

E (mV) (versus AgAgCl)

(a)

(b)

Figure 3 Cyclic voltammetry of charge transfer of ferricyanide ionson the electrode surface at bare Pt electrode (a) and Pt electrodemodified with s-BLM (b) in 10mmolL K

3[Fe(CN)

6]K4[Fe(CN)

6]

(1 1) mixture containing 01molL KCl Scan rate 50mVsdotsminus1

(a) (b)(c)

(d) (e)(f)

400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

Figure 4 Electrochemical data for FOSC action on s-BLM asa function of preincubation times Cyclic voltammetric responseof 10mmolL K

3[Fe(CN)

6]K4[Fe(CN)

6] (1 1) solution containing

01molL KCl at Pt electrode s-BLM after interaction with 50 gsdotL ofFOSC for different times (a) 0min (b) 5min (c) 10min (d) 15min(e) 30min and (f) 50min Scan rate 50mVsdotsminus1

1198772= 0988) revealing 477 plusmn 35 of the anodic peak current

obtained for FOS as compared to the unmodified electrode[13]These increased values found forKs suggest an increasedsurface area for charge transfer on the electrode surface withfructans binding [23] possibly inducing some active sites toFe(CN)

6

4minus3minus ions through the pores of s-BLM[23] Althoughthere are other mechanisms proposed in the literature toexplain membrane defects from electrochemical responsesof s-BLMs with active molecules the data of EIS and CVpresented in this work indicate a most probable formationof pores fructan-induced on the surface of s-BLM leading todecreased membrane resistivity to the probing molecules [14


400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

(A)

(a)

(b)(c)

(d)(e)

(f)

Figure 5 Electrochemical data for FOSC action on supported lipidbilayer membrane as a function of fructan concentration Cyclicvoltammetric response of 10mmolL K

3[Fe(CN)

6]K4[Fe(CN)

6]

(1 1) solution containing 01molL KCl at Pt-s-BLM after interac-tion with different concentrations of FOSC for 20min (a) 00 (b)25 (c) 50 (d) 75 (e) 100 and (f) 125 gL Scan rate 50mVsdotsminus1

23 25 28] These results can also discard stabilization on s-BLM induced by adsorption [25] as therewas no evidence forcurrent peak decrease simultaneously to the increase in ΔEpvalues [25] Moreover the similar ion permeability for theredox Fe(CN)

6

4minus3minus couple through s-BLM (unitary ipaipcratio Figure 5) also discards the possibility of an ionophore-like action and channel formation for the tested fructans [3536]Hence a surfactant-like effect [12 37 38] can be proposedto explain the mechanism of interaction between FOSC ands-BLMThis mechanism is similar to those reported for someactive peptides [39] and classical surfactants [37 39] Thisbinding model is also supported by the hydrogen bondingfound between the polyhydroxy groups of carbohydrates andthe phosphate head groups of phospholipids as studied fromIR spectroscopy [40 41] In this sense it is believed thatthe polyhydroxylated sugar replaces water and keeps thelateral spacing between lipid polar head groups in the drystate thereby minimizing van der Waals interactions of thehydrocarbon chains [41]

35 Action of FOSC on Prepared Liposome Aiming to testthe action of FOSC in another membrane model liposomescontaining dopamine (DA-liposomes) as a fluorescent probewere prepared and monitored against FOSC by fluorimetryandmicroscopyThe increase in fluorescence intensity whichis related to the dopamine release in liposome suspensions(DA-liposome) treated with SDS as control or FOS wasmeasured following the relation below [42]

119871 = 100 minus (119868119894minus 119868119887

119868119905minus 119868119887119905

) (3)

0

20

40

60

80

Lipo Lipo-DA

L (

)

Lipo-DA+ FOSC

Lipo-DA+ SDS

Figure 6 Relative changes in fluorescence intensity (119871) of mul-tilamellar liposomes and reconstituted DA-liposomes (Lipo-DA)challenged with SDS (5mmolL) and FOSC at 75mgmL

where 119871 is the percentage of the signal increase fromdopamine release 119868

119894is the fluorescence of liposome disper-

sions 119868119887is the background fluorescence 119868

119905is the fluorescence

after addition of SDS or FOSC and 119868119887119905

is the backgroundfluorescence after addition of these compounds Figure 6presents the results obtained after preincubation of DA-liposomes with 5mmolL SDS or 75mgmL FOSC The pre-biotic oligosaccharide was able to increase the fluorescenceintensity in solution up to 62 plusmn 2 from the basal level closeto the value presented for the surfactant SDS This findingsuggests a FOS-induced membrane rupture occurring withthe liposomes releasing dopamine molecules freely in thesolution

The direct action of FOSC on s-BLM structure canalso be corroborated with both fluorescence and phasecontrast photomicrography obtained for the treatment ofthe reconstituted liposomes as presented in Figure 7 Rho-damin B stained regular liposomes (arrowheads) beforeFOS treatment under fluorescence (a) and phase con-trast (b) microscopy After FOSC treatment the liposomesbecome irregularly shaped (arrowheads) and disrupted(arrows) under fluorescent Rhodamin B (c) and phase con-trast microscopy (d)

4 Conclusion

We have studied the interaction of fructooligosaccharidesfrom chicory with two biomimetic models of membranesupported bilayer membrane and reconstituted liposomesby means of electrochemical spectroscopy and microscopytechniques The overall results suggest that the mechanismof interaction seems to involve some hydrogen bondingbetween carbohydrate and the phosphate head group ofthe phospholipids leading to the appearance of progressivepinholes on the biomimetic membranes up to a com-plete disruption of the membrane structures This mech-anism of action directly on a membrane model could


7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)

Figure 7 Photomicrography of reconstituted liposomes before ((a) and (b)) challenged with FOSC ((c) and (d)) Liposomes were identifiedby Rhodamin B labeled probe ((a) and (c)) and phase contrast ((b) and (d)) The arrows are explained in the text

provide some thoughts to the biological activity of prebi-otics

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge the Minas Gerais State ResearchFoundationmdashFAPEMIG for the financial support of thiswork (Grant no CBB APQ 0192011)

References

[1] G R Gibson and M B Roberfroid ldquoDietary modulation ofthe human colonic microbiota introducing the concept ofprebioticsrdquo Journal of Nutrition vol 125 no 6 pp 1401ndash14121995

[2] M B Roberfroid ldquoChicory fructooligosaccharides and thegastrointestinal tractrdquo Nutrition vol 16 no 7-8 pp 677ndash6792000

[3] E Biedrzycka and M Bielecka ldquoPrebiotic effectiveness offructans of different degrees of polymerizationrdquo Trends in FoodScience amp Technology vol 15 no 3-4 pp 170ndash175 2004

[4] T Fukasawa A Kamei Y Watanabe J Koga and K AbeldquoShort-chain fructooligosaccharide regulates hepatic peroxi-some proliferator-activated receptor 120572 and farnesoid X receptortarget gene expression in Ratsrdquo Journal of Agricultural and FoodChemistry vol 58 no 11 pp 7007ndash7012 2010

[5] F R J Bornet F Brouns Y Tashiro and V Duvillier ldquoNutri-tional aspects of short-chain fructooligosaccharides naturaloccurrence chemistry physiology and health implicationsrdquoDigestive and Liver Disease vol 34 supplement 2 pp S111ndashS1202002

[6] A Nzeusseu D Dienst V Haufroid G Depresseux J-P Devogelaer and D-H Manicourt ldquoInulin and fructo-oligosaccharides differ in their ability to enhance the density ofcancellous and cortical bone in the axial and peripheral skeletonof growing ratsrdquo Bone vol 38 no 3 pp 394ndash399 2006

[7] W van denEndeD Peshev and L deGara ldquoDisease preventionby natural antioxidants and prebiotics acting as ROS scavengersin the gastrointestinal tractrdquoTrends in Food Science andTechnol-ogy vol 22 no 12 pp 689ndash697 2011

[8] N Matsukawa M Matsumoto A Shinoki M Hagio R Inoueand H Hara ldquoNondigestible saccharides suppress the bacterialdegradation of quercetin aglycone in the large intestine andenhance the bioavailability of quercetin glucoside in ratsrdquo


Journal of Agricultural and Food Chemistry vol 57 no 20 pp9462ndash9468 2009

[9] J M Laparra and Y Sanz ldquoInteractions of gut microbiota withfunctional food components and nutraceuticalsrdquo Pharmacolog-ical Research vol 61 no 3 pp 219ndash225 2010

[10] T Suzuki andHHara ldquoVarious nondigestible saccharides opena paracellular calcium transport pathway with the inductionof intracellular calcium signaling in human intestinal Caco-2cellsrdquo The Journal of Nutrition vol 134 no 8 pp 1935ndash19412004

[11] T Fukasawa K Murashima T Nemoto et al ldquoIdentificationof marker genes for lipid-lowering effect of a short-chainfructooligosaccharide by DNA microarray analysisrdquo Journal ofDietary Supplements vol 6 no 3 pp 254ndash262 2009

[12] W Huang Z Zhang X Han et al ldquoConcentration-dependentbehavior of nisin interaction with supported bilayer lipidmembranerdquo Biophysical Chemistry vol 99 no 3 pp 271ndash2792002

[13] Y Ma J Wang F Hui and S Zang ldquoThe reassembled behaviorof bilayer lipid membranes supported by Pt electroderdquo Journalof Membrane Science vol 286 no 1-2 pp 174ndash179 2006

[14] X Lu T Liao L Ding et al ldquoInteraction of quercetin withsupported bilayer lipid membranes on glassy carbon electroderdquoInternational Journal of Electrochemical Science vol 3 no 7 pp797ndash805 2008

[15] D Jiang P Diao R Tong D Gu and B Zhong ldquoCa2+ inducedFe(CN)

6

3minus4minus electron transfer at Pt supported BLM electroderdquoBioelectrochemistry and Bioenergetics vol 44 no 2 pp 285ndash288 1998

[16] S Basu and M Basu Liposome Methods and Protocols MethodsinMolecular Biology Humana Press New York NY USA 2002

[17] H Y Wang Y Sun and B Tang ldquoStudy on fluorescenceproperty of dopamine and determination of dopamine byfluorimetryrdquo Talanta vol 57 no 5 pp 899ndash907 2002

[18] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statisti cal ComputingVienna Austria 2012

[19] J J Harris and M L Bruening ldquoElectrochemical and in situellipsometric investigation of the permeability and stability oflayered polyelectrolyte filmsrdquo Langmuir vol 16 no 4 pp 2006ndash2013 2000

[20] P Diao D Jiang X Cui D Gu R Tong and B Zhong ldquoCyclicvoltammetry and ac impedance studies of Ca2+-induced ionchannels on Pt-BLMrdquo Bioelectrochemistry and Bioenergeticsvol 45 no 2 pp 173ndash179 1998

[21] D Pan J Chen W Tao L Nie and S Yao ldquoPhosphopoly-oxomolybdate absorbed on lipid membranescarbon nanotubeelectroderdquo Journal of Electroanalytical Chemistry vol 579 no 1pp 77ndash82 2005

[22] N Wilke and A M Baruzzi ldquoComparative analysis of thecharge transfer processes of the Ru(NH

3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus

6redox couples on glassy carbon electrodes

modified by different lipid layersrdquo Journal of ElectroanalyticalChemistry vol 537 no 1-2 pp 67ndash76 2002

[23] J Wang L Wang S Liu X Han W Huang and E WangldquoInteraction of K

7Fe3+P

2W17O62H2with supported bilayer lipid

membranes on platinum electroderdquo Biophysical Chemistry vol106 no 1 pp 31ndash38 2003

[24] X Liu W Huang and E Wang ldquoAn electrochemical study onthe interaction of surfactin with a supported bilayer lipid mem-brane on a glassy carbon electroderdquo Journal of ElectroanalyticalChemistry vol 577 no 2 pp 349ndash354 2005

[25] L Du X Liu W Huang and E Wang ldquoA study on theinteraction between ibuprofen and bilayer lipid membranerdquoElectrochimica Acta vol 51 no 26 pp 5754ndash5760 2006

[26] J Wang B Zeng C Fang and X Zhou ldquoInfluence of surfac-tants on the electron-transfer reaction at self-assembled thiolmonolayers modifying a gold electroderdquo Journal of Electroana-lytical Chemistry vol 484 no 1 pp 88ndash92 2000

[27] J-S Ye A Ottova H T Tien and F-S Sheu ldquoNanostructuredplatinum-lipid bilayer composite as biosensorrdquo Bioelectrochem-istry vol 59 no 1-2 pp 65ndash72 2003

[28] G Favero A DrsquoAnnibale L Campanella R Santucci and TFerri ldquoMembrane supported bilayer lipid membranes arraypreparation stability and ion-channel insertionrdquo AnalyticaChimica Acta vol 460 no 1 pp 23ndash34 2002

[29] K Asaka H Ti Tien and A Ottova ldquoVoltammetric study ofcharge transfer across supported bilayer lipid membranes (s-BLMs)rdquo Journal of Biochemical and BiophysicalMethods vol 40no 1-2 pp 27ndash37 1999

[30] V Kochev and M Karabaliev ldquoWetting films of lipids inthe development of sensitive interfaces An electrochemicalapproachrdquo Advances in Colloid and Interface Science vol 107no 1 pp 9ndash26 2004

[31] C G ZoskiHandbook of Electrochemistry vol 5 Elsevier NewYork NY USA 2007

[32] H Haas G Lamura and A Gliozzi ldquoImprovement of thequality of self assembled bilayer lipid membranes by using anegative potentialrdquo Bioelectrochemistry vol 54 no 1 pp 1ndash102001

[33] C Amatore J M Saveant and D Tessier ldquoCharge transfer atpartially blocked surfaces A model for the case of microscopicactive and inactive sitesrdquo Journal of Electroanalytical Chemistryvol 147 no 1-2 pp 39ndash51 1983

[34] N Yang Q Wan and X Wang ldquoVoltammetry of VitaminB12

on a thin self-assembled monolayer modified electroderdquoElectrochimica Acta vol 50 no 11 pp 2175ndash2180 2005

[35] H Sato H Hakamada Y Yamazaki M Uto M Sugawara andY Umezawa ldquoIonophore incorporated bilayer lipid membranesthat selectively respond to metal ions and induce membranepermeability changesrdquo Biosensors and Bioelectronics vol 13 no9 pp 1035ndash1046 1998

[36] W Huang Z Zhang X Han et al ldquoIon channel behavior ofAmphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayermodelmem-branes investigated by electrochemistry and spectroscopyrdquoBiophysical Journal vol 83 no 6 pp 3245ndash3255 2002

[37] S Schreier S V P Malheiros and E de Paula ldquoSurface activedrugs self-association and interaction with membranes andsurfactants Physicochemical and biological aspectsrdquo Biochim-ica et Biophysica Acta Biomembranes vol 1508 no 1-2 pp 210ndash234 2000

[38] X Liu H Bai W Huang L Du X Yang and E WangldquoConcentration and time dependant behavior of chlorpro-mazine interaction with supported bilayer lipid membranerdquoElectrochimica Acta vol 51 no 12 pp 2512ndash2517 2006

[39] Z Oren and Y Shai ldquoMode of action of linear amphipathic 120572-helical antimicrobial peptidesrdquo Biopolymers vol 47 no 6 pp451ndash463 1998

[40] L M Crowe J H Crowe and D Chapman ldquoInteractionof carbohydrates with dry dipalmitoylphosphatidylcholinerdquoArchives of Biochemistry and Biophysics vol 236 no 1 pp 289ndash296 1985

[41] J Grdadolnik and D Hadzi ldquoFT infrared and Raman inves-tigation of saccharide-phosphatidylcholine interactions usingnovel structure probesrdquo Spectrochimica Acta A Molecular andBiomolecular Spectroscopy vol 54 no 12 pp 1989ndash2000 1998


[42] L Paasonen T Laaksonen C Johans M Yliperttula K Kont-turi and A Urtti ldquoGold nanoparticles enable selective light-induced contents release from liposomesrdquo Journal of ControlledRelease vol 122 no 1 pp 86ndash93 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


membranes using reconstituted liposomes and supportedbilayer membranes (s-BLM) as target models

2 Material and Methods

21 Reagents L-120572-Phosphatidylcholine (PC) from fresh eggyolk fructooligosaccharides from chicory (FOSC) andcholesterol were of the highest obtainable purity and weresupplied by Sigma-Aldrich (St Louis MO USA) All otherchemicals were of the highest quality as possible and allchemicals were used without further purification The waterused in all experiments was twice distilled

22 Supported Lipid Bilayer Formation The formation of s-BLMs on a Pt electrode has been conducted as reportedbefore [12ndash14] Briefly L-120572-phosphatidylcholine (PC) wasdissolved in chloroform at 20mgmL of final concentrationand cholesterol (CH) at 7mgmL (BLM solution) A workingPt electrode (050mm diameter) was first polished with sandpaper followed by alumina slurry on polishing cloth Then itwas sonicated in pure water for 2min rinsed and immersedin a solution of 10molL H

2SO4 Cyclic voltammetry was

performed after Pt electrode activation in 1molL H2SO4

The Pt electrode was immediately taken out rinsed with purewater again and sonicated with a highly power supersonicwave generator inwater and ethanol bath for 5min Following5 120583L of BLM forming solution was dropped on the electrodesurface The electrode was then rinsed again with redistilledwater to remove chloroform traces and transferred into a01molL KCl solution for 20min in which the supportedlipid layer was formed spontaneously [15] All experimentswere conducted in triplicate

23 Liposome Preparation The procedure was done fol-lowing S Basu and M Basu [16] In short a chloroformsolution containing PC at 10mmolL was evaporated in testtubes to dry with argon following evacuation for 60minfor total remotion of the solvent Subsequently the film washydrated in sodium phosphate buffer 01molL pH 74 or10mmolL dopamine solution after complete dispersion atroom temperature (DA-liposome) Then the multilamellarvesicles produced were ultrasonicated in water bath for 1minand sized by extrusion through Sephadex G-50 This lastprocedure was conducted whenever 10mmolL dopaminewas mixed with PC solution for dopamine incorporation(DA-liposome) The eluted volume was measured by spec-trofluorimetry to avoid dopamine freely available in solutionThe liposome structure was confirmed by phase contrast andfluorescence microscopy with Rhodamin B with a NikonEclipse 80i (Nikon Co Tokyo Japan)

24 Electrochemical Assays Cyclic voltammetry (CV) wasperformed on a potentiostat-galvanostat instrument PG-39MCSV (Omni Metra Instr Cient Ltda RJ Brazil) Theapparatus used for electrochemical impedance spectroscopy(EIS) was composed of Autolab with potentiostatgalvanostatworking station 128N and the Frequency Response Analysis

System Software FRA (Metrohm Autolab BV The Nether-lands) Impedance measurements were performed from100 kHz to 420mHz with a signal amplitude of 10mVThe experiments were carried out with a three-electrodesystem composed of anAgAgCl (KCl-saturated) as referenceelectrode and a platinum wire (05mm diameter) as thecounter electrode The working electrode comprised a Ptdisc of 05mm diameter melted in a glass tube EIS runswere performed in the presence of 1mmolL Fe(CN)

6

3minus4minus

as a redox probe contained in 01molL KCl as supportsolution and at a system potential of E0 of 235mV Theworking electrode was cleaned before use with MaxiClean1400 (Unique Ind Com Ltda SP Brazil) The experimentswere conducted at room temperature

25 Spectrofluorimetry of Liposomes The action of FOSCagainst reconstituted liposomes containing dopamine wasalso monitored by spectrofluorimetry [17] The fluorescencemeasurements were performed with a Cary Eclipse spec-trofluorophotometer (Varian Australia) with a 10mm quartzcuvette at an excitation wavelength of 279 nm The fluores-cence emission spectra were recorded in the 300ndash400 nmwavelength range with a 5 nm bandwidth Spectrofluorimet-ric runs were carried out after 5min of preincubation ofliposomes with 5mmolL SDS (sodium dodecyl sulfate) orFOSC (up to 75mgmL)

26 Data Analysis All of the experiments were conducted intriplicateThe data are expressed as themeansplusmn SE Statisticalanalysis was done with the free computing environment R (RCore Development Team) [18]

3 Results and Discussion

31 Characterization of s-BLM and Study of Interaction ofFructans on the Pt Electrode for EIS Surface-modified elec-trodes can also be assessed by electrochemical approachesas EIS and CV [19] In this sense EIS was carried outaiming to get further information about s-BLM integrityupon the action of tested FOS The complex impedance canbe presented as the sum of the real 119885re and imaginary 119885imcomponents that are originated mainly from the resistanceand capacitance of the cell respectively (Nyquist plot [14])The resulting data can be analyzed by a relatively equivalentRandles circuit consisting of an ideal and nonideal electri-cal analogs to the real physical and chemical processes atheoretical abstraction of an interfacial system [20] Figure 1illustrates the results of impedance spectroscopy measure-ments on the bare electrode whereas Figure 2 presents theresults obtainedwith the electrode coatedwith s-BLM (a) andafter interaction for 10min with 50 gL of fructooligosaccha-rides from chicory (b) in 01molL KCl solution containing1mmolL Fe(CN)

6

4minus3minus measured at the formal potential ofthe system

For the untreated electrode a very small circle probed byhigh frequencies can be visualized near the origin followedby a Warburg-like mass transfer impedance in the low


700

600

500

400

300

200

100

0

7006005004003002001000

Z998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)


6


0 1000 2000 3000 4000 5000 6000 7000

0

1000

2000

3000

4000

5000

6000

7000

042 kHz480 kHz

W

R

(a)

(b)

195 kHzZ998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)

Rm

Cm Zw

CdlRsol

Rct


6






element



6



6

4minus3minus




(119885119908) In this model 119877


119898is


119898of 016 120583Fcm2 (00 error) 119877

119898






119862119898=

1205760sdot 120581

119889

(1)


0= 885times





6

4minus3minus


3[Fe(CN)

6]

K4[Fe(CN)




3[Fe(CN)

6]

K4[Fe(CN)




120579 = 1 minus

(06 sdot 119894lim sdot 119903)

119865119860119862119863

(2)



6



600

400

200

0

minus200

minus400

minus600

400 5003002001000minus100minus200

I(n

A)


(a)

(b)


3[Fe(CN)

6]K4[Fe(CN)

6]


(a) (b)(c)

(d) (e)(f)

400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)


3[Fe(CN)

6]K4[Fe(CN)





6



400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

(A)

(a)

(b)(c)

(d)(e)

(f)


3[Fe(CN)

6]K4[Fe(CN)

6]



6



119871 = 100 minus (119868119894minus 119868119887

119868119905minus 119868119887119905

) (3)

0

20

40

60

80

Lipo Lipo-DA

L (

)

Lipo-DA+ FOSC

Lipo-DA+ SDS









4 Conclusion



7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)





Acknowledgment


References


















6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


700

600

500

400

300

200

100

0

7006005004003002001000

Z998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)


6


0 1000 2000 3000 4000 5000 6000 7000

0

1000

2000

3000

4000

5000

6000

7000

042 kHz480 kHz

W

R

(a)

(b)

195 kHzZ998400998400

(Ohm

cm2)

Z 998400 (Ohm cm2)

Rm

Cm Zw

CdlRsol

Rct


6






element



6



6

4minus3minus




(119885119908) In this model 119877


119898is


119898of 016 120583Fcm2 (00 error) 119877

119898






119862119898=

1205760sdot 120581

119889

(1)


0= 885times





6

4minus3minus


3[Fe(CN)

6]

K4[Fe(CN)




3[Fe(CN)

6]

K4[Fe(CN)




120579 = 1 minus

(06 sdot 119894lim sdot 119903)

119865119860119862119863

(2)



6



600

400

200

0

minus200

minus400

minus600

400 5003002001000minus100minus200

I(n

A)


(a)

(b)


3[Fe(CN)

6]K4[Fe(CN)

6]


(a) (b)(c)

(d) (e)(f)

400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)


3[Fe(CN)

6]K4[Fe(CN)





6



400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

(A)

(a)

(b)(c)

(d)(e)

(f)


3[Fe(CN)

6]K4[Fe(CN)

6]



6



119871 = 100 minus (119868119894minus 119868119887

119868119905minus 119868119887119905

) (3)

0

20

40

60

80

Lipo Lipo-DA

L (

)

Lipo-DA+ FOSC

Lipo-DA+ SDS









4 Conclusion



7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)





Acknowledgment


References


















6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



6

4minus3minus


3[Fe(CN)

6]

K4[Fe(CN)




3[Fe(CN)

6]

K4[Fe(CN)




120579 = 1 minus

(06 sdot 119894lim sdot 119903)

119865119860119862119863

(2)



6



600

400

200

0

minus200

minus400

minus600

400 5003002001000minus100minus200

I(n

A)


(a)

(b)


3[Fe(CN)

6]K4[Fe(CN)

6]


(a) (b)(c)

(d) (e)(f)

400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)


3[Fe(CN)

6]K4[Fe(CN)





6



400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

(A)

(a)

(b)(c)

(d)(e)

(f)


3[Fe(CN)

6]K4[Fe(CN)

6]



6



119871 = 100 minus (119868119894minus 119868119887

119868119905minus 119868119887119905

) (3)

0

20

40

60

80

Lipo Lipo-DA

L (

)

Lipo-DA+ FOSC

Lipo-DA+ SDS









4 Conclusion



7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)





Acknowledgment


References


















6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


400 5003002001000minus100minus200


400

300

200

100

0

minus100

minus200

minus300

I(n

A)

(A)

(a)

(b)(c)

(d)(e)

(f)


3[Fe(CN)

6]K4[Fe(CN)

6]



6



119871 = 100 minus (119868119894minus 119868119887

119868119905minus 119868119887119905

) (3)

0

20

40

60

80

Lipo Lipo-DA

L (

)

Lipo-DA+ FOSC

Lipo-DA+ SDS









4 Conclusion



7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)





Acknowledgment


References


















6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


7120583m

(a)

7120583m

(b)

9120583m

(c)

9120583m

(d)





Acknowledgment


References


















6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










6









3)3+6Ru(NH

3)2+6

andFe(CN)3minus

6Fe(CN)4minus




7Fe3+P

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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