research article the potential health benefits of...

Research ArticleThe Potential Health Benefits of Polyphenol-Rich Extracts fromCichorium intybus L Studied on Caco-2 Cells Model

Elena Azzini Giuseppe Maiani Ivana Garaguso Angela PolitoMaria S Foddai Eugenia Venneria Alessandra Durazzo Federica Intorre Lara PalombaMaria L Rauseo Ginevra Lombardi-Boccia and Fabio Nobili

Council for Agricultural Research and Economics (CREA) Research Center for Food and Nutrition Via Ardeatina 54600178 Rome Italy

Correspondence should be addressed to Elena Azzini elenaazzinientecrait

Received 7 September 2015 Revised 28 October 2015 Accepted 28 October 2015

Academic Editor Gregor Drummen

Copyright copy 2016 Elena Azzini 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

Phytochemicals can exert their bioactivity without reaching the systemic circulation scarcely absorbed antioxidants might reachthe large bowel contributing to protection from oxidative damage-induced gastrointestinal diseases In the present work weaimed to study the relationship between potential activity of polyphenol-rich extracts from Cichorium intybus L and changes inmorphological characteristics on Caco-2 cells Phytochemicals content (carotenoids and flavonoids) and total antioxidant activityof Red Chicory of Treviso and Variegated Chicory of Castelfranco were evaluatedThe bioactivity of polyphenol-rich extracts fromchicories was studied in in vitroCaco-2 cell monolayersmodelMorphological characteristics changes to test the antioxidant andorprooxidant effect were verified by histological analysis and observed by Electronic Scansion Microscopy (SEM) On Caco-2 cellmodel the polyphenols fractions from chicories have indicated a moderate antioxidant behavior until 17120583M concentration while70 120583M and 34 120583M exert cytotoxic effects for Trevisorsquos and Castelfrancorsquos Chicory respectively highlighted by TEER decreasingincreased permeability and alteration of epithelium Our findings support the beneficial effects of these products in counteractingthe oxidative stress and cellular damage induced in vitro on Caco-2 cell model through interaction with the mucopolysaccharidecomplexes in the glycocalyx maintaining in vivo a healthy and effective intestinal barrier

1 Introduction

Chicory a plant genus typical ofMediterranean area is nativeto Europe Western Asia and North America and its colourvaries from white to red [1] ldquoRed Chicory of Trevisordquo andldquoVariegated Chicory of Castelfrancordquo with PGI according toEU rules are strongly linked to their territory and grownaccording to traditional cultivation techniques These prod-ucts have acquired great interest for their organoleptic andnutritional characteristics As well known the consumptionof phytochemicals from fruits and vegetables can improvethe prevention of several chronic degenerative pathologies[2 3] Phytochemicals content may be affected by severalfactors genetic characteristic environmental aspects agro-nomic practices and postharvest conditions [4 5] Geneticfactors exert great influence on nutritional and phytochemi-cals content between and within vegetables species Climate

condition light exposure temperature relative humidityand luminous intensity are specific parameters that affectfood quality In particular the choice of an appropriateagronomic practice could improve the levels and the profile ofbioactive compounds Among the vegetal crops red chicoriesare attractive because they may be consumed either rawor cooked In particular they are commonly eaten raw insalad during winter months when most vegetables are notin season In fact red chicories are particularly resistantto low temperatures [6] and their availability throughoutthe year is an important source of micronutrients duringthe coldest season The red color is caused in large part bythe presence of water-soluble pigments anthocyanins butseveral works show that the red-leaved varieties of Cichoriumintybus L have the highest content of polyphenols amongthe leafy vegetables that are consumed raw [7 8] Changesin phytochemicals content in agricultural production take on

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1594616 9 pageshttpdxdoiorg10115520161594616

2 Oxidative Medicine and Cellular Longevity

a particular importance in our dietThe bioactive compoundsin foods such as vitamins carotenoids and polyphenolsseem to be able tomodulate one ormoremetabolic processeswhich result in the promotion of better health [9] The mostaccepted explanation for the protective effect of foodprobablyderives from the observation that different plant phytochem-icals may act as ldquoscavengersrdquo of free radicals ldquoquenchersrdquosinglet oxygen ormetal chelators [10 11] and therefore induceprotection against oxidative damage through antibacterialanti-inflammatory hepatoprotective anticarcinogenic andvasodilator actions

Recently DrsquoEvoli et al [12] have shown that the highlevels of antioxidant anthocyanins present in Red Chicoryexert a direct scavenging effect against ROS formation interms of antioxidant and cytoprotective activities as well asantiproliferative activity in Caco-2 cell In the present workwe aimed to study the relationship between potential activityof polyphenol-rich extracts from chicories and morpholog-ical and chemicalphysical changes in Caco-2 cellular lineTo this purpose bioactive compounds content (carotenoidsand flavonoids) and total antioxidant activity were evaluatedin Early and Late Red Chicory of Treviso and VariegatedChicory of Castelfranco In addition the bioactivity ofpolyphenol-rich extracts from chicories in in vitroCaco-2 cellmonolayer model was studied Morphological characteristicschanges to test the antioxidant andor prooxidant effect wereverified by histological analysis and observed by ElectronicScansion Microscopy (SEM)

2 Materials and Methods

21 Reagents All solvents were purchased from Carlo Erba(Milan Italy) BDH (Poole England) andMerck (DarmstadtGermany) 246-Tri(2-pyridyl)-s-triazine (TPTZ) was fromFluka (Switzerland) Phosphate-buffered saline (PBS)6-hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) and ascorbic acid were provided by Sigma-AldrichSrl Commercial standards were also from Sigma-AldrichSrl (Milan Italy) AAPH (221015840-azobis (2-amidinopropane)dihydrochloride) (WACO Chem Richmond VA USA) wasused as a source of hydrophilic radicals Double distilledwater (Millipore Milan Italy) was used throughout thestudy

22 Cichorium intybus L Red Chicories of Treviso (twovarieties Early and Late) samples were grown in the regionincluding Quinto (Treviso) Zero Branco (Treviso) andScorze (Venezia) while PGI samples of Variegated Chicoryof Castelfranco come from Due Carrare (Padova) Mira(Venezia) and Monselice (Padova) A total weight of 5 kg ofeach variety for each locality was collected at harvesting timeand delivered to the laboratory Only the edible portion of thesamples was utilized for analysis according to the followingmethods

23 Analytical Methods The extraction of some flavonoids(quercetin kaempferol and apigenin) was performed asdescribed by Hertog et al [13] and the quantitative analysis

through a system-ESA HPLC with electrochemical detectoras reported by Azzini et al [14] Carotenoids were extractedfrom Trevisorsquos and Castelfrancorsquos chicory according to themethod described by Sharpless et al [15] For the quantifi-cation the samples were analyzed by high pressure liquidchromatography (HPLC) as reported by Maiani et al [16]Total Antioxidant Capacity (TAC) was evaluated using twodifferent assays Ferric Reducing Antioxidant Power (FRAP)[17] and Trolox Equivalent Antioxidant Capacity (TEAC)method [18 19] The results of each analysis have beenperformed in triplicate

24 Transepithelial Electrical Resistance (TEER) EvaluationFor TEER evaluation the polyphenol fractions from RedChicory of Treviso and Variegated Chicory of Castelfrancohydrolysed extracts have been studied on Caco-2 cell mono-layers to test the changes in tight-junction (TJ) permeabilityby TEER the phenol red passage and histological analysis

The cellular line was treated with increasing concentra-tion of two polyphenols extracts (02 13 10 17 34 and70 120583M) for 180 minutes to simulate in vivo physiologicalprocesses The variations of transepithelial potentials andphenol red permeability were recorded at time intervals (301015840)In the experiment the cells were seeded onto polycarbonatefilter cell culture chamber inserts (diameter 65mm area033 cm2 and pore diameter 04 120583m) at density of 15 sdot 105 cellsper filter and placed in a multiwell Falcon The filter dividedthe chamber into two parts apical and basal that representthe lumen and the basal area of the gastroenteric systemInto two chambers TEER measurement for assessment oftight-junction permeabilitywas performed using theMillicellERS apparatus (Millipore Bedford MA USA) according tothe manufacturerrsquos instruction After calibrating the resis-tance system the electrical resistance of the monolayer wasmeasured by placing one electrode on either side of thepolycarbonate filter [20 21] The results of each analysis havebeen performed in triplicate and expressed as OHM times cm2

Cells reached confluence and differentiation within 15ndash20days During this time cellular morphology was monitoredand checked with a Leitz Diavert inverted light microscope

25 Histological Analysis The cellular monolayer was iso-lated together with the filter and fixed in Bouin (an aqueoussolution composed of picric acid acetic acid and formalde-hyde) for about 12 hours then dehydrated using the alcoholascending scale (70 80 90 95 and 100) and finallyenclosed into resin blocks of polymerized epoxy (GMAJ134K polyscience Inc Warrington PA USA) Then thesamples were glued on embedded stubs and cut into 3 120583msections with a Micron rotary microtome (Zeiss Germany)appropriately assembled so as to use a crystal blade accordingto Ralphrsquos modification [22 23] The sections glued to themicroscope slides were stained with Harrisrsquo hematoxylin andeosin [24 25] The preparations were made permanent withslide covers and sealed with resin (Eukitt mountingmediumBDA Laboratories Supplies Pool England) All sections(3 120583m) were examined for histological changes by Diaplan 22light microscopy (Leitz Germany) as shown in Figure 1 All

Oxidative Medicine and Cellular Longevity 3

Table 1 Flavonoid identification from Red Chicory of Treviso and Variegated Chicory of Castelfranco extracts

Peak analyteRed Chicory of Treviso Variegated Chicory of Castelfranco

RT (min) Dominant channelheight Ratio accuracy RT (min) Dominant channel

height Ratio accuracy

Quercetin 5126 637 nA 0925 5119 227 nA 0947Kaempferol 5328 174 nA 0800 5313 123 nA 0807Apigenin 5561 373 nA 5552 364 nA

Treviso

Castelfranco

Resp

onse

(120583A

)

000

050

100

150

0

200

400

600

800

Resp

onse

(nA

)

100 200 300 400 50000Retention time (min)


Figure 1 Representative HPLC-ECD gradient array chromato-grams of polyphenol-rich extracts analyzed under specified condi-tions from Red Chicory of Treviso and Variegated of Castelfrancoextracts

pictures (microphotographies) have a magnification of 356xand were performed with Leica microscope Dialux 22

26 SEM Analysis At the end of each experiment (180minutes) the cellular monolayer was fixed for 12 hours informaldehyde (10) and glutaraldehyde (25) and thendried with alcohol solution Then the sample was dehy-drated by Critical Point Drying (Emitech K850 QuorumTechnologies West Sussex UK) The final dehydration wasachieved in CO

2 Finally the sample was sputtered with gold

for 120 s at 30mA in a modified atmosphere with 2 Argonand analyzed using SEM (EVO LS10 Carl Zeiss MicroscopyGmbH Jena Germany)

27 Statistics The results are expressed as mean plusmn SDStatistical data analysis was performed using the ANOVA testfollowed by Bonferroni post hoc test (significance at 119875 lt005)

3 Results

31 Phytochemical Characterization According to severalauthors flavonoid components could be used as potentialmarkers for the analysis of herbs and plants for humanconsumption [26ndash28]

With regard to the quantitative analysis of flavonoids(free plus conjugated forms) Figure 1 displays representa-tive HPLC-DAD gradient array chromatograms while theiridentification is reported in Table 1 The varied flavonoidscontent amongst different varieties and growing locations issummarized in Table 2 Comparing the growing locationstherewere no significant differences in the flavonoids contentwhile significant differences were recorded between varietiesQuercetin was found as the most abundant flavonoid in RedRadish of Treviso varieties (9004 plusmn 2516mgkg and 9788 plusmn3394mgkg resp in Late and Early Red Chicory) withrespect to the Variegated Chicory of Castelfranco (1420 plusmn451mgkg)

The kaempferol was significantly different among culti-vars and varieties Its content in the Late Red Chicory ofTreviso (2280 plusmn 584mgkg) variety was significantly higher(119875 lt 005) comparing to Early RedChicory of Treviso (1235plusmn765mgkg) and Variegated Chicory of Castelfranco (1180 plusmn364mgkg)

There were no significant differences in the apigenincontent between cultivar and production area on averagerange from 260 plusmn 140 to 358 plusmn 029mg kg respectivelyfor Early Red Chicory of Treviso and Variegated Chicory ofCastelfranco

Our data (Table 3) indicate that lutein and 120573-carotenewere the main carotenoids (ranging from 119 plusmn 024 to 240 plusmn061mgkg and from 019 plusmn 002 to 048 plusmn 015mgkg resp)A significant difference (119875 lt 005) was present between 120573-carotene content of Late Red Chicory of Treviso (038 plusmn008mgkg) and Early Red Chicory of Treviso (022 plusmn004mgkg) Variegated Chicory of Castelfranco showed amean of 120573-carotene content equal to 035 plusmn 014mgkg In theEarly Red Chicory of Treviso the mean lutein content was216 plusmn 028mgkg with significantly higher levels (119875 lt 005)than Late Red Chicory of Treviso (127 plusmn 020mg100 g) andVariegated Chicory of Castelfranco (120 plusmn 027mg100 g)

After evaluating the content of individual antioxidantsthe cooperative action of bioactive molecules in the differentspecimens of Cichorium intybus L was evaluated by totalantioxidant capacity (TAC) Figure 2 shows the synergisticeffects of various antioxidants measured by FRAP (mmolkg)and TEAC (mmol troloxkg) methods


Table 2 Flavonoids content amongst different varieties by growing locations (mgkg)

Quercetin (mgkg) Kaempferol (mgkg) Apigenin (mgkg)Late Red Chicory of TrevisoZero Branco 8392 plusmn 1508 2143 plusmn 219 210 plusmn 061Scorze 9589 plusmn 2651 2623 plusmn 1048 324 plusmn 082Quinto 9031 plusmn 339 2094 plusmn 486 305 plusmn 112

9004 plusmn 2516a 2280 plusmn 584a 280 plusmn 085Early Red Chicory of TrevisoZero Branco 10150 plusmn 4020 1442 plusmn 840 398 plusmn 181Scorze 10023 plusmn 424 1451 plusmn 980 151 plusmn 080Quinto 9189 plusmn 2922 811 plusmn 538 230 plusmn 160

9788 plusmn 3394a 1235 plusmn 765b 260 plusmn 140Variegated Chicory of CastelfrancoMonselice 1532 plusmn 354 868 plusmn 216 333 plusmn 044Mira 1447 plusmn 492 135 plusmn 381 378 plusmn 038Due Carrare 128 plusmn 508 1321 plusmn 497 362 plusmn 005

1420 plusmn 451b 1180 plusmn 364b 358 plusmn 029ANOVA 119875 lt 005 a versus b by column

Table 3 Carotenoids (lutein and 120573-carotene) means values (mgkg)by different cultivars ofChicorium intybusL and by production area

Lutein(mgkg)

beta-carotene(mgkg)

Late Red Chicory of TrevisoZero Branco 127 plusmn 017 027 plusmn 006Scorze 119 plusmn 024 020 plusmn 001Quinto 141 plusmn 007 019 plusmn 002

127 plusmn 020b 022 plusmn 004b

Early Red Chicory of TrevisoZero Branco 212 plusmn 089 045 plusmn 002Scorze 197 plusmn 011 035 plusmn 001Quinto 240 plusmn 061 034 plusmn 009

216 plusmn 028a 038 plusmn 008a

Variegated Chicory of CastelfrancoMonselice 128 plusmn 013 048 plusmn 015Mira 126 plusmn 036 035 plusmn 011Due Carrare 107 plusmn 04 023 plusmn 001

120 plusmn 027b 035 plusmn 014ab

ANOVA 119875 lt 005 a versus b by column

Our results highlighted the highest FRAP values of LateRed and Early Red Chicories of Treviso with respect toVariegatedChicory of Castelfranco (1170plusmn 192mmolkg and993 plusmn 311mmolkg versus 876 plusmn 446mmolkg resp) Nodifferences by varieties and production area were present inFRAP (mmol troloxkg) results

Significant differences were present in TEAC(mmol troloxkg) levels TEAC values of Late Red Chicory ofTreviso (454 plusmn 088 mmol troloxkg) and Early Red Chicory

0 5 10 15

Variegated Chicoryof Castelfranco

Late Red Chicory

Early Red Chicoryof Treviso

of Treviso

a

a

b

TEAC (mmolkg)FRAP (mmolkg)

ANOVA P lt 005 a versus b

Figure 2 Total antioxidant capacity FRAP (mmolkg) and TEAC(mmol troloxkg) values by different cultivars of Cichorium intybusL

of Treviso (532 plusmn 176mmol troloxkg) were higher (119875 lt005) compared with Variegated Chicory of Castelfranco(212 plusmn 074mmol troloxkg)

32 Cell-Based Assays Differently from other food compo-nents phytochemicals can exert their bioactivity withoutreaching the systemic circulation Scarcely absorbed antiox-idants might reach the large bowel contributing to protec-tion fromoxidative damage-induced gastrointestinal diseases[29]There are several reports about pharmacological actionsand anti-inflammatory effects of chicory [30 31]

TEER measurements are routinely used to characterizemonolayer integrity in the context of cell monolayer per-meability experiments or to quantify permeability changesfor example as a consequence of paracellular permeabilityenhancers [32 33]


0 30 60 90 120 150 180(min)

(120583M)ControlRT 02RT 13RT 10

RT 17RT 34RT 70

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(a)

0 30 60 90 120 150 180(min)

ControlVC 02VC 13VC 10

VC 17VC 34VC 70

(120583M)

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(b)

Figure 3 TEER changes at different concentration of polyphenol extract (a) Red Chicory of Treviso (RT) and (b) Variegated Chicory ofCastelfranco (VC) polyphenol extract

(a) (b)

Figure 4 Histological analysis (a) and scanning electron micrographs (b) of normal Caco-2 pattern treated with polyphenolics extract ofTreviso Red (17 120583M)

Utilizing polyphenol fractions from Red Trevisorsquos andCastelfrancorsquos Chicory the changes in TJs permeability weretested on Caco-2 cell line in monolayers culture TEER mea-surements at increasing phenolic concentrations are shownin Figures 3(a) and 3(b) respectively for Treviso Red Chicoryand Variegated Chicory of Castelfranco

Upon treatment with Red Chicory of Trevisorsquos and Var-iegated Chicory of Castelfrancorsquos polyphenolics extract asindicated by TEER values the results showed a monolayerequilibrium model (healthy monolayer cells) at 02-13-10-17 120583M extract concentration Until 17120583M polyphenolicextracts concentration the TEER measurements hold out aplateau indicating the tightness of TJs and the absence ofdirect interaction between epithelial Caco-2 cells and chicoryextracts as confirmed by histological analyses Figures 4(a)and 4(b) display the 17 120583M effect upon both treatments Theultrastructural cytological analysis by SEM highlighted andconfirmed the absence of morphological change or extractsactivity on the cellular monolayer (Figures 5(a) and 5(b))

The concentrations of 70 120583M and 34 120583M showed a hightoxicity respectively for Trevisorsquos and Castelfrancorsquos Chicoryextracts tested These treatments produce lowering of TEERvalues (Figures 3(a) and 3(b)) highlighted by increased per-meability of TJs (phenol red) and by alteration of epitheliumThese concentrations promote cellular necrosis in Caco-2 cells monolayer as shown in histological analysis andconfirmed by SEM observations (Figures 6(a) 6(b) and 7(a)7(b) for Trevisorsquos and Castelfrancorsquos Chicory resp)

To better understand their bioactivity and attempting todemonstrate the probable prebiotic role of these extractsCaco-2 oxidative stress was induced by adding 221015840azobis (2-amidinopropane) dihydrochloride (AAPH) to the cell culturemedium The interactions between polyphenol-rich RedChicory of Treviso extracts against AAPH-induced oxidativestress are reported in Figure 8(a) The progressive TEERincreases indicated that upon 02 13 and 10120583M chicoryextracts treatment displayed a strong antioxidant activity thatappeared to be able to counteract the peroxidative trigger


(a) (b)

Figure 5 Histological analysis (a) and scanning electron micrographs (b) of normal Caco-2 pattern treated with polyphenolics extract ofVariegated Chicory of Castelfranco (17120583M)

(a) (b)

Figure 6 Histological analysis (a) and scanning electron micrographs (b) showing Caco-2 necrotic pattern upon treatment with poly-phenolics extract Treviso Red extract (70 120583M)

(a) (b)

Figure 7 Histological analysis (a) and scanning electron micrographs (b) showing Caco-2 necrotic pattern upon treatment with poly-phenolics extract Variegated Chicory of Castelfranco extract (34120583M)

induced by AAPH suggesting a cellular membrane integrityand confluency restoring Only 02 120583M Variegated Chicoryof Castelfranco extract exhibited a low antioxidant effect(Figure 8(b)) that promotes a cell damage recovery resultingby slight TEER increase

4 Discussion

The antioxidant properties of several varieties of Cicho-rium genus vegetables have been attributed in part tothe presence of phytochemicals including hydroxycinnamicacid derivatives mono- and diglycosides of flavonoids andanthocyanins As reported by Rossetto et al [6] the presence

of these phenolics confers to red chicories an exceptionallyhigh peroxyl radical scavenging activity in terms of bothcapacity and efficiency particularly in their early stage ofgrowth The lower carotenoid values observed in the presentstudy could be due to the limited exposure to sunlight andthe lower temperature during winter because carotenoidsbiosynthesis is not stimulated as in vegetables in openfields As reported by Niizu and Rodriguez-Amaya [34] thegreen chicory showed a higher average content of 537 plusmn83 (mgkg) and 353 plusmn 50 (mgkg) for lutein and 120573-carotenerespectively As discussed elsewhere [35] green chicory fromLazio exhibited higher carotenoids content In particularlutein and 120573-carotene values were 1410 plusmn 330mgkg and


(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180

Control RT 10 + AAPHRT 13 + AAPHRT 02 + AAPHControl + AAPH

RT 70 + AAPHRT 34 + AAPHRT 17 + AAPH

(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180

Control VC 10 + AAPHVC 13 + AAPHVC 02 + AAPHControl + AAPH

VC 70 + AAPHVC 34 + AAPHVC 17 + AAPH

(b)

Figure 8 TEER changes at different concentration of polyphenol-rich extract afterAAPH-induced oxidative stress (a) RedChicory of Treviso(RT) extract and (b) Variegated Chicory of Castelfranco (VC) polyphenol extract

4212 plusmn 599mgkg respectively in wild type while theaverage content was 1551 plusmn 328mgkg for lutein and 4062plusmn 215mgkg for 120573-carotene in cultivated chicory

The total antioxidant capacity is a parameter stronglyinfluenced by type of food (ie species and varieties for thesame species) but also by growing conditions environmentalfactors and preservation techniques Variegated Chicoryof Castelfranco exhibited the lowest TAC values for theFRAP and TEAC methods respectively Red Chicory ofTreviso (Early and Late) results analyzed by the FRAP andTEAC assays were according to Pellegrini et al [36] Asreported elsewhere [35] changes in antioxidant compositionof several vegetables were linked to agricultural practices andenvironmental factors Regarding cell-based assays TEERmeasurement represents a physical measure to evaluate thephysiological state of cell cultures detecting the early intesti-nal barrier function in vivo damage As known defect inepithelial permeability caused by alteration of TJs is seenin several inflammatory bowel diseases Different elementsincluding robust innate immune responses epithelial para-cellular permeability and epithelial cell integrity as wellas the production of mucus contribute to the integrity ofthe intestinal barrier [37] Our experimental system wasthe confluent monolayer of Caco-2 cells at equilibrium Inthis condition the epithelial culture is more susceptible tovariations of the chicory extracts concentration So smallrelative changes in concentrations of extracts correspondto chemical and physical altered cellular patterns and dra-matic cell morphological changes The tightness of TJs asconfirmed by histological analyses (Figures 4(a) and 5(a))and the absence of morphological change or extracts activityon the cellular monolayer by SEM (Figures 4(b) and 5(b))does not specify if these extracts maintain the protectiveeffect of the antioxidant mixture but indicates the absenceof direct interaction between epithelial Caco-2 cells and

chicory extracts These extracts could be compartmentalizedin the mucopolysaccharides of the glycocalyx like prebioticmixture As known the glycocalyx is a meshwork of glyco-protein molecules that binds to mucus largely composed ofmucopolysaccharides produced by goblet cells The glyco-calyx and mucus form a flexible coat which provides cellsprotection frommechanical and chemical damage Nutrientscan diffuse into the mucosa be acted upon by the enzymesin the glycocalyx and create an area at high concentrationof more easily adsorbed molecules by concentration gradient[38] At the moment our research is focused on clustering ofthe food extracts by glycocalyx interaction

The greatest protective effect on cell cytotoxicity derivingfrom Treviso Red radish extract (70 120583M) with respect toVariegated Chicory of Castelfranco extract (34120583M) could bedue to specific composition in polyphenol-rich fractionsThemain flavonoids in above-mentioned extracts are reportedin Table 1 and the TJs respond to various naturally plant-derived and food extracts TEERmeasurements present dose-response curve patterns indicating the absence of alterationin Caco-2 cell monolayer as underlined by our results from02 to 17 120583Mchicory extracts treatments (Figure 3)MoreoverCichorium intybus L seems to counteract and improve theoxidative stress and cellular damage induced by AAPH invitro Caco-2 cell model (Figure 8) As previously reported[39] when the prooxidant (AAPH) is added the TEERmeasurements dramatically decrease until cellular necrosisin addition as reported by Finotti et al [40] the presenceof oxidant (AAPH) induces an increase in the mucopolysac-charides secretion located at microvilli glycocalyx Overall abetter healthy action by Red Chicory of Treviso polyphenol-rich extract on Caco-2 (at 02-13-10 120583M concentrations)could be assumed which does not interact with monolayercells while as exogenous substance it helps to maintainoptimal cellular functions by its strong antioxidant activity


On the other hand Red Chicory is characterized by a highcontent of anthocyanin pigments [41] that could exert severalbeneficial health or nutraceutical effects [12 42 43]

5 Conclusion

It could be concluded that the TJs response depends onthe dose exposure and particular chemical composition offood extracts by synergic and interdependent antioxidanteffects (enzymatic and nonenzymatic) with the epithelialglycocalyx Even if the redox balance does not originate froma single cause our study suggests that the interaction betweenantioxidant extracts and the mucopolysaccharide complexesin the glycocalyx could protect the in vivo lining of gut fromdamage maintaining a healthy and effective intestinal barrier

Abbreviations

Caco-2 Human colon carcinoma cell lineFRAP Ferric Reducing Antioxidant PowerTEAC Trolox Equivalent Antioxidant CapacityTEER Transepithelial Electrical ResistanceTJ Tight-junctionSEM Scanning Electron Microscopy

Conflict of Interests

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

Acknowledgments

The present study was performed within the ProjectsBIOVITA and TERRAVITA funded by the Italian Ministryof Agriculture Food and Forestry Policies

References

[1] H P Bais and G A Ravishankar ldquoCichorium intybus Lmdashcultivation processing utility value addition and biotechnol-ogy with an emphasis on current status and future prospectsrdquoJournal of the Science of Food and Agriculture vol 81 no 5 pp467ndash484 2001

[2] D A Evans J B Hirsch and S Dushenkov ldquoPhenolicsinflammation and nutrigenomicsrdquo Journal of the Science of Foodand Agriculture vol 86 no 15 pp 2503ndash2509 2006

[3] M Gerber M-C Boutron-Ruault S Hercberg E Riboli AScalbert and M-H Siess ldquoFood and cancer state of the artabout the protective effect of fruits and vegetablesrdquo Bulletin duCancer vol 89 no 3 pp 293ndash312 2002

[4] N Hounsome B Hounsome D Tomos and G Edwards-JonesldquoPlantmetabolites and nutritional quality of vegetablesrdquo Journalof Food Science vol 73 no 4 pp R48ndashR65 2008

[5] M Schreiner ldquoVegetable crop management strategies toincrease the quantity of phytochemicalsrdquo European Journal ofNutrition vol 44 no 2 pp 85ndash94 2005

[6] M Rossetto A Lante P Vanzani P Spettoli M Scarpa andA Rigo ldquoRed chicories as potent scavengers of highly reactiveradicals a study on their phenolic composition and peroxyl

radical trapping capacity and efficiencyrdquo Journal of Agriculturaland Food Chemistry vol 53 no 21 pp 8169ndash8175 2005

[7] A Papetti M Daglia and G Gazzani ldquoAnti- and pro-oxidantactivity of water soluble compounds in Cichorium intybus varsilvestre (Treviso red chicory)rdquo Journal of Pharmaceutical andBiomedical Analysis vol 30 no 4 pp 939ndash945 2002

[8] G Gazzani M Daglia A Papetti and C Gregotti ldquoIn vitro andex vivo anti- and prooxidant components ofCichorium intybusrdquoJournal of Pharmaceutical and Biomedical Analysis vol 23 no1 pp 127ndash133 2000

[9] L Hooper and A Cassidy ldquoA review of the health care potentialof bioactive compoundsrdquo Journal of the Science of Food andAgriculture vol 86 no 12 pp 1805ndash1813 2006

[10] W Kalt C F Forney A Martin and R L Prior ldquoAntioxidantcapacity vitamin C phenolics and anthocyanins after freshstorage of small fruitsrdquo Journal of Agricultural and Food Chem-istry vol 47 no 11 pp 4638ndash4644 1999

[11] L R Fukumoto and G Mazza ldquoAssessing antioxidant andprooxidant activities of phenolic compoundsrdquo Journal of Agri-cultural and Food Chemistry vol 48 no 8 pp 3597ndash3604 2000

[12] L DrsquoEvoli F Morroni G Lombardi-Boccia et al ldquoRed chicory(Cichorium intybus L cultivar) as a potential source of antioxi-dant anthocyanins for intestinal healthrdquoOxidativeMedicine andCellular Longevity vol 2013 Article ID 704310 8 pages 2013

[13] M G L Hertog P C H Hollman and D P Venema ldquoOpti-mization of a quantitative HPLC determination of potentiallyanticarcinogenic flavonoids in vegetables and fruitsrdquo Journal ofAgricultural and Food Chemistry vol 40 no 9 pp 1591ndash15981992

[14] E Azzini A Durazzo M S Foddai et al ldquoPhytochemicalscontent in Italian garlic bulb (Allium sativum L) varietiesrdquoJournal of Food Research vol 3 no 4 pp 26ndash32 2014

[15] K E Sharpless M Arce-Osuna and J Brown-ThomasldquoValue assignment of retinol retinyl palmitate tocopherol andcarotenoid concentrations in Standard Reference Material 2383(baby food composite)rdquo Journal of AOAC International vol 82no 2 pp 288ndash296 1999

[16] G Maiani G Pappalardo A Ferro-Luzzi et al ldquoAccumulationof 120573minuscarotene in normal colorectal mucosa and colonic neo-plastic lesions in humansrdquo Nutrition and Cancer vol 24 no 1pp 23ndash31 1995

[17] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[18] N Pellegrini D Del Rio B Colombi M Bianchi andF Brighenti ldquoApplication of the 221015840-azinobis(3-ethylbenzot-hiazoline-6-sulfonic acid) radical cation assay to a flow injec-tion system for the evaluation of antioxidant activity of somepure compounds and beveragesrdquo Journal of Agricultural andFood Chemistry vol 51 no 1 pp 260ndash264 2003

[19] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[20] S Ferruzza G Ranaldi M Di Girolamo and Y SambuyldquoThe transport of lysine across monolayers of human cul-tured intestinal cells (Caco-2) depends on Na+-dependent andNa+-independent mechanisms on different plasma membranedomainsrdquo Journal of Nutrition vol 125 no 10 pp 2577ndash25851995

[21] S Ferruzza Y Sambuy A Onetti-Muda F Nobili and ML Scarino ldquoCopper toxicity to tight junctions in the human


intestinal Caco-2 cell linerdquo in Handbook of Copper Pharmacol-ogy and Toxicology E J Massaro Ed pp 397ndash416 HumanaPress Totowa NJ USA 2002

[22] T M Szczesny ldquoHolder assembly for lsquoRalphrsquo type glass knivesrdquoStain Technology vol 53 no 1 pp 50ndash51 1978

[23] R Semba ldquoContributions to semithin sectioning on a conven-tional rotary microtomerdquo Stain Technology vol 54 no 5 pp251ndash255 1979

[24] L G LunaManual of Histologic Staining Methods of the ArmedForces Institute of Pathology McGrawHill Book New York NYUSA 1968

[25] H S Bennett A D Wyrick S W Lee and J H Mcneil JrldquoScience and art in preparing tissues embedded in plastic forlight microscopy with special reference to glycol methacrylateglass knives and simple stainsrdquo Stain Technology vol 51 no 2pp 71ndash97 1976

[26] S Salvatore N Pellegrini O V Brenna et al ldquoAntioxidantcharacterization of some Sicilian edible wild greensrdquo Journal ofAgricultural and Food Chemistry vol 53 no 24 pp 9465ndash94712005

[27] C E Lewis J R L Walker J E Lancaster and K H SuttonldquoDetermination of anthocyanins flavonoids and phenolic acidsin potatoes II wild tuberous Solanum speciesrdquo Journal of theScience of Food and Agriculture vol 77 no 1 pp 58ndash63 1998

[28] R Chirinos D Campos C Arbizu et al ldquoEffect of geno-type maturity stage and post-harvest storage on phenoliccompounds carotenoid content and antioxidant capacity ofAndeanmashua tubers (Tropaeolum tuberosumRuiz amp Pavon)rdquoJournal of the Science of Food and Agriculture vol 87 no 3 pp437ndash446 2007

[29] A Bhattacharyya R Chattopadhyay S Mitra and S E CroweldquoOxidative stress an essential factor in the pathogenesis ofgastrointestinal mucosal diseasesrdquo Physiological Reviews vol94 no 2 pp 329ndash354 2014

[30] C Cavin M Delannoy A Malnoe et al ldquoInhibition of theexpression and activity of cyclooxygenase-2 by chicory extractrdquoBiochemical and Biophysical Research Communications vol 327no 3 pp 742ndash749 2005

[31] H A Hassan and M I Yousef ldquoAmeliorating effect of chicory(Cichorium intybus L)-supplemented diet against nitrosamineprecursors-induced liver injury and oxidative stress in maleratsrdquo Food and Chemical Toxicology vol 48 no 8-9 pp 2163ndash2169 2010

[32] C Masungi J Mensch B Willems et al ldquoUsefulness of a novelCaco-2 cell perfusion system II Characterization of monolayerproperties and peptidase activityrdquo Pharmazie vol 64 no 1 pp36ndash42 2009

[33] C Jonker-Venter D Snyman C Janse van Rensburg et al ldquoLowmolecular weight quaternised chitosan (11) in vitro assessmentof absorption enhancing propertiesrdquo Pharmazie vol 61 no 4pp 301ndash305 2006

[34] P Y Niizu and D B Rodriguez-Amaya ldquoNew data on thecarotenoid composition of raw salad vegetablesrdquo Journal of FoodComposition and Analysis vol 18 no 8 pp 739ndash749 2005

[35] E Azzini A Durazzo M S Foddai E Venneria A Raguzziniand G Maiani ldquoBiodiversity and local food products in ItalyrdquoJournal of Agriculture and Biodiversity Research vol 1 no 1 pp1ndash10 2012

[36] N Pellegrini M Serafini B Colombi et al ldquoTotal antioxidantcapacity of plant foods beverages and oils consumed in Italyassessed by three different in vitro assaysrdquo Journal of Nutritionvol 133 no 9 pp 2812ndash2819 2003

[37] L Pastorelli C De Salvo J R Mercado M Vecchi andT T Pizarro ldquoCentral role of the gut epithelial barrier inthe pathogenesis of chronic intestinal inflammation lessonslearned from animal models and human geneticsrdquo Frontiers inImmunology vol 4 article 280 2013

[38] C A Lewis Enteroimmunology A Guide to Prevention andTreatment of Chronic Disease C A Lewis 602 Riverview DrPsy Press Carrabelle Fla USA 2013

[39] F Nobili E Finotti L Martinoli I Garaguso and M AudisioldquoAntioxidant capacity andmorphological changes inmonolayerCaco-2 cells systemby treatmentwith polyphenolic compoundsextracted from virgin olive oil and an oxidative compoundrdquoActa Physiologica vol 188 p 199 2006

[40] E Finotti R Gezzi F Nobili I Garaguso and M FriedmanldquoEffect of apple baobab red-chicory and pear extracts oncellular energy expenditure and morphology of a Caco-2 cellsusing transepithelial electrical resistance (TEER) and scanningelectron microscopy (SEM)rdquo RSC Advances vol 5 no 29 pp22490ndash22498 2015

[41] C Carazzone D Mascherpa G Gazzani and A PapettildquoIdentification of phenolic constituents in red chicory salads(Cichorium intybus) by high-performance liquid chromatog-raphy with diode array detection and electrospray ionisationtandem mass spectrometryrdquo Food Chemistry vol 138 no 2-3pp 1062ndash1071 2013

[42] T Tsuda ldquoDietary anthocyanin-rich plants biochemical basisand recent progress in health benefits studiesrdquo MolecularNutrition and Food Research vol 56 no 1 pp 159ndash170 2012

[43] T C Wallace ldquoAnthocyanins in cardiovascular diseaserdquoAdvances in Nutrition vol 2 no 1 pp 1ndash7 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


a particular importance in our dietThe bioactive compoundsin foods such as vitamins carotenoids and polyphenolsseem to be able tomodulate one ormoremetabolic processeswhich result in the promotion of better health [9] The mostaccepted explanation for the protective effect of foodprobablyderives from the observation that different plant phytochem-icals may act as ldquoscavengersrdquo of free radicals ldquoquenchersrdquosinglet oxygen ormetal chelators [10 11] and therefore induceprotection against oxidative damage through antibacterialanti-inflammatory hepatoprotective anticarcinogenic andvasodilator actions

Recently DrsquoEvoli et al [12] have shown that the highlevels of antioxidant anthocyanins present in Red Chicoryexert a direct scavenging effect against ROS formation interms of antioxidant and cytoprotective activities as well asantiproliferative activity in Caco-2 cell In the present workwe aimed to study the relationship between potential activityof polyphenol-rich extracts from chicories and morpholog-ical and chemicalphysical changes in Caco-2 cellular lineTo this purpose bioactive compounds content (carotenoidsand flavonoids) and total antioxidant activity were evaluatedin Early and Late Red Chicory of Treviso and VariegatedChicory of Castelfranco In addition the bioactivity ofpolyphenol-rich extracts from chicories in in vitroCaco-2 cellmonolayer model was studied Morphological characteristicschanges to test the antioxidant andor prooxidant effect wereverified by histological analysis and observed by ElectronicScansion Microscopy (SEM)

2 Materials and Methods

21 Reagents All solvents were purchased from Carlo Erba(Milan Italy) BDH (Poole England) andMerck (DarmstadtGermany) 246-Tri(2-pyridyl)-s-triazine (TPTZ) was fromFluka (Switzerland) Phosphate-buffered saline (PBS)6-hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) and ascorbic acid were provided by Sigma-AldrichSrl Commercial standards were also from Sigma-AldrichSrl (Milan Italy) AAPH (221015840-azobis (2-amidinopropane)dihydrochloride) (WACO Chem Richmond VA USA) wasused as a source of hydrophilic radicals Double distilledwater (Millipore Milan Italy) was used throughout thestudy

22 Cichorium intybus L Red Chicories of Treviso (twovarieties Early and Late) samples were grown in the regionincluding Quinto (Treviso) Zero Branco (Treviso) andScorze (Venezia) while PGI samples of Variegated Chicoryof Castelfranco come from Due Carrare (Padova) Mira(Venezia) and Monselice (Padova) A total weight of 5 kg ofeach variety for each locality was collected at harvesting timeand delivered to the laboratory Only the edible portion of thesamples was utilized for analysis according to the followingmethods

23 Analytical Methods The extraction of some flavonoids(quercetin kaempferol and apigenin) was performed asdescribed by Hertog et al [13] and the quantitative analysis

through a system-ESA HPLC with electrochemical detectoras reported by Azzini et al [14] Carotenoids were extractedfrom Trevisorsquos and Castelfrancorsquos chicory according to themethod described by Sharpless et al [15] For the quantifi-cation the samples were analyzed by high pressure liquidchromatography (HPLC) as reported by Maiani et al [16]Total Antioxidant Capacity (TAC) was evaluated using twodifferent assays Ferric Reducing Antioxidant Power (FRAP)[17] and Trolox Equivalent Antioxidant Capacity (TEAC)method [18 19] The results of each analysis have beenperformed in triplicate

24 Transepithelial Electrical Resistance (TEER) EvaluationFor TEER evaluation the polyphenol fractions from RedChicory of Treviso and Variegated Chicory of Castelfrancohydrolysed extracts have been studied on Caco-2 cell mono-layers to test the changes in tight-junction (TJ) permeabilityby TEER the phenol red passage and histological analysis

The cellular line was treated with increasing concentra-tion of two polyphenols extracts (02 13 10 17 34 and70 120583M) for 180 minutes to simulate in vivo physiologicalprocesses The variations of transepithelial potentials andphenol red permeability were recorded at time intervals (301015840)In the experiment the cells were seeded onto polycarbonatefilter cell culture chamber inserts (diameter 65mm area033 cm2 and pore diameter 04 120583m) at density of 15 sdot 105 cellsper filter and placed in a multiwell Falcon The filter dividedthe chamber into two parts apical and basal that representthe lumen and the basal area of the gastroenteric systemInto two chambers TEER measurement for assessment oftight-junction permeabilitywas performed using theMillicellERS apparatus (Millipore Bedford MA USA) according tothe manufacturerrsquos instruction After calibrating the resis-tance system the electrical resistance of the monolayer wasmeasured by placing one electrode on either side of thepolycarbonate filter [20 21] The results of each analysis havebeen performed in triplicate and expressed as OHM times cm2

Cells reached confluence and differentiation within 15ndash20days During this time cellular morphology was monitoredand checked with a Leitz Diavert inverted light microscope

25 Histological Analysis The cellular monolayer was iso-lated together with the filter and fixed in Bouin (an aqueoussolution composed of picric acid acetic acid and formalde-hyde) for about 12 hours then dehydrated using the alcoholascending scale (70 80 90 95 and 100) and finallyenclosed into resin blocks of polymerized epoxy (GMAJ134K polyscience Inc Warrington PA USA) Then thesamples were glued on embedded stubs and cut into 3 120583msections with a Micron rotary microtome (Zeiss Germany)appropriately assembled so as to use a crystal blade accordingto Ralphrsquos modification [22 23] The sections glued to themicroscope slides were stained with Harrisrsquo hematoxylin andeosin [24 25] The preparations were made permanent withslide covers and sealed with resin (Eukitt mountingmediumBDA Laboratories Supplies Pool England) All sections(3 120583m) were examined for histological changes by Diaplan 22light microscopy (Leitz Germany) as shown in Figure 1 All







Treviso

Castelfranco

Resp

onse

(120583A

)

000

050

100

150

0

200

400

600

800

Resp

onse

(nA

)









3 Results














Lutein(mgkg)

beta-carotene(mgkg)










0 5 10 15


Late Red Chicory


of Treviso

a

a

b








0 30 60 90 120 150 180(min)


RT 17RT 34RT 70

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(a)

0 30 60 90 120 150 180(min)


VC 17VC 34VC 70

(120583M)

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(b)


(a) (b)







(a) (b)


(a) (b)


(a) (b)



4 Discussion




(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Treviso

Castelfranco

Resp

onse

(120583A

)

000

050

100

150

0

200

400

600

800

Resp

onse

(nA

)









3 Results














Lutein(mgkg)

beta-carotene(mgkg)










0 5 10 15


Late Red Chicory


of Treviso

a

a

b








0 30 60 90 120 150 180(min)


RT 17RT 34RT 70

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(a)

0 30 60 90 120 150 180(min)


VC 17VC 34VC 70

(120583M)

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(b)


(a) (b)







(a) (b)


(a) (b)


(a) (b)



4 Discussion




(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Lutein(mgkg)

beta-carotene(mgkg)










0 5 10 15


Late Red Chicory


of Treviso

a

a

b








0 30 60 90 120 150 180(min)


RT 17RT 34RT 70

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(a)

0 30 60 90 120 150 180(min)


VC 17VC 34VC 70

(120583M)

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(b)


(a) (b)







(a) (b)


(a) (b)


(a) (b)



4 Discussion




(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 30 60 90 120 150 180(min)


RT 17RT 34RT 70

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(a)

0 30 60 90 120 150 180(min)


VC 17VC 34VC 70

(120583M)

0

300

600

900

1200

1500

1800

(Ohm

cm

2)

(b)


(a) (b)







(a) (b)


(a) (b)


(a) (b)



4 Discussion




(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)


(a) (b)


(a) (b)



4 Discussion




(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(min)

(120583M)

0

500

1000

1500

2000

2500(O

hmc

m2)

0 30 60 90 120 150 180



(a)

(min)

(120583M)

0

500

1000

1500

2000

2500

(Ohm

cm

2)

0 30 60 90 120 150 180



(b)








5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion


Abbreviations




Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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