Red chicory (Cichorium intybus L. cultivar) as a potential source of antioxidant anthocyanins for intestinal health

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 704310 8 pageshttpdxdoiorg1011552013704310

Research ArticleRed Chicory (Cichorium intybus L cultivar) as a PotentialSource of Antioxidant Anthocyanins for Intestinal Health

Laura Drsquoevoli1 Fabiana Morroni2 Ginevra Lombardi-Boccia1 Massimo Lucarini1

Patrizia Hrelia2 Giorgio Cantelli-Forti3 and Andrea Tarozzi3

1 National Research Institute on Food and Nutrition Via Ardeatina 546 00178 Rome Italy2 Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Via Irnerio 48 40126 Bologna Italy3 Department for Life Quality Studies Alma Mater Studiorum University of Bologna Corso drsquoAugusto 237 47921 Rimini Italy

Correspondence should be addressed to Andrea Tarozzi andreatarozziuniboit

Received 13 June 2013 Accepted 26 July 2013

Academic Editor Cristina Angeloni

Copyright copy 2013 Laura Drsquoevoli 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

Fruit- and vegetable-derived foods have become a very significant source of nutraceutical phytochemicals Among vegetables redchicory (Cichorium Intybus L cultivar) has gained attention for its content of phenolic compounds such as the anthocyanins Inthis study we evaluated the nutraceutical effects in terms of antioxidant cytoprotective and antiproliferative activities of extractsof the whole leaf or only the red part of the leaf of Treviso red chicory (a typical Italian red leafy plant) in various intestinal modelssuch as Caco-2 cells differentiated in normal intestinal epithelia and undifferentiated Caco-2 cells The results show that the wholeleaf of red chicory can represent a good source of phytochemicals in terms of total phenolics and anthocyanins as well as theability of these phytochemicals to exert antioxidant and cytoprotective effects in differentiated Caco-2 cells and antiproliferativeeffects in undifferentiated Caco-2 cells Interestingly compared to red chicory whole leaf extracts the red part of leaf extracts hada significantly higher content of both total phenolics and anthocyanins The same extracts effectively corresponded to an increaseof antioxidant cytoprotective and antiproliferative activities Taken together these findings suggest that the red part of the leaf ofTreviso red chicory with a high content of antioxidant anthocyanins could be interesting for development of new food supplementsto improve intestinal health

1 Introduction

Many degenerative diseases of the gastrointestinal tract suchas colorectal cancer or inflammatory bowel disease are asso-ciated with a persistent state of oxidative stress which resultsfrom an imbalance in the production of reactive oxygenspecies (ROS) and cell antioxidant defenses at intestinalepithelial level [1 2] In particular various exogenous andendogenous sources including dietary oxidants and inflam-matory processes respectively together with the disruptionof the glutathione antioxidant defense system support oxida-tive stress within intestinal cells [3 4] Recent studies haveshown that the induction of oxidative shift in the cellularredox status induces epigenetic events and cellular mitogenicor apoptotic responses which contribute to progression ofcolorectal cancer [5 6]

In this context there is increasing interest in food as asource of antioxidant phytochemicals which can provide aninexpensive readily applicable and easily accessible approachfor cancer control and prevention [7 8] Several studiessupport this interest showing that consumption of fruitsand vegetables is associated with a decreased risk of severalcancers particularly colorectal cancer possibly linked to theirphytochemical content which is of interest due to several pro-posed health benefits including antioxidant and anticanceractivities [9] Preserving the redox status of intestinal cells aswell as preventing the early events of cellular oxidative dam-age involved in the carcinogenesis processes by administrat-ing dietary phytochemicals therefore provides an importantstrategy for colorectal cancer chemoprevention [10]

Among vegetables chicory (Cichorium intybus) a typicalvegetable indigenous to Europe and North and Western

2 Oxidative Medicine and Cellular Longevity

America has gained attention for its content of phytochem-icals with potential nutraceutical effects such as phenolicacids flavonoids and anthocyanins [11] Using analyticalmethods various studies demonstrate the ability of differentchicory varieties to counteract various free radicals as wellas a linear correlation between phytochemical content andantioxidant capacity of this vegetable [12ndash14] A more recentstudy shows an interesting antioxidant activity of the redchicory variety against the oxidative stress response in aeukaryotic model system suggesting its health activity atcellular level [15]

By contrast to other chicory varieties red chicory is char-acterized by a high content of anthocyanin pigments [16]Thepresence of anthocyanins in red chicory is of special interestbecause several studies have describedmany beneficial healthor nutraceutical effects of anthocyanins on visual capacitybrain cognitive function obesity cardiovascular risk andcancer prevention [7 17ndash19] The anticancer properties ofdietary anthocyanins have been widely evaluated in in vitrostudies and some animal gastrointestinal cancer models [7]Although many studies have evaluated the anticancer effectsmainly as antiproliferative activity of various berry fruitextracts or of anthocyanins from berries in human coloncancer cells the ability of these dietary phytochemicals toalso prevent oxidative events underlying colon cancer inappropriate intestinal models still remains unanswered Anintegrated experimental approach with in vitro colon cancerand normal intestinal models is needed to characterize thepotential nutraceutical effects including both the preventionand the control of colon cancer of food or part of a food

The present study was planned to investigate the lev-els of phytochemicals such as total phenolics and antho-cyanins and total antioxidant activity (TAA) as well asthe in vitro bioactivity of red chicory of Treviso a typi-cal Italian red leafy vegetables which has been attributedto Protected Geographical Indication status according toEuropean Union rules [20] The in vitro bioactivity interms of antioxidant cytoprotective and antiproliferativeactivities of red chicory extracts was studied using differentintestinal models including human colon carcinoma (Caco-2) cells differentiated in normal intestinal epithelia andundifferentiated Caco-2 cells In particular both antioxidantand cytoprotective activities were evaluated in differentiatedCaco-2 cells which provide a suitable model for assess-ment of the physiological response of intestinal epitheliato oxidative injury [21] To mimic oxidative damage tointestinal epithelia we used tert-butyl hydroperoxide (t-BuOOH) due to its ability to generate peroxyl and alkoxylradicals that catalyze the peroxidation of membrane lipids[22]

2 Materials and Methods

21 Chemicals Gallic acid tetrazolium salt (MTT) t-BuOOH 221015840-azino-bis-(3-ethylbenzothiazoline-6-sulphon-ic acid) diammonium salt (ABTS) 21015840-71015840dichlorodihydroflu-orescein diacetate (DCFH-DA) and Folin-Ciocalteursquos phenolreagent were obtained from Sigma Chemical Co (St Louis

MO USA) All other reagents were of analytical grade puritycommercial available

22 Sample Selection Treviso red chicory (Cichorium inty-bus L cultivar) from certified producers operating in Veneto(Italy)was purchased froma single retail outlet Four differentlots of red chicory at comparable ripening stages wereanalyzed All red chicory samples were processed within 48ndash72 h of purchase

23 Sample Extraction Briefly 100 g of weight of the wholeleaf (WL) or only the red part of the leaf (RL) withoutthe white side of Treviso red chicory was mixed with coldmethanol (HCl 01) andhomogenized using anUltra Turraxhomogenizer for 5min The mixture was filtered through aWhatman paper under vacuum and the methanol in thefiltrate was evaporated at 35∘C Subsequently the residue wasdiluted to 10mL with methanol and stored at minus20∘C untilphytochemical content analysis Subsequently some samplesof residue in methanol were dried completely and resus-pended in appropriate culture mediums for the bioactivitydeterminations

24 Total Phenolic Content Analysis Total phenolic concen-trations were measured using the Folin Ciocalteau assay [23]Briefly appropriate dilutions of extracts were treated withFolin Ciocalteau reagent and the reaction was neutralizedwith 7 sodium carbonate The absorbance of the resultingblue color was measured spectrophotometrically at 750 nmusing a Beckman DU 7400 spectrophotometer Gallic acidwas used as standard and results expressed as milligrams ofgallic acid equivalents (GAE) per 100 g of the fresh edible partof the red chicory

25 Total Anthocyanin Content Analysis Total anthocyanincontent of the extracts of Treviso red radicchio was deter-mined spectrophotometrically by the pH differential methodof Rapisarda et al [24]

26 Determination of TAA The TAA of the extracts derivedfrom Treviso red chicory was measured as reported by Re etal [25]This method is based on the ability of the antioxidantmolecules in the vegetable extracts to reduce the radicalcation of the ABTS determined by the decolorization ofABTS∙+ andmeasured as quenching of absorbance at 740 nmValues obtained for each sample were compared with theconcentration-response curve of a standard Trolox solutionand expressed asmicromoles of trolox equivalent antioxidantactivity (TEAA)100 g of fresh vegetable

27 Cell Cultures of Human Colon Carcinoma Cells Humancolon carcinoma (Caco-2) cells were routinely grown at37∘C in a humidified incubator with 5 CO

2in Dulbeccorsquos

Modified Eaglersquos Medium (DMEM) supplemented with20 (FCS) 2mmolL glutamine 50UmL penicillin and50120583gmL streptomycin To evaluate intracellular antioxidantand cytoprotective activities as well as antioxidant activity inmembrane and cytosolic fractions of theWL and RL extracts

Oxidative Medicine and Cellular Longevity 3

Caco-2 cells were seeded at a density of 8 times 104 cellscm2 inmulti-well dishes once the cells were confluent the mediumwas changed every 48 h using DMEM20 FCS Experimentswere performed using completely differentiated cultures at12ndash14 days after seeding To evaluate the antiproliferativeactivity of the same extracts undifferentiated Caco-2 cellswere seeded in 96-well microliter plates at a density of 15 times104 cellscm2 Experiments were performed after 24 h ofincubation at 37∘C in 5 CO

2

28 Determination of Intracellular Antioxidant and Cytopro-tective Activities We evaluated the antioxidant and cyto-protective activities of WL and RL extracts of Treviso redchicory against both formation of intracellular ROS andcytotoxicity in differentiated Caco-2 cells after treatment witht-BuOOH Formation of intracellular ROS was determinedusing a fluorescent probe DCFH-DA as described by Wangand Joseph [26] Briefly differentiated Caco-2 cells wereincubated for 4 h with different concentrations of the extractsderived from Treviso red chicory samples correspondingto 5ndash30mg vegetablemL Cells were washed with PBS andthen incubated with 5 120583M DCFH-DA in phosphate bufferedsaline (PBS) in 5 CO

2at 37∘C for 30min After removal

of DCFH-DA and further washing the cells were incubatedwith 05mM t-BuOOH in PBS for 30min At the end ofincubation the fluorescence of the cells from each well wasmeasured (wavelength 485535 nm) with a spectrofluorome-ter (SpectraMaxGeminiMolecular DevicesMNUSA)Thevalues are expressed as percentage of increase of intracellularROS evoked by exposure to t-BuOOH

Cytotoxicity was monitored by trypan blue uptake aspreviously described [27] Briefly differentiated Caco-2 cellswere incubated for 4 h with extracts of WL and RL (cor-responding to 5ndash30mg vegetablemL) washed with PBSand then incubated with 05mM t-BuOOH in DMEM 20FBS at 37∘C in 5 CO

2 After 24 h of incubation the cells

were collected by gentle scraping in PBS and dispersed byrepeated gentle pipetting An aliquot of cell suspension wasthen diluted 1 1 with 05 trypan blue in 10mM sodiumphosphate buffer (pH 72) and placed on a hemocytometerwith a cover slip Percentages of viable cells were recorded onat least three separate counts

29 Determination of Antioxidant Activity in Membraneand Cytosolic Fractions Cytosolic and membrane-enrichedfractions were separated as we previously reported [28]Briefly after 4 h of incubation with WL and RL extracts(corresponding to 5ndash30mg vegetablemL) at 37∘C in 5CO

2

differentiated Caco-2 cells were washed 3 times with coldPBS Cells were subsequently collected in 1mL of PBS andcentrifuged for 10min at 10000 rpm at 4∘C after which thesupernatant was removed and the cells were washed with1mL of PBS This was repeated further 2 times and thepellet was finally reconstituted in 600 120583L of 005 TritonX-100 Cells were then homogenized and allowed to standat 4∘C for 30min Cytosolic and membrane fractions weresubsequently separated by centrifugation at 14000 rpm for15min at 4∘C Membrane and cytosolic fractions were stored

at minus20∘C Small amounts were removed for determination ofthe protein concentration using the Bradford method TAAwas then measured on cytosolic and membrane fractionsusing ABTS method as previously reported [25] This exper-imental approach makes it possible to determine the cellularuptake of bioactive molecules and their ability to counteractthe free radicals at different subcellular levels Values obtainedfor each cellular fraction sample were expressed as 120583mol ofTrolox equivalent antioxidant activity per mg of protein

210 Determination of Antiproliferative Activity The antipro-liferative activity of WL and RL extracts of Treviso redchicory was determined in undifferentiated Caco-2 cells invitro as we previously reported [21] Briefly after 96 h ofincubation with WL and RL extracts (corresponding to 1ndash50mg vegetablemL) Caco-2 cells were washed with PBSand then incubated with MTT (5mgmL) in PBS for 4 hAfter removal of MTT and further washing the formazancrystals were dissolved with isopropanol The amount offormazan was measured (405 nm) with a spectrophotometer(TECAN Spectra model Classic Salzburg Austria) The cellviability was expressed as a percentage of control cells Atleast three independent dose-response curves were plottedand the concentration of red chicory extracts resulting in 50inhibition of cell proliferation (IC

50) was calculated

211 Statistical Analysis Data are reported as mean plusmn SD ofat least 3 independent experiments Compositional data werestatistically processed utilizing the Studentrsquos t-test Statisticalanalysis of biological data was performed using one-wayANOVA with Dunnettrsquos Post Hoc Test and Studentrsquos t-test asappropriate and Pearsonrsquos correlation coefficient for relationsamong variables Differences were considered significant at119875 lt 005 Analyses were performed using PRISM 3 softwareon a Windows platform

3 Results and Discussion

We first determined the phytochemical contents such as totalphenolics and anthocyanins in extracts obtained from ediblesamples of WL or RL of Treviso red chicory As reportedin Table 1 the extracts of RL had a significantly higher totalphenolic and anthocyanin content than the correspondingextracts ofWL (all119875 lt 005) In parallel the TAA of the sameextracts was measured by ABTS radical cation decolorizationassay and expressed as 120583mol of trolox equivalent antioxidantactivity (TEAA)100 g edible sample (Table 1) On the basisof weight the TAA of the RL extracts was significantly higherthan the activity of WL extracts (119875 lt 005)

Taken together the high levels of total phenolic andanthocyanin content found in WL extracts are in the rangealready reported in the literature for other varieties of redchicory [13 14 16 29] In this regard we also confirmedthe low levels of other antioxidant components such astotal ascorbic acid in both WL and RL extracts of redchicory (data not shown) These findings are supported by arecent antioxidant characterization of Cichorium intybus that


Table 1 Total phenolics total anthocyanins and TAA of edible samples of WL or RL of Treviso red chicory1

Treviso red chicory Total phenolics2 Total anthocyanins TAA3

mg of GAE100 g samples mg of anthocyanins100 g samples 120583mol TEAA100 g samplesWL

3116 plusmn 126 1108 plusmn 82 5067 plusmn 356

RL3704 plusmn 144

lowast1426 plusmn 75


lowast

1Values are means plusmn SD of at least four determinations (RL versus WL lowast119875 lt 005 at Studentrsquos 119905-test)2Values were expressed as gallic acid equivalents (GAE) in milligrams per 100 g of edible sample3Values were expressed as micromoles of trolox equivalent antioxidant activity (TEAA) per 100 g of edible sample

0 5 10 20 300

50

100

150

200

WL

RL

Red chicory concentration (mgmL)

ROS

form

atio

n (

of i

ncre

ase)

lowast

lowastlowast

lowastlowastlowastlowast

∘

∘

Figure 1 Antioxidant activity of WL and RL extracts of Treviso redchicory in Caco-2 cells differentiated in normal intestinal epitheliaThe cells were treated with various concentrations of extracts for 4 hand then treated with t-BuOOH (05mM) for 30min At the endof incubation intracellular ROS formation was determined using afluorescence probe DCFH-DA as described in the Materials andMethods sectionThe values are expressed as percentage of increaseof intracellular ROS formation evoked by exposure to t-BuOOHThevalues are shown asmeanplusmn SDof four independent experiments( lowast119875

lt 005 lowastlowast119875

lt 001 versus untreated cells at ANOVA withDunnettrsquos Post Hoc Test ∘

119875

lt 005 versus cells treated with WLextracts at Studentrsquos t-test)

recorded the marginal role of total ascorbic acid in the totalantioxidant activity of this vegetable [30]

Interestingly the levels of the same phenolic compoundsas well as TAA of RL extracts were significantly higherthan WL extracts supporting the hypothesis that phenoliccompounds may be important antioxidant components toaccount for the observed antioxidant activity In particularrecent studies on the phytochemical composition of redchicories recorded a large amount of hydroxybenzoic andhydroxycinnamic acids as well as of red anthocyanins whichgive red chicories an exceptionally high peroxyl radical scav-enging activity in terms of both capacity and efficiency [16]Although we did not characterize the phenolic compoundcontent it is reasonable to suppose that the combination ofthese compounds mainly the red anthocyanins could be

0

20

40

60

80

100

Cel

l via

bilit

y (

)

0 5 10 20 30

WL

RL


lowastlowast

lowastlowast

lowastlowast

∘

∘

Figure 2 Cytoprotective activity of WL and RL extracts of Trevisored chicory in Caco-2 cells differentiated in normal intestinalepithelia The cells were treated with various concentrations ofextracts for 4 h and then treated with t-BuOOH (05mM) for 24 hAt the end of incubation cytotoxicity was determined using trypanblue assay as described in the Materials and Methods section Thevalues are expressed as percentage of cell viability after exposure tot-BuOOHThe values are shown as mean plusmn SD of four independentexperiments ( lowast

119875


lt 001 versus untreated cells atANOVAwithDunnettrsquos Post Hoc Test ∘

119875

lt 005 versus cells treatedwith WL extracts at Studentrsquos t-test)

important due to the higher TAA observed in RL extracts andto confer a potential health value to this part of red chicory

We then assessed the nutraceutical effects in terms of theantioxidant and cytoprotective activities of theseWL and RLextracts of Treviso red chicory against both the intracellularROS formation and cytotoxicity in differentiated Caco-2cells after treatment with t-BuOOH As shown in Figures1 and 2 treatment of differentiated Caco-2 cells with bothWL and RL extracts (5ndash30mgmL) showed a decrease in adose-dependent manner of intracellular ROS formation andcytotoxicity elicited by t-BuOOH The ability to counteractthe ROS formation and cytotoxicity was significantly higherwith RL than WL extracts for the concentrations 20 and30mgmL (all 119875 lt 005)

To confirm the antioxidant and cytoprotective activitiesof WL and RL extracts at cellular level we evaluated TAA(expressed as 120583mol TEAAmg protein) at two different


0

100

200

300

400

500

600

700

800 C

aco-

2 m

embr

ane T

AA

0 5 10 20 30Concentration (mgmL)

WL

RL

(120583m

ol T

EAA

mg

of p

rote

in)

lowast


∘

(a)

0 5 10 20 300

500

1000

1500

2000

2500

Concentration (mgmL)

WL

RL

Cac

o-2

mem

bran

e TA

A(120583

mol

TEA

Am

g of

pro

tein

)

(b)

Figure 3 Effects of WL and RL extracts of Treviso red chicory on TAA of membrane (a) and cytosolic (b) fractions of Caco-2 cellsdifferentiated in normal intestinal epithelia The cellular fractions were submitted to the ABTS methods after 4 h of incubation with variousconcentrations of extracts as described in Section 2 The results obtained for each cellular fraction sample were expressed as 120583mol of TEAAper mg of protein The values represent the mean plusmn SD of three independent experiments ( lowast

119875


lt 001 versus untreated cells atANOVA with Dunnettrsquos Post Hoc Test ∘

119875

lt 005 versus cells treated with WL extracts at Studentrsquos t-test)

subcellular levels membrane and cytosol As depicted inFigure 3 the membrane and cytosolic fractions obtainedfrom differentiated Caco-2 cells treated withWL (30mgmL)and RL (20 and 30mgmL) extracts for 4 h showed a signif-icant increase of TAA in comparison with untreated cellsRemarkably the recorded increase of membrane TAA wassignificantly higher in Caco-2 cells treated with RL than WLextracts at the concentrations of 30mgmL (119875 lt 005)By contrast both RL and WL extracts did not modify thebasal levels of differentiated Caco-2 cytosolic fraction TAAInterestingly a highly significant linear correlation was foundbetween Caco-2 cell membrane TAA and cytoprotectiveactivity for both the WL (119903 = minus093 119875 lt 0001 at Pearsonrsquoscorrelation coefficient) and RL (119903 = minus091 119875 lt 0001)extracts

Taken together these results show that RL extracts led togreater increases in antioxidant and cytoprotective activitiesin the differentiated Caco-2 cells than WL extracts Inparticular the highest TAA observed at Caco-2 membranelevel could be attributed to higher TAA levels of the antho-cyanin fraction of the RL extracts Our previous studiessuggest an accumulation and antioxidant activity of thesephenolic compounds present in anthocyanin-rich fruits suchas red orange and strawberry in the membrane of varioushuman epithelial cells including intestinal cells [26 2731] There is evidence that the hydrophobic nature of thering structure of anthocyanins determines their interactionswith the hydrophobic component of the cell membraneinfluencing the membrane fluidity and preventing lipid per-oxidation and oxidative damage [32] A recent study supports

these scientific considerations showing the ability of variousCichorium intybus extracts to prevent lipid peroxidation andintracellular ROS formation using neuron cell-based assays[33]

Last we measured a nutraceutical effect as the antiprolif-erative activity of the WL and RL extracts in undifferentiatedCaco-2 cells using MTT assay To compare the antiprolifera-tive activity of the extracts we also used the IC

50(concentra-

tion of the extract resulting in 50 inhibition of colon cancercell proliferation) extrapolated by a wide range of extractconcentrations from 1 to 50mgmL As reported in Figure 4treatment of Caco-2 cells with both WL and RL extractsinduced a decrease of cell proliferation in a concentration-dependent manner The inhibitory effects on Caco-2 cellproliferation were significantly higher with RL than WLextracts for the 5 and 10mgmL concentrations (all 119875 lt001) At higher concentrations the inhibitory effects weresaturated for both extracts Considering the concentration-inhibitor effect relationship the IC

50was significantly lower

with RL thanWL extracts (1485plusmn026versus 2138plusmn047119875 lt 005 at Studentrsquos t-test)

These findings could be ascribed to a greater amountof bioactive molecules with anticarcinogenicity propertiespresent in red chicory In this context it is interesting to notethat the Cichorium intybus extracts at low concentrations (5and 10mgmL) show antiproliferative effects in the absence ofantioxidant and cytoprotective effects There is an emergingview that phenolic compounds could exert anticarcinogenic-ity effects not only through their antioxidant potential butalso through the modulation of signaling cascades gene


1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast

Figure 4 Effects of WL and RL extracts of Treviso red chicory oncell proliferation of undifferentiated Caco-2 cells The cell prolifera-tion was determined by the MTT assay after 96 h of incubation withvarious concentrations of extracts The results were expressed as apercentage of control cells The values represent the mean plusmn SD ofthree independent experiments ( lowastlowast

119875

lt 001 versus cells treatedwithWL extracts at Studentrsquos t-test)

expression involved in the regulation of cell proliferationdifferentiation and apoptosis as well as the suppression ofchronic inflammation metastasis and angiogenesis [34]

In particular among phenolic compounds it has beendemonstrated that phenolic acids such as hydroxybenzoicand hydroxycinnamic acids can induce strong antiprolifer-ative effects and apoptosis in human colon cancer cells [9]Further several studies also indicate that anthocyanins areable to inhibit the growth of different cancer cells suggestingtheir possible role as chemopreventive agents [35 36] Inparticular it has been suggested that the inhibitory effects ofanthocyanin-rich fruit and vegetable extracts are based on theconcentration rather than the composition of anthocyanins[37ndash39]

4 Conclusions

Our results showed that Treviso red chicory can representa good source of nutraceutical phytochemicals for intestinalhealth In particular the high levels of antioxidant antho-cyanins present in red chicorymight exert a direct scavengingeffect against ROS formationwithin the gastrointestinal tractThe anthocyanins andor their metabolites could furthercontribute to intestinal health through their ability to spreadout in internal intestinal tissue Recent studies recorded thepresence of glycoside aglycone and both methylated andglucuronide derivatives of anthocyanins in tissues includingthe stomach and small intestine of animal models fed eithera single anthocyanin or berry extracts [7]

We also demonstrated that the red part of the leaf of redchicory could exert an interesting additional nutraceuticalvalue in terms of antioxidant and cytoprotective activities

as well as antiproliferative activity with respect to the wholeleaf of red chicory These findings from an industrial pointof view indicate that the revaluation of some parts of redchicory with a high content of antioxidant anthocyaninscould be interesting for the production of new commercialproducts such as food supplements of high quality and lowcost However further clinical studies are needed to confirmwhether the whole and the red part of the leaf of Treviso redchicory are likely to improve intestinal health

Abbreviations

ABTS 221015840-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt

Caco-2 Human colon carcinoma cellsDCFH-DA 21015840-71015840Dichlorodihydrofluorescein diacetateGAE Gallic acid equivalentsMTT Tetrazolium saltRL Red part of leafROS Reactive oxygen speciesTAA Total antioxidant activityt-BuOOH tert-Butyl hydroperoxideTEAA Trolox equivalent antioxidant activityWL Whole leaf

Authorsrsquo Contribution

LauraDrsquoevoli andFabianaMorroni contributed equally to thiswork

Acknowledgments

This work was supported by MiPAAF (BIOVITA ResearchProject) and ARSIAL (Regional Agency for the Developmentand the Innovation of the Agriculture of Lazio Region)

References

[1] T A Ullman and S H Itzkowitz ldquoIntestinal inflammation andcancerrdquo Gastroenterology vol 140 no 6 pp 1807ndash1816 2011

[2] M L Circu andT Y Aw ldquoIntestinal redox biology and oxidativestressrdquo Seminars in Cell and Developmental Biology vol 23 no7 pp 729ndash737 2012

[3] T-G Wang Y Gotoh M H Jennings C A Rhoads and T YAw ldquoLipid hydroperoxide-induced apoptosis in human colonicCaCo-2 cells is associated with an early loss of cellular redoxbalancerdquoThe FASEB Journal vol 14 no 11 pp 1567ndash1576 2000

[4] M L Circu and T Y Aw ldquoRedox biology of the intestinerdquo FreeRadical Research vol 45 no 11-12 pp 1245ndash1266 2011

[5] T Inokuma M Haraguchi F Fujita Y Tajima and T Kane-matsu ldquoOxidative stress and tumor progression in colorectalcancerrdquo Hepato-Gastroenterology vol 56 no 90 pp 343ndash3472009

[6] K A Kang R Zhang G Y Kim S C Bae and J W HyunldquoEpigenetic changes induced by oxidative stress in colorectalcancer cells methylation of tumor suppressor RUNX3rdquoTumourBiology vol 33 no 2 pp 403ndash412 2012


[7] J C Espın M T Garcıa-Conesa and F A Tomas-BarberanldquoNutraceuticals facts and fictionrdquo Phytochemistry vol 68 no22ndash24 pp 2986ndash3008 2007

[8] G Paliyath M Bakovic and K Shetty Functional FoodsNutraceuticals and Degenerative Disease Prevention Wiley-Blackwell Oxford UK 1st edition 2011

[9] E M Brown C I R Gill G J McDougall and D StewartldquoMechanisms underlying the anti-proliferative effects of berrycomponents in in vitro models of colon cancerrdquo CurrentPharmaceutical Biotechnology vol 13 no 1 pp 200ndash209 2012

[10] A Acharya I Das D Chandhok and T Saha ldquoRedox reg-ulation in cancer a double-edged sword with therapeuticpotentialrdquoOxidative Medicine and Cellular Longevity vol 3 no1 pp 23ndash34 2010

[11] 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

[12] R Llorach F A Tomas-Barberan and F Ferreres ldquoLettuce andchicory byproducts as a source of antioxidant phenolic extractsrdquoJournal of Agricultural and Food Chemistry vol 52 no 16 pp5109ndash5116 2004

[13] D Heimler L Isolani P Vignolini S Tombelli and A RomanildquoPolyphenol content and antioxidative activity in some speciesof freshly consumed saladsrdquo Journal of Agricultural and FoodChemistry vol 55 no 5 pp 1724ndash1729 2007

[14] V Lavelli ldquoAntioxidant activity of minimally processed redchicory (Cichorium intybus L) evaluated in xanthine oxidase-myeloperoxidase- and diaphorase-catalyzed reactionsrdquo Journalof Agricultural and Food Chemistry vol 56 no 16 pp 7194ndash7200 2008

[15] A Lante T Nardi F Zocca A Giacomini and V CorichldquoEvaluation of red chicory extract as a natural antioxidant bypure lipid oxidation and yeast oxidative stress response asmodelsystemsrdquo Journal of Agricultural and Food Chemistry vol 59 no10 pp 5318ndash5324 2011

[16] 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 peroxylradical trapping capacity and efficiencyrdquo Journal of Agriculturaland Food Chemistry vol 53 no 21 pp 8169ndash8175 2005

[17] 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

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

[19] J P E Spencer D Vauzour and C Rendeiro ldquoFlavonoidsand cognition the molecular mechanisms underlying theirbehavioural effectsrdquo Archives of Biochemistry and Biophysicsvol 492 no 1-2 pp 1ndash9 2009

[20] httpeur-lexeuropaeu[21] A Tarozzi A Marchesi G Cantelli-Forti and P Hrelia ldquoCold-

storage affects antioxidant properties of apples in Caco-2 cellsrdquoJournal of Nutrition vol 134 no 5 pp 1105ndash1109 2004

[22] W Chamulitrat ldquoNitric oxide inhibited peroxyl and alkoxylradical formation with concomitant protection against oxidantinjury in intestinal epithelial cellsrdquoArchives of Biochemistry andBiophysics vol 355 no 2 pp 206ndash214 1998

[23] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998

[24] P Rapisarda F Fanella and E Maccarone ldquoReliability ofanalytical methods for determining anthocyanins in bloodorange juicesrdquo Journal of Agricultural and Food Chemistry vol48 no 6 pp 2249ndash2252 2000

[25] 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

[26] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[27] A Tarozzi S Hrelia C Angeloni et al ldquoAntioxidant effective-ness of organically and non-organically grown red oranges incell culture systemsrdquo European Journal of Nutrition vol 45 no3 pp 152ndash158 2006

[28] L DrsquoEvoli A Tarozzi P Hrelia et al ldquoInfluence of cultivationsystem on bioactive molecules synthesis in strawberries spin-off on antioxidant and antiproliferative activityrdquo Journal of FoodScience vol 75 no 1 pp C94ndashC99 2010

[29] V Mulabagal H Wang M Ngouajio and M G Nair ldquoCharac-terization and quantification of health beneficial anthocyaninsin leaf chicory (Cichorium intybus) varietiesrdquo European FoodResearch and Technology vol 230 no 1 pp 47ndash53 2009

[30] 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

[31] A Tarozzi A Marches S Hrelia et al ldquoProtective effects ofcyanidin-3-O-szlig-glucopyranoside against UVA-induced oxida-tive stress in human keratinocytesrdquo Photochemistry and Photo-biology vol 81 no 3 pp 623ndash629 2005

[32] J K Jacob K Tiwari J Correa-Betanzo A Misran R Chan-drasekaran and G Paliyath ldquoBiochemical basis for functionalingredient design from fruitsrdquo Annual Review of Food ScienceTechnology vol 3 pp 79ndash104 2012

[33] S Schaffer S Schmitt-Schillig W E Muller and G P EckertldquoAntioxidant properties of Mediterranean food plant extractsgeographical differencesrdquo Journal of Physiology and Pharmacol-ogy vol 56 supplement 1 pp 115ndash124 2005

[34] M-H Pan C-S Lai J-C Wu and C-T Ho ldquoMolecular mech-anisms for chemoprevention of colorectal cancer by naturaldietary compoundsrdquo Molecular Nutrition and Food Researchvol 55 no 1 pp 32ndash45 2011

[35] F Tramer S Moze A O Ademosun S Passamonti and JCvorovic ldquoDietary anthocyanins impact on colorectal cancerand mechanisms of activityrdquo in Colorectal CancermdashFrom Pre-vention to Patient Care R Ettarh Ed pp 110ndash120 In Tech 2012

[36] Y Zhang N P Seeram R Lee L Feng and D Heber ldquoIsolationand identification of strawberry phenolics with antioxidantand human cancer cell antiproliferative propertiesrdquo Journal ofAgricultural and Food Chemistry vol 56 no 3 pp 670ndash6752008

[37] E M Coates G Popa C I R Gill et al ldquoColon-availableraspberry polyphenols exhibit anti-cancer effects on in vitromodels of colon cancerrdquo Journal of Carcinogenesis vol 6 article4 2007


[38] J L Johnson J A Bomser J C Scheerens and M MGiusti ldquoEffect of black raspberry (Rubus occidentalis L) extractvariation conditioned by cultivar production site and fruitmaturity stage on colon cancer cell proliferationrdquo Journal ofAgricultural and Food Chemistry vol 59 no 5 pp 1638ndash16452011

[39] Q KWu JMKoponenHMMykkanen andA R TorronenldquoBerry phenolic extracts modulate the expression of p21WAF1and Bax but Not Bcl-2 in HT-29 colon cancer cellsrdquo Journal ofAgricultural and Food Chemistry vol 55 no 4 pp 1156ndash11632007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch


OphthalmologyJournal of


ISRN Allergy


BioMed Research International


ObesityJournal of


ISRN Addiction



Computational and Mathematical Methods in Medicine

ISRN AIDS


Clinical ampDevelopmentalImmunology

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2013

Diabetes ResearchJournal of


Evidence-Based Complementary and Alternative Medicine

Volume 2013Hindawi Publishing Corporationhttpwwwhindawicom


Gastroenterology Research and Practice


ISRN Biomarkers


MEDIATORSINFLAMMATION

of


America has gained attention for its content of phytochem-icals with potential nutraceutical effects such as phenolicacids flavonoids and anthocyanins [11] Using analyticalmethods various studies demonstrate the ability of differentchicory varieties to counteract various free radicals as wellas a linear correlation between phytochemical content andantioxidant capacity of this vegetable [12ndash14] A more recentstudy shows an interesting antioxidant activity of the redchicory variety against the oxidative stress response in aeukaryotic model system suggesting its health activity atcellular level [15]

By contrast to other chicory varieties red chicory is char-acterized by a high content of anthocyanin pigments [16]Thepresence of anthocyanins in red chicory is of special interestbecause several studies have describedmany beneficial healthor nutraceutical effects of anthocyanins on visual capacitybrain cognitive function obesity cardiovascular risk andcancer prevention [7 17ndash19] The anticancer properties ofdietary anthocyanins have been widely evaluated in in vitrostudies and some animal gastrointestinal cancer models [7]Although many studies have evaluated the anticancer effectsmainly as antiproliferative activity of various berry fruitextracts or of anthocyanins from berries in human coloncancer cells the ability of these dietary phytochemicals toalso prevent oxidative events underlying colon cancer inappropriate intestinal models still remains unanswered Anintegrated experimental approach with in vitro colon cancerand normal intestinal models is needed to characterize thepotential nutraceutical effects including both the preventionand the control of colon cancer of food or part of a food

The present study was planned to investigate the lev-els of phytochemicals such as total phenolics and antho-cyanins and total antioxidant activity (TAA) as well asthe in vitro bioactivity of red chicory of Treviso a typi-cal Italian red leafy vegetables which has been attributedto Protected Geographical Indication status according toEuropean Union rules [20] The in vitro bioactivity interms of antioxidant cytoprotective and antiproliferativeactivities of red chicory extracts was studied using differentintestinal models including human colon carcinoma (Caco-2) cells differentiated in normal intestinal epithelia andundifferentiated Caco-2 cells In particular both antioxidantand cytoprotective activities were evaluated in differentiatedCaco-2 cells which provide a suitable model for assess-ment of the physiological response of intestinal epitheliato oxidative injury [21] To mimic oxidative damage tointestinal epithelia we used tert-butyl hydroperoxide (t-BuOOH) due to its ability to generate peroxyl and alkoxylradicals that catalyze the peroxidation of membrane lipids[22]

2 Materials and Methods

21 Chemicals Gallic acid tetrazolium salt (MTT) t-BuOOH 221015840-azino-bis-(3-ethylbenzothiazoline-6-sulphon-ic acid) diammonium salt (ABTS) 21015840-71015840dichlorodihydroflu-orescein diacetate (DCFH-DA) and Folin-Ciocalteursquos phenolreagent were obtained from Sigma Chemical Co (St Louis

MO USA) All other reagents were of analytical grade puritycommercial available

22 Sample Selection Treviso red chicory (Cichorium inty-bus L cultivar) from certified producers operating in Veneto(Italy)was purchased froma single retail outlet Four differentlots of red chicory at comparable ripening stages wereanalyzed All red chicory samples were processed within 48ndash72 h of purchase

23 Sample Extraction Briefly 100 g of weight of the wholeleaf (WL) or only the red part of the leaf (RL) withoutthe white side of Treviso red chicory was mixed with coldmethanol (HCl 01) andhomogenized using anUltra Turraxhomogenizer for 5min The mixture was filtered through aWhatman paper under vacuum and the methanol in thefiltrate was evaporated at 35∘C Subsequently the residue wasdiluted to 10mL with methanol and stored at minus20∘C untilphytochemical content analysis Subsequently some samplesof residue in methanol were dried completely and resus-pended in appropriate culture mediums for the bioactivitydeterminations

24 Total Phenolic Content Analysis Total phenolic concen-trations were measured using the Folin Ciocalteau assay [23]Briefly appropriate dilutions of extracts were treated withFolin Ciocalteau reagent and the reaction was neutralizedwith 7 sodium carbonate The absorbance of the resultingblue color was measured spectrophotometrically at 750 nmusing a Beckman DU 7400 spectrophotometer Gallic acidwas used as standard and results expressed as milligrams ofgallic acid equivalents (GAE) per 100 g of the fresh edible partof the red chicory

25 Total Anthocyanin Content Analysis Total anthocyanincontent of the extracts of Treviso red radicchio was deter-mined spectrophotometrically by the pH differential methodof Rapisarda et al [24]

26 Determination of TAA The TAA of the extracts derivedfrom Treviso red chicory was measured as reported by Re etal [25]This method is based on the ability of the antioxidantmolecules in the vegetable extracts to reduce the radicalcation of the ABTS determined by the decolorization ofABTS∙+ andmeasured as quenching of absorbance at 740 nmValues obtained for each sample were compared with theconcentration-response curve of a standard Trolox solutionand expressed asmicromoles of trolox equivalent antioxidantactivity (TEAA)100 g of fresh vegetable

27 Cell Cultures of Human Colon Carcinoma Cells Humancolon carcinoma (Caco-2) cells were routinely grown at37∘C in a humidified incubator with 5 CO

2in Dulbeccorsquos

Modified Eaglersquos Medium (DMEM) supplemented with20 (FCS) 2mmolL glutamine 50UmL penicillin and50120583gmL streptomycin To evaluate intracellular antioxidantand cytoprotective activities as well as antioxidant activity inmembrane and cytosolic fractions of theWL and RL extracts



2








2




50) was calculated










RL3704 plusmn 144



lowast


0 5 10 20 300

50

100

150

200

WL

RL


ROS

form

atio

n (

of i

ncre

ase)

lowast

lowastlowast


∘

∘




119875




0

20

40

60

80

100

Cel

l via

bilit

y (

)

0 5 10 20 30

WL

RL


lowastlowast

lowastlowast

lowastlowast

∘

∘


119875



119875






0

100

200

300

400

500

600

700

800 C

aco-

2 m

embr

ane T

AA


WL

RL

(120583m

ol T

EAA

mg

of p

rote

in)

lowast


∘

(a)

0 5 10 20 300

500

1000

1500

2000

2500


WL

RL

Cac

o-2

mem

bran

e TA

A(120583

mol

TEA

Am

g of

pro

tein

)

(b)


119875



119875






50(concentra-






1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast


119875




4 Conclusions




Abbreviations





Acknowledgments


References

















































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of



2








2




50) was calculated










RL3704 plusmn 144



lowast


0 5 10 20 300

50

100

150

200

WL

RL


ROS

form

atio

n (

of i

ncre

ase)

lowast

lowastlowast


∘

∘




119875




0

20

40

60

80

100

Cel

l via

bilit

y (

)

0 5 10 20 30

WL

RL


lowastlowast

lowastlowast

lowastlowast

∘

∘


119875



119875






0

100

200

300

400

500

600

700

800 C

aco-

2 m

embr

ane T

AA


WL

RL

(120583m

ol T

EAA

mg

of p

rote

in)

lowast


∘

(a)

0 5 10 20 300

500

1000

1500

2000

2500


WL

RL

Cac

o-2

mem

bran

e TA

A(120583

mol

TEA

Am

g of

pro

tein

)

(b)


119875



119875






50(concentra-






1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast


119875




4 Conclusions




Abbreviations





Acknowledgments


References

















































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of






RL3704 plusmn 144



lowast


0 5 10 20 300

50

100

150

200

WL

RL


ROS

form

atio

n (

of i

ncre

ase)

lowast

lowastlowast


∘

∘




119875




0

20

40

60

80

100

Cel

l via

bilit

y (

)

0 5 10 20 30

WL

RL


lowastlowast

lowastlowast

lowastlowast

∘

∘


119875



119875






0

100

200

300

400

500

600

700

800 C

aco-

2 m

embr

ane T

AA


WL

RL

(120583m

ol T

EAA

mg

of p

rote

in)

lowast


∘

(a)

0 5 10 20 300

500

1000

1500

2000

2500


WL

RL

Cac

o-2

mem

bran

e TA

A(120583

mol

TEA

Am

g of

pro

tein

)

(b)


119875



119875






50(concentra-






1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast


119875




4 Conclusions




Abbreviations





Acknowledgments


References

















































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of


0

100

200

300

400

500

600

700

800 C

aco-

2 m

embr

ane T

AA


WL

RL

(120583m

ol T

EAA

mg

of p

rote

in)

lowast


∘

(a)

0 5 10 20 300

500

1000

1500

2000

2500


WL

RL

Cac

o-2

mem

bran

e TA

A(120583

mol

TEA

Am

g of

pro

tein

)

(b)


119875



119875






50(concentra-






1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast


119875




4 Conclusions




Abbreviations





Acknowledgments


References

















































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of


1 10 1000

20

40

60

80

100

120

WLRL


Cell

pro

lifer

atio

n (

ver

sus c

ontro

l)

lowastlowast

lowastlowast


119875




4 Conclusions




Abbreviations





Acknowledgments


References

















































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of











































Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of











Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of








Volume 2013

ISRN Anesthesiology


OncologyJournal of


PPARRe sea rch




ISRN Allergy




ObesityJournal of


ISRN Addiction




ISRN AIDS




Volume 2013








ISRN Biomarkers



of

Documents

Red chicory (Cichorium intybus L. cultivar) as a potential source of antioxidant anthocyanins for intestinal health