Food Chemistry 157 (2014) 540–552

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Phenolic composition, antioxidant properties, and endothelial cellfunction of red and white cranberry fruits

http://dx.doi.org/10.1016/j.foodchem.2014.02.0470308-8146/Published by Elsevier Ltd.

⇑ Corresponding author. Tel.: +1 708 924 0646; fax: +1 708 924 0690.E-mail address: artemio.tulio@fda.hhs.gov (A.Z. Tulio Jr.).

Artemio Z. Tulio Jr. a,⇑, Joseph E. Jablonski a, Lauren S. Jackson a, Claire Chang a,b, Indika Edirisinghe c,Britt Burton-Freeman c

a U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 6502 South Archer Road, Bedford Park, IL 60501, United Statesb Oak Ridge Institute for Science and Education, Oak Ridge, TN 37830, United Statesc Institute for Food Safety and Health, Illinois Institute of Technology, 6502 South Archer Road, Bedford Park, IL 60501, United States

a r t i c l e i n f o

Article history:Received 7 August 2013Received in revised form 6 February 2014Accepted 12 February 2014Available online 22 February 2014

Keywords:CranberryPhenolic compoundsAntioxidantsAnthocyaninsProanthocyanidinsFlavonolsPhenolic acidsEndothelial cell functionp-AktCell migrationTube formation

a b s t r a c t

The effects of phenolic constituents in red cranberry extracts (RCE) and white cranberry extracts (WCE)on the endothelial cell function were investigated. Peonidin-3-O-galactoside, cyanidin-3-O-arabinoside,and cyanidin-3-O-galactoside were the predominant anthocyanins characterized, whereas a procyanidintetramer was the predominant proanthocyanidin identified. The antioxidant properties of RCE and WCEwere not significantly different regardless of antioxidant assays (DPPH, FRAP, and TEAC) used. Both RCEand WCE induced the phosphorylation of Akt in vitro in human umbilical endothelial cells (HUVEC),resulting in the phosphorylation of endothelial nitric oxide synthase, cell migration, and tube formation.The enhanced phosphorylation of PI3/Akt kinase in HUVEC, endothelial cell wound healing, and tubeformation elicited by RCE and WCE suggest that overall phenolic constituents rather than individualphenolic compounds within the cranberry matrix may be responsible for these biological effects.

Published by Elsevier Ltd.

1. Introduction Bourquin, 2001). Anthocyanins are primarily responsible for the

Naturally-occurring secondary metabolites present in fruits andvegetables have received widespread attention due to their pur-ported health-promoting properties. Epidemiological studies haveconsistently shown a direct correlation between consumption offruits and vegetables with lower risk for developing chronic ill-nesses such as cardiovascular diseases (Mckay & Blumberg, 2007;Reed, 2002) and certain types of cancer (Neto, 2011). These healthbenefits from consumption of fruits and vegetables have been lar-gely attributed to the plant metabolites known as phytochemicals.

Cranberries (Vaccinium macrocarpon Aiton), are a rich source ofphytochemicals, particularly phenolic compounds (Reed, 2002).These compounds, further divided into a subclass of compoundscalled flavonoids, include flavonols, flavones, flavan-3-ols,flavanones, isoflavones, and anthocyanins (Fig. 1). Among theseflavonoids, anthocyanins have been widely studied due to theirpotential to act as an antioxidant (Seeram, Momin, Nair, &

red, pink, and purple color of cranberries. Cranberry fruits are alsoknown for their proanthocyanidin compounds, particularly thetype A-proanthocyanidin trimer (Fig. 1C), which is associated withhaving potent anti-bacterial and anti-adhesive properties (Foo, Lu,Howell, & Vorsa, 2000b).

A recent consumer trend is the introduction of cranberryproducts from immature (white) berry stage, since they tend tobe less tart and tangy than their red counterparts, being marketedspecifically for children. Hence, not only cranberries from mature(red) but also immature (white) stages are recently being marketedcommercially. Most of the studies examining the health-promotingbenefits of cranberries have been with red or mature berries.Although consumption of white cranberries is substantially lessthan with the red cranberries, more information is needed onhow white cranberries compare to red cranberries with regardsto chemical composition, their biological effects, and their poten-tial health-promoting benefits.

Therefore, this work was conducted to provide information thatwill further enhance our understanding of the phenolic composi-tion, antioxidant properties, biological activities, and other

anthocyanidin R1 R2------------------------------------------------------------cyanidin OH O-galactoside

O-glucosideO-arabinoside

peonidin OCH3 O-galactosideO-glucosideO-arabinoside

O

OHOH

HO

OHOH

O

O

OHR1

OH

OHR2

O

OH

OH

OH

O

OH

OH

OH

O

OH

OH

OH

OH

COOH

OHH

protocatechuic acid

OH

OCH3H3CO

sinapic acid

COOH

H

condensed tannins(n indicates number of subunits) O

OHOH

HO

OHOH

**

catechin

A B

C D

E

OH

OH

OH

OH

OH

OH O

OHOHO

HOOH

n

quercetin

O

OHOH

HO

OHOH

O

OH

myricetin

Fig. 1. Structures of (A) anthocyanins, (B) flavonols, (C) proanthocyanidins, (D) phenolic acids, and (E) flavan-3-ols found in cranberries.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 541

potential health benefits of mature (red) and immature (white)cranberries. The specific objective of this study was to characterizethe differences between the phenolic profiles of red and whitecranberries using LC–MS/MS and spectrophotometric assays. Fur-thermore, this study was conducted to evaluate and compare theeffects of red and white cranberry extracts on the activation of pro-tein kinase B (Akt) via the redox-sensitive phosphatidylinositol-3kinase-mediated signaling pathway in cultured human umbilicalendothelial cells (HUVEC). Endothelial cell migration and tube for-mation, known to modulate via Akt/PI3 kinase pathway, were alsoused for assessing the bioactivity of the polyphenol-rich extracts inin vitro.

2. Materials and methods

2.1. Cranberry samples and preparation of extracts

Red and white cranberries (Vaccinium macrocarpon Aiton) ofthe ‘‘Stevens’’ cultivar were obtained from a grower in Wisconsin,USA. Red cranberry fruits were dark red-colored berries harvestedat a ripe, mature stage, whereas white cranberries were light

pinkish-colored berries, still unripe and immature, that wereharvested along with the red berries After harvest, white berrieswere visually sorted and segregated from red berries, packed sep-arately, transported to U.S. Food and Drug Administration (FDA)laboratory in Bedford Park, IL, USA and immediately stored at�20 �C.

2.2. Preparation of cranberry extracts

Frozen cranberry fruits were lyophilized using Millrock Bench-Top Freeze-Dryer (Kingston, NY), milled with coffee grinder, andthe powder was stored at �20 �C. The freeze-dried material(2.5 g) was extracted with 50 ml of acetone/water/acetic acid(70/29.5/0.5, v/v/v) solution. After 1 h of vigorous shaking in thedark, the mixture was centrifuged at 10,000�g for 20 min at 4 �Cand then filtered by vacuum filtration with Whatman #2 filter pa-per. The resulting supernatant was divided into 6 equal portions(�5 ml/tube). The extract in each tube was freeze-dried followingremoval of the solvent mixture using N2 gas stream. Freeze-driedred cranberry extracts (RCE) and white cranberry extracts (WCE)were weighed and stored at �20 �C.

542 A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552

2.3. Reagents

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 4-dimethylamino-cinnamaldehyde(DMAC), Folin–Ciocalteau phenol reagent, sodium acetate (C2H3-

NaO2�3H2O), iron (III) chloride hexahydrate (FeCl3�6H2O), alumi-num chloride (AlCl3�6H2O), sodium nitrite (NaNO2), gallic acid,catechin, (�)-epicatechin gallate, quercetin-3-O-galactoside, quer-cetin-3-O-rhamnoside, myricetin, quercetin, p-coumaric acid,chlorogenic acid, and sinapic acid were supplied by Sigma Chemi-cal Co. (St. Louis, MO, USA). Ethyl acetate was purchased from Al-drich Chemical Co. (Milwaukee, WI, USA). HPLC-grade water,methanol, acetonitrile, glacial acetic acid, and formic acid werepurchased from Fisher Scientific Co. (Fairlawn, NJ, USA). Cyanidin3-O-glucoside chloride (Cy-3-glc), cyanidin 3-O-arabinoside chlo-ride (Cy-3-ara), cyanidin 3-O-galactoside chloride (Cy-3-gal),peonidin 3-O-glucoside chloride (Pn-3-glc), peonidin 3-O-arabino-side chloride (Pn-3-ara), procyanidin A2, B1, B2, and C1 wereobtained from ChromaDex, Inc. (Irvine, CA, USA). Phosphate buf-fered-saline (PBS) solution was purchased from Fisher Bioreagents(Fair Lawn, NJ, USA).

2.4. Chemical analysis

2.4.1. Determination of total phenolic contentThe total phenolic content was estimated colorimetrically using

the Folin–Ciocalteau phenol reagent according to the modified pro-cedure of (Slinkard & Singleton, 1977). Briefly, RCE and WCE sam-ples were diluted with de-ionized water prior to mixing withFolin–Ciocalteau phenol reagent. The mixture was allowed to standfor 5 min at room temperature before adding 7% sodium carbonatesolution followed by de-ionized water. Solutions were mixed andallowed to stand for 2 h at room temperature. The total phenolconcentration was determined from gallic acid standard calibra-tion curve (0, 100, 200, 300, 400, 500 lg/ml) using a Cary 100 ConcUV–Visible spectrophotometer (Varian Inc., Walnut Creek, CA,USA) at 765 nm. Results were expressed as mg gallic acid equiva-lents (GAE) per 100 g fresh weight (FW).

2.4.2. Determination of total monomeric anthocyanin contentThe total monomeric anthocyanin content in cranberry extracts

was determined colorimetrically using the pH differential methoddescribed previously (Giusti & Wrolstad, 2005). The absorbancewas measured at 510 nm and 700 nm in PBS with pH 1.0 and 4.5using a Cary 100 Conc UV–Visible spectrophotometer. A molarextinction coefficient of 29,600 for Cy-3-glc was used to calculatethe total monomeric anthocyanin contents. Results were expressedas mg Cy-3-glc equivalents (C3GE) per 100 g FW.

2.4.3. Determination of total flavonoid contentThe total flavonoid content was determined by the aluminum

chloride colorimetric method described by (Meyers, Watkins,Pritts, & Liu, 2003) and (Zhishen, Mengcheng, & Jianming, 1999)with modifications. In brief, RCE and WCE samples were dilutedwith de-ionized water before adding 5% NaNO2 solution. After6 min, 10% AlCl3�6H2O solution was added. After another 5 minhad lapsed, 1 M NaOH solution was added into the mixture. Theabsorbance at 510 nm was measured immediately against the pre-pared blank using a Cary 100 Conc UV–Visible spectrophotometerand total flavonoid concentrations were calculated based on thecatechin standard calibration curve (0, 100, 200, 300, 400, and500 lg/ml). Results were expressed as mg catechin equivalents(CE) per 100 g FW.

2.4.4. Determination of total procyanidin contentThe total procyanidin content was determined using 4-dimeth-

ylaminocinnamaldehyde (DMAC) colorimetric assay (Prior et al.,2010). Procyanidin A2 standard calibration curve (0, 10, 20,30 lg/ml) was prepared and the absorbance at 640 nm was mea-sured after 20 min against the prepared blank using a Cary 100Conc UV–Visible spectrophotometer. Results were expressed asmg procyanidin A2 equivalents (PA2) per 100 g FW.

2.5. LC–MS/MS analysis

2.5.1. Standard preparationStock standard solutions (1 mg/ml) were prepared in 50:50

methanol/water (v/v) and stored at �80 �C. Calibration standardswere diluted from stock solutions. Mixed anthocyanin calibrationstandards containing Cy-3-gal, Cy-3-glc, Cy-3-ara, Pn-3-gal, Pn-3-glc, and Pn-3-ara were prepared at concentrations of 0.1, 0.5, 1.0,5.0, and 10.0 lg/ml. Mixed phenolic calibration standards contain-ing sinapic acid, p-coumaric acid, (�) epicatechin gallate, querce-tin, quercetin-3-galactoside, quercetin-3-rhamnoside, myricetin,procyanidin B1, B2, and C1 were prepared at concentrations of 1,10 and 25 lg/ml. Standards were stored in the freezers at �20 �C.

2.5.2. Sample preparation for HPLC analysisPrior to HPLC analysis, cranberry extracts (RCE and WCE) were

suspended in de-ionized water for anthocyanin analysis and PBSfor the analyses of flavonols, phenolic acids, and procyanidins.RCE and WCE samples were brought to room temperature and thenvortexed to thoroughly mix and re-suspend the extract powder.Samples were then centrifuged and/or filtered through 0.22 lmnylon syringe filters to produce a clear supernatant. An aliquot ofthe supernatant was transferred to an autosampler HPLC vial andthen diluted with de-ionized water to ensure responses were inthe calibration range. Samples were stored in the freezers at�20 �C.

2.5.3. LC–PDA–MS/MS analysis of anthocyaninsA Waters Alliance 2795 HPLC system (Milford, MA, USA)

equipped with a Waters 996 photodiode array detector (PDA),Waters 2475 fluorescence detector (FLD), Quattro Micromass triplequadrupole mass spectrometer, and MassLynx (V.4.1) softwarewas used to identify and quantify anthocyanins in the red andwhite cranberry extracts. Separations were performed on a SynergiMax-RP column (150 � 3.0 mm, 4 lm packing; Phenomenex, Tor-rance, CA, USA). The mobile phase consisted of 1% formic acid inwater (solvent A) and 1% formic acid in acetonitrile (solvent B).The gradient elution program was set as follows: 2% B from 0 to4 min, 2–20% B linear from 4 to 20 min, 20–80% B from 20 to24 min, 80% B from 24 to 30 min, and then returning to initial con-centration of 2% from 30 to 35 min to re-equilibrate the system.The flow rate was 0.4 ml/min and the injection volume was setat 20 ll. The column temperature was maintained at 40 �C andthe autosampler was cooled at 15 �C. The PDA was set at 520 nmto monitor the UV–visible absorption of anthocyanins, and theUV–visible spectra were recorded from 200 to 600 nm with a res-olution of 1.2 nm and acquisition of 1 spectra/s. After passingthrough the PDA, the column eluate was split, and 0.2 ml/minwas diverted to a mass spectrometer fitted with electrospray ion-ization (ESI) interface. ESI positive mode was utilized for the detec-tion of anthocyanins. The mass spectrometer conditions were setup as follows: source temperature, 100 �C; desolvation tempera-ture, 300 �C; nitrogen desolvation flow, 800 l/h; capillary, 3000 V;cone, 30 V; and MS–MS collision energy, 15 V.

2.5.4. LC–PDA–MS/MS analysis of flavonols and phenolic acidsQualitative and quantitative analysis of flavonols and phenolic

acids were performed using the LC–PDA–MS/MS parameters

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 543

described above. The elution conditions were as follows: elutionsolvent A (0.1% or 2% formic acid in water) and B (0.1% or 2% formicacid in methanol) using a gradient program of 5% B from 0 to 4 min,5–50% B linear in 4–34 min, 50% from 34 to 40 min, and returnedto 5% B at 40–45 min for re-equilibration of the column. The flowrate was set at 0.5 ml/min and 50% of the column eluate was di-verted to the mass spectrometer. The injection volume was set at10 ll, column temperature was set at 40 �C, and peak quantitationwas accomplished by acquiring PDA data at 340 nm. The negativeionization mode (ESI–) of the mass spectrometer was used for thedetection of flavonols and phenolic acids. The mass spectrometerparameters were also similar to the above procedure except forthe capillary voltage at 2500 V, cone voltage at either 20 or 40 V,and MS–MS collision energy at 25 V.

2.5.5. LC–FLD–MS/MS analysis of procyanidinsThe conditions for the qualitative and quantitative analyses of

procyanidins were similar to the analyses of flavonols and phenolicacids. In addition, fluorescence detection (FLD) was conducted withan excitation wavelength of 285 nm and an emission wavelengthof 315 nm. The emission unit full scale (EUFS) was set at 5000and gain at 1.

2.6. Antioxidant activity assays

2.6.1. DPPH-free radical scavenging assayThe free radical scavenging assay was performed according to

the method of Brand-Williams, Cuvelier, and Berset (1995). Briefly,2,2-diphenyl-1-picrylhydrazyl (DPPH) dissolved in methanol, wasused to estimate the free radical scavenging abilities of variousantioxidants. The loss of DPPH radical absorbance was determinedusing a Cary 100 Conc UV–Visible spectrophotometer at 517 nmafter keeping the sample in the dark for 1 h (Ozgen, Reese, Tulio,Scheerens, & Miller, 2006). Results were expressed as lM TE/g FW.

2.6.2. Ferric reducing antioxidant power (FRAP) assayThe FRAP assay was performed as previously described by Benzie

and Strain (1996). In brief, the reaction mixtures were prepared bycombining 300 mM C2H3NaO2�3H2O, buffer (pH 3.6), 20 mM ofFeCl3�6H2O, and 10 mM TPTZ in 40 mM HCl, in a 10:1:1 ratio.The analysis was carried out at 25–30 �C and the absorbance wasread at 593 nm after 1 h in the dark using a Cary 100 Conc UV–Visible spectrophotometer. Results were expressed as lM TE/g FW.

2.6.3. Trolox equivalent antioxidant capacity assayThe trolox equivalent antioxidant power was determined using

2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) fol-lowing the method described by Ozgen et al. (2006). All assay reac-tions were kept in the dark for 1 h and the levels of reduced ABTSreactants were measured at 734 nm using a Cary 100 Conc UV–Vis-ible spectrophotometer. Results were expressed as lM TE/g FW.

2.7. In vitro cell culture assays

2.7.1. Cell viability and apoptosis assaysHuman umbilical vein endothelial cells or HUVECs were

purchased from Lonza (Washington, DC, USA) and grown in endo-thelial cell growth medium 2 (EGM-2, Lonza) with 4% fetal bovineserum (FBS). Cell viability assay was determined in response todifferent concentrations and time factors using the Trypan blueexclusion test. Apoptosis assay was determined using Caspase 9colorimetric activity assay kit (Millipore, Waltham, MA, USA). Cellculture assays were investigated with HUVEC cells having apassage number of 2.

2.7.2. AlphaScreen� SureFire� phosphorylated kinase b (p-Akt) assayThe ability of RCE and WCE samples to phosphorylate p-Akt

in vitro in HUVEC was performed following the method describedpreviously (Tulio et al., 2012). In brief, HUVECs were grown inEGM-2 supplemented with 10% FBS. Cells were grown to confluentmonolayer (�90%) and then transferred into 6-well plates. HUVECwere starved for 6 h in 2% serum media before the cells were trea-ted with different concentrations of RCE and WCE (0.1, 0.2, 0.5 and1.0 mg/ml). Wortmannin (Ascent Scientific LLC, Princeton, NJ,USA), a PI3 kinase inhibitor, was used to confirm PI3 kinaseinvolvement. A dose of 30 nmol/l wortmannin, which does not in-duce toxicity in HUVECs, was used to treat HUVECs for 30 min be-fore exposure of RCE and WCE and then incubated at 37 �C in ahumidified atmosphere containing 5% CO2 for 10 min. The reactionwas stopped immediately by adding ice cold PBS (pH 7.4) and thecells were washed twice with the same PBS buffer. Cell lysateswere prepared in cell lysis buffer containing protease inhibitors(Calbiochem, Rockland, MA, USA). Relative phosphorylated-Aktlevels in the cell lysates were measured by AlphaScreen� SureFire�

assay (PerkinElmer, Waltham, MA, USA) at an excitation wave-length of 680 nm and emission wavelength of 520–620 nm and ex-pressed as p-Akt/Total Akt. PBS buffer (pH 7.4) was used as thecontrol. Protein levels were measured using Pierce bicinchoninicacid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA).

2.7.3. Endothelial cell migration assayEndothelial cell migration assay was performed following the

Tulio et al. protocol (Tulio et al., 2012). In brief, HUVECs weregrown in a cell culture flask in a 5% CO2 incubator at 37 �C untilconfluent and then the cells were transferred into 6-well plates.After cell growth reached �90% confluence, the bottom of eachwell containing a HUVEC monolayer was scraped in a straight linewith a 200 ll sterile pipet tip. Cell debris was removed by washingthe plate twice with PBS (pH 7.4). Cells were exposed to differentconcentrations of RCE and WCE samples (0.05, 0.1, and 0.2 mg/ml) in a medium containing 2% serum and the cells were incubatedat 37 �C in 5% CO2 with for 24 h. Half of the plates were treatedwith wortmannin (30 nmol/l) for 30 min before the addition ofthe cranberry extracts to confirm the involvement of PI3/Akt sig-naling pathway. Cells treated with PBS were used as control inthe study.

2.7.4. Endothelial cell tube formation assayEndothelial cell tube formation experiments were carried out

following the method described previously (Tulio et al., 2012). Inbrief, HUVECs were starved for 1 h in endothelial growth mediumcontaining 2% serum media. The cells were washed with PBS, andthen plated in 48-well plates that had been pre-coated with100 ll of growth factor-reduced Matrigel Matrix (BD Bioscience,Franklin Lakes, NJ, USA). The cells were incubated for 24 h withRCE and WCE samples (0.05, 0.1, and 0.2 mg/ml) at 37 �C in ahumidified incubator containing 5% CO2. Some cell plates weretreated with 30 nmol/l wortmannin and cranberry extracts to con-firm involvement of the PI3/Akt signaling pathway in capillary-liketubular formation. Capillary-like tube formations were photo-graphed after 0, 5, 12, and 24 h under 40� magnification levelusing an Olympus CKX41 microscope (Center Valley, PA, USA).

2.8. Statistical analysis

Means, standard errors, regression analysis, and significantdifferences between treatments by single factor-analysis of vari-ance (ANOVA) at 95% confidence level were calculated using SASv.9.2 (Cary, NC, USA). All values were reported as means ± SE forthree replications.

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3. Results

3.1. Estimation of total phenolics, total monomeric anthocyanins, totalflavonoids, and total procyanidins

The total phenolic, total monomeric anthocyanin, total flavo-noid, and total procyanidin contents of RCE and WCE were com-pared in this study using spectrophotometric assays (Table 1).The total phenolic contents of RCE (341.9 ± 2.1 mg GAE/100 g freshweight) and WCE (WCE had 339.4 ± 4.5 mg GAE/100 g freshweight) were not significantly different (p > 0.05). In contrast, theamount of estimated total monomeric anthocyanins, total flavo-noids, and total procyanidins between RCE and WCE were signifi-cantly different (p < 0.05). The total monomeric anthocyanincontent of RCE calculated based on the molar extinction coefficientof Cy-3-gluc was 21-times greater than that of WCE. The estimatedtotal flavonoid content of WCE in terms of catechin equivalentswas 1.2 times compared with RCE. In addition, the total procyani-din content of WCE estimated as procyanidin A2 equivalents, wasalmost twice than that of RCE.

3.2. LC–PDA–MS/MS analysis of anthocyanins

Fig. 2a.a shows the anthocyanins detected by PDA at 520 nm inthe standard mixtures, WCE, and RCE, respectively. Five majoranthocyanin peaks were identified in the chromatograms of theRCE samples (Fig. 2a.c), whereas the same anthocyanins wereeither non-existent or found in negligible amounts in WCE(Fig. 2a.b). The retention time of four of these peaks correspondedto retention times of the authentic anthocyanin standards injected.These major peak anthocyanins in RCE were identified as follows:1 = Cy-3-gal, 3 = Cy-3-ara, 4 = Pn-3-gal, 5 = Pn-3-glc, and 6 = Pn-3-ara (Fig. 2a.c). A fifth peak eluted at 17.33 min for which therewas not a corresponding standard peak but the spectrum corre-sponds to that of a peonidin glycoside. The ESI + MS scan base peakwas 463 amu (Fig. 3a.a). An MS–MS analysis of the parent 463 amuion showed a major daughter fragment ion base peak at 301 amu.Based on these observations and the retention time of 17.33 min,this peak was assigned as Pn-3-gal. The retention time of17.3 min for this peak in the reversed-phase separation of anthocy-anins at acidic pH is consistent with previous report which showPn-3-gal elutes after Cy-3-arab and Pn-3-arab (Brown & Shipley,2011). Pn-3-gal was the most abundant anthocyanin in red berriesfollowed by Cy-3-ara and Cy-3-gal, while the least abundantanthocyanin was Pn-3-glc (Table 2). The sum of individualanthocyanins in RCE was estimated at 68.27 mg per 100 g of fresh

Table 1Total phenolic, total anthocyanin, total flavonoid, total procyanidin contents and tospectrophometric assays.

Sample Total phenoliccontent (mgGAE)a,b

Total anthocyanincontent (mgC3GE)a,c

Total procyacontent (mgPA2E)a,d

Red cranberry extracts 341.6 ± 2.1 a 88.3 ± 3.98 a 115.5 ± 2.51White cranberry extracts 339.4 ± 4.5 a 3.9 ± 0.87 b 184.1 ± 13.34

a Expressed per 100 g FW. Means ± standard errors (n = 3). Columns with different letb GAE, gallic acid equivalentsc C3GE, Cyanidin 3-O-glucoside equivalents.d PA2E, procyanidin A2 equivalents.e CE, catechin equivalents.f Expressed as lmol Trolox equivalents/g fresh weight.g Ferric reducing antioxidant power.h Trolox equivalent antioxidant capacity.i 2,2-Diphenyl-1-picrylhydrazyl.

berries, whereas WCE contained a total of 0.81 mg per 100 g offresh berries.

3.3. LC–PDA–MS/MS analysis of flavonols and phenolic acids

Flavonols and phenolic acids in RCE and WCE were separatedand detected by PDA at 340 nm. Analyte peak identity was as-signed by co-elution with authentic reference standards whereavailable, and/or with MS full scan, and MS–MS daughter scanspectra. LC–MS analyses were conducted with two sets of condi-tions in an effort to obtain suitable separation and ionization inESI negative mode. Quantitative analysis of flavonols at 340 nmabsorbance detection was conducted using 2% formic acid in mo-bile phase (Fig. 2b). The peak profile of the WCE (Fig. 2b.b) andRCE (Fig. 2b.c) appear very similar. The chromatogram of the stan-dard (Fig. 2b.a) shows matching retention times for some peakspresent in the WCE (Fig. 2b.b) and RCE (Fig. 2b.c) samples. Peaksshown in Fig. 2b.b and c. were identified as follows: 1 = caffeic aciddihexose, 2 = caffeic acid mono-hexose, 3 = chlorogenic acid,4 = sinapic acid mono-hexose, 5/6 = epicatechin gallate/p-couma-ric acid, 7 = myricetin 3-hexoside, 8 = sinapic acid, 9 = myricetin3-pentoside, 10 = quercetin 3-galactoside, 11 = myricetin,12 = quercetin 3-pentoside, 13 = quercetin 3-rhamnoside, and14 = quercetin. MS-full scan spectra of caffeic acid dihexose (peak1) in WCE samples in 0.1% formic acid, CV = 20 V and 2% formicacid, CV = 40 V is shown in Fig. 3b, whereas the MS–MS daughterscans of 503 amu parent of caffeic acid dihexose (peak 1) in flavo-nol chromatograms is shown in Fig 3c. The flavonols were moreabundant in RCE than in WCE (Table 2). The major flavonols/phe-nolic acids in RCE were quercetin-3-galactoside, myricetin 3-hexo-side, and quercetin 3-rhamnoside. The total flavonol and phenolicacid contents of RCE from HPLC analysis was 35.99 mg/100 g offresh berries, whereas WCE had only 22.30 mg/100 g of freshberries (Table 2).

3.4. LC–FLD–MS/MS analysis of procyanidins

Procyanidins and flavanol monomers were separated, identi-fied, and quantitated using fluorescence detection with excitationand emission wavelengths of 285 and 315 nm, respectively. Thefluorescence peak profiles of the WCE (Fig. 2c.b) and RCE(Fig. 2c.c) were very similar. In addition, the chromatogram of amixture of analytical standards (Fig. 2c.a) showed matching reten-tion times for some peaks present in the WCE and RCE samples.The chromatograms in Fig. 2c were from the HPLC separation with0.1% formic acid in mobile phase and cone voltage of 20 V. Fig. 2c.band c show the peaks identified as follows: 1 = protocatechuic acid

tal antioxidant capacity of red and white cranberry extracts as determined by

nidin Total flavonoidcontent (mgCE)a,e

Total antioxidant capacityf

FRAPg TEACh DPPHi

a 47.5 ± 0.66 a 12.96 ± 0.13 a 13.04 ± 0.61 a 6.28 ± 0.13 bb 58.3 ± 2.62 b 13.89 ± 0.15 a 13.78 ± 0.35 a 6.13 ± 0.13 b

ters are significantly different (p < 0.05).

Time8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

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0.0

2.5e-2

5.0e-2

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AU

0.0

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5.0e-2

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

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(b)

(a)

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1

1

Fig. 2a. Anthocyanin profiles of (a) 10 ppm mixed anthocyanin standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected by photodiodearray at 520 nm. Peak ID, 1 = cyanidin 3-galactoside, 2 = cyanidin 3-glucoside, 3 = cyanidin 3-arabinoside, 4 = peonidin 3-galactoside, 5 = peonidin 3-glucoside, and6 = peonidin 3-arabinoside.

Table 2Summary of the anthocyanins, flavonols, phenolic acids, and proanthocyanidins quantified in red and white cranberry extracts as determined by LC–MS/MS.a

Figure Peak Compound Red cranberry extracts (mg/100 g FWb) White cranberry extracts (mg/100 g FW)

2a Anthocyanins1 Cyanidin 3-galactoside 13.75 ± 1.03 0.17 ± 1.032 Cyanidin 3-glucoside ndc nd3 Cyanidin 3-arabinoside 18.71 ± 0.68 0.35 ± 0.684 Peonidin 3-galactoside 28.39 ± 0.87 0.23 ± 0.875 Peonidin 3-glucoside 1.97 ± 0.46 nd6 Peonidin 3-arabinoside 5.45 ± 1.57 0.06 ± 1.57

Total Anthocyanins 68.27 0.81

2b Flavonols and phenolic acids1 Caffeic acid dihexose 0.30 ± 1.05 0.28 ± 0.102 Caffeic acid mono-hexose 2.09 ± 2.26 1.69 ± 6.593 Chlorogenic acidd 2.10 ± 1.67 2.81 ± 1.624 Sinapic acid mono-hexose 0.50 ± 0.34 0.62 ± 1.615 Epicatechin gallate/p-coumaric acid nd nd6 Epicatechin gallate/p-coumaric acid nd nd7 Myricetin 3-hexoside 6.48 ± 0.82 2.02 ± 1.038 Sinapic acid nd nd9 Myricetin 3-pentoside 2.27 ± 0.68 1.15 ± 1.1910 Quercetin 3-galactoside 12.82 ± 4.32 6.89 ± 1.2511 Myricetin nd nd12 Quercetin 3-pentoside 3.75 ± 0.34 2.33 ± 0.8313 Quercetin 3-rhamnoside 2.39 ± 0.40 1.52 ± 1.8414 Quercetin nd nd

2c 1 Protocatechuic acid hexoside 3.29 ± 0.21 2.99 ± 0.10Total Flavonols and Phenolic Acids 35.99 22.30

2c Proanthocyanidins2 Procyanidin B1 0.37 ± 0.00 0.50 ± 0.003 Catechin 0.87 ± 0.57 2.11 ± 2.384 Procyanidin B2 1.56 ± 0.24 1.46 ± 0.325 Procyanidin C1 4.27 ± 1.69 6.55 ± 0.826 Procyanidin trimer type A 1.10 ± 7.33 2.38 ± 14.777 Procyanidin tetramer type A 7.93 ± 0.07 17.21 ± 0.09

Total proanthocyanidins 16.10 30.21

a Means ± relative standard deviations (n = 3).b Fresh weight.c Not detected.d Including unidentified compounds.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 545

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

AU

0.0

5.0e-2

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

AU

0.0

2.0e-2

4.0e-2

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

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0.0

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14

13

13

13

12

12

11

10

10

9

9

8

7

7

5/6

4

4

3

3

2

2

1

1

(c)

(b) 10

Fig. 2b. Flavonols and phenolic acid profiles of (a) 10 ppm mixed flavonol standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected byphotodiode array at 340 nm. Peak ID, 1 = caffeic acid dihexose, 2 = caffeic acid mono-hexose, 3 = chlorogenic acid, 4 = sinapic acid mono-hexose, 7 = myricetin hexoside,9 = myricetin pentoside, 10 = quercetin galactoside, 12 = quercetin pentoside, 13 = quercetin rhamnoside.

Time8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

%

0

100

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

%

0

100

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

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100

(c)

(b)

(a)

7

7

6

6

5

5

5

4

4

4

3

3

2

1

1

Fig. 2c. Procyanidin profiles of (a) 10 ppm mixed procyanidin standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected by fluorescence atexcitation and emission wavelengths of 285 and 315 nm. Peak ID, 1 = protocatechuic acid hexoside, 2 = procyanidin B1, 3 = catechin, 4 = procyanidin B2, 5 = procyanidin C1,6 = procyanidin trimer type A, 7 = procyanidin tetramer type A.

546 A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552

hexoside, 2 = procyanidin B1, 3 = catechin, 4 = procyanidin B2,5 = procyanidin C1, 6 = procyanidin trimer type A, and 7 = procy-anidin tetramer type A. Based on matching retention times andMS spectral data, fluorescence peaks for procyanidin B1, B2 andC1 were identified in both the WCE and RCE. A procyanidin trimertype A, and tetramer type A were identified based on MS data.Mass spectral data indicate that the peak eluting at 10.83 min inthe sample extracts is likely protocatechuic acid hexoside (3,4dihydroxybenzoic acid hexoside) (Fig. 3d). This compound exhibitssignificant fluorescence at excitation/emission wavelengths used

for the analysis. The fluorescent profile of peaks in Fig. 2c.b and cshows the largest peak at retention time at 22.9 min. The full scanspectrum showed at distinct peak at 1151.5 amu which corre-sponds to a Type A tetramer. The MS-MS daughters of 1151.5 spec-tra of this compound gave a major fragment at 575.3 indicative of aprocyanidin trimer. This peak was therefore assigned as a procy-anidin tetramer type A. Quantitation of the tetramer was madeusing an RRF of 1 from the calibration curve of procyanidin C1.

Procyanidin contents were higher in WCE compared with RCE(Table 2). The procyanidin tetramer values were 17.21 mg/100 g

m/z100 150 200 250 300 350 400 450 500 550 600 650 700

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%

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100Cranberry_Samples_03272010_011 408 (17.396) 2: Scan ES+

1.03e7463.3

439.2301.3

464.3

465.2

Cranberry_Samples_03292010_002 199 (17.360) 4: Daughters of 463ES+ 1.30e6301.2

463.3

(a)

(b)

Fig. 3a. Full scan mass spectrum of peonidin 3-galactoside (peak 4 in Fig. 2a) eluting at 17.4 min in RCE, and (b) daughters of 463 MS-MS spectrum of peonidin 3-galactoside(peak 4) eluting at 17.4 min in RCE.

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600

%

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m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600

%

0

100 341.3

113.2

165.1153.1 179.1

503.3

342.3375.2 409.3 504.3

571.3

161.2117.1

146.1

131.2

503.2

341.1

179.2 191.1236.1

196.1 293.2240.2 325.1 353.0 425.1 440.0 469.8556.1504.3 577.2

578.2

(a)

(b)

Fig. 3b. MS- full scan spectra of caffeic acid dihexose (peak 1 in Fig. 3b) in white cranberry extract (WCE) with (a) 0.1% formic acid, CV = 20 V and (b) 2% formic acid, CV = 40 V.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 547

fresh weight in white cranberries. In contrast, red cranberries had7.93 mg/100 g of fresh berries. The procyanidin tetramer was themost abundant procyanidin quantified and detected by mass spec-trometry. The total procyanidin content based upon the sum ofindividual values from HPLC analysis of the white cranberry sam-ples was 30.21 mg/100 g of fresh fruit, whereas red cranberrieshad only 16.10 mg/100 g of fresh berries (Table 2).

3.5. Estimation of antioxidant capacity

The total antioxidant capacities of RCE and WCE were estimatedbased on three antioxidant assays including free radical scavenging(DPPH), ferric reducing antioxidant power (FRAP), and troloxequivalent antioxidant capacity (TEAC) assays (Table 1). Results

showed that the antioxidant activities of RCE were not significantlydifferent (p > 0.05) from WCE regardless of the antioxidant assayused. In addition, the assays that showed high antioxidant capacitymeasured in terms of lM trolox equivalents in RCE and WCE wereABTS and FRAP, whereas the least antioxidant capacity was ob-served from the free radical scavenging assay.

3.6. Estimation of phosphorylated Akt in vitro in HUVEC

The relative phosphorylated Akt concentrations expressed asp-Akt/total p-Akt were estimated in response to exposure ofHUVEC to RCE and WCE, and compared with PBS control(Fig. 4a). Total Akt levels did not change in response to differenttreatments. Results showed that at concentrations of 0.2 and

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

%

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100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

%

0

100 179.3161.3

135.3

341.4

323.1221.4

161.4

149.4

179.3

341.2

263.8199.2 246.9 286.5

Fig. 3c. MS–MS Daughter scans of 503 amu parent of caffeic acid dihexose (peak 1 in flavonol chromatograms in Fig 3a) in mobile phase containing (a) 0.1% formic acid,CV = 20 V and (b) 2.0% formic acid, CV = 40 V.

m/z100 150 200 250 300 350 400 450 500 550 600 650 700

%

0

100

m/z100 150 200 250 300 350 400 450 500 550 600 650 700

%

0

100cb_procy_05182010_005 365 (10.752) 1: Scan ES-

6.10e5315.3

153.3112.8 181.1

316.3 383.3351.2 451.2

cb_flav_msms_05142010_006 291 (10.777) 2: Daughters of 315ES- 3.22e5153.3

123.3

(b)

(a)

Fig. 3d. (a) Full scan mass spectrum of protocatechuic acid hexoside (peak 1) in fluorescence chromatogram of white cranberry extract (WCE) and (b) daughters of 315 amuMS-MS spectrum of protocatechuic acid hexoside (peak 1) in fluorescence chromatogram of WCE.

548 A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552

1.0 mg/ml, both RCE and WCE significantly increased the phos-phorylation of protein kinase B (Akt) via redox sensitive phospha-tidylinositol-3-kinase (PI3K) in vitro in HUVEC relative to the basalp-Akt level (i.e., PBS-control). In addition, no significant difference(p > 0.05) was observed in the relative phosphorylated Akt be-tween RCE and WCE.

3.7. Endothelial cell migration

The effects of RCE and WCE on endothelial cell migration in vitroin HUVEC were determined using a wound healing assay (Fig. 4b.).Cell migration increased when HUVEC was treated with RCE andWCE at a lower concentration of 0.2 mg/ml. Treatment with wort-mannin, a PI3 K inhibitor, along with RCE and WCE, attenuated the

cell migration compared to the cells treated with RCE or WCEalone. Microscopic images from cell migration also showed 0% cellsurvival in HUVEC after 5 h exposure to a higher concentration(1 mg/ml) of RCE and WCE (images not shown) indicating possibletoxicity at higher concentrations. Therefore, the higher concentra-tions were not chosen in cell migration assay.

3.8. Endothelial cell tube formation

The effects of RCE and WCE on tube formation in vitro in HUVECwere determined by the endothelial cell tube formation assay(Fig. 4c.). Results of the tube formation microscopic images indi-cated that angiogenesis increased after treatment of HUVEC with0.2 mg/ml of RCE and WCE compared to PBS-control. Primary

0

5000

10000

15000

20000

25000

Control RCE WCESample

Rel

ativ

e p-

Akt

/mg

prot

ein

0.2 mg/ml1.0 mg/ml

Basal p-Akt

Fig. 4a. Relative phosphorylated Akt concentrations in response to red cranberry(RCE) and white cranberry extracts (WCE) in vitro in human umbilical endothelialcells (HUVEC) using phosphate-buffered saline (PBS) as control. Data shown aremeans ± standard errors (n = 3). Bars with different letters are significantly different(p < 0.05).

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 549

endothelial cells developed tubular-like networks in between 5and 36 h of exposure. At higher concentration such as 1 mg/ml, celldeath occurred within 6 h (images not shown). The microscopicimages also showed that angiogenesis was inhibited when cellswere treated with wortmannin and cranberry extracts, confirmingthe involvement of PI3 kinase pathway. Moreover, RCE showed en-hanced tube formation effect compared with WCE (p < 0.05).

4. Discussion

Cranberries have been widely studied due to their putativehealth-promoting benefits. These fruits are rich in phenolic com-pounds with high antioxidant properties such as anthocyanins,proanthocyanidins, flavonols, flavanols, and phenolic acids(Fig. 1). In this study, the total phenolic content of RCE was similar

(a)

(b)

(c)

(d)

(e)

(f)

(

(

(

Fig. 4b. Effects of RCE and WCE on cell migration in in vitro HUVEC examined by wound h0.2 mg/ml of RCE or WCE. Photographs of PBS-control (a, b, and c), PI3 K inhibitor (d, e,respectively.

to that of WCE. However, the total monomeric anthocyanincontent of RCE was significantly higher than that of WCE andwas similar to the value of 78 mg per 100 g reported by (Mazza& Miniati, 1993) for cranberries. In contrast, significantly highertotal proanthocyanidin content was found in WCE than RCE. Thetotal procyanidin values estimated for RCE and WCE were withinthe reported range of powdered cranberry samples (63–1770 mg/100 g) (Prior et al., 2010). In addition the total flavonoid contentof WCE was higher than that of RCE.

Although there is a wide difference in the color or maturity ofred and white cranberries used in this study, the lack of anthocya-nins in white cranberries may have been compensated by the highamount of total procyanidins present in the same berries. This mayaccount for the similarities in the amount of overall RCE and WCEphenolics observed in this study. Similarity in the overall phenolicsobserved between RCE and WCE has been reported in other studies(Celik, Ozgen, Serce, & Kaya, 2008). The concentration of totalphenolics in light red berries was similar to dark red-colored cran-berries, although the green or earliest stage of cranberry maturitycontained much higher phenolic values than either light or darkred cranberries (Celik et al., 2008).

As expected, the individual anthocyanin content in the RCEsamples was greater than that in WCE; only traces amount of indi-vidual anthocyanins were detected by HPLC in the WCE sample.The individual anthocyanins that contribute to the total anthocya-nin contents of RCE were identified as Pn-3-gal, followed by Cy-3-ara, and Cy-3-gal. Forty-two percent of the cranberry anthocyaninswere represented by Pn-3-gal alone. Likewise, Cy-3-ara contrib-uted approximately 27% of the anthocyanins while 20% of the totalanthocyanin composition was attributed to Cy-3-gal. Pn-3-glc andPn-3-ara accounted for the rest of the total anthocyanins in cran-berries. The sum of all individual anthocyanins quantified in RCE(68 mg/100 g fresh weight) was within the range of published

(j)

(k)

(l)

g)

h)

i)

ealing assay. Endothelial cell monolayers were wounded at time 0 and cultured withand f), RCE (g, h, i), and WCE (j, k, l) were taken after 0, 5, and 24 h of incubation,

550 A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552

values (Timberlake, 1988) and comparable to the value estimatedfrom the spectrophometric monomeric anthocyanin assay(Table 1).

Aside from anthocyanins, red cranberries are also a good sourceof flavonols and phenolic acids such as quercetin 3-galactoside,myricetin 3-hexoside, quercetin 3-pentoside and caffeic acids.Approximately 36% of the total flavonols in red cranberries are inthe form of quercetin 3-galactoside. The values for quercetin andmyricetin were in similar range as reported for total quercetinand myricetin from hydrolyzed extracts of cranberry (Bilyk &Sapers, 1986; Hakkinen, Karenlampi, Heinonen, Mykkanen, &Torronen, 1999). Bilyk and Sapers (1986) found that the darker col-ored berries contained higher levels of flavonols compared withthe lighter colored berries, as reported in this study.

Individual procyanidin compounds such as procyanidin tetra-mer type A, procyanidin C1, and catechins accounted for most ofthe total procyanidins in white cranberries. Procyanidin tetramervalues were 2.2-fold greater in WCE compared with RCE, and thiscompound contributes 57% to the overall procyanidins observedin white berries. In contrast, the tetramer was the largest molecu-lar weight procyanidin detected in these extracts based on the re-sult of mass spectrometer scan range (upper limit of 1200) andchromatographic conditions. Procyanidin pentamers, hexamers,and heptamers were found in cranberries as described in previousreports (Foo, Lu, Howell, & Vorsa, 2000a; Prior, Lazarus, Cao, Mucci-telli, & Hammerstone, 2001). The tetramer was the largest procy-anidin found in these extracts as result of mass spectrometerscan range and chromatographic conditions. It is possible thatthe tetramer is the largest procyanidin detected in our study dueto solubility considerations. PBS was the dissolution solvent usedfor procyanidin analysis in this study. Most studies of cranberryprocyanidin content employ fractionation schemes and dissolutionof the final sample in organic/aqueous solvent mixtures which en-hance solubility of the higher molecular weight procyanidins (Fooet al., 2000b; Prior et al., 2001, 2010). The decline in the individualprocyanidin content of RCE compared with WCE was also observedin other studies which measured procyanidins in cranberries as afunction of ripening (Vvedenskaya & Vorsa, 2004).

It is known that cranberries rank high among fruits in terms oftheir antioxidant contents. The antioxidant capacity of cranberriesin both red and white samples were observed to be similar in thethree antioxidant assays (FRAP, free radical scavenging, and TEAC)used in this study. These results may be due to the similarities inthe overall phenolic contents observed in both berries. The antiox-idant effects of cranberries are mainly due to the high levels ofphenolic compounds present in the fruit (Sun, Chu, Wu, & Liu,

(a)

(e)

(b)

(f)

(c

(g

Fig. 4c. Effects of RCE and WCE on vessel formation in in vitro HUVEC examined by tubeand WCE. Photographs of RCE-treated vessels: (a) with PI3K inhibitor, (b) with LY294002(e) with PI3K inhibitor, (f) with LY294002, (g) after 5 h, and (h) after 36 h of incubation

2002). Mazza and Miniati also attributed the high antioxidantproperties of cranberries to anthocyanins and other phenolic com-pounds (Mazza & Miniati, 1993). Prior et al. claimed that procyani-din fraction accounted for 54% of the total antioxidant capacitymeasured in cranberries (Prior et al., 2001). The combination ofphytochemicals and synergistic mechanisms of these compoundsin the fruit matrix may be responsible for the antioxidant proper-ties of cranberries.

4.1. Endothelial cell function

The ability of the cranberry extracts to act as in vitro antioxi-dants may be associated with the phenolic contents of the berries.Polyphenolic compounds are known to change the redox sensitiv-ity of the cells. PI3 is a redox sensitive kinase and therefore PI3kinase-mediated phosphorylated Akt levels in vitro in HUVEC weredetermined upon exposure to red and white cranberry extracts.Our findings indicate that the abundant anthocyanins in red cran-berries and the abundant procyanidins in white cranberries mayhave partly contributed to the activation of Akt via redox sensitivePI3-kinase-mediated signaling pathway in cultured HUVEC. Thephosphorylation of Akt is known to activate downstream signalingthat results in endothelial nitric oxide synthase (eNOS) activation.eNOS is an important regulator of cardiovascular homeostasis be-cause it is the major catalyst for nitric oxide (NO) production invascular endothelial cells and plays a crucial role in the state ofblood vessel vasodilatation, and hence, maintain the vascularintegrity and regulate the blood pressure.

Grape-seed extract was used in this study as a positive controlsince it is known to induce endothelium-dependent relaxationthrough PI3 kinase/Akt signaling pathway (data not shown). Ediri-singhe et al. demonstrated that in grape-seed extract, polyphenoliccompounds caused an endothelium-dependent relaxation of bloodvessels and that it was mediated by the activation of the PI3 K/Aktsignaling pathway through a redox-sensitive mechanism, resultingin the phosphorylation of eNOS (Edirisinghe, Burton-Freeman, &Kappagoda, 2008). The polyphenolic compounds in grape-seed ex-tract are mostly proanthocyanidins which occur as mixtures ofoligomers and polymers of catechin and epicatechin. In addition,the grape-seed extract consists mainly of dimers and trimers with-out gallic acid residues. Tetramers and pentamers, including largeroligomers of proanthocyanidins, were responsible for the endothe-lium-dependent vasodilator effects demonstrated by (Fitzpatrick,Bing, Maggi, Fleming, & O’Malley, 2002). In cranberry extracts,these oligomeric procyanidins were also abundant, thus, they

(d)

(h)

)

)

formation assay. Endothelial cell monolayers were cultured with 0.2 mg/ml of RCE, (c) after 5 h, and (d) after 36 h of incubation. Photographs of WCE-treated vessels:

. The PI3K inhibitor was 35 nmol/l wortmannin.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540–552 551

may have similar action in HUVEC as those found in grape-seedextract.

The p-Akt activity observed in red and white cranberry extractsmay also be attributed to the high anthocyanin and proanthocyani-din components, respectively. Red wine- and grape-derived prod-ucts are found to be potent vasodilators in vitro wherein theeffect have been mainly attributed to the high content of polyphe-nols in these products. Specific anthocyanins from grapes andother red fruits may be able to induce vasorelaxation (Edirisinghe,Lu, Nalbandyan, & Kappagoda, 2007; Mullen et al., 2002). Edirisin-ghe et al. reported that strawberry extracts rich in phenolic com-pounds caused an endothelial relaxation on both aortic rings andHUVECs through activation of PI3 kinase/Akt and attributed theseeffects to the antioxidant properties of phenolic compounds (Ediri-singhe, Burton-Freeman, Varelis, & Kappagoda, 2008). Their studiessuggest that the anthocyanin-rich strawberry extracts may resultin improving the functionality of endothelium and thus, reducingblood pressure and the risk of cardiovascular disease. Recently,we demonstrated that berry fruits such as wild blueberry, cran-berry, and strawberry which contain significant quantities ofanthocyanins induced the activation of PI3/Akt/eNOS in HUVEC(Tulio et al., 2012).

The results of cell migration and tube formation assays in thisstudy indicate a possible PI3-kinase/Akt-dependent mechanismthat modulate endothelial function induced by phenoliccompounds in RCE and WCE when present at physiological concen-trations (0.2 mg/ml). Tube formation assay is used as a quantitativemodel for studying inhibitors and activators of angiogenesis.Angiogenesis is the formation of new blood vessels from existingvasculature and it is an integral part of both normal and patholog-ical processes. Endothelial cells are the key cell type involved inthis process. During angiogenesis, these cells disrupt the surround-ing basement membrane, migrate toward an angiogenic stimulus,proliferate to provide additional cells that make up a new vessel,and re-organize to form the necessary three-dimensional vesselstructures.

Exposure of HUVEC to RCE and WCE at physiological concentra-tion enhanced the formation of the three dimensional vessels.However at higher concentrations, cell deaths were observedsuggesting possible toxicity effects. Anthocyanin compounds arebioavailable and generally found in nmol/l concentration after eat-ing fruits rich in anthocyanins. Therefore, our data in the presentin vitro study is in agreement with in vivo setup. The phenolic con-stituents in red and white cranberries may also be responsible forthe induction of the endothelial cells to form three-dimensionalcapillary-like tubular structures and provides evidence for aphenolic-/redox-mediated contribution to angiogenesis, woundhealing, and cell death. This is in agreement with our previousstudy that polyphenolic-rich berry fruits not only activated PI3/Akt but also enhanced cell migration and tube formation inendothelial cells (Tulio et al., 2012).

5. Conclusion

This study provides evidence that phenolic constituents such asanthocyanins, proanthocyanidins, flavonols and phenolic acids,contribute to the antioxidant and biological properties of red andwhite cranberries. Our data suggest that similarities in the overallphenolic profiles and total antioxidant properties of red and whitecranberries may likely explain why the activation of p-Akt did notdiffer between red and white cranberry extract-treated HUVECs. Inaddition, polyphenolic compounds derived from red and whitecranberry extracts may have caused the induction of redox-sensitive PI3 kinase/Akt pathway and activated downstreamsignals resulting in endothelial cell migration and vessel formation.

In summary, the enhanced phosphorylation of PI3/Akt kinase, cellwound healing, and tube vessel formation in HUVEC elicited byRCE and WCE suggest that overall phenolic constituents (anthocy-anins, proanthocyanidins, flavonols, phenolic acids, and otherphenolic compounds) rather than individual compounds withinthe cranberry matrix may be responsible for these biological ef-fects. Furthermore, the results in this study suggest that red andwhite cranberries may have vasodilator effects in vivo. Clinicalstudies are warranted to confirm the potential cardioprotectivebenefits being offered by these fruits.

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