Food Chemistry 157 (2014) 540552

Contents lists available at ScienceDirect

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

Phenolic composition, antioxidant properties, and endothelial cell

function of red and white cranberry fruits

b
c

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

a b s t r a c t

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

1. Introduction

AC

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 procyanidin
tetramer was the predominant proanthocyanidin identied. The antioxidant properties of RCE and WCE
were not signicantly different regardless of antioxidant assays (DPPH, FRAP, and TEAC) used. Both RCE
and 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 tube
formation elicited by RCE and WCE suggest that overall phenolic constituents rather than individual
phenolic compounds within the cranberry matrix may be responsible for these biological effects.
Published by Elsevier Ltd.

ET

Keywords:
Cranberry
Phenolic compounds
Antioxidants
Anthocyanins
Proanthocyanidins
Flavonols
Phenolic acids
Endothelial cell function
p-Akt
Cell migration
Tube formation

TE

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

Naturally-occurring secondary metabolites present in fruits and

vegetables have received widespread attention due to their purported health-promoting properties. Epidemiological studies have
consistently shown a direct correlation between consumption of
fruits and vegetables with lower risk for developing chronic illnesses such as cardiovascular diseases (Mckay & Blumberg, 2007;
Reed, 2002) and certain types of cancer (Neto, 2011). These health
benets from consumption of fruits and vegetables have been largely attributed to the plant metabolites known as phytochemicals.
Cranberries (Vaccinium macrocarpon Aiton), are a rich source of
phytochemicals, particularly phenolic compounds (Reed, 2002).
These compounds, further divided into a subclass of compounds
called avonoids, include avonols, avones, avan-3-ols,
avanones, isoavones, and anthocyanins (Fig. 1). Among these
avonoids, anthocyanins have been widely studied due to their
potential to act as an antioxidant (Seeram, Momin, Nair, &
Corresponding author. Tel.: +1 708 924 0646; fax: +1 708 924 0690.
E-mail address: artemio.tulio@fda.hhs.gov (A.Z. Tulio Jr.).
http://dx.doi.org/10.1016/j.foodchem.2014.02.047
0308-8146/Published by Elsevier Ltd.

Bourquin, 2001). Anthocyanins are primarily responsible for the

red, pink, and purple color of cranberries. Cranberry fruits are also
known for their proanthocyanidin compounds, particularly the
type A-proanthocyanidin trimer (Fig. 1C), which is associated with
having potent anti-bacterial and anti-adhesive properties (Foo, Lu,
Howell, & Vorsa, 2000b).
A recent consumer trend is the introduction of cranberry
products from immature (white) berry stage, since they tend to
be less tart and tangy than their red counterparts, being marketed
specically for children. Hence, not only cranberries from mature
(red) but also immature (white) stages are recently being marketed
commercially. Most of the studies examining the health-promoting
benets of cranberries have been with red or mature berries.
Although consumption of white cranberries is substantially less
than with the red cranberries, more information is needed on
how white cranberries compare to red cranberries with regards
to chemical composition, their biological effects, and their potential health-promoting benets.
Therefore, this work was conducted to provide information that
will further enhance our understanding of the phenolic composition, antioxidant properties, biological activities, and other

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A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

R1

OH

OH
OH

OH

HO
R2

OH

OH

OH

quercetin

anthocyanidin
R1
R2
-----------------------------------------------------------cyanidin
OH
O-galactoside
O-glucoside
O-arabinoside
peonidin
OCH3
O-galactoside
O-glucoside
O-arabinoside

OH
OH

HO

OH
OH

OH

myricetin

OH

COOH
O

OH
OH

OH
OH

OH

OH

OH

OH
OH

OH
OH

TE

OH

n
OH

O
OH O

OH

HO
HO

OH

OH

ET

condensed tannins
(n indicates number of subunits)

COOH

AC

OH

protocatechuic acid

OCH3

H3 CO
OH
sinapic acid

OH
OH

HO

O
*
*

OH

OH
catechin

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

potential health benets of mature (red) and immature (white)

cranberries. The specic objective of this study was to characterize
the differences between the phenolic proles of red and white
cranberries using LCMS/MS and spectrophotometric assays. Furthermore, this study was conducted to evaluate and compare the
effects of red and white cranberry extracts on the activation of protein kinase B (Akt) via the redox-sensitive phosphatidylinositol-3
kinase-mediated signaling pathway in cultured human umbilical
endothelial cells (HUVEC). Endothelial cell migration and tube formation, known to modulate via Akt/PI3 kinase pathway, were also
used for assessing the bioactivity of the polyphenol-rich extracts in
in vitro.

2. Materials and methods

2.1. Cranberry samples and preparation of extracts
Red and white cranberries (Vaccinium macrocarpon Aiton) of
the Stevens cultivar were obtained from a grower in Wisconsin,
USA. Red cranberry fruits were dark red-colored berries harvested
at a ripe, mature stage, whereas white cranberries were light

pinkish-colored berries, still unripe and immature, that were

harvested along with the red berries After harvest, white berries
were visually sorted and segregated from red berries, packed separately, 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 BenchTop Freeze-Dryer (Kingston, NY), milled with coffee grinder, and
the 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 the
dark, the mixture was centrifuged at 10,000g for 20 min at 4 C
and then ltered by vacuum ltration with Whatman #2 lter paper. The resulting supernatant was divided into 6 equal portions
(5 ml/tube). The extract in each tube was freeze-dried following
removal of the solvent mixture using N2 gas stream. Freeze-dried
red cranberry extracts (RCE) and white cranberry extracts (WCE)
were weighed and stored at 20 C.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),

2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl1-picrylhydrazyl
(DPPH),
4-dimethylamino-cinnamaldehyde
(DMAC), FolinCiocalteau phenol reagent, sodium acetate (C2H3NaO23H2O), iron (III) chloride hexahydrate (FeCl36H2O), aluminum chloride (AlCl36H2O), sodium nitrite (NaNO2), gallic acid,
catechin, ()-epicatechin gallate, quercetin-3-O-galactoside, quercetin-3-O-rhamnoside, myricetin, quercetin, p-coumaric acid,
chlorogenic acid, and sinapic acid were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Ethyl acetate was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). HPLC-grade water,
methanol, acetonitrile, glacial acetic acid, and formic acid were
purchased from Fisher Scientic Co. (Fairlawn, NJ, USA). Cyanidin
3-O-glucoside chloride (Cy-3-glc), cyanidin 3-O-arabinoside chloride (Cy-3-ara), cyanidin 3-O-galactoside chloride (Cy-3-gal),
peonidin 3-O-glucoside chloride (Pn-3-glc), peonidin 3-O-arabinoside chloride (Pn-3-ara), procyanidin A2, B1, B2, and C1 were
obtained from ChromaDex, Inc. (Irvine, CA, USA). Phosphate buffered-saline (PBS) solution was purchased from Fisher Bioreagents
(Fair Lawn, NJ, USA).
2.4. Chemical analysis

2.5. LCMS/MS analysis

2.5.1. Standard preparation
Stock standard solutions (1 mg/ml) were prepared in 50:50
methanol/water (v/v) and stored at 80 C. Calibration standards
were diluted from stock solutions. Mixed anthocyanin calibration
standards containing Cy-3-gal, Cy-3-glc, Cy-3-ara, Pn-3-gal, Pn-3glc, 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 containing sinapic acid, p-coumaric acid, () epicatechin gallate, quercetin, 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 analysis
Prior to HPLC analysis, cranberry extracts (RCE and WCE) were
suspended in de-ionized water for anthocyanin analysis and PBS
for the analyses of avonols, phenolic acids, and procyanidins.
RCE and WCE samples were brought to room temperature and then
vortexed to thoroughly mix and re-suspend the extract powder.
Samples were then centrifuged and/or ltered through 0.22 lm
nylon syringe lters to produce a clear supernatant. An aliquot of
the supernatant was transferred to an autosampler HPLC vial and
then diluted with de-ionized water to ensure responses were in
the calibration range. Samples were stored in the freezers at 20 C.

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AC

2.4.1. Determination of total phenolic content

The total phenolic content was estimated colorimetrically using
the FolinCiocalteau phenol reagent according to the modied procedure of (Slinkard & Singleton, 1977). Briey, RCE and WCE samples were diluted with de-ionized water prior to mixing with
FolinCiocalteau phenol reagent. The mixture was allowed to stand
for 5 min at room temperature before adding 7% sodium carbonate
solution followed by de-ionized water. Solutions were mixed and
allowed to stand for 2 h at room temperature. The total phenol
concentration was determined from gallic acid standard calibration curve (0, 100, 200, 300, 400, 500 lg/ml) using a Cary 100 Conc
UVVisible spectrophotometer (Varian Inc., Walnut Creek, CA,
USA) at 765 nm. Results were expressed as mg gallic acid equivalents (GAE) per 100 g fresh weight (FW).

2.4.4. Determination of total procyanidin content

The total procyanidin content was determined using 4-dimethylaminocinnamaldehyde (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 measured after 20 min against the prepared blank using a Cary 100
Conc UVVisible spectrophotometer. Results were expressed as
mg procyanidin A2 equivalents (PA2) per 100 g FW.

2.3. Reagents

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542

2.4.2. Determination of total monomeric anthocyanin content

The total monomeric anthocyanin content in cranberry extracts
was determined colorimetrically using the pH differential method
described previously (Giusti & Wrolstad, 2005). The absorbance
was measured at 510 nm and 700 nm in PBS with pH 1.0 and 4.5
using a Cary 100 Conc UVVisible spectrophotometer. A molar
extinction coefcient of 29,600 for Cy-3-glc was used to calculate
the total monomeric anthocyanin contents. Results were expressed
as mg Cy-3-glc equivalents (C3GE) per 100 g FW.
2.4.3. Determination of total avonoid content
The total avonoid content was determined by the aluminum
chloride colorimetric method described by (Meyers, Watkins,
Pritts, & Liu, 2003) and (Zhishen, Mengcheng, & Jianming, 1999)
with modications. In brief, RCE and WCE samples were diluted
with de-ionized water before adding 5% NaNO2 solution. After
6 min, 10% AlCl36H2O solution was added. After another 5 min
had lapsed, 1 M NaOH solution was added into the mixture. The
absorbance at 510 nm was measured immediately against the prepared blank using a Cary 100 Conc UVVisible spectrophotometer
and total avonoid concentrations were calculated based on the
catechin standard calibration curve (0, 100, 200, 300, 400, and
500 lg/ml). Results were expressed as mg catechin equivalents
(CE) per 100 g FW.

2.5.3. LCPDAMS/MS analysis of anthocyanins

A Waters Alliance 2795 HPLC system (Milford, MA, USA)
equipped with a Waters 996 photodiode array detector (PDA),
Waters 2475 uorescence detector (FLD), Quattro Micromass triple
quadrupole mass spectrometer, and MassLynx (V.4.1) software
was used to identify and quantify anthocyanins in the red and
white cranberry extracts. Separations were performed on a Synergi
Max-RP column (150  3.0 mm, 4 lm packing; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 1% formic acid in
water (solvent A) and 1% formic acid in acetonitrile (solvent B).
The gradient elution program was set as follows: 2% B from 0 to
4 min, 220% B linear from 4 to 20 min, 2080% B from 20 to
24 min, 80% B from 24 to 30 min, and then returning to initial concentration of 2% from 30 to 35 min to re-equilibrate the system.
The ow rate was 0.4 ml/min and the injection volume was set
at 20 ll. The column temperature was maintained at 40 C and
the autosampler was cooled at 15 C. The PDA was set at 520 nm
to monitor the UVvisible absorption of anthocyanins, and the
UVvisible spectra were recorded from 200 to 600 nm with a resolution of 1.2 nm and acquisition of 1 spectra/s. After passing
through the PDA, the column eluate was split, and 0.2 ml/min
was diverted to a mass spectrometer tted with electrospray ionization (ESI) interface. ESI positive mode was utilized for the detection of anthocyanins. The mass spectrometer conditions were set
up as follows: source temperature, 100 C; desolvation temperature, 300 C; nitrogen desolvation ow, 800 l/h; capillary, 3000 V;
cone, 30 V; and MSMS collision energy, 15 V.
2.5.4. LCPDAMS/MS analysis of avonols and phenolic acids
Qualitative and quantitative analysis of avonols and phenolic
acids were performed using the LCPDAMS/MS parameters

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A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

2.6. Antioxidant activity assays

2.7.3. Endothelial cell migration assay

Endothelial cell migration assay was performed following the
Tulio et al. protocol (Tulio et al., 2012). In brief, HUVECs were
grown in a cell culture ask in a 5% CO2 incubator at 37 C until
conuent and then the cells were transferred into 6-well plates.
After cell growth reached 90% conuence, the bottom of each
well containing a HUVEC monolayer was scraped in a straight line
with a 200 ll sterile pipet tip. Cell debris was removed by washing
the plate twice with PBS (pH 7.4). Cells were exposed to different
concentrations of RCE and WCE samples (0.05, 0.1, and 0.2 mg/
ml) in a medium containing 2% serum and the cells were incubated
at 37 C in 5% CO2 with for 24 h. Half of the plates were treated
with wortmannin (30 nmol/l) for 30 min before the addition of
the cranberry extracts to conrm the involvement of PI3/Akt signaling pathway. Cells treated with PBS were used as control in
the study.

AC

2.6.1. DPPH-free radical scavenging assay

The free radical scavenging assay was performed according to
the method of Brand-Williams, Cuvelier, and Berset (1995). Briey,
2,2-diphenyl-1-picrylhydrazyl (DPPH) dissolved in methanol, was
used to estimate the free radical scavenging abilities of various
antioxidants. The loss of DPPH radical absorbance was determined
using a Cary 100 Conc UVVisible spectrophotometer at 517 nm
after keeping the sample in the dark for 1 h (Ozgen, Reese, Tulio,
Scheerens, & Miller, 2006). Results were expressed as lM TE/g FW.

2.5.5. LCFLDMS/MS analysis of procyanidins

The conditions for the qualitative and quantitative analyses of
procyanidins were similar to the analyses of avonols and phenolic
acids. In addition, uorescence detection (FLD) was conducted with
an excitation wavelength of 285 nm and an emission wavelength
of 315 nm. The emission unit full scale (EUFS) was set at 5000
and gain at 1.

2.7.2. AlphaScreen SureFire phosphorylated kinase b (p-Akt) assay

The ability of RCE and WCE samples to phosphorylate p-Akt
in vitro in HUVEC was performed following the method described
previously (Tulio et al., 2012). In brief, HUVECs were grown in
EGM-2 supplemented with 10% FBS. Cells were grown to conuent
monolayer (90%) and then transferred into 6-well plates. HUVEC
were starved for 6 h in 2% serum media before the cells were treated with different concentrations of RCE and WCE (0.1, 0.2, 0.5 and
1.0 mg/ml). Wortmannin (Ascent Scientic LLC, Princeton, NJ,
USA), a PI3 kinase inhibitor, was used to conrm PI3 kinase
involvement. A dose of 30 nmol/l wortmannin, which does not induce toxicity in HUVECs, was used to treat HUVECs for 30 min before exposure of RCE and WCE and then incubated at 37 C in a
humidied atmosphere containing 5% CO2 for 10 min. The reaction
was stopped immediately by adding ice cold PBS (pH 7.4) and the
cells were washed twice with the same PBS buffer. Cell lysates
were prepared in cell lysis buffer containing protease inhibitors
(Calbiochem, Rockland, MA, USA). Relative phosphorylated-Akt
levels in the cell lysates were measured by AlphaScreen SureFire
assay (PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 680 nm and emission wavelength of 520620 nm and expressed as p-Akt/Total Akt. PBS buffer (pH 7.4) was used as the
control. Protein levels were measured using Pierce bicinchoninic
acid (BCA) protein assay (Thermo Scientic, Rockford, IL, USA).

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described above. The elution conditions were as follows: elution

solvent A (0.1% or 2% formic acid in water) and B (0.1% or 2% formic
acid in methanol) using a gradient program of 5% B from 0 to 4 min,
550% B linear in 434 min, 50% from 34 to 40 min, and returned
to 5% B at 4045 min for re-equilibration of the column. The ow
rate was set at 0.5 ml/min and 50% of the column eluate was diverted to the mass spectrometer. The injection volume was set at
10 ll, column temperature was set at 40 C, and peak quantitation
was accomplished by acquiring PDA data at 340 nm. The negative
ionization mode (ESI) of the mass spectrometer was used for the
detection of avonols and phenolic acids. The mass spectrometer
parameters were also similar to the above procedure except for
the capillary voltage at 2500 V, cone voltage at either 20 or 40 V,
and MSMS collision energy at 25 V.

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2.6.2. Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed as previously described by Benzie
and Strain (1996). In brief, the reaction mixtures were prepared by
combining 300 mM C2H3NaO23H2O, buffer (pH 3.6), 20 mM of
FeCl36H2O, and 10 mM TPTZ in 40 mM HCl, in a 10:1:1 ratio.
The analysis was carried out at 2530 C and the absorbance was
read 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 assay

The trolox equivalent antioxidant power was determined using
2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) following the method described by Ozgen et al. (2006). All assay reactions were kept in the dark for 1 h and the levels of reduced ABTS
reactants were measured at 734 nm using a Cary 100 Conc UVVisible spectrophotometer. Results were expressed as lM TE/g FW.
2.7. In vitro cell culture assays
2.7.1. Cell viability and apoptosis assays
Human umbilical vein endothelial cells or HUVECs were
purchased from Lonza (Washington, DC, USA) and grown in endothelial cell growth medium 2 (EGM-2, Lonza) with 4% fetal bovine
serum (FBS). Cell viability assay was determined in response to
different concentrations and time factors using the Trypan blue
exclusion test. Apoptosis assay was determined using Caspase 9
colorimetric activity assay kit (Millipore, Waltham, MA, USA). Cell
culture assays were investigated with HUVEC cells having a
passage number of 2.

2.7.4. Endothelial cell tube formation assay

Endothelial cell tube formation experiments were carried out
following the method described previously (Tulio et al., 2012). In
brief, HUVECs were starved for 1 h in endothelial growth medium
containing 2% serum media. The cells were washed with PBS, and
then plated in 48-well plates that had been pre-coated with
100 ll of growth factor-reduced Matrigel Matrix (BD Bioscience,
Franklin Lakes, NJ, USA). The cells were incubated for 24 h with
RCE and WCE samples (0.05, 0.1, and 0.2 mg/ml) at 37 C in a
humidied incubator containing 5% CO2. Some cell plates were
treated with 30 nmol/l wortmannin and cranberry extracts to conrm involvement of the PI3/Akt signaling pathway in capillary-like
tubular formation. Capillary-like tube formations were photographed after 0, 5, 12, and 24 h under 40 magnication level
using an Olympus CKX41 microscope (Center Valley, PA, USA).
2.8. Statistical analysis
Means, standard errors, regression analysis, and signicant
differences between treatments by single factor-analysis of variance (ANOVA) at 95% condence level were calculated using SAS
v.9.2 (Cary, NC, USA). All values were reported as means SE for
three replications.

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A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

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

fresh berries.

The total phenolic, total monomeric anthocyanin, total avonoid, and total procyanidin contents of RCE and WCE were compared in this study using spectrophotometric assays (Table 1).
The total phenolic contents of RCE (341.9 2.1 mg GAE/100 g fresh
weight) and WCE (WCE had 339.4 4.5 mg GAE/100 g fresh
weight) were not signicantly different (p > 0.05). In contrast, the
amount of estimated total monomeric anthocyanins, total avonoids, and total procyanidins between RCE and WCE were signicantly different (p < 0.05). The total monomeric anthocyanin
content of RCE calculated based on the molar extinction coefcient
of Cy-3-gluc was 21-times greater than that of WCE. The estimated
total avonoid content of WCE in terms of catechin equivalents
was 1.2 times compared with RCE. In addition, the total procyanidin content of WCE estimated as procyanidin A2 equivalents, was
almost twice than that of RCE.

3.2. LCPDAMS/MS analysis of anthocyanins

Flavonols and phenolic acids in RCE and WCE were separated

and detected by PDA at 340 nm. Analyte peak identity was assigned by co-elution with authentic reference standards where
available, and/or with MS full scan, and MSMS daughter scan
spectra. LCMS analyses were conducted with two sets of conditions in an effort to obtain suitable separation and ionization in
ESI negative mode. Quantitative analysis of avonols at 340 nm
absorbance detection was conducted using 2% formic acid in mobile phase (Fig. 2b). The peak prole of the WCE (Fig. 2b.b) and
RCE (Fig. 2b.c) appear very similar. The chromatogram of the standard (Fig. 2b.a) shows matching retention times for some peaks
present in the WCE (Fig. 2b.b) and RCE (Fig. 2b.c) samples. Peaks
shown in Fig. 2b.b and c. were identied as follows: 1 = caffeic acid
dihexose, 2 = caffeic acid mono-hexose, 3 = chlorogenic acid,
4 = sinapic acid mono-hexose, 5/6 = epicatechin gallate/p-coumaric acid, 7 = myricetin 3-hexoside, 8 = sinapic acid, 9 = myricetin
3-pentoside,
10 = quercetin
3-galactoside,
11 = myricetin,
12 = quercetin 3-pentoside, 13 = quercetin 3-rhamnoside, and
14 = quercetin. MS-full scan spectra of caffeic acid dihexose (peak
1) in WCE samples in 0.1% formic acid, CV = 20 V and 2% formic
acid, CV = 40 V is shown in Fig. 3b, whereas the MSMS daughter
scans of 503 amu parent of caffeic acid dihexose (peak 1) in avonol chromatograms is shown in Fig 3c. The avonols were more
abundant in RCE than in WCE (Table 2). The major avonols/phenolic acids in RCE were quercetin-3-galactoside, myricetin 3-hexoside, and quercetin 3-rhamnoside. The total avonol and phenolic
acid contents of RCE from HPLC analysis was 35.99 mg/100 g of
fresh berries, whereas WCE had only 22.30 mg/100 g of fresh
berries (Table 2).

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Fig. 2a.a shows the anthocyanins detected by PDA at 520 nm in

the standard mixtures, WCE, and RCE, respectively. Five major
anthocyanin peaks were identied in the chromatograms of the
RCE samples (Fig. 2a.c), whereas the same anthocyanins were
either non-existent or found in negligible amounts in WCE
(Fig. 2a.b). The retention time of four of these peaks corresponded
to retention times of the authentic anthocyanin standards injected.
These major peak anthocyanins in RCE were identied as follows:
1 = Cy-3-gal, 3 = Cy-3-ara, 4 = Pn-3-gal, 5 = Pn-3-glc, and 6 = Pn-3ara (Fig. 2a.c). A fth peak eluted at 17.33 min for which there
was not a corresponding standard peak but the spectrum corresponds to that of a peonidin glycoside. The ESI + MS scan base peak
was 463 amu (Fig. 3a.a). An MSMS analysis of the parent 463 amu
ion 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 of
17.3 min for this peak in the reversed-phase separation of anthocyanins at acidic pH is consistent with previous report which show
Pn-3-gal elutes after Cy-3-arab and Pn-3-arab (Brown & Shipley,
2011). Pn-3-gal was the most abundant anthocyanin in red berries
followed by Cy-3-ara and Cy-3-gal, while the least abundant
anthocyanin was Pn-3-glc (Table 2). The sum of individual
anthocyanins in RCE was estimated at 68.27 mg per 100 g of fresh

3.3. LCPDAMS/MS analysis of avonols and phenolic acids

3.1. Estimation of total phenolics, total monomeric anthocyanins, total

avonoids, and total procyanidins

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3. Results

3.4. LCFLDMS/MS analysis of procyanidins

Procyanidins and avanol monomers were separated, identied, and quantitated using uorescence detection with excitation
and emission wavelengths of 285 and 315 nm, respectively. The
uorescence peak proles of the WCE (Fig. 2c.b) and RCE
(Fig. 2c.c) were very similar. In addition, the chromatogram of a
mixture of analytical standards (Fig. 2c.a) showed matching retention times for some peaks present in the WCE and RCE samples.
The chromatograms in Fig. 2c were from the HPLC separation with
0.1% formic acid in mobile phase and cone voltage of 20 V. Fig. 2c.b
and c show the peaks identied as follows: 1 = protocatechuic acid

Table 1
Total phenolic, total anthocyanin, total avonoid, total procyanidin contents and total antioxidant capacity of red and white cranberry extracts as determined by
spectrophometric assays.
Sample

Red cranberry extracts

White cranberry extracts
a
b
c
d
e
f
g
h
i

Total phenolic
content (mg
GAE)a,b

Total anthocyanin
content (mg
C3GE)a,c

Total procyanidin
content (mg
PA2E)a,d

Total avonoid
content (mg
CE)a,e

Total antioxidant capacityf

FRAPg

TEACh

DPPHi

341.6 2.1 a
339.4 4.5 a

88.3 3.98 a
3.9 0.87 b

115.5 2.51 a
184.1 13.34 b

47.5 0.66 a
58.3 2.62 b

12.96 0.13 a
13.89 0.15 a

13.04 0.61 a
13.78 0.35 a

6.28 0.13 b
6.13 0.13 b

Expressed per 100 g FW. Means standard errors (n = 3). Columns with different letters are signicantly different (p < 0.05).
GAE, gallic acid equivalents
C3GE, Cyanidin 3-O-glucoside equivalents.
PA2E, procyanidin A2 equivalents.
CE, catechin equivalents.
Expressed as lmol Trolox equivalents/g fresh weight.
Ferric reducing antioxidant power.
Trolox equivalent antioxidant capacity.
2,2-Diphenyl-1-picrylhydrazyl.

545

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

1
5.0e-2
AU

2
5

(a)

2.5e-2
0.0

8.00

5.0e-2

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

AU

(b)

0.0

8.00

4
1

AU

5.0e-2

(c)

2.5e-2

8.00

10.00

12.00

TE

0.0

6
5

2.5e-2

14.00

16.00

18.00

20.00

22.00

24.00

Time

AC

Fig. 2a. Anthocyanin proles of (a) 10 ppm mixed anthocyanin standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected by photodiode
array 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, and
6 = peonidin 3-arabinoside.

Table 2
Summary of the anthocyanins, avonols, phenolic acids, and proanthocyanidins quantied in red and white cranberry extracts as determined by LCMS/MS.a
Red cranberry extracts (mg/100 g FWb)

White cranberry extracts (mg/100 g FW)

13.75 1.03
ndc
18.71 0.68
28.39 0.87
1.97 0.46
5.45 1.57
68.27

0.17 1.03
nd
0.35 0.68
0.23 0.87
nd
0.06 1.57
0.81

Flavonols and phenolic acids

Caffeic acid dihexose
Caffeic acid mono-hexose
Chlorogenic acidd
Sinapic acid mono-hexose
Epicatechin gallate/p-coumaric acid
Epicatechin gallate/p-coumaric acid
Myricetin 3-hexoside
Sinapic acid
Myricetin 3-pentoside
Quercetin 3-galactoside
Myricetin
Quercetin 3-pentoside
Quercetin 3-rhamnoside
Quercetin

0.30 1.05
2.09 2.26
2.10 1.67
0.50 0.34
nd
nd
6.48 0.82
nd
2.27 0.68
12.82 4.32
nd
3.75 0.34
2.39 0.40
nd

0.28 0.10
1.69 6.59
2.81 1.62
0.62 1.61
nd
nd
2.02 1.03
nd
1.15 1.19
6.89 1.25
nd
2.33 0.83
1.52 1.84
nd

Protocatechuic acid hexoside

Total Flavonols and Phenolic Acids

3.29 0.21
35.99

2.99 0.10
22.30

Proanthocyanidins
Procyanidin B1
Catechin
Procyanidin B2
Procyanidin C1
Procyanidin trimer type A
Procyanidin tetramer type A
Total proanthocyanidins

0.37 0.00
0.87 0.57
1.56 0.24
4.27 1.69
1.10 7.33
7.93 0.07
16.10

0.50 0.00
2.11 2.38
1.46 0.32
6.55 0.82
2.38 14.77
17.21 0.09
30.21

Peak

Compound

1
2
3
4
5
6

Anthocyanins
Cyanidin 3-galactoside
Cyanidin 3-glucoside
Cyanidin 3-arabinoside
Peonidin 3-galactoside
Peonidin 3-glucoside
Peonidin 3-arabinoside
Total Anthocyanins

2a

2b

ET

1
2
3
4
5
6
7
8
9
10
11
12
13
14

Figure

2c

2c
2
3
4
5
6
7
a
b
c
d

Means relative standard deviations (n = 3).

Fresh weight.
Not detected.
Including unidentied compounds.

546

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

8
11

2.0e-2

AU

5.00

10.00

15.00

(b)

5.00

25.00

10.00

30.00

20.00

25.00

30.00

12
9
4

10.00

40.00

15.00

13

TE

AU

2
1
5.00

35.00

13

10

0.0

40.00

12
7

15.00

(c)

35.00

10

0.0

20.00

2.0e-2

5.0e-2

14

5/6

0.0

4.0e-2

13

10

AU

(a)

20.00

25.00

30.00

Time
40.00

35.00

2
(a)

8.00

10.00

12.00

100

(b)

8.00

(c)

10.00

12.00

8.00

16.00

14.00

10.00

12.00

18.00

20.00

22.00

24.00

7
5
6

16.00

18.00

20.00

22.00

24.00

100

14.00

ET

100

AC

Fig. 2b. Flavonols and phenolic acid proles of (a) 10 ppm mixed avonol standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected by
photodiode 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.

4
3

14.00

16.00

18.00

20.00

22.00

24.00

Time

Fig. 2c. Procyanidin proles of (a) 10 ppm mixed procyanidin standards, (b) white cranberry extract (WCE), and (c) red cranberry extract (RCE) detected by uorescence at
excitation 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.

hexoside, 2 = procyanidin B1, 3 = catechin, 4 = procyanidin B2,

5 = procyanidin C1, 6 = procyanidin trimer type A, and 7 = procyanidin tetramer type A. Based on matching retention times and
MS spectral data, uorescence peaks for procyanidin B1, B2 and
C1 were identied in both the WCE and RCE. A procyanidin trimer
type A, and tetramer type A were identied based on MS data.
Mass spectral data indicate that the peak eluting at 10.83 min in
the sample extracts is likely protocatechuic acid hexoside (3,4
dihydroxybenzoic acid hexoside) (Fig. 3d). This compound exhibits
signicant uorescence at excitation/emission wavelengths used

for the analysis. The uorescent prole of peaks in Fig. 2c.b and c
shows the largest peak at retention time at 22.9 min. The full scan
spectrum showed at distinct peak at 1151.5 amu which corresponds to a Type A tetramer. The MS-MS daughters of 1151.5 spectra of this compound gave a major fragment at 575.3 indicative of a
procyanidin trimer. This peak was therefore assigned as a procyanidin tetramer type A. Quantitation of the tetramer was made
using 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

547

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

Cranberry_Samples_03272010_011 408 (17.396)

2: Scan ES+
1.03e7

463.3

100

(a)
464.3
301.3
0
100
150
200
250
300
Cranberry_Samples_03292010_002 199 (17.360)
301.2
100

350

400

465.2

450

500

(b)

150

200

250

300

350

400

600

m/z
650
700
4: Daughters of 463ES+
1.30e6

TE

463.3

0
100

550

439.2

450

500

550

600

m/z
700

650

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.

(a)

113.2

375.2

125

150

175

200

161.2
117.1

225

250

275

300

325

100

503.3

342.3

153.1 165.1 179.1

0
100

AC

341.3

100

350

375

504.3

409.3

400

425

571.3
450

475

ET

525

550

575

600

m/z

503.2

(b)

341.1

146.1

179.2 191.1

131.2

196.1

125

150

175

200

236.1
240.2

225

250

293.2

275

300

325.1
325

425.1 440.0

353.0
350

0
100

500

375

400

425

450

504.3

556.1

469.8
475

577.2
578.2

500

525

550

575

600

m/z

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.

fresh weight in white cranberries. In contrast, red cranberries had

7.93 mg/100 g of fresh berries. The procyanidin tetramer was the
most abundant procyanidin quantied and detected by mass spectrometry. The total procyanidin content based upon the sum of
individual values from HPLC analysis of the white cranberry samples was 30.21 mg/100 g of fresh fruit, whereas red cranberries
had only 16.10 mg/100 g of fresh berries (Table 2).

showed that the antioxidant activities of RCE were not signicantly

different (p > 0.05) from WCE regardless of the antioxidant assay
used. In addition, the assays that showed high antioxidant capacity
measured in terms of lM trolox equivalents in RCE and WCE were
ABTS and FRAP, whereas the least antioxidant capacity was observed from the free radical scavenging assay.

3.6. Estimation of phosphorylated Akt in vitro in HUVEC

3.5. Estimation of antioxidant capacity
The total antioxidant capacities of RCE and WCE were estimated
based on three antioxidant assays including free radical scavenging
(DPPH), ferric reducing antioxidant power (FRAP), and trolox
equivalent antioxidant capacity (TEAC) assays (Table 1). Results

The relative phosphorylated Akt concentrations expressed as

p-Akt/total p-Akt were estimated in response to exposure of
HUVEC to RCE and WCE, and compared with PBS control
(Fig. 4a). Total Akt levels did not change in response to different
treatments. Results showed that at concentrations of 0.2 and

548

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

179.3
161.3

100

341.4

323.1
135.3
0
100 120

221.4

140 160

180 200

220 240 260

280 300

320 340 360

380 400

420 440

460 480 500

520 540

m/z

161.4
179.3

100

341.2

140 160

180 200

263.8 286.5

246.9

199.2

220 240 260

TE

149.4
0
100 120

280 300

320 340 360

380 400

420 440

460 480 500

520 540

m/z

Fig. 3c. MSMS Daughter scans of 503 amu parent of caffeic acid dihexose (peak 1 in avonol 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.

cb_procy_05182010_005 365 (10.752)

(a)

153.3
112.8

AC

315.3

100

316.3

181.1

383.3

351.2

451.2

350

400

450

500

550

600

350

400

450

500

550

600

m/z
650
700
2: Daughters of 315ES3.22e5

(b)

150

200

250

300

0
100

ET

0
100
150
200
250
300
cb_flav_msms_05142010_006 291 (10.777)
153.3
100
123.3

1: Scan ES6.10e5

650

700

m/z

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

1.0 mg/ml, both RCE and WCE signicantly increased the phosphorylation of protein kinase B (Akt) via redox sensitive phosphatidylinositol-3-kinase (PI3K) in vitro in HUVEC relative to the basal
p-Akt level (i.e., PBS-control). In addition, no signicant difference
(p > 0.05) was observed in the relative phosphorylated Akt between RCE and WCE.

cell migration compared to the cells treated with RCE or WCE

alone. Microscopic images from cell migration also showed 0% cell
survival in HUVEC after 5 h exposure to a higher concentration
(1 mg/ml) of RCE and WCE (images not shown) indicating possible
toxicity at higher concentrations. Therefore, the higher concentrations were not chosen in cell migration assay.

3.7. Endothelial cell migration

3.8. Endothelial cell tube formation

The effects of RCE and WCE on endothelial cell migration in vitro

in HUVEC were determined using a wound healing assay (Fig. 4b.).
Cell migration increased when HUVEC was treated with RCE and
WCE at a lower concentration of 0.2 mg/ml. Treatment with wortmannin, a PI3 K inhibitor, along with RCE and WCE, attenuated the

The effects of RCE and WCE on tube formation in vitro in HUVEC

were determined by the endothelial cell tube formation assay
(Fig. 4c.). Results of the tube formation microscopic images indicated that angiogenesis increased after treatment of HUVEC with
0.2 mg/ml of RCE and WCE compared to PBS-control. Primary

549

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

1.0 mg/ml
15000
10000

Basal p-Akt

5000
0
Control

RCE
Sample

WCE

Fig. 4a. Relative phosphorylated Akt concentrations in response to red cranberry

(RCE) and white cranberry extracts (WCE) in vitro in human umbilical endothelial
cells (HUVEC) using phosphate-buffered saline (PBS) as control. Data shown are
means standard errors (n = 3). Bars with different letters are signicantly different
(p < 0.05).

endothelial cells developed tubular-like networks in between 5

and 36 h of exposure. At higher concentration such as 1 mg/ml, cell
death occurred within 6 h (images not shown). The microscopic
images also showed that angiogenesis was inhibited when cells
were treated with wortmannin and cranberry extracts, conrming
the involvement of PI3 kinase pathway. Moreover, RCE showed enhanced tube formation effect compared with WCE (p < 0.05).

AC

4. Discussion

0.2 mg/ml

20000

to that of WCE. However, the total monomeric anthocyanin

content of RCE was signicantly higher than that of WCE and
was similar to the value of 78 mg per 100 g reported by (Mazza
& Miniati, 1993) for cranberries. In contrast, signicantly higher
total proanthocyanidin content was found in WCE than RCE. The
total procyanidin values estimated for RCE and WCE were within
the reported range of powdered cranberry samples (631770 mg/
100 g) (Prior et al., 2010). In addition the total avonoid content
of WCE was higher than that of RCE.
Although there is a wide difference in the color or maturity of
red and white cranberries used in this study, the lack of anthocyanins in white cranberries may have been compensated by the high
amount of total procyanidins present in the same berries. This may
account for the similarities in the amount of overall RCE and WCE
phenolics observed in this study. Similarity in the overall phenolics
observed between RCE and WCE has been reported in other studies
(Celik, Ozgen, Serce, & Kaya, 2008). The concentration of total
phenolics in light red berries was similar to dark red-colored cranberries, although the green or earliest stage of cranberry maturity
contained much higher phenolic values than either light or dark
red cranberries (Celik et al., 2008).
As expected, the individual anthocyanin content in the RCE
samples was greater than that in WCE; only traces amount of individual anthocyanins were detected by HPLC in the WCE sample.
The individual anthocyanins that contribute to the total anthocyanin contents of RCE were identied as Pn-3-gal, followed by Cy-3ara, and Cy-3-gal. Forty-two percent of the cranberry anthocyanins
were represented by Pn-3-gal alone. Likewise, Cy-3-ara contributed approximately 27% of the anthocyanins while 20% of the total
anthocyanin composition was attributed to Cy-3-gal. Pn-3-glc and
Pn-3-ara accounted for the rest of the total anthocyanins in cranberries. The sum of all individual anthocyanins quantied in RCE
(68 mg/100 g fresh weight) was within the range of published

TE

Relative p-Akt/mg protein

25000

(g)

(j)

(e)

(h)

(k)

(f)

(i)

(l)

(d)

ET

(a)

Cranberries have been widely studied due to their putative

health-promoting benets. These fruits are rich in phenolic compounds with high antioxidant properties such as anthocyanins,
proanthocyanidins, avonols, avanols, and phenolic acids
(Fig. 1). In this study, the total phenolic content of RCE was similar

(b)

(c)

Fig. 4b. Effects of RCE and WCE on cell migration in in vitro HUVEC examined by wound healing assay. Endothelial cell monolayers were wounded at time 0 and cultured with
0.2 mg/ml of RCE or WCE. Photographs of PBS-control (a, b, and c), PI3 K inhibitor (d, e, and f), RCE (g, h, i), and WCE (j, k, l) were taken after 0, 5, and 24 h of incubation,
respectively.

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

2002). Mazza and Miniati also attributed the high antioxidant

properties of cranberries to anthocyanins and other phenolic compounds (Mazza & Miniati, 1993). Prior et al. claimed that procyanidin fraction accounted for 54% of the total antioxidant capacity
measured in cranberries (Prior et al., 2001). The combination of
phytochemicals and synergistic mechanisms of these compounds
in the fruit matrix may be responsible for the antioxidant properties of cranberries.

4.1. Endothelial cell function

The ability of the cranberry extracts to act as in vitro antioxidants may be associated with the phenolic contents of the berries.
Polyphenolic compounds are known to change the redox sensitivity of the cells. PI3 is a redox sensitive kinase and therefore PI3
kinase-mediated phosphorylated Akt levels in vitro in HUVEC were
determined upon exposure to red and white cranberry extracts.
Our ndings indicate that the abundant anthocyanins in red cranberries and the abundant procyanidins in white cranberries may
have partly contributed to the activation of Akt via redox sensitive
PI3-kinase-mediated signaling pathway in cultured HUVEC. The
phosphorylation of Akt is known to activate downstream signaling
that results in endothelial nitric oxide synthase (eNOS) activation.
eNOS is an important regulator of cardiovascular homeostasis because it is the major catalyst for nitric oxide (NO) production in
vascular endothelial cells and plays a crucial role in the state of
blood vessel vasodilatation, and hence, maintain the vascular
integrity and regulate the blood pressure.
Grape-seed extract was used in this study as a positive control
since it is known to induce endothelium-dependent relaxation
through PI3 kinase/Akt signaling pathway (data not shown). Edirisinghe et al. demonstrated that in grape-seed extract, polyphenolic
compounds caused an endothelium-dependent relaxation of blood
vessels and that it was mediated by the activation of the PI3 K/Akt
signaling pathway through a redox-sensitive mechanism, resulting
in the phosphorylation of eNOS (Edirisinghe, Burton-Freeman, &
Kappagoda, 2008). The polyphenolic compounds in grape-seed extract are mostly proanthocyanidins which occur as mixtures of
oligomers and polymers of catechin and epicatechin. In addition,
the grape-seed extract consists mainly of dimers and trimers without gallic acid residues. Tetramers and pentamers, including larger
oligomers of proanthocyanidins, were responsible for the endothelium-dependent vasodilator effects demonstrated by (Fitzpatrick,
Bing, Maggi, Fleming, & OMalley, 2002). In cranberry extracts,
these oligomeric procyanidins were also abundant, thus, they

ET

AC

values (Timberlake, 1988) and comparable to the value estimated

from the spectrophometric monomeric anthocyanin assay
(Table 1).
Aside from anthocyanins, red cranberries are also a good source
of avonols and phenolic acids such as quercetin 3-galactoside,
myricetin 3-hexoside, quercetin 3-pentoside and caffeic acids.
Approximately 36% of the total avonols in red cranberries are in
the form of quercetin 3-galactoside. The values for quercetin and
myricetin were in similar range as reported for total quercetin
and myricetin from hydrolyzed extracts of cranberry (Bilyk &
Sapers, 1986; Hakkinen, Karenlampi, Heinonen, Mykkanen, &
Torronen, 1999). Bilyk and Sapers (1986) found that the darker colored berries contained higher levels of avonols compared with
the lighter colored berries, as reported in this study.
Individual procyanidin compounds such as procyanidin tetramer type A, procyanidin C1, and catechins accounted for most of
the total procyanidins in white cranberries. Procyanidin tetramer
values were 2.2-fold greater in WCE compared with RCE, and this
compound contributes 57% to the overall procyanidins observed
in white berries. In contrast, the tetramer was the largest molecular weight procyanidin detected in these extracts based on the result of mass spectrometer scan range (upper limit of 1200) and
chromatographic conditions. Procyanidin pentamers, hexamers,
and heptamers were found in cranberries as described in previous
reports (Foo, Lu, Howell, & Vorsa, 2000a; Prior, Lazarus, Cao, Muccitelli, & Hammerstone, 2001). The tetramer was the largest procyanidin found in these extracts as result of mass spectrometer
scan range and chromatographic conditions. It is possible that
the tetramer is the largest procyanidin detected in our study due
to solubility considerations. PBS was the dissolution solvent used
for procyanidin analysis in this study. Most studies of cranberry
procyanidin content employ fractionation schemes and dissolution
of the nal sample in organic/aqueous solvent mixtures which enhance solubility of the higher molecular weight procyanidins (Foo
et al., 2000b; Prior et al., 2001, 2010). The decline in the individual
procyanidin content of RCE compared with WCE was also observed
in other studies which measured procyanidins in cranberries as a
function of ripening (Vvedenskaya & Vorsa, 2004).
It is known that cranberries rank high among fruits in terms of
their antioxidant contents. The antioxidant capacity of cranberries
in both red and white samples were observed to be similar in the
three antioxidant assays (FRAP, free radical scavenging, and TEAC)
used in this study. These results may be due to the similarities in
the overall phenolic contents observed in both berries. The antioxidant effects of cranberries are mainly due to the high levels of
phenolic compounds present in the fruit (Sun, Chu, Wu, & Liu,

TE

550

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Fig. 4c. Effects of RCE and WCE on vessel formation in in vitro HUVEC examined by tube formation assay. Endothelial cell monolayers were cultured with 0.2 mg/ml of RCE
and WCE. Photographs of RCE-treated vessels: (a) with PI3K inhibitor, (b) with LY294002, (c) after 5 h, and (d) after 36 h of incubation. Photographs of WCE-treated vessels:
(e) with PI3K inhibitor, (f) with LY294002, (g) after 5 h, and (h) after 36 h of incubation. The PI3K inhibitor was 35 nmol/l wortmannin.

551

A.Z. Tulio Jr. et al. / Food Chemistry 157 (2014) 540552

In summary, the enhanced phosphorylation of PI3/Akt kinase, cell

wound healing, and tube vessel formation in HUVEC elicited by
RCE and WCE suggest that overall phenolic constituents (anthocyanins, proanthocyanidins, avonols, phenolic acids, and other
phenolic compounds) rather than individual compounds within
the cranberry matrix may be responsible for these biological effects. Furthermore, the results in this study suggest that red and
white cranberries may have vasodilator effects in vivo. Clinical
studies are warranted to conrm the potential cardioprotective
benets being offered by these fruits.

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TE

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may have similar action in HUVEC as those found in grape-seed

extract.
The p-Akt activity observed in red and white cranberry extracts
may also be attributed to the high anthocyanin and proanthocyanidin components, respectively. Red wine- and grape-derived products are found to be potent vasodilators in vitro wherein the
effect have been mainly attributed to the high content of polyphenols in these products. Specic anthocyanins from grapes and
other red fruits may be able to induce vasorelaxation (Edirisinghe,
Lu, Nalbandyan, & Kappagoda, 2007; Mullen et al., 2002). Edirisinghe et al. reported that strawberry extracts rich in phenolic compounds caused an endothelial relaxation on both aortic rings and
HUVECs through activation of PI3 kinase/Akt and attributed these
effects to the antioxidant properties of phenolic compounds (Edirisinghe, Burton-Freeman, Varelis, & Kappagoda, 2008). Their studies
suggest that the anthocyanin-rich strawberry extracts may result
in improving the functionality of endothelium and thus, reducing
blood pressure and the risk of cardiovascular disease. Recently,
we demonstrated that berry fruits such as wild blueberry, cranberry, and strawberry which contain signicant quantities of
anthocyanins induced the activation of PI3/Akt/eNOS in HUVEC
(Tulio et al., 2012).
The results of cell migration and tube formation assays in this
study indicate a possible PI3-kinase/Akt-dependent mechanism
that modulate endothelial function induced by phenolic
compounds in RCE and WCE when present at physiological concentrations (0.2 mg/ml). Tube formation assay is used as a quantitative
model for studying inhibitors and activators of angiogenesis.
Angiogenesis is the formation of new blood vessels from existing
vasculature and it is an integral part of both normal and pathological processes. Endothelial cells are the key cell type involved in
this process. During angiogenesis, these cells disrupt the surrounding 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 vessel
structures.
Exposure of HUVEC to RCE and WCE at physiological concentration enhanced the formation of the three dimensional vessels.
However at higher concentrations, cell deaths were observed
suggesting possible toxicity effects. Anthocyanin compounds are
bioavailable and generally found in nmol/l concentration after eating fruits rich in anthocyanins. Therefore, our data in the present
in vitro study is in agreement with in vivo setup. The phenolic constituents in red and white cranberries may also be responsible for
the induction of the endothelial cells to form three-dimensional
capillary-like tubular structures and provides evidence for a
phenolic-/redox-mediated contribution to angiogenesis, wound
healing, and cell death. This is in agreement with our previous
study that polyphenolic-rich berry fruits not only activated PI3/
Akt but also enhanced cell migration and tube formation in
endothelial cells (Tulio et al., 2012).

5. Conclusion
This study provides evidence that phenolic constituents such as
anthocyanins, proanthocyanidins, avonols and phenolic acids,
contribute to the antioxidant and biological properties of red and
white cranberries. Our data suggest that similarities in the overall
phenolic proles and total antioxidant properties of red and white
cranberries may likely explain why the activation of p-Akt did not
differ between red and white cranberry extract-treated HUVECs. In
addition, polyphenolic compounds derived from red and white
cranberry extracts may have caused the induction of redoxsensitive PI3 kinase/Akt pathway and activated downstream
signals resulting in endothelial cell migration and vessel formation.

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