The importance of polymerization and galloylation for
the antiproliferative properties of procyanidin-rich
natural extracts
D. Lizarraga
1
, C. Lozano
2
, J. J. Briede
´
3
, J. H. van Delft
3
, S. Tourin
˜
o
2
, J. J. Centelles
1
,
J. L. Torres
2
and M. Cascante
1,2
1 Biochemistry and Molecular Biology Department, Biology Faculty, University of Barcelona, Biomedicine Institute from University of Barcelona
(IBUB) and Centre for Research in Theoretical Chemistry, Scientific Park of Barcelona (CeRQT-PCB), Associated Unit to CSIC, Spain
2 Institute for Chemical and Environmental Research (IIQAB-CSIC), Barcelona, Spain
3 Department of Health Risk Analysis and Toxicology, Maastricht University, the Netherlands
Colorectal cancer is the third most commonly diagnosed
cancer in the world and is one of the major causes of
cancer-associated mortality in the USA [1,2]. Epidemio-
logical studies indicate that colon cancer incidence is

M. Cascante Serratosa, Department of
Biochemistry and Molecular Biology,
University of Barcelona, Biology Faculty,
Av. Diagonal 645, 08028 Barcelona, Spain
Fax: +34 934021219
Tel: +34 934021593
E-mail: [email protected]
(Received 2 May 2007, revised 3 July 2007,
accepted 18 July 2007)
doi:10.1111/j.1742-4658.2007.06010.x
Grape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widely
used as nutritional supplements. Procyanidin-rich fractions from grape and
pine bark extract showing different mean degrees of polymerization, per-
centage of galloylation (percentage of gallate esters) and reactive oxygen
species-scavenging capacity were tested on HT29 human colon cancer cells.
We observed that the most efficient fractions in inhibiting cell proliferation,
arresting the cell cycle in G
2
phase and inducing apoptosis were the grape
fractions with the highest percentage of galloylation and mean degree of
polymerization. Additionally, the antiproliferative effects of grape fractions
were consistent with their oxygen radical-scavenging capacity and their
ability to trigger DNA condensation–fragmentation.
Abbreviations
DMPO, 5,5-dimethyl-1-pyrolline-N-oxide; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; MTT,
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide.
4802 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS
catechins with some galloylation and mainly poly-
merized procyanidins [20]. In contrast, procyanidin
fractions from pine bark extracts do not contain gallo-

sulfates and methyl esthers, as described for the small
intestine [28]. They are also, in part, extensively metab-
olized to phenolic acids such as 3-hydroxyphenylvaleric
acid and 3-hydroxyphenylpropionic acid, which are
then absorbed as glucuronates and sulfates [27,29].
The gallate esters are more stable than the simple cate-
chins upon being metabolized [30] and may be more
bioavailable in the colon. Gallates have been reported
to inhibit cell growth, trigger cell cycle arrest in tumor
cell lines and induce apoptosis [31,32]. Furthermore,
studies have shown that they also offer protection by
scavenging reactive oxygen species such as superoxide
anion, hydrogen peroxide and hydroxyl radicals, which
cause destruction of biochemical components that are
important in physiological metabolism [33,34]. This
capacity to prevent the imbalance between high-level
oxidant exposure and antioxidant capacity, which
leads to several pathological processes, may contribute
to the chemopreventive effect of the gallic acid deriva-
tives. Because grape is a rich source of procyanidins
and contains some galloylation, procyanidin fractions
from grape could be potential antiproliferative com-
pounds of interest in the prevention of colon cancer.
In the present study, we investigated the relationship
of different structural factors of procyanidins, such as
the mean degree of polymerization and percentage of
galloylation, with their antiproliferative potential and
their scavenging capacity for hydroxyl and superoxide
anion radicals.
Results and Discussion

50
and IC
80
values that were approximately half
those of the homologous pine fractions. Moreover, as
was observed for pine fractions, the grape oligomers
were much more efficient than the monomers.
These results clearly show that both polymerization
and galloylation enhance the antiproliferative capacity
of polyphenolic fractions, which suggests that natural
polyphenolic extracts with a high degree of galloyla-
tion and containing oligomers are more suitable as
potential antiproliferative agents than those containing
monomers.
Cell cycle analysis
To examine the effects of grape and pine fractions on
the cell cycle pattern at concentrations equal to their
IC
50
and IC
80
values (Table 1), HT29 cells were treated
with each fraction for 72 h and then analyzed with a
fluorescence-activated cell sorter (FACS) (Fig. 2). The
cell cycle distribution pattern induced after grape poly-
phenolic treatments showed that, at IC
50
, the fractions
with the highest mean degree of polymerization and
percentage of galloylation (VIIIG and IVG) induced a

HT29 cell incubations with polyphenolic fractions
were performed at the concentrations described in
Experimental procedures. As show in Fig. 3A, at
IC
50
, the grape polyphenolic fractions VIIIG and
IVG induced significant percentages of apoptosis in
HT29 cells (approximately 25% and 17%, respec-
tively) as measured by FACS analysis. Fraction VI-
IIG also induced a significant percentage of necrosis
(approximately 5%), which could be due to a pro-
oxidant effect at high concentration [35,36]. More-
over, this percentage is negligible in comparison to
the apoptotic effect induced by fraction VIIIG on
HT29 cells. At a concentration equal to IC
80
, frac-
tions VIIIG and IVG induced significant percentages
of apoptosis in HT29 cells (approximately 24% and
18%, respectively) and fraction VIG also displayed
a significant effect (approximately 22%) (Fig. 3A).
Fraction VIG is chemically classified in Table 1 as
having the third highest mean degree of polymeriza-
tion and galloylation, situated below fractions VIIIG
and IVG, respectively.
The pine fractions VIIIP and IVP were analyzed
to determine whether galloylation enhanced the apop-
totic induction observed; a significant percentage of
apoptosis was induced, but the percentages were
Table 1. Comparative chemical characteristics and HT29 cell growth inhibition of grape and pine fractions. Percentage of galloylation (%G),

revealed early membrane alterations at the beginning
of the apoptotic process. Chromatin condensation was
also seen, and confirmed the induction of apoptosis by
fractions VIIIG and IVG (Fig. 4A). Finally, DNA
fragmentation was detected as a late marker of apop-
tosis by observing the pattern of DNA laddering at
IC
50
and IC
80
(Fig. 4B).
Oxygen radical scavenging activity as detected
by ESR spectroscopy
The next series of experiments used ESR spectroscopy
to test the radical-scavenging capacity of the fractions.
The results show that the oligomeric fractions (VIIIG,
IVG, VIIIP and IVP), which were the most effective in
the previous assays using HT29 cells, were also the
most efficient as hydroxyl radical and superoxide scav-
engers at 50 lm (Fig. 5A). Fraction VIIIG was the
most potent radical scavenger, followed by fraction
IVG and the pine fractions VIIIP and IVP. The same
levels of efficiency were also observed in the induction
of cell cycle arrest and apoptosis. When fractions were
tested at their respective IC
50
values, fractions VIIIG,
IVG, VIIIP and IVP were again the most effective
(Fig. 5B). There is a clear relationship between high
scavenger capacity ⁄ lower IC

VIIIG
VIIIG
G1SG2 G1SG2
IEC-6 IEC-18
Cell cycle at IC50 (Grape fractions)
A
BC
0 10203040506070
ct
VIIIG
IVG
VIG
OWG
VG
ct
VIIIG
IVG
VIG
OWG
VG
ct
VIIIG
IVG
VIG
OWG
VG
G1 S G2
G1 S G2
Cell cycle stages
Cell cycle stages

VIIIG
IVG
VIG
OWG
VG
ct
VIIIG
IVG
VIG
OWG
VG
ct
VIIIG
IVG
VIG
OWG
VG
*
*
*
*
*
*
*
*
Fig. 2. Cell cycle analysis of HT29, IEC-6 and IEC-18 cells treated with grape and pine polyphenolic fractions. (A) HT29 cells at their respec-
tive grape IC
50
and IC
80

natural antioxidant extracts, each of which has been
claimed to exert chemopreventive activity in cellular
models of cancer [43,44]. Recent publications have sta-
ted that the antiproliferative activity of flavonoids is
dependent on particular structure motifs, such as gal-
late groups and degree of polymerization [45,46].
Our results suggest that polymerization plays a
greater role than galloylation in cell cycle arrest in
HT29 cells. Interestingly, galloylation appears to be
more influential than polymerization in the biological
apoptosis activities tested and in the hydroxyl and
superoxide anion radical-scavenging capacity of the
fractions when compared at the same concentration of
50 lm (Fig. 5A). The galloylated and polymerized
grape procyanidins were the most effective hydroxyl
radical scavengers and also triggered cell cycle arrest
and apoptosis, and although this does not necessarily
indicate that both effects are mechanistically related,
such as relationship cannot be ruled out. The present
results are in general agreement with previously
reported data for pure compounds [47]. Essentially, the
induction of apoptosis seems to be related to the elec-
tron transfer capacity of the phenolic extracts. Other
antioxidants with anti-inflammatory and anticancer
activities have been reported, such as edaravone [48]
and the flavonoid silydianin [49], both of which induce
apoptosis and act as radical scavengers.
It was also observed that the most efficient procyani-
din fraction, VIIIG, which induced approximately
Apoptosis at IC50 (Pine fractions)

OWG
VG
ct
VIIIG
IVG
VIG
OWG
VG
Early Late Necrotic
Cell stage
% Cell distribution (HT29)
0
5
10
15
20
% Cell distribution (HT29)
0
5
10
15
20
% Cell distribution (HT29)
0
5
10
15
20
% Cell distribution
**

Apoptosis at IC50 (Grape fraction)
ct
VIIIG
ct
VIIIG
ct
VIIIG
ct
VIIIG
ct
VIIIG
ct
VIIIG
Early Late Necrotic Early Late Necrotic
IEC-6 IEC-18
Cell stage
*
Fig. 3. Apoptosis was induced in HT29 tumor cells and did not affect normal epithelial cells. (A) HT29 cells after treatment with grape poly-
phenolic fractions at their respective IC
50
and IC
80
values. (B) HT29 cells after treatment with pine polyphenolic fractions at their respective
IC
50
values. (C) IEC-6 and IEC-18 cells treated with grape fraction VIIIG at HT29 IC
50
. Percentages of cells in different cell stages are shown
(cell stages shown on the x-axis). (% cells ± SEM, *P < 0.05, **P < 0.001). Experiments were performed in triplicate.
Antiproliferative properties of natural extracts D. Lizarraga et al.

purchased from Biological Industries (Kibbutz Beit Ha-
emet, Israel). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetra-
zolium bromide (MTT), dimethylsulfoxide, propidium
iodide (PI) and Igepal CA-630 were obtained from Sigma
Chemical Co. NADH disodium salt (grade I) was supplied
by Boehringer (Mannheim, Germany). RNase and agarose
MP were obtained from Roche Diagnostics (Mannheim,
Germany). Iron(II) sulfate heptahydrate was obtained from
Merck (Darmstadt, Germany) a-a-a-Tris(hydroxymeth-
yl)aminomethane was obtained from Aldrich-Chemie
(Steinheim, Germany) and moviol from Calbiochem (La
Jolla, CA, USA). The annexin V ⁄ fluorescein isothiocyanate
(FITC) kit was obtained from Bender System (Vienna, Aus-
tria), the Realpure DNA extraction kit, including protein-
ase K, was obtained from Durviz S.L. (Paterna, Spain),
and Blue ⁄ Orange Loading dye and the 1 kb DNA ladder
were purchased from Promega (Madison, WI, USA).
5,5-Dimethyl-1-pyrolline-N-oxide (DMPO), hydrogen per-
oxide, phenazine methosulfate and Hoescht were obtained
from Sigma (St Louis, MO). DMPO was further purified
by charcoal treatment.
Fractions
The polyphenolic mixtures were obtained previously in our
laboratories [26,50] and contain mainly procyanidins.
OWG and OWP are composed of species that are soluble
in both ethyl acetate and water, and the rest of the frac-
tions (G for grape, P for pine) were generated by a combi-
nation of preparative RP-HPLC and semipreparative
chromatography on a Toyopearl TSK HW-40F column
(TosoHass, Tokyo, Japan), which separated the compo-

Control
48H (VIIIG)
72H (VIIIG)
48H (IVG)
72H (IVG)
Control
A
B
A= IC50
B= IC80
Fig. 4. Induction of apoptosis by grape fractions VIIIG and IVG in
HT29 cells. (A) Nuclear condensation of HT29 cells. Arrows indicate
the apoptotic cells with condensed and fragmented nuclei. (B) DNA
laddering induced in both treatments.
D. Lizarraga et al. Antiproliferative properties of natural extracts
FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS 4807
Cells were cultured and passaged in DMEM supplemented
with 10% heat-inactivated fetal bovine serum and 0.1%
streptomycin ⁄ penicillin.
Cell growth inhibition
HT29, IEC-6 and IEC-18 cells were seeded densities
of 3 · 10
3
cells per well, 5 · 10
3
cells per well and
1 · 10
3
cells per well, respectively, in 96-well flat-bottomed
plates. After 24 h of incubation at 37 °C, the polyphenolic

3
cells per well and 29.1 · 10
3
cells per well,
respectively. The number of cells was determined as cells
per area of well, as used in the cell growth inhibition assay.
The culture was incubated for 72 h in the absence or pres-
ence of the polyphenolic mixture at its respective IC
50
values. The cells were then trypsinized, pelleted by centri-
fugation [371 g for 3 min at room temperature (RT) using
a 5415D centrifuge (Eppendorf, Hamburg, Germany) and a
24-place fixed angle rotor] and stained in Tris-buffered
saline (NaCl ⁄ Tris) containing 50 lgÆmL
)1
PI, 10 lgÆmL
)1
RNase free of DNase and 0.1% Igepal CA-630 in the dark
for 1 h at 4 °C. Cell cycle analysis was performed with a
FACS (Epics XL flow cytometer; Coulter Corporation,
Hialeah, FL, USA) at 488 nm. All experiments were
performed in triplicate, as described previously [47].
Apoptosis analysis by FACS
Annexin V ⁄ FITC and PI staining were measured by FACS.
Cells were seeded, treated and collected as described in
Superoxide anion radical scavenger capacity
** **
**
**
**

Ct
VIIIG
IVG
OWG
VG
VIIIP
IVP
OWP
VP
Polyphenolic fractions at 50 µ
M

Percentage hydroxyl radical system
0
20
40
60
80
100
120
Ct
VIIIG
IVG
OWG
VG
VIIIP
IVP
OWP
VP
Percentage hydroxyl radical system

Ct
VIIIG
IVG
OWG
VG
VIIIP
IVP
OWP
VP
Polyphenolic fractions at IC50
**
**
**
**
**
**
**
**
Fig. 5. Scavenging activity of OH and O
ÁÀ
2
analyzed by ESR. Grape and pine fractions were evaluated at: (A) 50 lM and (B) IC
50
in HT29 cells
in hydroxyl radical- and superoxide anion radical-generating systems, as described in Experimental procedures. Experiments were performed
in duplicate (*P < 0.05, **P < 0.001).
Antiproliferative properties of natural extracts D. Lizarraga et al.
4808 FEBS Journal 274 (2007) 4802–4811 ª 2007 The Authors Journal compilation ª 2007 FEBS
the previous section. Following centrifugation [371 g for
3 min at RT using a 5415D centrifuge (Eppendorf) with

amounts of DNA (20 lg), estimated by measuring absorp-
tion at 260 ⁄ 280 nm, were electrophoretically separated on
1% TAE agarose gel and viewed under a UV transillumi-
nator (Vilber Lourmat, Marne-la-Valle
´
e, France).
Apoptosis detection by Hoescht staining
Apoptotic induction was also studied using Hoescht stain-
ing. Samples were incubated with grape fractions VIIIG
and IVG at 0, 48 and 72 h. After incubation, cells were
trypsinized and fixed with cold methanol for 1 h at ) 20 °C.
After being rinsed with NaCl ⁄ P
i
three times, cells were
stained in the dark with Hoescht (50 ngÆmL
)1
in NaCl ⁄ P
i
)
for 50 min. Finally, cells were rinsed, suspended in NaCl ⁄ P
i
and diluted 1 : 2 with moviol. The samples were mounted
on a slide and observed with a fluorescent microscope at an
excitation wavelength of 334 nm and an emission wave-
length of 365 nm.
ESR spectroscopy
ESR measurements were performed at concentrations that
caused 50% cell growth inhibition (IC
50
) and 50 lm grape

2,
and hydroxyl radicals generated in this system
were trapped by DMPO, forming a spin adduct detected by
the ESR spectrometer. The typical 1 : 2 : 2 : 1 ESR signal
of DMPO-OH was observed. The superoxide radical genera-
tion system used performed using 50 lm of the reduced form
of b-NADH and 3.3 lm phenazine methosulfate, and the
superoxide radicals generated in this system were trapped by
DMPO, forming a spin adduct detected by the ESR spec-
trometer. The typical ESR signal of DMPO-OOH ⁄ DMPO-
OH was observed. The OH and O
2
-scavenging activity was
calculated on the basis of decreases in the DMPO-OH or
DMPO-OOH ⁄ DMPO-OH signals, respectively, in which the
coupling constant for DMPO-OH was 14.9 G.
Data presentation and statistical analysis
Assays were analyzed using the Student’s t-test and
were considered statistically significant at P<0.05 and
P<0.001. The data shown are representative of three
independent experiments, with the exception of ESR experi-
ments, which were performed in duplicate. ESR experi-
ments were analyzed separately by radicals, Two-way
anova was applied (day was a block factor; due to the
nonsignificant effect of the day factor, we reanalyzed with a
one-way anova), and finally, a multicomparison between
compounds with respect to the control was performed.
anova with Bonferroni and Scheffe post hoc test was per-
formed in ESR experiments.
Acknowledgements

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