Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - Studies in platelets, monocytes, and human endothelial cells

Article abstract of DOI:10.1021/jf035360v

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used, Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-α (TNF-α) secretion in lipopolysaccharide plus interferon-γ-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-α-induced monocyte chemoattractant protein production in primary human endothelial cells.

Full text of DOI:10.1021/jf035360v

3378 J. Agric. Food Chem. 2004, 52, 3378−3384
Antioxidant and Cellular Activities of Anthocyanins and Their
Corresponding Vitisins AsStudies in Platelets, Monocytes, and
Human Endothelial Cells
MARIÄA GARCIÄA-ALONSO,† GERALD RIMBACH,‡,§ JULIAÄ N C. RIVAS-GONZALO,† AND
SONIA DE PASCUAL-TERESA*,‡,|
Unidad de Nutricio´n y Bromatolog´ıa, Facultad de Farmacia, University of Salamanca, Campus Miguel
de Unamuno, 37007 Salamanca, Spain, and School of Food Biosciences, Hugh Sinclair Human
Nutrition Unit, University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom
During red wine aging, there is a loss of anthocyanins and the formation of various other pigments,
so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins
with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most
abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and
their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2′-azinobis
(3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding
vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the
assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation
and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production
and tumor necrosis factor-R (TNF-R) secretion in lipopolysaccharide plus interferon-γ-activated
macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet
aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects
against TNF-R-induced monocyte chemoattractant protein production in primary human endothelial
cells.
KEYWORDS: Anthocyanin; vitisin A; antioxidant activity; nitric oxide production; TNF-r secretion; MCP-1
production; platelet aggregation
INTRODUCTION
through the interaction of the original anthocyanins with pyruvic
acid (6). The structure of vitisin A is based on the anthocyanidin-
3-glucoside with an additional C3O2 between position C4 and
the 5-hydroxyl group of the molecule (7). The chemical
interaction between anthocyanins and pyruvic acid has been
shown to influence the color of red wine and may also impact
the antioxidant and biological properties of anthocyanins.
Although there is evidence that anthocyanins provide bene-
ficial effects to human health, their cellular mechanism of action
remains largely unknown. The purpose of the present study was
2-fold: to analyze the antioxidant activity of the most abundant
anthocyanins present in red wine, Dp-3-glu, Pet-3-glu, and Mv-
3-glu, in comparison to their corresponding vitisins A and to
study the effect of anthocyanins and vitisins on NO production
and TNF-R secretion in macrophages, collagen-induced platelet
aggregation, and MCP-1 production in primary HUVEC. We
have chosen these three cell types since they play a key role in
the pathogenesis of atherosclerosis.
Anthocyanins are a group of phenolic compounds responsible
for the red-blue color of many fruits and vegetables. They are
glycosylated polyhydroxy or polymethoxy derivatives of 2-
phenylbenzopyrylium or flavilium salts (1). The main dietary
sources of anthocyanins include red-colored fruits and vegetables
and red wine. The intake of anthocyanins in humans has been
estimated to be 180-215 mg/day (2) in the Unites States, which
is considerably higher than the intake of other flavonoids (23
mg/day) including quercetin, kaempferol, myricetin, apigenin,
and luteolin (3). Epidemiological studies suggest that a moderate
consumption of anthocyanins may be, at least partly, associated
with a protection against coronary heart disease (4, 5). During
red wine aging, there is a loss of anthocyanins and it appears
that other pigments, so-called vitisins A (Figure 1), are formed
* To whom correspondence should be addressed. Tel: +34 91 549 23
00. Fax: +34 91 549 36 27. E-mail: soniapt@if.csic.es.
† University of Salamanca.
‡ University of Reading.
MATERIALS AND METHODS
§ Current address: Insitute of Human Nutrition and Food Science,
Christian Albrechts University, D-24111 Kiel, Germany.
| Current address: Department of Plant Food Science and Technology,
Instituto del Frio, CSIC, Jose Antonio Novais 10, E-28040 Madrid, Spain.
Isolation and Purification of Anthocyanins. Anthocyanins (Dp-
3-glu, Pet-3-glu, and Mv-3-glu) were isolated from a methanol-acid
red grape skin extract by semipreparative high-performance liquid
10.1021/jf035360v CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/11/2004

Activities of Anthocyanins and Vitisins A
J. Agric. Food Chem., Vol. 52, No. 11, 2004 3379
Figure 1. Reaction of anthocyanins (Dp-3-glu, Pt-3-glu, and Mv-3-glu) and pyruvic acid to form vitisins A (Vitdp-3-glu, Vitpt-3-glu, and Vitmv-3-glu), which
occur during red wine aging.
chromatography (HPLC) using a Waters 600 chromatograph. The
column was an Ultracarb ODS, 5 µm, 250 mm × 10.00 mm
(Phenomenex). The solvents were 5% acetic acid (A) and methanol
(B) with the following gradient: from 10 to 15% B over 15 min,
isocratic 15% B for 5 min, from 15 to 20% B over 10 min, from 20 to
25% B over 10 min, from 25 to 30% B over 10 min, from 30 to 45%
B over 10 min, and from 45 to 10% B over 10 min at a flow rate of
3 mL/min. Detection was carried out at 520 nm.
with distilled water as a blank. Data were expressed relative to values
obtained for ascorbic acid (200 µM) and expressed as ascorbic acid
equivalents.
TEAC. Sixteen hours prior to the assay being performed, the ABTS
radical was prepared by adding 5 mL of a 4.9 mM potassium persulfate
solution to 5 mL of a 14 mM ABTS solution (10). This solution was
diluted in distilled water to yield an absorbance of 0.70 at 734 nm
(Perkin-Elmer UV/Vis Lambda Bio 20). The final reaction mixture
contained 10 µL of standard or test compound in 1 mL of ABTS
solution. The samples were vortexed for 10 s, and 6 min after the
addition, the absorbance was recorded and compared to the ABTS
radical solution plus distilled water. A standard curve was obtained by
using Trolox as an internal standard (range, 0-100 µM). Different
dilutions of the test components were assayed in order to express the
TEAC values as the µmols of Trolox with the antioxidant capacity
corresponding to 1.0 µmol of the test substance (11).
ORAC. The procedure was based on a previous report of Cao et al.
(12). In the final assay mixture (3.35 mL total volume), sodium
fluorescein (7.0 × 10-8 M) was used as a target of free radical damage,
AAPH (17 mM) was used as a peroxyl radical generator, and the Trolox
was used as a control antioxidant standard. The test components were
added to the assay mixture at 5, 10, and 15 µM in every case. The
fluorescent filters were set to pass the light with an excitation
wavelength of 493 nm and an emission wavelength of 513 nm, and
the fluorescence was measured every 5 min after the addition of AAPH.
The final ORAC values were calculated by using a regression equation
between the Trolox concentration and the net area under the fluorescein
decay curve and were expressed as Trolox equivalents. The relative
ORAC values (Trolox equivalents) were calculated as relative ORAC
value ) [(AUCsample - AUCBlank)/(AUCTrolox - AUCblank) × (molarity
of Trolox/molarity of sample).
Macrophages. RAW 264.7 cells, a murine cell line of monocyte
macrophages (obtained from the European Collection of Cell Culture,
Salisbury, U.K.) were maintained at 37 °C in 5% CO2 according to
standard protocols (13). The medium consisted of Dulbecco’s modified
Eagle’s medium containing 10% heat-inactivated fetal bovine serum,
2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and
100 µg/mL streptomycin (Sigma, Poole, U.K.). The cells were plated
in six well plates at a density of 5 × 105 in 3 mL of medium, cultured
for 48 h until the cells reached 80% confluence, and then treated as
follows. After pretreatment with anthocyanins, vitisins A, both up to
50 µM, or DMSO for 6 h, cells were stimulated with 500 ng/mL LPS
plus 100 U/mL IFN-γ (Sigma) for 24 h.
Endothelial Cells. Primary HUVEC (Biowhittaker, Wokingham,
U.K.) were grown in cell basal medium (HEPES and bicarbonate-
buffered medium), which was supplemented with cell growth supple-
ment (containing heparin, hydrocortisone, human epidermal growth
factor, and fetal bovine serum) and antibiotics (gentamicin and
amphotericin B) (Large Vessel Endothelial Cell Growth Medium
Package, TCS Cell Works Ltd., Liverpool, U.K.). Passages were
performed according to standardized protocols using trypsin/ethylene-
Vitisin A Synthesis. The synthesis of vitisins A was performed as
described previously by Romero et al. (7). Pyruvic acid was added to
pure anthocyanin and dissolved in potassium hydrogen tartrate buffer
containing 10% ethanol, in a molar ratio of pyruvic acid to pure
anthocyanins of 300:1. The pH was adjusted to 3.7 by the addition of
Na2CO3, and the solution was incubated at 32 °C in the dark in the
presence of air. The identity of the vitisins A was confirmed in the
liquid chromatography-mass spectrometry (LC-MS) analysis, and the
kinetics of formation was followed by HPLC analysis as described
below.
The purity of anthocyanins and vitisins A was tested by HPLC
analysis following the method of de Pascual-Teresa et al. (8) with slight
modifications. Briefly, Hewlett-Packard 1100 HPLC equipment was
used with a quaternary pump and a photodiode array detector. The
column was an Aqua C18, 5 µm (150 mm × 4.6 mm) (Phenomenex),
thermostated at 35 °C. Solvents were 0.1% trifluoroacetic acid (A) and
acetonitrile (B) with the following gradient: isocratic 10% B for 5
min, from 10 to 15% B over 15 min, isocratic 15% B for 5 min, from
15 to 18% B over 5 min, and from 18 to 35% B over 20 min at a flow
rate of 0.5 mL/min. The detection was made in the photodiode
apparatus, selecting 520 nm as the preferred wavelength.
The identity of the vitisins A synthesized was confirmed by HPLC
using a dual on-line detection by diode array spectrophotometry and
MS. MS was performed using a Finningan LCQ equipped with an API
surface, using an electrospray ionization interface. The HPLC system
was connected to the probe of the mass spectrometer via the diode
array detector cell outlet, using polyethyl etherketone tubing. Both the
auxiliary and the sheath gas were a mixture of nitrogen and helium at
flow rates of 1.2 and 6 L/min, respectively. The capillary temperature
was 195 °C, and the capillary voltage was 4 V. The MS detector was
programmed to perform a series of three consecutive scans: a full scan
from 120 to 1500 amu, an MS2 scan of the most abundant ion in the
full mass, and an MS3 scan of the most abundant ion in the MS2. The
normalized energy of collision was 45%. The spectra were recorded in
the positive ion mode.
Antioxidant Activity. FRAP. The FRAP assay was performed as
previously described (9). The FRAP reagent was freshly prepared each
day by mixing together 10 mM TPTZ and 20 mM iron(III) chloride in
0.25 M acetate buffer, pH 3.6. A 100 µL amount of test components
(100 µM) was added to 3 mL of FRAP reagent, and the absorbance
was read at 593 nm (Perkin-Elmer UV/Vis Lambda Bio 20) after
incubation at room temperature for 6 min, using the FRAP reagent

3380 J. Agric. Food Chem., Vol. 52, No. 11, 2004
Garc´ıa-Alonso et al.
diaminetetraacetic acid, buffered saline rising solution, and trypsin
blocking solution (Passage PacksZHR-9941, TCS Cell Works Ltd.)
and by diluting the cells 1:4. HUVEC cells, within passages 2-4, were
seeded in 3 mL of medium in six well plates and cultured until they
reached 80-90% apparent confluence. After pretreatment with 50 µM
anthocyanins, vitisins A, or DMSO for 24 h, cells were stimulated with
40 ng/mL of TNF-R (Sigma) for 24 h.
Cell Viability. The uptake of neutral red dye was used to measure
cell viability as described previously (14). Macrophages and HUVEC
were pretreated with anthocyanins and vitisins A for 6 or 24 h,
respectively. After activation of RAW264.7 macrophages with LPS
plus INF-γ and HUVEC with TNF-R for 24 h, the culture medium
was removed and replaced with fresh medium containing 60 µg/mL of
neutral red for 3 h at 37 °C. Following incubation with the neutral red
dye, the medium was removed and the cells were extracted using a
solution comprising 50:49:1 (v/v/v) ethanol, water, and glacial acetic
acid. The absorbance was recorded at 540 nm using a microplate reader.
Neither anthocyanins nor vitisins A exhibited any cytotoxic effect in
macrophages and HUVEC up to a concentration of 100 µM.
RESULTS
Test Compounds. Purities of the all anthocyanins and vitisins
A used in this work, which were tested by HPLC analysis, were
93-94%. The identities of the vitisins A synthesized, Vitdp-
3-glu, Vitpt-3-glu, and Vitmv-3-glu, were confirmed by LC-
MS, which showed (Figure 2), for their peaks, molecular ions
at m/z 533, 547, and 561, respectively. In their MS-MS spectra,
major fragments appeared at m/z 371, 385, and 399, respectively
(-162 amu, loss of a glucose moiety), corresponding to the
aglycons of the corresponding glycosides of vitisins A.
For all experiments, the anthocyanins as well as the vitisins
A were dissolved in DMSO and stored at -80 °C. When test
compounds in DMSO were added to the medium, the final
DMSO concentration was e0.1% (v/v), and in every case, a
control containing the same amount of DMSO was done.
FRAP, TEAC, and ORAC Values. FRAP values indicate
the ability of anthocyanins and vitisins A to reduce Fe3+ to Fe2+
.
NO Production. The NO production was assessed by measurement
of nitrite concentration (NO2-) in the medium using the Griess reaction.
Supernatants of cultured macrophages were collected and deproteinized
with 0.3 M NaOH and 0.3 M ZnSO4. An equal volume of the Griess
reagent (1% sulfanilamide/0.1% N-(1-naphthyl)ethylenediamine di-
hydrochloride/2.5% H3PO4) and the deproteinized samples was incu-
bated for 10 min at room temperature protected from light. The nitrite
concentration was determined by measuring the absorbance at 548 nm
against a standard curve for sodium nitrite (15).
As summarized in Figure 3A, both anthocyanins and vitisins
A showed a higher iron reducing activity than the reference
ascorbic acid. Among the anthocyanins tested, dp-3-gluc was
the most potent test component, followed by pt-3-glu and mv-
3-glu. Vitmv-3-glu showed a slightly higher, although not
statistically or significantly different, FRAP value than Vitdp-
3-glu and Vitpt-3-glu.
The TEAC assay measures the ability of an antioxidant to
scavenge the ABTS free radical. The results are compared to
the ABTS scavenging activity of Trolox, giving a Trolox
equivalent value. In the TEAC assay, all test compounds showed
a higher antioxidant activity than the standard Trolox (Figure
3B). Dp-3-glu was the most potent scavenger of the free ABTS
radical (four times greater than that of Trolox), followed by
mv-3-glu with a TEAC value of 3.7. The other test compounds
exhibited an about 3-fold higher ABTS scavenging activity than
Trolox.
TNF-r Secretion in RAW 264.7. Supernatants collected for NO
production assessment were also used to determine TNF-R secretion
in RAW 264.7. Upon collection, samples were centrifuged at 13 000
rpm for 10 min and the supernatants were kept at -80 °C until they
were analyzed. The TNF-R secretion was measured using a com-
mercially available enzyme-linked immunosorbent assay (ELISA) kit
(Quantine M mouse TNF-R immunoassay, R&D Systems Europe,
Abingdon, U.K.).
MCP-1 Secretion in HUVEC. Upon collection of the supernatant
from HUVEC cells, samples were centrifuged at 13 000 rpm for 10
min and the supernatants were kept at -80 °C until analyzed. The
secretion of MCP-1 by HUVEC was measured by using a commercially
available ELISA kit (R&D Systems Europe).
The ORAC assay determines the potency of antioxidant to
scavenge peroxyl free radicals. The anthocyanins as well as
Vitdp-3-glu were about 5-fold more potent, and Vitpt-3-glu and
Vitmv-3-glu were about 2-fold more potent than Trolox in
scavenging the peroxyl free radical (Figure 3C).
Platelet Isolation and Platelet Aggregation Measurement. Venous
blood samples were obtained from volunteers within the Hugh Sinclair
Unit of Human Nutrition at the University of Reading. Blood for platelet
aggregation studies was drawn into 4.5 mL sodium citrate vacutainer
tubes (Becton Dickinson, Plymouth, U.K.) and kept at room temperature
until analysis. Platelet aggregation studies were performed in a two
channel whole blood impedance aggregometer (model 590, Chronolog
Corporation, Labmedics Ltd., Cheshire, U.K.). Whole blood was diluted
(500 µL), 1:1, with PBS, placed in cuvettes with a stir bar, and warmed
to 37 °C. Two microliters of the anthocyanins, vitisins A, or DMSO
controls was then added, and the sample was stirred for 10 s. The blood
samples were incubated with the test compound for 10 min. The
aggregometer’s impedance probe was placed into the warmed blood,
and the baseline impedance was set to zero on the chart recorders.
Collagen (1.5 µg/mL) was added to the cuvette at time zero, and the
impedance increased proportionally to the amount of platelet aggrega-
tion on the impedance probe. The maximum aggregation was measured
at 6 min with the extent of aggregation expressed in ohms of the
impedance using Aggrolink software (Chronolog Corporation, Lab-
medics Ltd.). Control aggregation curves were obtained in the absence
of anthocyanins and vitisins A and were compared with the curves
obtained in the presence of the test compound. The results were used
to calculate the percentage inhibition (100 - percentage of aggregation).
Genistein (100 µM) was used as a positive control for this assay.
NO Production and TNF-r Secretion in Monocytes.
Following 24 h of activation of RAW264.7 macrophages with
INF-γ plus LPS, levels of nitrite and TNF-R increased from
basal levels to 80 µM/106 cells and 2.7 ng/mL, respectively.
Pretreatment of RAW 264.7 with anthocyanins and vitisins A
did not affect nitrite production or TNF-R secretion. In contrast,
50 µM genistein was found to reduce TNF-R and NO production
by 55 and 49%, respectively, comparable to previously published
data (16).
MCP-1 Secretion in Endothelial Cells. A statistically
significant inhibitory effect on MCP-1 secretion was found when
HUVEC were pretreated with anthocyanins; this inhibition was
not evident to the same extent in the case of the corresponding
visitins A (Figure 4). The anthocyanin pt-3-glu (∼70% inhibi-
tion) was the most potent inhibitor of MCP-1 secretion followed
by dp-3-glu (∼50% inhibition) and mv-3-glu (∼40%). Pretreat-
ment of HUVEC with Vitdp-3-glu inhibited MCP-1 production
by 35%, whereas Vitpt-3-glu and Vitmv-3-glu did not affect
endothelial MCP-1 secretion.
Platelet Aggregation. Neither anthocyanins nor vitisins A
inhibited collagen-induced platelet aggregation at concentrations
up to 50 µM. However, a significant inhibitory effect was
evident for genistein (29%), used as a positive control (16).
Statistical Analysis. Data are presented as means ( standard
deviations (STDEV) of between three and five independent experiments
performed in duplicate. Statistical analysis was carried out using analysis
of variance and Student’s t-test (SPSS for Windows version 10.0).

Activities of Anthocyanins and Vitisins A
J. Agric. Food Chem., Vol. 52, No. 11, 2004 3381
Figure 2. MS and MS/MS spectra of Vitdp-3-glu (A), Vitpt-3-glu (B), and Vitmv-3-glu (C).
DISCUSSION
monocytes, and endothelial cells. Here, we investigate both the
antioxidant and the cellular effects of anthocyanins and vitisins
A.
Anthocyanins are thought to play a role in the prevention of
coronary heart disease, but the mechanisms by which they and
their corresponding vitisins A mediate these effects have not
been fully established. Antioxidant properties are an obvious
candidate. However, although antioxidant properties of antho-
cyanins have been described, the free radical scavenging and
iron chelating activities of vitisins A in comparison to antho-
cyanins are largely unknown. Furthermore, little is known about
the cellular effects of anthocyanins and vitisins A in platelets,
Antioxidant Properties of Anthocyanins and Vitisins A.
FRAP, TEAC, and ORAC data indicate that both anthocyanins
and vitisins A have the potency to reduce Fe3+ to Fe2+ and to
scavenge the ABTS and peroxyl free radicals. The FRAP and
TEAC values of anthocyanins decreased in the order dp-3-glu
> mv-3-glu ) pt-3-glu. The structural difference among the
anthocyanins tested is the substitution in the 3′- and 5′-position
in the B ring. Dp-3-glu is the only compound studied that

3382 J. Agric. Food Chem., Vol. 52, No. 11, 2004
Garc´ıa-Alonso et al.
Figure 3. FRAP (A), TEAC (B), and ORAC (C) values for the anthocyanins and vitisins A. Data are the means ± STDEV of the three independent
experiments performed in triplicate. Different lower case letters indicate significant (P < 0.05) differences between anthocyanins or vitisins A. Different
upper case letters indicate significant (P < 0.05) differences between each anthocyanin and its corresponding vitisin A.
Figure 4. Effect of 50 µM anthocyanins and vitisins A on MCP-1 secretion in TNF-R-activated HUVEC. Results were expressed as a percentage of
MCP-1 secretion. Data were compared to activated controls not treated with anthocyanins or vitisins A.
contains the 3′-, 4′-, and 5′-hydroxy group (gallocatechol
structure) in the B ring. Mv-3-glu and pt-3-glu with one and
two hydroxyl groups, respectively, in the B ring exhibited a
lower antioxidant activity than dp-3-glu indicating that the
gallocatechol group is an important determinant for the radical
scavenging potential of polyphenols. Furthermore, our data
demonstrate that anthocyanins exhibit a higher antioxidant
activity than their corresponding vitisins A. Thus, the incorpora-
tion of an additional C3O2 group in positions 4 and 5 of the
molecule decreases the ability of vitisins A to scavenge ABTS
and peroxyl free radicals. This decrease in antioxidant activity
may be due to the lack of the 5-hydroxyl group in the vitisin A
molecule, which together with the 7-hydroxyl group contributes
to the antioxidant activity of anthocyanins (17).
Cellular Activity of Anthocyanins and Vitisins A. Platelets,
macrophages, and endothelial cells were chosen as model
systems since these cell types play a critical role in the
pathogenesis of thrombosis and atherosclerosis. Endothelial cells
appear to be involved from the earliest stages. At sites of
endothelial injury, perhaps induced by initial accumulation and
modification of lipids, these cells produce chemokines such as
MCP-1, which attract monocytes. Subsequently, the monocytes
are activated to produce proinflammatory molecules such as
TNF-R and NO and accumulate lipids to become the foam cells
characteristic of fatty streaks. Platelets may also be involved in
these early stages of atherosclerosis, in response to the endo-
thelial damage, and are clearly important in the aetiology of
thrombogenesis (18).

Activities of Anthocyanins and Vitisins A
J. Agric. Food Chem., Vol. 52, No. 11, 2004 3383
Monocyte Function. Monocytes are the principal mediators
in inflammation and are involved in the development of
atheromatous plaques. Macrophages themselves, once activated,
secrete a variety of biologically active molecules, including NO
and TNF-R (19).
NO produced by macrophages is a cytotoxic mediator and
contributes to the events initiated during host defense (20). It
has also been shown that the production of TNF-R is crucial
for the synergistic induction of NO synthesis in IFN-γ- and/or
LPS-stimulated macrophages (15).
fraction) from red wine suppressed ADP-induced platelet
aggregation. It should be taken into account that aqueous extracts
from red wine contain, beside anthocyanins, other water soluble
compounds, which might be responsible for the inhibitory effect
on platelet aggregation.
Current data indicate that the chemical interaction between
anthocyanins and pyruvic acid as it occurs during red wine aging
slightly decreases the antioxidant activity of anthocyanins.
Furthermore, our findings indicate that anthocyanins, and to a
much lesser extent vitisins A, have protective effects against
TNF-R-induced MCP-1 secretion in primary human endothelial
cells. Interestingly, neither anthocyanins nor vitisins A did affect
monocyte function and platelet aggregation whereas the endo-
thelium was highly sensitive to changes in anthocyanin con-
centration. It should be noted that the concentrations of
anthocyanins used in the present study are higher than their
circulating plasma levels in humans. However, cellular exposure
to the test components in the present study was acute, i.e., 24
h. Therefore, it would be interesting to assess the effects of long-
term exposure of lower, physiologically achievable concentra-
tions of anthocyanins and vitisins A and their metabolites on
endothelial cell function.
However, overproduction of NO has been associated with
oxidative stress and with the pathophysiology of numerous
diseases such as septic shock, chronic inflammation, and
atherosclerosis (21). In the present study, both anthocyanins and
vitisins A did not inhibit NO production or TNF-R in activated
macrophages. In contrast to our findings, Wang et al. (22) and
Hu et al. (23) reported an inhibitory effect of anthocyanins on
LPS-induced NO production in macrophages. However, the
coincubation of LPS and anthocyanins may have given rise to
this effect since polyphenols suppress LPS activity due to a
chemical interaction (24); inhibition of NO production might
have been caused by a direct interaction of anthocyanins with
LPS, rather than a direct effect on the cell. In the present
investigation, macrophages were preincubated with anthocyanins
for 24 h and were then washed twice with PBS before the
addition of IFN-γ and LPS in order to avoid any direct chemical
interaction.
Endothelium Function and Platelet Aggregation. The
MCP-1 is known to mediate the recruitment of macrophages to
sites of infection or inflammation. A direct role of MCP-1 on
atherogenesis has been established. MCP-1 mRNA has been
detected in atherosclerotic lesions by in situ hybridization (25,
26). Furthermore, a decrease in atherosclerotic lesion size is
observed in mice deficient of the MCP-1 receptor CCR-2, and
fewer macrophages and monocytes are present in their aortas
(27).
In the present study, the pretreatment of primary human
endothelial cells with anthocyanins substantially decreased
MCP-1 secretion. Our results are in accordance with data by
Youdim and co-workers (28), who found that anthocyanin
extracts obtained from blueberries and cranberries were protec-
tive against TNF-R-induced MCP-1 expression in human
microvascular endothelial cells. It is known that the expression
of MCP-1 is regulated at the transcriptional level. MCP-1
transcription is mediated by several transcription factors among
which are activator protein-1 and NF-κB (29). Therefore, it is
hypothesized that anthocyanins may regulate TNF-R-induced
MCP-1 expression through these transcription factors. Interest-
ingly, Atalay et al. (30) demonstrated that the pretreatment of
endothelial cells with two different extracts of berry, containing
high amounts of anthocyanins, significantly decreased the NF-
κB DNA binding activity.
The fact that anthocyanins are more potent than vitisins A in
inhibiting TNF-R-induced MCP-1 expression may be, at least
partly, related to their higher antioxidant potential. Furthermore,
stereochemical differences and potential differences in the
cellular uptake between anthocyanins and vitisins A should also
be taken into account when comparing their biological and
cellular properties.
ABBREVIATIONS USED
ABTS, 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulfonate);
TNF-R, tumor necrosis factor-R; MCP-1, monocyte chemo-
attractant protein; NO, nitric oxide; HUVEC, human umbilical
vein endothelial cells; Dp-3-glu, delphinidin-3-glucoside; Pet-
3-glu, petunidin-3-glucoside; Mv-3-glu, malvidin-3-glucoside;
Vitdp-3-glu, vitisin A of delphinidin-3-glucoside; Vitpt-3-glu,
vitisin A of petunidin-3-glucoside; Vitmv-3-glu, vitisin A of
malvidin-3-glucoside; DMSO, dimethyl sulfoxide; FRAP, ferric
reducing ability of plasma; TEAC, Trolox equivalent antioxidant
capacity; ORAC, oxygen radical absorbing capacity; TPTZ,
2,4,6-tripridyl-s-triazine; AAPH, 2,2′-azobis(2-amidinopropane)-
dihydrochloride; LPS, lipopolysaccharide; IFN-γ, interferon-
γ; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB;
ADP, adenosine diphosphate.
ACKNOWLEDGMENT
We thank M. P. Mart´ın-Lopez and C. Alcalde-Eon for their
assistance in the synthesis of the visitisins A.
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Received for review November 19, 2003. Revised manuscript received
March 26, 2004. Accepted April 2, 2004. Financial support provided
by MCYT and FEDER program (Spain) and BBSRC (U.K.). The Ph.D.
studentship of M.G.A. is supported by a grant from the Ministerio de
Educacio´n, Cultura y Deporte (FPU grant), Spain.
JF035360V