Original article 109
Fractions of aqueous and methanolic extracts from tomato
(Solanum lycopersicum L.) present platelet antiaggregant
activity
Eduado J. Fuentesa,b,d, Luis A. Astudillob,d, Margarita I. Gutiérrezb,d,
Samuel O. Contrerasa, Luis O. Bustamantea, Pia I. Rubioa,
Rodrigo Moore-Carrascoa,b, Marcelo A. Alarcóna,b, Jaime A. Fuentesa,
Daniel E. Gonzálezc and Iván F. Palomoa,b
Cardiovascular disease (CVD) is the leading cause of
death worldwide. Its prevention emphasizes three aspects:
not smoking, physical activity and a healthy diet.
Recently, we screened the antithrombotic activity of a
selected group of fruits and vegetables. Among them,
tomato showed an important effect. The aim of this study
was to evaluate and characterize the platelet
antiaggregatory activity of tomato (Solanum lycopersicum
L.). For this, we obtained aqueous and methanolic tomato
extracts and evaluated the effect of pH (2 and 10) and
temperature (22, 60 and 100-C) on this activity.
Furthermore, in order to isolate the antiaggregant principle,
we separated tomato extracts into several fractions (A–D)
by size exclusion chromatography. In addition, we evaluated
the platelet antiaggregating activity ex vivo in Wistar rats.
Aqueous and methanolic extracts of tomato treated at 22, 60
and 100-C and pH 2 and 10 still inhibited platelet
aggregation (in vitro). Moreover, it was noted that one of the
fractions (fraction C), from both aqueous and methanolic
extracts, presented the highest activity (
70% inhibition of
platelet aggregation) and concentration dependently
inhibited platelet aggregation significantly compared
with control (P < 0.05). These fractions did not contain
lycopene but presented two peaks of absorption, at 210
and 261 nm, compatible with the presence of nucleosides.
Introduction
The current lifestyle of the population contributes
to the development of risk factors for cardiovascular
disease (CVD), such as hypertension, diabetes, smoking
and hypercholesterolemia [1–3]. These states are associ-
ated with endothelial dysfunction, triggering a cascade
of inflammatory reactions, with complex interactions
between monocytes, platelets, T cells and smooth muscle
cells [4]. The erosion or rupture of these lesions causes
the interaction between platelets and endothelium,
favouring the process of atherosclerosis [5]. From a public
health viewpoint, efforts should be directed at primary
prevention, namely, to reduce the above-mentioned
cardiovascular risk factors [6,7]. In relation to food,
regular consumption of fruits and vegetables is part
of the so-called Mediterranean diet [8]. Fruits and
0957-5235 ß 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
In rats treated with tomato macerates, a mild platelet
antiaggregating effect ex vivo was observed. Further
studies are required to identify the molecules
with platelet antiaggregating activity and
antiplatelet mechanisms of action. Blood Coagul
Fibrinolysis 23:109–117 ß 2012 Wolters Kluwer Health |
Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2012, 23:109–117
Keywords: cardiovascular diseases, platelet antiaggregant activity,
tomatoes
a
Departamento de Bioquı́mica Clı́nica e Inmunohematologı́a, Facultad de
Ciencias de la Salud, Programa de Investigación en Factores de Riesgo de
Enfermedades Cardiovasculares (PIFRECV), Universidad de Talca, bCentro de
Estudio en Alimentos Procesados (CEAP), Conicyt-Regional, Gore Maule, Talca,
c
Departamento de Ciencias Básicas Biomédicas, Facultad de Ciencias de la
Salud, Programa de Investigación en Factores de Riesgo de Enfermedades
Cardiovasculares (PIFRECV), Universidad de Talca and dLaboratorio de Sı́ntesis,
Instituto de Quı́mica de los Recursos Naturales, Universidad de Talca, Talca,
Chile
Correspondence to Iván F.P. González, PhD, Departamento de Bioquı́mica e
Inmunohematologı́a Clı́nica, Facultad de Ciencias de la Salud, Universidad de
Talca, P.O. Box 747, Talca, Chile
Tel: +56 71 200493; fax: +56 71 200488; e-mail: ipalomo@utalca.cl
Received 21 June 2011 Revised 24 August 2011
Accepted 15 September 2011
vegetables are known for their antioxidant content [9],
but less is known about their antithrombotic activity.
Tomato (Solanum lycopersicum L.), in addition to its
antioxidant content [10,11], presents some platelet
antiaggregant activity [12–16]. Previous studies by
Dutta-Roy et al. [12], Yamamoto et al. [16] and Lazarus
and Garg [15] described the platelet antiaggregant
activity of aqueous extract of tomato, highlighting the
presence of adenosine, thermal stability and the absence
of changes in basal cAMP levels. Moreover, O’Kennedy
et al. [13] showed a significant reduction in ex-vivo
platelet aggregation after 3 h of tomato supplementation.
The purpose of this research was to advance the
characterization of the platelet antiaggregant activity of
tomato and evaluate its thermal and extreme pH stability.
DOI:10.1097/MBC.0b013e32834d78dd
 110 Blood Coagulation and Fibrinolysis 2012, Vol 23 No 2
We also attempted to obtain an active principle of tomato
using size exclusion chromatography.
Materials and methods
Plant material
Cluster tomatoes, fresh and ripe, obtained from the
Centro Regional de Abastecimiento from Talca, were
washed and then separated into skin and pulp.
Maceration of tomato pulp
Tomato pulp was ground in a blender (Somela BL1500)
and filtered through gauze to remove large pieces.
The macerate was centrifuged (Rotofix 32; Hettich,
Tuttlingen, Germany) at 650g for 10 min. A portion of
the precipitate was autoclaved. Hence, three fractions
were obtained (supernatant, precipitate fresh and precipi-
tate autoclaved), which were frozen at 808C (Ultra Low;
Sanyo Electric Co. Ltd., Moriguchi, Japan) until use.
Chemical composition analysis
The chemical composition of tomato pulp was analyzed
as follows: water content (by evaporation), protein
(Kjeldahl method), fat (Soxhlet method), ash (oven
drying at 5508C), crude fiber (sequential acid/alkaline
hydrolysis with 1.25% H2SO4 and 1.25% NaOH) and
carbohydrates (remainder after all other components
were measured) [8]. Each measurement was performed
in triplicate. Lycopene content was measured by high-
performance liquid chromatography (HPLC) using lyco-
pene standard from Sigma.
Preparation of aqueous and methanolic extracts
To prepare aqueous extract, small pieces of pulp were
macerated in a blender and the product was filtered
twice through gauze. The yellow liquid obtained was
lyophilized (Freezone 6; Labconco, Kansas, USA) and
stored at 808C until use. To prepare methanolic extract,
tomato skin was macerated in a blender and the product
was mixed with methanol (Archimedes, Santiago, Chile)
at a ratio of 1 : 1. The mixture was subjected to sonication
(Transsonic 700/H; Elma-Hans Schmidbauer, Germany)
for 5 min and then filtered twice through gauze. The
filtrate was concentrated using a rotary evaporator
(Labo 4001; Heidolph, Germany or RE 111-B461;
BUCHI Labortechnik AG, Holland); once concentrated,
the product was lyophilized and stored in the same way as
the aqueous extract.
To evaluate the stability of aqueous and methanol
extracts at different pH and temperatures, they were
resuspended at pH 2 and 10, subjected to 22, 60 and
1008C for 1 h, kept at 48C for 5 min, and then used in the
studies of platelet aggregation in vitro.
Separation of fractions
A portion of the aqueous extract was reconstituted in
distilled water until solubilization, whereas a portion of
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
the methanolic extract was dissolved in methanol :
distilled water (1 : 1, v/v). Both extracts were fractionated
according to molecular size in a Sephadex LH-20 column
(length 50 cm, inner diameter 2 cm; Pharmacia Fine
Chemicals, Piscataway, New Jersey, USA). Different
fractions obtained were subjected to thin-layer chroma-
tography (Merck, Darmstadt, Germany), revealed (reader
ultraviolet), allowed us to join fractions with the same
retention factor, yielding four fractions from each extract
according to Hostettmann et al. [17].
Molecular fractions that showed higher percentage of
platelet antiaggregant activity were studied by HPLC
(Merck-Hitachi, LaChrom, Tokyo, Japan) to determine
the presence of lycopene. For this, 300 ml of fraction was
dissolved in n-hexane (solvent extraction of lycopene) at
a ratio of 1 : 1 (v/v) and stirred for 10 min. The upper organic
layer was analyzed by HPLC using an isocratic mobile
phase consisting of methanol : acetonitrile (9 : 1, v/v)
(Merck). The flow rate was 1 ml/min. Lycopene was
determined at a wavelength of 475 nm [18].
Human platelets
Venous blood was collected from the volunteers (healthy
university students), who had previously signed informed
consent, by phlebotomy using a vacuum tube system
(Becton Dickinson Vacutainer Systems, Franklin Lakes,
New Jersey, USA), in 3.2% citrate tubes (9 : 1, v/v).
The protocol was authorized by the ethics committee
of the Universidad de Talca in accordance with the
Declaration of Helsinki. The samples were homogenized
gently by inversion five times and allowed to stand for
5 min. Then they were centrifuged (DCS-16 Centrifugal
Presvac RV) at 240 g for 10 min and 1 ml of platelet-rich
plasma (PRP) was collected from each tube. Platelets
were counted (in triplicate) using hematologic counter
(ACT Diff; Beckman Coulter Inc., USA). The original
tubes were centrifuged at 650g for 10 min to obtain
platelet-poor plasma (PPP); 500 ml of this was centrifuged
at 12 000 g for 5 min and the supernatant so obtained was
used as control in the studies of platelet aggregation.
Finally, the PRP was adjusted to 2  105 platelets/ml
with PPP.
Tomato supplementation in rats
Wistar rats (age 8–9 weeks; weight 200–300 g) were
obtained from the Public Health Institute (Santiago,
Chile). The number of animals was determined on
the basis of statistical power calculations as indicated
in the methodology by other authors [19,20]. The animals
were maintained at 22  28C, with water and solid food
(Champion SA, Santiago de Chile). The protocols
used were approved by the Institutional Committee
for Bioethics at the University of Talca and were in
accordance with the norms of the Canadian Council on
Animal Care [21]. Acute treatment consisted of admin-
istration of 3 ml of tomato paste, using intragastric

gavages (20  1.50 Popper and Sons, Inc., New York, USA).
To this end, rats were anaesthetized with ethyl ether
(Equilab, New York, USA). The effect was evaluated at
3-h posttreatment. Chronic treatment consisted of daily
administration of 2 ml of macerated tomato for 15 days, by
gavage. After each treatment, animals were anaesthetized
with a mixture of ketamine (90 mg/kg) and xylazine
(10 mg/kg) before blood withdrawal. Blood was collected
from the aorta and mixed with 3.2% sodium citrate
(9 : 1 v/v) for preparation of PRP. The mixture was
centrifuged at 240 g for 10 min (room temperature).
Aliquots of 1 ml of PRP were used for platelet count
(in triplicate). Then the rest of the PRP was centrifuged
at 650 g for 10 min to obtain PPP; 500 ml of this were
centrifuged at 12000 g for 5 min and the supernatant was
used as the control. The count of PRP was adjusted to
5  105/ml, to be used in the platelet aggregation studies.
To evaluate the bleeding time in rats, an incision was made
on the ventral surface of the tail to 2 mm from the tip [22].
Bleeding time was measured until the end of bleeding.
Platelet aggregation
The platelet aggregation method was used for study both
in vitro (human platelets) and ex vivo (rat platelets).
Assay of platelet aggregation
For this study, the method described by Born and Cross
[23] was employed. A screening aggregometer was used
(Cecil Instruments, Cambridge, UK) to measure the
transmitted light and a coagulometer (Clot1; RAL SA,
Barcelona, Spain) to maintain platelets with stirring at
378C. Four hundred and eighty mcrioliter of adjusted
PRP was added (2  105/ml human, 5  105/ml rats) to
the reaction cuvette of the coagulometer for 1 min. In
the case of human platelets, 20 ml of extract was added
(negative control: 0.9% saline; positive control: Prosta-
glandin E1 250 mg/ml) and stirred for 1 min. Then, 20 ml of
8 mmol/l ADP [adenosine 50 -diphosphate bis (cyclohexy-
lammonium) salt, from bacterial source, Sigma Chemical
Co., St. Louis, USA] was added and aggregation was
measured for 5 min. Readings at 630 nm and 378C were
obtained. The instrument was calibrated with PRP
and PPP, giving 0 and 100% transmittance, respectively.
All measurements were performed in triplicate. Platelet
aggregation data were expressed as the following: percen-
tage of inhibition of maximum platelet aggregation: 100,
[(%AgX  100)/% AgC] (%AgX, percentage of aggrega-
tion of the component under study; %AgC, percentage
aggregation of control); area under the curve (AUC): t0 Rt1
A(1  eKt)dt ¼ At0 þ A/K(1  eKt0); and slope: y ¼
A(1  eKt) (A, percentage of maximum aggregation; t,
time (seconds); K, constant of proportionality; e, log).
Statistical analysis
The percentage of maximal platelet aggregation
inhibition is expressed as mean  SD. To study platelet
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tomato: a cardioprotective functional food Fuentes et al. 111
aggregation, Student’s t-test was used (SPSS 17.0;
SPSS Inc., Chicago, Illinois, USA). The analysis of
variance (ANOVA) with Newman–Keuls as post-hoc test
was used to compare treatments. P < 0.05 was considered
as the limit of significance [16].
Results
Platelet antiaggregant activity in vitro
Antiaggregation activity of extracts
Both aqueous and methanolic tomato extracts (1 mg/ml)
inhibited maximum platelet aggregation induced by
8 mmol/l ADP, by 26.2  2% and 25.2  3%, respectively.
The AUC for the aqueous and methanolic extracts
was 171.5 and 180.6, respectively. The slope of platelet
aggregation for aqueous and methanolic extracts was
18.03 and 19.53, respectively. In all cases, the inhibition
of maximum platelet aggregation was statistically signifi-
cant compared with negative control (P < 0.05).
Effect of pH and temperature
We studied the effect of pH on the ability of aqueous and
methanolic tomato extracts to inhibit platelet aggregation
(Fig. 1). The activity was retained at pH 2 and 10 by
28  2% and 24  2% for aqueous extract, and 34  1%
and 29  3% for methanolic extract, respectively.
The AUC at pH 2 and 10 was 180.6 and 195.7 for the
aqueous extract, and 187.8 and 180.8 for the methanolic
extract, respectively. The slope of aggregation at pH 2
and 10 was 14.0 and 11.9 for the aqueous extract, and
9.5 and 14.9 for the methanolic extract, respectively. In all
cases the inhibition of maximal platelet aggregation was
statistically significant compared with negative control
(P < 0.05). Both types of extracts, after being subjected to
22, 60 and 1008C (Fig. 2), retained their ability to inhibit
maximal platelet aggregation by 36  2%, 31  6% and
33  2% (for the aqueous extract) and by 38  2%,
37  3% and 44  3% (for the methanolic extract),
respectively. The AUC at 22, 60 and 1008C was 208.6,
189.5 and 178.3 for the aqueous extract, and 161.9, 169.5
and 159.9 for the methanolic extract, respectively.
The slope at 22, 60 and 1008C was 17.5, 9.67 and 16.6
for the aqueous extract, and 14.5, 13.1 and 15.5 for the
methanolic extract, respectively. In all cases, the inhi-
bition of platelet aggregation maximum was statistically
significant compared with negative control (P < 0.05).
Antiaggregation activity of fractions
Aqueous and methanolic tomato extracts were submitted
to fractionation by size exclusion chromatography. The
fractions obtained, in order of elution, were designated:
A, B, C and D. Fig. 3a shows that inhibition of maximal
platelet aggregation for fractions A, B, C and D of the
aqueous extract was 34.6  4%, 5.8  5%, 66.7  11% and
32.3  3%, respectively. The AUC for each fraction was
119.9, 200.6, 86.9 and 79.5, and the slope 11.2, 19.6, 6.0
and 9.6, respectively. The inhibition of maximal platelet
aggregation by fraction C was statistically significant
 112 Blood Coagulation and Fibrinolysis 2012, Vol 23 No 2

Fig. 1

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
(a) Aqueous extract pH = 2 (1 mg/ml)
80 Aqueous extract pH = 10 (1 mg/ml)
Platelet aggregation (%)

60

40

20

100
0
0 50 100 150 200 250 300

Platelet aggregation
80
Time (s)

inhibition (%)
60

(b) Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
40
Methanolic extract pH = 2 (1 mg/ml)
80 Methanolic extract pH = 10 (1 mg/ml)
20
Platelet aggregation (%)

60 0
pH 2 pH 10 pH 2 pH 10 PGE1

40 Aqueous MeOH

20

0
0 50 100 150 200 250 300
Time (s)

Effect of pH (2 and 10) on platelet aggregation inhibitory activity of (a) aqueous and (b) methanolic tomato extracts. Platelet aggregation was
induced by 8 mmol/l ADP. The graph depicts the average  SD of n ¼ 3 experiments. All values are statistically significant vs. control (P < 0.05).

compared with negative control (P ¼ 0.006). Figure 3b concentrations (0–1000 mg/ml) of each fraction (Fig. 4).
shows that the inhibition of maximum platelet aggre- Both C fractions inhibited platelet aggregation concen-
gation for fractions A, B, C and D of the methanolic tration dependently.
extract was 7  9%, 23  7%, 68  8% and 56  12%,
respectively. The AUC was 132.7, 147.5, 49.7 and 24.9, High-performance liquid chromatography and spectral
and the slope 17.5, 13.5, 2.8 and 6.7, respectively. For the scanning of the fractions C
methanolic fraction C, the inhibition of maximum plate- Aqueous and methanolic fractions C were submitted to
let aggregation was statistically significant compared with HPLC to determine the presence of lycopene (Fig. 5).
negative control (P ¼ 0.014). The chromatograms obtained from fractions C, both
Of all the fractions studied, the aqueous and methanolic aqueous and methanolic, did not show the characteristic
fractions C showed the highest inhibition of maximal absorption maximum of carotenoids with a retention time
platelet aggregation (66.7  11 and 68  8%, respectively). of 3.84 min. Moreover, a spectral scanning between 200
and 500 nm for both fractions C (Fig. 6) showed two
To evaluate the effect of high temperature on the platelet
absorption maxima at 210 and 261 nm.
aggregating activity of aqueous and methanolic fractions
C, they were autoclaved 1 h before use. It was noted that
Platelet antiaggregant activity ex vivo and in vivo
the fractions inhibited the maximum platelet aggregation
Acute treatment
by 50.4  3 and 60.2  5%, respectively.
In ex-vivo platelet aggregation assay, significant decrease
Next, we assessed the concentration response of the was observed in the percentage of maximum platelet
inhibitory activity of these fractions. For this, we assayed aggregation (49.1  6 vs. 66.8  4% control; P < 0.05) and
platelet aggregation induced by ADP, using increasing AUC (101.3  3 vs. 168.2  1 control; P < 0.05).

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 Tomato: a cardioprotective functional food Fuentes et al. 113

Fig. 2

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
(a) Aqueous extract 22°C (1 mg/ml)
80 Aqueous extract 60°C (1 mg/ml)
Aqueous extract 100°C (1 mg/ml)
Platelet aggregation (%)

60

40
H2O Methanolic PGE1
100
20
80

Platelet aggregation
0

inhibition (%)
0 50 100 150 200 250 300
60
Time (s)

40
Negative control (saline 0.9%)
(b) Positive control (PGE1 250 µg/ml)
Methanolic extract 22°C (1 mg/ml)
80 Methanolic extract 60°C (1 mg/ml)
Methanolic extract 100°C (1 mg/ml)
20
Platelet aggregation (%)

60 0
22 60 100
40 Temperature (°C)

20

0
0 50 100 150 200 250 300
Time (s)

Effect of temperature (22, 60 and 1008C) on platelet aggregation inhibitory activity of (a) aqueous and (b) methanolic tomato extracts. Platelet
aggregation was induced by 8 mmol/l ADP. The graphs depict the average  SD of n ¼ 3 experiments. All values are statistically significant vs. control
(P < 0.05).

Chronic treatment maximum platelet aggregation induced by ADP, at

In addition to the usual diets, rats received macerated a concentration of 1 mg/ml. Previous studies have
tomato pulp (water 25.3  1.5%; protein 9.1  0.04%; fat also demonstrated the platelet antiaggregant activity
0.44  0.04%; ash 8.9  0.11%; carbohydrate 56.5  0.4%; of aqueous extract of tomato [12–16]. When collagen
crude fiber 0.4  0.03% and lycopene 152.7  1.45 mg/g) was used as agonist, inhibition was lower, whereas
for 15 days. Platelet function ex vivo showed that there when arachidonic acid and thrombin receptor activator
was a decrease in the slope and AUC in case of rats peptide were used, tomato showed no inhibitory
treated with macerated tomato (Table 1). effect [24]. These results rule out the platelet anti-
aggregant activity of salicylic acid or a related species
Bleeding time [25].
There was no platelet antiaggregant effect in vivo
(using bleeding time) in case of both acute (3.7  2 vs. To further study the mechanism of antiplatelet activity of
4.3  0.5 min control; P > 0.05) and chronic (3.5  0.6 vs. these extracts, they were analysed for thermal and pH
3.6  0.8 min control; P > 0.05) treatments with macer- stability. When aqueous and methanolic extracts, in 0.9%
ated tomato pulp. physiological saline at pH 4.5, were resuspended in
more acidic (pH 2) and basic (pH 10) solutions, they
Discussion maintained their platelet aggregation inhibitory activity.
Although the antioxidant effect of fruits and vegetables Because it is known that carotenoids are unstable at
is well known, the antithrombotic activity has been extreme pH, it is reasonable to conclude that these
less studied. This research showed that both aqueous antioxidant compounds are not responsible for platelet
and methanolic extracts of cluster tomatoes inhibited antiaggregant activity [26,27]. In addition, platelet

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 114 Blood Coagulation and Fibrinolysis 2012, Vol 23 No 2

Fig. 3

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/mL)
Aqueous fraction A (1 mg/ml)
Aqueous fraction B (1 mg/ml)
(a) Aqueous fraction C (1 mg/ml)
Aqueous fraction D (1 mg/ml) 100
80
*

Inhibition of maximal
Platelet aggregation (%)

80

aggregation (%)
60
60

40 40

20 20

0
0 A B C D
0 50 100 150 200 250 300
Aqueous fractions
Time (s)

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
(b) Methanolic fraction A (1 mg/ml)
Methanolic fraction B (1 mg/ml) 80 *
Methanolic fraction C (1 mg/ml)
Methanolic fraction D (1 mg/ml)

Inhibition of maximal
80

aggregation (%)
60
Platelet aggregation (%)

60 40

40 20

20 0
A B C D
0 Methanolic fractions
0 50 100 150 200 250 300
Time (s)

Inhibitory effect on platelet aggregation of fractions from (a) aqueous and (b) methanolic tomato extracts obtained by size exclusion chromatography.
Aggregation was induced by 8 mmol/l ADP. The graphs depict the average  SD of n ¼ 3 experiments. , P < 0.05.

antiaggregant activity decreases during tomato matu- currently used in the antithrombotic therapy show high
ration, whereas the lycopene content increases [16]. instability under environmental conditions [28].
Aqueous and methanolic extracts subjected to different After obtaining molecular fractions from both extracts
temperatures (22, 60 and 1008C) maintained their plate- using size exclusion chromatography, the platelet anti-
let antiaggregant activity, suggesting that the active aggregating activity concentrated in the fractions C,
components with platelet antiaggregating activity in both unlike the results of O’Kennedy et al. [13] who obtained
extracts are not affected by heat treatment. These results three fractions by semipreparative HPLC, all of them
confirm those by Yamamoto et al. [16], the difference showing platelet antiaggregant activity. Because polar
being that our extracts were submitted to different stress solvents were used (methanol and water), the bioactive
conditions and longer exposure time (60 min). compounds (given their molecular size) in C fractions
would be similar in polarity to one of the fractions
These results allow us to categorize tomato as a functional
obtained by O’Kennedy et al. [13] who used 0.05%
food, having one or more bioactive components with
trifluoroacetic acid and acetonitrile with gradient elution.
pH and thermal stability to exert its cardioprotective
activity (platelet antiaggregating activity). Owing to The inhibitory activity is concentration dependent, for
these properties, the molecular structure of the active both aqueous and methanolic C fractions. Studies by
principle contained in tomato, and hence, its platelet O’Kennedy et al. [13] showed an IC50 of 60 mg/ml. The
antiaggregating activity, is kept intact during processing, HPLC results show the absence of lycopene in those
storage, transport and handling. Most of the drugs fractions that present higher degree of aggregation

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 Tomato: a cardioprotective functional food Fuentes et al. 115

Fig. 4

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
Aqueous fraction C ( 1000 µg/ml)
Aqueous fraction C (700 µg/ml)
(a) Aqueous fraction C (500 µg/ml)
Aqueous fraction C (300 µg/ml)

80
Platelet aggregation (%)

60

40
80
20

0 60

aggregation (%)
0 50 100 150 200 250 300

Max. platelet
Time (s)
40

Negative control (saline 0.9%)

Positive control (PGE1 250 µg/ml)
Aqueous C
Methanolic fraction C ( 1000 µg/ml) 20
Methanolic fraction C (700 µg/ml) Methanol C
Methanolic fraction C (500 µg/ml)
(b) Methanolic fraction C (300 µg/ml)
80 0
Platelet aggregation (%)

0 200 400 600 800 1000

60 Fraction concentration
(mg/ml)
40

20

0
0 50 100 150 200 250 300
Time (s)

Concentration–response effect. Inhibitory effect on platelet aggregation in vitro. ADP 8 mmol/l in the presence of (a) aqueous fraction C and
(b) methanolic fraction C, both at different concentrations. Aggregation was induced by ADP 8 mmol/l. The graph depicts the concentration–
response effect of n ¼ 3 experiments for each fraction.

inhibition. The absence of lycopene is in line with the
results of Lazarus and Garg [15]. Others would be active
compounds with platelet aggregation inhibitory activity
in these fractions. To identify the presence of a com-
pound in the fractions C (aqueous and methanolic)
spectral scanning was performed, highlighting the
presence of two absorption maxima at wavelengths of
210 and 261 nm in both fractions. These wavelengths are
absorbed by aromatic amino acids, nitrogenated bases
and nucleosides (adenosine, cytidine, inosine, guanosine,
adenosine monophosphate and guanosine monophos-
phate) [29].
Finally, the ex-vivo platelet antiaggregant effect of
tomato confirmed the studies by Yamamoto et al. [16]
and by other groups in vitro [12–15]. It has been
suggested that adenosine or other nucleosides may be
responsible for this inhibition, apparently by a mechan-
ism independent of cAMP generation and thromboxane
(cyclooxygenase) pathway [15]. However, the possibility
that nucleoside derivatives exert their action ex vivo is
limited by their short half-life in the gut or the blood-
stream [13].
Despite these results, there is still controversy regarding
the mechanism of antiaggregant activity of tomato
fractions, given that adenosine binding to its receptors
(A2a and A2b) is associated with increases in intracellular
cAMP [30].
Moreover, there is a wide range of compounds in
tomatoes with platelet antiaggregating activity, some of
which are known (derivatives of flavonoids) and others
that have not been identified [31].
It is also necessary to determine which anatomical site
of tomato (skin, pulp, seeds) contains the compounds with
platelet antiaggregating activity [32]. Our results show that
these bioactive compounds have thermal and extreme pH

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 116 Blood Coagulation and Fibrinolysis 2012, Vol 23 No 2

Fig. 5

(a)

2.59
0.02
Lycopene
Intensity (AU)

0.01

2.83
0.0

0 1 2 3 4
Retention time (min)

(b) 2.59
(c)

2.59
0.02
0.02
Intensity (AU)

Intensity (AU)
0.01 0.01
0.83

83
0.0 0.0

0 1 2 3 4 0 1 2 3 4
Retention time (min) Retention time (min)

Lycopene analysis. (a) HPLC chromatogram of lycopene standard (retention time 3.84 min, absorption at 475 nm). The peak around 2.7 min
represents an artefact due to solvent change. (b) Chromatogram of aqueous fraction C. (c) Chromatogram of methanolic fraction C. Note the
absence of the lycopene peak in both fractions.

stability, absence of lycopene, and a low molecular weight Hence, it is possible to establish that tomato extracts,
(<1000 Da). We also postulate that these compounds both aqueous and methanolic, present more than one
exert their platelet antiaggregating activity via two platelet compound with platelet aggregation inhibitory activity,
receptors: glycoprotein VI (GPVI) (using collagen as with variations in their chemical structures, and thus with
agonist), and P2Y1 and P2Y12 (using ADP as agonist). different action in intraplatelet signalling pathways.

Fig. 6

(a)
1.5 1,427
Absorbance

1.0
0,665
0.5

0.0
200 250 300 350 400 450
(b) Wavelength (nm)

1.0 0.903
Absorbance

0.5

0.0
200 250 300 350 400 450
Wavelength (nm)

(a) Spectral scanning of aqueous C (b) and methanolic C fractions. Peaks at 210 and 261 nm are characteristic of nucleosides or related species.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Table 1 Study of platelet aggregation ex vivo in rats treated
chronically with macerated tomato pulp
Tomato pulp Control
Maximum platelet aggregation (%) 57.8  4.5 60.3  0.95
Slope (s) 0.020  0.005M 0.031  0.002
Area under the curve 10470  494M 12336  305
M
Tomato pulp vs. negative control (physiological saline 0.9%). P < 0.05.
Conclusion
Advance in the knowledge of platelet antiaggregating
activity of tomato could contribute to the prevention of
CVD. This study showed that bioactive compounds with
platelet antiaggregating activity, using ADP as agonist,
absorb at 210 and 261 nm, resist high temperatures and
extreme pH, and have effects ex vivo. Extracts or fractions
of these could be used in the preparation of functional
foods or supplements with platelet antiaggregating
activity. Further studies are needed to identify the mole-
cule(s) with platelet antiaggregating activity and under-
stand the underlying mechanisms for these actions.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 World Health Organization. Informe sobre la salud en el mundo. Technical
Report. Series ISBN 924356207X. WHO: Geneva, 2002.
2 Bustos P, Amigo H, Arteaga A, Acosta A, Rona R. Factores de riesgo de
enfermedad cardiovascular en adultos jóvenes. Rev Méd Chile 2003;
131:973–980.
3 Mathers C, Loncar D. Projections of global mortality and burden of disease
from 2002 to 2030. PLoS Med 2006; 3:2011–2020.
4 Palomo I, Icaza G, Mujica V, Núñez L, Leiva E, Vásquez M, et al. Prevalencia
de factores de riesgo cardiovascular clásicos en población adulta de Talca,
Chile, 2005. Rev Med Chile 2007; 135:904–912.
5 Badimon L, Vilahur G. Platelets, arterial thrombosis and cerebral ischemia.
Cerebrovascular Diseases 2007; 24:30–39.
6 Estruch R, Martı́nez-González M, Corella D, Salas-Salvadó J, Ruiz-Gutiérrez
V, Covas M, et al. Effects of a Mediterranean-style diet on
cardiovascular risk factors: a randomized trial. Ann Intern Med 2006;
145:1–11.
7 Pearson T, Blair S, Daniels S, Eckel R, Fair J, Fortmann S, et al. AHA
guidelines for primary prevention of cardiovascular disease and stroke:
2002 update: consensus panel guide to comprehensive risk reduction for
adult patients without coronary or other atherosclerotic vascular diseases.
Circulation 2002; 106:388–391.
8 Palomo I, Leiva E, Vásquez M. Dieta mediterránea: prevención de las
enfermedades cardiovasculares. Talca, Chile: Universidad de Talca Press,
2007.
9 Carlsen M, Halvorsen B, Holte K, Bohn S, Dragland S, Sampson L, et al.
The total antioxidant content of more than 3100 foods, beverages, spices,
herbs and supplements used worldwide. Nutr J 2010; 9:1–11.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tomato: a cardioprotective functional food Fuentes et al. 117
10 Palomo I, Fuentes E, Carrasco G, González D, Moore-Carrasco R.
Actividad antioxidante, hipolipemiante y antiplaquetaria del tomate
(Solanum lycopersicum L.), y el efecto de su procesamiento y almacenaje.
Rev Chil Nutr 2010; 37:524–533.
11 Capanoglu E, Beekwilder J, Boyacioglu D, De Vos R, Hall R. The effect of
industrial food processing on potentially health-beneficial tomato
antioxidants. Crit Rev Food Sci Nutr 2010; 50:919–930.
12 Dutta-Roy A, Crosbie L, Gordon M. Effects of tomato extract on human
platelet aggregation in vitro. Platelets 2001; 12:218–227.
13 O’Kennedy N, Crosbie L, Van Lieshout M, Broom J, Webb D, Duttaroy A.
Effects of antiplatelet components of tomato extract on platelet function in
vitro and ex vivo: a time-course cannulation study in healthy humans. Am J
Clin Nutr 2006; 84:570–579.
14 O’Kennedy N, Crosbie L, Whelan S, Luther V, Horgan G, Broom J, et al.
Effects of tomato extract on platelet function: a double-blinded crossover
study in healthy humans. Am J Clin Nutr 2006; 84:561–569.
15 Lazarus S, Garg M. Tomato extract inhibits human platelet aggregation in
vitro without increasing basal cAMP levels. Int J Food Sci Nutr 2004;
55:237–247.
16 Yamamoto J, Taka T, Yamada K, Ijiri Y, Murakami M, Hirata Y, et al. Tomatoes
have natural antithrombotic effects. Br J Nutr 2003; 90:1031–1038.
17 Hostettmann K, Marston A, Hostettmann M. Preparative Chromatography
techniques: applications in natural product isolation. Berlin: Springer;
1997.
18 Olives A, Cámara M, Sánchez M, Fernández V, López M. Application of a
UV-vis detection-HPLC method for a rapid determination of lycopene and
[beta]-carotene in vegetables. Food Chemistry 2006; 95:328–336.
19 Gadi D, Bnouham M, Aziz M, Ziyyat A, Legssyer A, Legrand C. Parsley
extract inhibits in vitro and ex vivo platelet aggregation and prolongs
bleeding time in rats. J Ethnopharmacol 2009; 125:170–174.
20 Akagi Y, Nio Y, Shimada S, Aoyama T. Influence of nonsteroidal anti-
inflammatory drugs on the antiplatelet effects of aspirin in rats. Biol Pharm
Bull 2011; 34:233–237.
21 Olfert E, Cross B, McWilliam A. Guide to the care and use of experimental
animals. Ottawa: Canadian Council on Animal Care; 1993.
22 De C, Goossens J, Reneman R. Effects of anti-inflammatory, anticoagulant
and vasoactive compounds on tail bleeding time, whole blood coagulation
time and platelet retention by glass beads in rats. Thromb Res 1976;
8:179–193.
23 Born G, Cross M. The aggregation of blood platelets. J Physiol 1963;
168:178–195.
24 Torres-Urrutia C, Guzmán L, Schmeda-Hirschmann G, Moore-Carrasco R,
Alarcón M, Astudillo L, et al. Antiplatelet, anticoagulant, and fibrinolytic
activity in vitro of extracts from selected fruits and vegetables. Blood
Coagul Fibrinolysis 2011; 22:197–205.
25 Arazi HC, Doiny DG, Torcivia RS, Grancelli H, Waldman SV, Nojek C, et al.
Impaired antiplatelet effect of aspirin, inflammation and platelet turnover in
cardiac surgery. Interact Cardiovasc Thorac Surg 2010; 10:863–867.
26 Nguyen M, Schwart S. Lycopene: chemical and biological properties. Food
Technol 1999; 53:38–45.
27 Shi J, Le Maguer M. Lycopene in tomatoes: chemical and physical
properties affected by food processing. Crit Rev Biotechnol 2000;
20:293–334.
28 Agrawal H, Kaul N, Paradkar AR, Mahadik KR. Stability indicating HPTLC
determination of clopidogrel bisulphate as bulk drug and in pharmaceutical
dosage form. Talanta 2003; 61:581–589.
29 Devlin T. Bioquı́mica. Libro de texto con aplicaciones clı́nicas. Barcelona:
Reverte; 2004.
30 Johnston-Cox HA, Yang D, Ravid K. Physiological implications of adenosine
receptor-mediated platelet aggregation. J Cell Physiol 2011; 226:46–51.
31 Murphy K, Chronopoulos A, Singh I, Francis M, Moriarty H, Pike M, et al.
Dietary flavanols and procyanidin oligomers from cocoa (Theobroma
cacao) inhibit platelet function. Am J Clin Nutr 2003; 77:1466–1473.
32 Nuez F. El cultivo del tomate. Barcelona: Mundi-Prensa; 1995.