Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia xanthocarpa.

Abstract

In a previous work based on popular belief, Campomanesia xanthocarpa Berg., popularly known as "guavirova", showed to have a potential effect in the control of a number of conditions associated with cardiovascular diseases. The aim of the present work was to investigate the effects of C. xanthocarpa extract (CXE) on antiplatelet, antithrombotic and fibrinolytic activities in mice and in human blood. Mice were treated orally for 5 days with CXE or acetylsalicylic acid and at the end of the treatment period animals were challenged for bleeding, acute thromboembolism and ulcerogenic activity. In addition, we have assessed the prothrombin time and activated partial thromboplastin time (aPTT) after oral administration. In in vitro assays, antiplatelet effects of CXE was evaluated on platelet aggregation, and fibrinolytic activity of the extract was observed by mice or human artificial blood clot degradation. Platelet citotoxicity of the extract was also determined by the LDH assay. Results demonstrated that CXE has a significant protective effect on thrombosis. It also inhibits platelet aggregation without demonstrating cytotoxicity on platelets. CXE slightly prolonged aPTT and showed no ulcerogenic activity after oral administration. In addition, CXE showed a fibrinolytic activity. Thus, C. xanthocarpa showed antiplatelet, antithrombotic and fibrinolytic activities in mice.

Full text

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Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 954748, 8 pages
doi:10.1155/2012/954748
Research Article
Antiplatelet, Antithrombotic,and FibrinolyticActivities of
Campomanesiaxanthocarpa
Jonatas ZeniKlafke,1,2MarianeArnoldi daSilva,1,2Mateus Fortes Rossato,1
GabrielaTrevisan,1CristianiIsabelBander´ o Walker,1Cl´ audioAlbertoMartins Leal,1
Diego Olschowsky Borges,2MariaRosa ChitolinaSchetinger,1RafaelNoal Moresco,3
Marta MariaMedeirosFrescuraDuarte,4AdairRoberto Soaresdos Santos,5
Paulo Ricardo Naz´ ario Viecili,2andJulianoFerreira1
1Programa de P´ os-Graduac ¸˜ ao em Ciˆ encias Biol´ ogicas: Bioqu´ ımica Toxicol´ ogica, Universidade Federal de Santa Maria,
97105-900 Santa Maria, RS, Brazil
2Grupo Multidisciplinar de Sa´ ude, Universidade de Cruz Alta, 98020-290 Cruz Alta, RS, Brazil
3Programa de P´ os-Graduac ¸˜ ao em Ciˆ encias Farmacˆ euticas, Centro de Ciˆ encias da Sa´ ude, Universidade Federal de Santa Maria,
97105-900 Santa Maria, RS, Brazil
4Departamento de Ciˆ encias da Sa´ ude, Universidade Luterana do Brasil, 97020-001 Santa Maria, RS, Brazil
5Department of Physiological Sciences, Universidade Federal de Santa Catarina, 88040-970 Florian´ opolis, SC, Brazil
Correspondence should be addressed to Jonatas Zeni Klafke, jonzeni@hotmail.com
Received 7 February 2011; Revised 8 July 2011; Accepted 8 July 2011
Academic Editor: Ann Gill Taylor
Copyright © 2012 Jonatas Zeni Klafke et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
In a previous work based on popular belief, Campomanesia xanthocarpa Berg., popularly known as “guavirova”, showed to have a
potential effect in the control of a number of conditions associated with cardiovascular diseases. The aim of the present work was
to investigate the effects of C. xanthocarpa extract (CXE) on antiplatelet, antithrombotic and fibrinolytic activities in mice and in
human blood. Mice were treated orally for 5 days with CXE or acetylsalicylic acid and at the end of the treatment period animals
were challenged for bleeding, acute thromboembolism and ulcerogenic activity. In addition, we have assessed the prothrombin
time and activated partial thromboplastin time (aPTT) after oral administration. In in vitro assays, antiplatelet effects of CXE
was evaluated on platelet aggregation, and fibrinolytic activity of the extract was observed by mice or human artificial blood
clot degradation. Platelet citotoxicity of the extract was also determined by the LDH assay. Results demonstrated that CXE has
a significant protective effect on thrombosis. It also inhibits platelet aggregation without demonstrating cytotoxicity on platelets.
CXE slightly prolonged aPTT and showed no ulcerogenic activity after oral administration. In addition, CXE showed a fibrinolytic
activity. Thus, C. xanthocarpa showed antiplatelet, antithrombotic and fibrinolytic activities in mice.
1.Introduction
The interaction between platelets and blood vessels is impor-
tant in the development of thrombosis and cardiovascular
diseases [1]. Platelets are essential in the maintenance of car-
diovascular integrity and in the control of bleeding through
forming blood clot. However, they are also implicated in
the pathological progression of atherosclerotic lesions and
arterial vascular thrombosis [2]. Uncontrolled platelet aggre-
gation is critical in arterial thrombosis and may cause life
threatening disorders [3]. Antiplatelet agents are therefore
considered as a key tool in the treatment and/or prevention
of cardiovascular thrombotic diseases [4]. Although it is well
established that aspirin still provides an effective secondary
prevention of ischemic cardiovascular disorders, this drug
can produce hemorrhagic events and upper gastrointestinal
bleeding as major drawbacks [5].
During the past decade, several trials have led to an effort
in the search for novel compounds or sources to suppress the
platelet aggregation [6, 7]. As a consequence, a number of
protective effects of plants against the serious health risks
have been summarized due to thrombotic diseases, such

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2Evidence-Based Complementary and Alternative Medicine
as coronary thrombosis and atherosclerosis, and numerous
experimental studies have been carried out both in vivo and
in vitro [8, 9].
In Brazil, the edible plant Campomanesia xanthocarpa
Berg. (Myrtaceae), popularly known as “guavirova,” is
present in the Southern region, and is likewise found
in Argentina, Paraguay, and Uruguay [10]. Studies have
shown that C. xanthocarpa possesses a wide spectrum of
physiological effects: the leaves of this plant are used as
infusion in folk medicine to treat inflammatory diseases
and hypercholesterolemia [11]. Moreover, C. xanthocarpa is
empirically used for weight loss and for the control of a
number of conditions associated with obesity [12]. One of
themostrecentstudiesdemonstratedthattheC.xanthocarpa
produced an effect parallel with the mechanism of oral
hypolipemiants, and that this plant showed intense presence
of saponins [13], which are widely distributed in plants and
have many biological activities, such as antiplatelet activity
[14, 15]. Since hypolipemiants also exert antithrombotic
effects [16, 17], and although C. xanthocarpa has reduced
the blood cholesterol levels in hypercholesterolemic patients
[13], until now no information has been available about the
antithrombotic effect of C. xanthocarpa.
C. xanthocarpa has been used in the prevention and
treatment of cardiovascular diseases based on popular belief
and has recently received considerable attention, but the
antithrombotic and fibrinolytic activities of this plant still
remain unknown. Thus, the aim of the present work was to
investigate the effects of C. xanthocarpa on antithrombotic
and fibrinolytic activities in mice.
2.MaterialsandMethods
2.1. Drugs and Reagents. Drugs used in the present study
were acetylsalicylic acid (ASA), adenosine diphosphate
(ADP), epinephrine and calf collagen type III used for
experiments provided from Sigma-Aldrich (St. Louis, USA).
Furthermore, streptokinase used in in vitro experiments was
obtained from Bergamo (S˜ ao Paulo, Brazil).
2.2. Plant and Extract Preparation. Leaves were collected in
May, 2010 from a Campomanesia xanthocarpa tree in the
city of Cruz Alta, RS, Brazil. A Voucher specimen number
1088 was deposited at the Herbarium of University of Cruz
Alta. The material collected underwent a cleaning process
involving 1h in a diluted solution of 20% hypochlorite
made from a 2% stock solution (final concentration of
hypochlorite in diluted solution of 0.4%), immediately
followed by washing in running potable water for 15min.
Then, the material was dried at 40–45◦C and triturated to
a fine powder [13].
In order to carry out the tests, an extract of C. xan-
thocarpa leaves was prepared. Initially, 500mg of dry leaves
was added to 30mL of a water solution at 37◦C under
constant agitation for 30min. After, the solution was filtered
and evaporated to determine total dry content. The final
powder was diluted in water and then adjusted to the desired
concentration to perform the tests.
2.3. Animal and Human Participants. Male Swiss mice
(30–40g) were used and all animals were housed under
standard conditions, with constant temperature (22–24◦C)
and humidity (55–65%) levels, a 12h dark-light cycle, and
free access to food and water. Animals were acclimatized to
the laboratory for at least 1h before testing. The present
study was conducted in accordance with current guidelines
for the care of laboratory animals and all procedures were
approved by our Institutional Ethics Committee of the Fed-
eral University of Santa Maria (number 80/2010). Besides,
human blood was collected of six healthful individuals, with
showed 30±7 years of age to evaluate in vitro assays, and our
research followed guidelines of the Declaration of Helsinki
and Tokyo for humans and informed consent was obtained
for all the six subjects.
2.4. In Vitro Assays
2.4.1. Blood Manipulation. Blood samples were collected
from mice or subjects into Vacutainer (BD Diagnostics,
Plymouth, UK) tubes with sodium citrate 3.8% (1 part of
citrate:9 parts of blood). The samples of blood from mice
were collected from the hepatic vein. The blood samples
were centrifuged for 15min at 180×g to obtain platelet-rich
plasma (PRP) or 10min at 2000×g to obtain platelet-poor
plasma (PPP).
2.4.2. Platelet Aggregation Test. To evaluate the platelet ag-
gregation, the test was performed by turbidimetric mea-
surement with a Chrono-log optical aggregometer, with
AGGRO/LINK Model 810-CA software for Windows version
5.1 [18]. After calibration of the aggregometer, the sample
data concerning the assays and reagents were entered on
a computer coupled to the equipment, and the sample
test was then performed. Aggregation was recorded as the
percent change in light transmission: the baseline value
was set using PRP and maximal transmission using PPP.
PRP was preincubated at 37◦C for 4min with vehicle or
the C. xanthocarpa before addition of the platelet agonist,
adenosine diphosphate (ADP). Maximal aggregation was
obtained stimulating platelet with ADP 10μM.
2.4.3. Determination of Cytotoxicity. To verify the possible
effect of cytotoxicity of C. xanthocarpa on platelets, we
analyzed the leakage of lactate dehydrogenase (LDH) from
platelets of human. After incubation with test drugs at 37◦C
for 3min, aliquots were collected and centrifuged at room
temperature for 2min at 12.000×g. A 50μL aliquot of the
resultant supernatant was used to measure the LDH leakage
using enzymatic methods with (Labtest, Lagoa Santa-MG,
Brazil). The extent of LDH leakage was expressed as % of
total enzyme activity measured in platelets completely lysed
with 10% sodium dodecyl sulphate (SDS final concentration
was 0.05%).
2.4.4. Fibrinolytic Activity. The fibrinolytic activity of the
C. xanthocarpa extract was observed by artificial blood clot
degradation [19]. An artificial blood clot was made by

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Evidence-Based Complementary and Alternative Medicine3
spontaneous coagulation of 50μL of fresh mice or human
blood in a glass test tube. One hour later, the artificial blood
clot was rinsed out repeatedly. The artificial blood clot was
dipped in 1, 3, 10, 30, and 100μg/mL of C. xanthocarpa
extract or streptokinase at room temperature. Saline was
used as a control. The degradation of the clot was estimated
by color development after 1h at room temperature. Then,
red blood cells were lysed by adding 20μL of triton 5% and
then the absorbance of the final solution was read at 560nm.
The amount of color was estimated by linear regression
analysis of a standard curve obtained from samples of mice
blood properly diluted.
2.5. In Vivo Assays
2.5.1. Animal Treatment. The mice were randomly assigned
to distinct groups subjected to the following treatments: 10,
30, or 100mg/Kg of C. xanthocarpa extract or 100mg/Kg of
ASA.Controlanimalsreceivedonlyvehicle.Pharmacological
treatments were given orally once a day for 5 consecutive
days. The last treatment was performed 3h before the
experiment. Animal body weights and general behavior were
recorded at the beginning and at the end of the subacute
treatment to assess the tolerability of repeated adminis-
trations.
2.5.2.Bleeding. Thetailtransectionbleedingwasdetermined
applying the method modified [20]. Briefly, tails of mice
under light isofluorane anesthesia were transected at 2mm
from the tip and immersed in 1mL of 37◦C saline for 2min.
Red blood cells were lysed by adding 20μL of triton 5% and
absorbance of the solution was read at 560nm. The amount
of hemorrhage was estimated by linear regression analysis
of a standard curve obtained from samples of mice blood
properly diluted.
2.5.3. Acute Pulmonary Thromboembolism. To verify the
acute pulmonary thromboembolism, a modification of
method [21] was used. Pulmonary acute thromboembolism
was induced in mice by rapid intravenous injection in the
tail vein of a mixture of 12mg/Kg collagen and 1mg/Kg
epinephrine in order to induce about 85% of paralysis in
the control group. The loss of the righting reflex for 30s
was considered as indication of paralysis. The occurrence of
paralyzed animals was recorded for 15min after thrombotic
mixture injection. Immediately after the last test, mice were
euthanized by CO2chamber.
2.5.4. Ulcerogenic Activity. To evaluate the gastric tolerability
of animals after oral administration of C. xanthocarpa
extract, they were fasted for 18h prior to the last extract
exposure (water ad libitum) and three hours after they were
euthanized. The stomachs were opened by cutting along the
greater curvature and washed with saline 4◦C. Immediately
after that, the development of lesions was assessed with
support of a magnifying glass. The quantification of gastric
mucosal lesions was scored according to their number
and size in a scale from 0 up to 8 points, adapted from
previous method [22], as follows: (0) without injury, (1)
color modification, (2) few petechia/alterations of villous,
(3) 1–3 small injuries (≤1mm length), (4) 1–3 big injuries
(≤1mmlength),(5)1–3biginjuries(>1mm),(6)morethan
three small injuries, (7) more than three big injuries, and (8)
more than three deep injuries.
2.6. Effect on Prothrombin Time (PT) and Activated Partial
Thromboplastin Time (aPTT). For the measure of aPTT,
citrate plasma (0.1mL) was mixed with 0.1mL of human
placenta lipid extract (Pathrombin; Behringwerke), and the
mixture was incubated for 2min at 37◦C. Coagulation
was initiated by the addition of 0.1mL calcium chloride
(25mM), at which point the coagulometer was started and
time to clot formation was recorded [23].
For the measure of PT, citrated plasma (0.1mL) was
incubatedfor1minat37◦C,atwhichpoint0.2mLofhuman
thromboplastin (Thromborel; Behringwerke) was added.
The coagulometer was started, and time to clot formation
was recorded [23].
2.7. Statistical Analysis. Results were expressed as means ±
SEM, except the gastric lesion scores that were expressed
as median. Values of effective concentration that induce
50% of effect (EC50) were reported as geometric means
accompanied by their respective 95% confidence limits.
Emax (maximal effect) and Imax(maximal inhibition) were
calculated based on response of control groups. Data were
analyzed by one-way analysis of variance (ANOVA), or t-test
when appropriate. Post hoc tests (Student-Newman-Keuls
test-SNK) were carried out when appropriate. Differences
between groups were analyzed by χ2test for pulmonary
thromboembolism. Nonparametric Kruskal-Wallis followed
by Dunn’s test was used to analyze gastric lesion scores.
The EC50 values were determined by nonlinear regression
analysis using a sigmoidal concentration-response equation
of individual experiments using GraphPad Software 5.0
(GraphPad, USA). P < 0.05 was considered indicative of
significant differences between groups.
3.Results
3.1. In Vitro Assays. We firstly evaluated the antiplatelet
effectsofC.xanthocarpaextractusingfreshlyisolatedhuman
platelets against agonist-induced platelet aggregation. C.
xanthocarpa extract showed a concentration-dependent
inhibition of ADP-induced platelet aggregation. The Imax
observed was 36 ± 5% for the concentration of 1000μg/mL
and the EC50value of the C. xanthocarpa extract was 35 (15–
84)μg/mL compared with the control group (Figure 1(a)).
To examine the cytotoxicity of C. xanthocarpa extract, we
measured the LDH release from platelet for the determina-
tion of cell lysis. No concentration tested of C. xanthocarpa
extract induced LDH release, while the positive control, SDS
10%, significantly increased the LDH release (Figure 1(b))
reflecting that C. xanthocarpa does not affect cell integrity.
In the evaluation of the fibrinolytic effect, blood clot
degradation was observed in all the test tubes of C.

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4 Evidence-Based Complementary and Alternative Medicine
10 301003001000
0
30
60
90
120
ADP (10µM)
∗
∗
Vehicle
C. xanthocarpa (µg/mL)
Agreggation (control %)
(a)
Vehicle10 30 1003001000 10% SDS
0
25
50
75
100
125
∗
LDH release (total activity %)
C. xanthocarpa (µg/mL)
(b)
Figure 1: Effects of Campomanesia xanthocarpa extract on ADP-
inducedplateletaggregation(a)andonLDHreleaseinplatelets(b).
Data are shown as mean ± S.E.M. (n = 4–6).∗P < 0.05 versus
vehicle control (one-way ANOVA, followed by Student Newman-
Keuls’ test).
xanthocarpa extract or streptokinase, with spreads of red
blood cells trapped by multiple fibrins in mice and human
blood. In mice blood, the EC50 value to C. xanthocarpa
extract and streptokinase was 21 (5–87)μg/mL and 24 (9–
64)μg/mL, and the Emax observed was 56 ± 9% and 60 ±
14%, respectively, for the concentration of 100μg/mL of
both (Figure 2(a)). In human blood, the EC50 value to C.
xanthocarpa extract and streptokinase was 11 (3–33)μg/mL
and 4 (1–10)μg/mL, and the Emaxobserved was 62 ± 7% and
70 ± 6%, respectively, for the concentration of 100μg/mL of
both (Figure 2(b)).
3.2. In Vivo Assays. The mean amount of blood lost from
control animals 2min after tail transaction was 5.8 ± 3.1μL.
Mice treated with antithrombotic doses C. xanthocarpa,
30 and 100mg/Kg/day, showed significant difference from
control, losing 24.3 ± 6.6 and 43.4 ± 14.5μL of blood,
respectively. ASA-treated animals lost 51.9 ± 12.9μL of
−6.5
−6
−5.5
Log (µg/mL)
−5
−4.5
−4
0
25
50
75
100
extract
Streptokinase
Vehicle
C. xanthocarpa
µL blood mL−1(control%)
(a)
−6.5
−6
−5.5
Log (µg/mL)
−5
−4.5
−4
0
25
50
75
100
extract
Streptokinase
Vehicle
C. xanthocarpa
µL blood mL−1(control%)
(b)
Figure 2: Effects of Campomanesia xanthocarpa extract or strep-
tokinase on fibrinolytic activity. Concentration-response curve to
mice (a) or human (b) blood. Each point represents the mean of 3
experiments performed in duplicate and the vertical lines represent
SEM (nonlinear regression analysis).
blood, showing a significant difference when compared to
the control group (Figure 3).
C. xanthocarpa had highly significant antithrombotic
activity when administered at 100mg/Kg day since it pre-
vented paralysis induced by events in 80% compared with
control. ASA displayed lower efficacy since it showed no
significant difference compared to the control to reduce
paralysis (Table 1).
We also evaluated the possible ulcerogenic activity of
C. xanthocarpa extract. None of the doses of the extract
were capable of inducing ulcerogenic activity, while aspirin
(100mg/Kg/day, positive control) induced the formation of

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Evidence-Based Complementary and Alternative Medicine5
VehicleASA10
C. xanthocarpa
30100
1
10
100
(mg/Kg)
∗
∗
∗
Bleeding (µL blood 2min−1)
Figure 3: Effects of Campomanesia xanthocarpa extract or ASA on
bleeding in mice. Data are shown as mean ± SEM (n = 4).∗P <
0.05 versus vehicle control (one-way ANOVA, followed by Student
Newman-Keuls’ test).
Table 1: Effects of C. xanthocarpa extract on pulmonary thrombo-
sis in mice.
Treatment
Dose
(mg/Kg)
—
100
30
10
100
No. of paralysed/
no. of treated mice
9/10
2/10a
4/9a
7/9
5/9a
Protection
(%)
10
80
66.6
33.3
55.5
Vehicle
C. xanthocarpa
Asprin
aP < 0.05 versus vehicle control. Data are shown as mean ± SEM (n = 9-10)
(χ2test).
gastric lesions (the medians (25–75 percentiles)) with lesion
scores of 0 (0-1); 4 (3-4); 0 (0-1), 1 (0-1), and 2 (1-2) for
vehicle; aspirin: 10mg/Kg, 30mg/Kg, and 100mg/Kg day−1
of C. xanthocarpa extract, respectively.
3.3. Effect on Ex Vivo aPTT and PT. As shown in Table 2,
the aPTT and the PT in the vehicle group were 30.90 ±
0.25sec and 11.33 ± 0.08sec, respectively. In the C. xan-
thocarpa-treated groups, the aPPT increased significantly to
32.30 ± 0.91sec and 33.13 ± 0.63sec, at the doses of 30
and 100mg/Kg, respectively, although remaining in normal
limits when compared to the vehicle group. There were no
significant increases of PT when compared to the vehicle
group (Table 2).
4.DiscussionandConclusions
Platelets are blood cells that participate in the human
primary hemostatic mechanism. Platelet-platelet interaction
has the final purpose to produce a platelet thrombus that
constitutes the primary hemostatic plug [24]. In addition,
platelet adhesion and aggregation on blood vessel walls
contribute to the occurrence of thrombosis and emboli
formation, and have relation with other cardiovascular
Table 2: Effect of C. xanthocarpa extract on ex vivo aPTT and PT in
mice.
Treatment
Vehicle
Dose (mg/Kg)
—
10
30
100
aPTT (sec)
30.90 ± 0.25
30.75 ± 0.08
32.30 ± 0.91a
33.13 ± 0.63a
PT (sec)
11.33 ± 0.08
11.30 ± 0.10
11.43 ± 0.13
11.35 ± 0.08
C. xanthocarpa
aP < 0.05 versus vehicle control (one-way ANOVA, followed by Student
Newman-Keuls’ test). Data are shown as mean ± SEM (n = 6).
diseases [25–27]. Thus, it is important to evaluate platelet
function in the thrombosis. Several studies have been carried
out to develop antithrombotic agents with improved efficacy
for preventing or treating arterial or venous thrombosis [28,
29]. Here, we tested the effect of C. xanthocarpa on platelet
aggregation, as well as its antithrombotic and fibrinolytic
activities that are still unknown. The results of the present
study demonstrated that C. xanthocarpa extract has a
significant protective effect in thrombosis once it is capable
of inhibiting platelet aggregation without demonstrating
cytotoxicity. Furthermore, C. xanthocarpa extract slightly
prolonged aPTT, showed a fibrinolytic activity, and did not
show ulcerogenic activity after oral administration.
The interactions between platelets and various adhesive
proteins, such as collagen, and soluble agonists, such as ADP,
provide potential targets for developing antiplatelet agents
[30, 31]. The effect of C. xanthocarpa extract on haemostasis
in in vivo assays was studied using a model of pulmonary
thromboembolism induced by the intravenous injection
of collagen plus epinephrine. This model is characterized
by the massive activation of circulating platelets and the
widespread formation of platelet thrombi in the microcir-
culation of the lungs leading to disseminated pulmonary
microembolism and paralysis of the animal [32]. The oral
administration of C. xanthocarpa extract, once a day for 5
days, markedly reduced the percentage of paralysis in a fairly
dose-dependent manner. C. xanthocarpa extract resulted
to be more effective than ASA in preventing paralysis.
During thrombus formation, ADP plays a key role inducting
the interaction of membrane receptor glycoprotein IIb–
IIIa with fibrinogen, being the most important aggregating
agent [26, 33, 34]. Thus, ADP was chosen as agonist to
induce platelet aggregation in the study. Results showed
that during platelet aggregation, the C. xanthocarpa extract
significantly inhibited ADP-induced platelet aggregation.
All results suggest that the antithrombotic activities of C.
xanthocarpa extract were related at least in part to its ability
to inhibit platelet aggregation.
Blood coagulation is not only the result of a complex
process initiated by the intrinsic system or the extrinsic
systemand/oracommonpathway,butalsoahighlyregulated
process involving interactions between platelets, plasma
coagulation factors, and the vessel wall [35]. Inhibitors
(anticoagulants) and activators (procoagulant) of blood
coagulation may affect any of the factors. The PT test and
the aPTT test are used for distinguishing between the effects
of test agents on the extrinsic and intrinsic pathways. In

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6Evidence-Based Complementary and Alternative Medicine
the clinical tests of blood coagulation, aPTT is used to
evaluate the intrinsic clotting index. A prolonged aPTT
usually represents a deficiency in factors VIII, IX, XI, XII,
and V or Willebrand’s factor. PT is used to evaluate the
extrinsic clotting pathway. A prolonged weak PT indicates
a deficiency in coagulation factors V, VII, and X [36]. The
results reported here show that while the C. xanthocarpa did
not have anticlotting effect when examined by the PT test, a
slight anticoagulant effect is portrayed by the aPTT test. This
indicatesthattheC.xanthocarpamightnotinhibitafactoror
factors in the intrinsic pathway of blood coagulation. At this
stage, there is no indication of the presence of anticoagulants
or procoagulants components in the C. xanthocarpa, since it
did not affect the PT. Although C. xanthocarpa has shown
slightly anticoagulant effect on aPTT test, this effect did not
show clinical significance because the values remained in
normal limits.
To further assess the possible effect of C. xanthocarpa
on platelet activity, the bleeding in mice was measured.
The evaluation of bleeding is a crude test of haemostasis,
which is a useful tool to estimate how well platelets interact
with blood vessel walls to form blood clots [37]. Here, we
demonstratedthatC.xanthocarpaextracttreatmentandASA
affect blood loss. The transactional bleeding time in mice
model is sensitive not only to platelet-active compounds, but
also to drugs that are capable of influencing the fibrinolytic
system [38]. Moreover, C. xanthocarpa extract displayed
favorable gastric effects and did not induce gastric lesions
after oral administration up to 100mg/Kg, demonstrating
to be safer than ASA. Our results are in accordance with
a previous preliminary preclinical study that showed that
C. xanthocarpa proved to be effective in preventing gastric
ulceration in rats and did not produce toxic symptoms in
mice[39].Besides,thetreatmentwithC.xanthocarpaextract
was well tolerated since the treated mice grew similarly to
control mice (data not shown).
We also investigated the fibrinolytic activity of the C.
xanthocarpa extract, which showed fibrinolytic activity in
human and in mice compared to streptokinase. To further
investigate the possible mechanism of action of C. xan-
thocarpa, streptokinase was used as standard. Probably, C.
xanthocarpashowasamemechanismsimilartothestreptok-
inase. We suggest that the primary role of C. xanthocarpa is
theconversionofplasminogentoplasmin.Activatedplasmin
enzymatically cleaves fibrin, which, with platelets and other
hemostatic elements, underlies the pathological processes of
the acute occlusive disorders [40]. Besides, when plasmin
breaks down fibrin, a number of soluble parts are produced
as the D-dimer, that is, a small protein fragment present
in the blood after a blood clot is degraded by fibrinolysis
[41]. Preliminary results of our group demonstrated that
both C. xanthocarpa and streptokinase produced D-dimer
during fibrinolytic activity. These optimistic results point to
the value of further studies in this field. Then with the results
presented in the paper we could only assume that C. xantho-
carpa might have a similar mechanism of streptokinase, and
the result suggested that the C. xanthocarpa extract, besides
inhibiting platelet aggregation, also exerted its antithrom-
botic activity through fibrinolytic activity. Antithrombotic
agents acting in only one pathway of thrombosis formation
have limited efficacy in treating arterial thrombotic diseases
[42]. Hence, the C. xanthocarpa extract with a combination
of antiplatelet aggregation, antithrombotic, and fibrinolytic
activities may be effective in preventing thrombus formation
through several pathways.
Saponins are widely distributed in plants and have many
biological activities, such as antiplatelet activity [14, 15].
The phytochemical components responsible for C. xantho-
carpa antiplatelet effect are still unknown, but preliminary
studies demonstrated the presence of tannins and flavonoids
(quercetin,myricetin,quercitrin,andrutin)inthisplant[39,
43]. Moreover, recent preliminary phytochemical analysis of
C. xanthocarpa by our group indicated the intense presence
of saponins, tannins, and terpenes and small presence
of flavonoids [13]. Thus, we suggest that C. xanthocarpa
bioactive compounds may be inhibiting platelet aggregation.
In conclusion, the C. xanthocarpa extract demonstrated
antiplatelet, antithrombotic, and fibrinolytic activities in
mice. The antithrombotic activity of C. xanthocarpa extract
derived probably from antiplatelet aggregation and fibri-
nolytic activities. The present research showed quite opti-
mistic results pointing to the value of further studies in this
field.
Conflicts of Interests
The authors declared that there is no conflict of interests.
Acknowledgments
This study was supported by Conselho Nacional de Desen-
volvimentoCient´ ıfico(CNPq)(Brazil).Thefellowshipsfrom
CNPq and CAPES are also acknowledged.
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