Journal of Ethnopharmacology 89 (2003) 101–105

Coagulant and anticoagulant activities in Jatropha curcas latex

Omolaja Osoniyi, Funmi Onajobi∗
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife 22005, Nigeria
Received 4 December 2002; accepted 9 July 2003

Abstract

Jatropha curcas Linn. (Euphorbiaceae), a medicinal plant commonly grown in the Tropics, is traditionally used as a haemostatic. Investiga-
tion of the coagulant activity of the latex of Jatropha curcas showed that whole latex significantly (P < 0.01) reduced the clotting time of human
blood. Diluted latex, however, prolonged the clotting time: at high dilutions, the blood did not clot at all. This indicates that Jatropha curcas la-
tex possesses both procoagulant and anticoagulant activities. Prothrombin time (PT) and activated partial thromboplastin time (APTT) tests on
plasma confirm these observations. Solvent partitioning of the latex with ethyl acetate and butanol led to a partial separation of the two opposing
activities: at low concentrations, the ethyl acetate fraction exhibited a procoagulant activity, while the butanol fraction had the highest anticoag-
ulant activity. The residual aqueous fraction had no significant effect on the clotting time of blood and the PT but slightly prolonged the APTT.
© 2003 Published by Elsevier Ireland Ltd.

Keywords: Jatropha curcas; Latex; Blood; Coagulant; Anticoagulant

1. Introduction
Jatropha curcas Linn. (Euphorbiaceae) is a shrub, which
is widely distributed in tropical areas, especially in Tropical
Africa. Different parts of this plant have been tradition-
ally used for various purposes including therapeutic uses
(Dalziel, 1955; Duke, 1988). Some of the ethnomedical uses
of the extracts of Jatropha curcas leaves and roots include
use as a remedy for cancer, as an abortifacient, antiseptic,
diuretic, purgative and haemostatic (Dalziel, 1955). The nut
of the plant has also been used traditionally for the treatment
of many ailments including burns, convulsions, fever and
inflammation. In spite of the myriad of ethnomedical uses to
which various parts of the Jatropha curcas plant have been
put, it is important to note that toxic properties have also been
adduced to parts of the plant, especially the seeds (Watt and
Breyer-Brandwijk, 1962; el Badwi et al., 1995). For exam-
ple, the seed contains curcin, a toxalbumin which is highly
irritant and produces deleterious effects on blood. The latex
is acrid and irritable to the skin (Watt and Breyer-Brandwijk,
1962). Curcain, a protease has been isolated from the latex
of Jatropha curcas (Nath and Dutta, 1991).
Some of the ethnomedical uses of Jatropha curcas have
received support from the results of scientific investigations
in recent times. For example, some compounds with antitu-
mor activities were reportedly found in this plant (Van den
Berg et al., 1995). Furthermore Goonasekera et al. (1995)
showed that various solvent extracts of Jatropha curcas have
an abortive effect.
One of the reported traditional uses of Jatropha curcas
leaves and latex is as a haemostatic or styptic: for exam-
ple, when the latex or the crushed leaf of this plant is
applied directly to cuts and bleeding wounds, the bleed-
ing soon stops (Dalziel, 1955; Watt and Breyer-Brandwijk,
1962; Neuwinger, 1996). Furthermore, Villegas et al. (1997)
have demonstrated a significant wound-healing activity in
Jatropha curcas extracts.
These observations indicate the presence of procoagulant
activity in this plant. We were interested in investigating
the nature of the coagulant activity of the latex of Jatropha
curcas as this has great medical potentials. In the course of
this investigation, we discovered that the Jatropha curcas
latex also exhibits anticoagulant activity when diluted. This
paper reports on these activities. A preliminary report on
the effect of Jatropha curcas latex on blood coagulation was
previously given at a conference (Osoniyi et al., 1996).
2. Materials and methods
2.1. Plant material

∗ Corresponding author. The latex of the plant, Jatropha curcas Linn. (Euphor-
E-mail address: fonajobi@oauife.edu.ng (F. Onajobi). biaceae) was used. The plant was grown from seeds in the

0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd.

doi:10.1016/S0378-8741(03)00263-0
102 O. Osoniyi, F. Onajobi / Journal of Ethnopharmacology 89 (2003) 101–105
experimental plants garden of the Biological Sciences Unit
of the Obafemi Awolowo University, Ile-Ife, Nigeria. The
seeds were planted at the onset of the rainy season in April
1994 under normal environmental conditions without any
artificial fertilizers. The plants were authenticated at the
herbarium of the Botany Department of Obafemi Awolowo
University, Ile-Ife, Nigeria.
2.2. Collection and solvent partitioning of Jatropha
curcas latex
Fresh latex was collected into glass vials and immediately
assayed for its effect on blood clotting. The latex, which was
to be subjected to solvent extraction, was collected into vials
containing a few drops of 95% ethanol to prevent brown-
ing and oxidation. It was extracted with two volumes of
ethyl acetate three times and the extracts combined to ob-
tain an ethyl acetate fraction Fe . The aqueous layer was fur-
ther extracted three times with two volumes of butanol and
the extracts combined to obtain a butanol fraction Fb . The
residual aqueous layer constituted the aqueous fraction, Fa .
The three fractions were evaporated in vacuo at 35 ◦ C in a
rotary evaporator. Latex (96 ml = 98.2 g) when partitioned
with ethyl acetate followed by butanol, yielded 0.289 g Fe ,
2.067 g Fb and 9.32 g Fa . Thus, the percentage yields were
0.29, 2.11 and 9.50 for Fe , Fb and Fa , respectively.
The fractions dissolved in 0.1 M phosphate buffered saline
(PBS) as well as whole latex or latex which had been di-
luted with PBS were all assayed for their effect on clotting
of human blood. Prothrombin time (PT) test and activated
partial thromboplastin time (APTT) were also carried out.
2.3. Assay of blood clotting time
A modified method of Lee and White, as reported by
Wintrobe (1967) was used for the assay of blood clotting
time. Glass test tubes containing 0.1–1 ml each of undiluted
latex, diluted latex, PBS, or acid–citrate–dextrose (antico-
agulant) were equilibrated in a water bath at 37 ◦ C. Blood
was collected from healthy adult volunteers by venipuncture
into sterile plastic disposable syringes. The blood donors
had been screened for the study by a medical practitioner to
ensure that they had not taken any medications for at least 1
week before the blood was collected. Starting a stopwatch,
1 ml of this blood was immediately transferred into each of
the equilibrated test tubes by carefully allowing the blood to
run down the side of the tube. At intervals of 30 s, the tubes,
still in the water bath, were gently tilted to an angle of 45◦
to check for blood clot formation. This was continued until
the tubes could be inverted without blood flowing; the stop-
watch was immediately stopped and the blood clotting time
was recorded.
The latex fractions (Fe , Fb and Fa ) were dissolved in 0.1 M
PBS to give 100 mg/ml stock solutions. The stock solutions
were then diluted serially with the PBS solution to give
solutions of 50, 25, 12.5, 6.25 and 3.125 mg/ml. Aliquots
(0.1 ml) of these solutions were tested for their effects on
the clotting time of blood as described above.
2.4. Assay for prothrombin time (PT) and activated partial
thromboplastin time (APTT)
The method described by Brown (1988) was used for the
determination of PT. Plasma was obtained by centrifuging
citrated blood for 15 min at 1500 × g. Thromboplastin-
calcium reagent (Sigma Diagnostics, St. Louis, MO)
was reconstituted with distilled water according to the
manufacturer’s instructions. It was then prewarmed by plac-
ing it in a water bath at 37 ◦ C for at least 10 min before com-
mencement of the test. One hundred microliters of plasma
was placed in a test tube and incubated in the water bath
for 180 s. For the controls, 100 ␮l of prewarmed PBS, fol-
lowed by 200 ␮l of the prewarmed thromboplastin-calcium
reagent was rapidly pipetted into the plasma while simulta-
neously starting a timer. The test tube was then gently tilted
back and forth, until a clot formed, at which time the timer
was stopped and the clotting time recorded. For the tests,
100 ␮l of the prewarmed sample solution in PBS was mixed
with the plasma, just before adding the thromboplastin-
calcium reagent. All experiments were carried out in at
least, duplicates.
The method described by Brown (1988), suitably modi-
fied, was used for the APTT tests. Alexin® (Sigma Diag-
nostics), which is the partial thromboplastin with activator,
and calcium chloride (0.02 M) were prewarmed to 37 ◦ C
separately in a water bath. Fifty microliters of plasma was
placed in a test tube. After incubating for 180 s in the wa-
ter bath, 50 ␮l of Alexin® was added, and the contents were
mixed rapidly. The mixture was then incubated for another
180 s, after which 50 ␮l of PBS, then 50 ␮l of the prewarmed
calcium chloride solution was added while simultaneously
starting a timer. The tube was then allowed to remain in the
water bath while gently tilting the test tube every 5 s. At the
end of 20 s, the test tube was removed from the water, wiped
clean with dry gauze, and gently tilted back and forth until
a clot was seen and the time recorded. For the test samples,
50 ␮l of the material was added to the contents of the test
tube just prior to the addition of calcium chloride, and read-
ings taken as before.
3. Results
3.1. Effect of Jatropha curcas whole latex on human
blood coagulation time
The blood coagulation time of 24 adult volunteers varied
between 4 and 8 min, with a mean of 5.83 ± 1.25 min. In the
presence of 1 ml (1.05 g) of whole latex, the mean of clotting
time was significantly (P < 0.01) reduced to 3.83±1.01 min
with a range of 2–5 min. The use of 1 ml PBS instead of
latex, as a control, gave a mean clotting time of 6.29 ±
 O. Osoniyi, F. Onajobi / Journal of Ethnopharmacology 89 (2003) 101–105 103

Table 1
Effect of Jatropha curcas latex fractions on the clotting time of blood
Additions to blooda Clotting time

Latex fractionb Amount PBS Mean S.D.

(mg) (ml) (min) (min)
Control – – 4.41 a 0.220
Control (PBS) – 0.1 4.44 a 0.313
Fe 0.313 – 3.30 b 0.312
Fe 0.625 – 2.94 c 0.472
Fb 0.313 – 5.43 d 0.638
Fb 0.625 – 6.74 e 1.026
Fa 0.313 – 4.27 a 0.342
Fa 0.625 – 4.50 a 0.318
Values are mean ± S.D. (n = 12). The letters (a, b, c, d, e) denote values
which are significantly different from one another using Student’s t-test.
The letters (b, c, d, e) are each significantly different from letter (a) at
P < 0.001. Letter (b) is significantly different from letter (c) at P < 0.05.
Letter (d) is significantly different from letter (e) at P < 0.01.
a All tubes had 1 ml blood.
b Each latex fraction was contained in 0.1 ml of a solution made up

in PBS.
Fig. 1. Effect of Jatropha curcas latex on the clotting time of blood.
Data represent the means and standard deviation of the means (n = 16).
Each assay tube contained 1 ml of blood and 1 ml of diluted latex. Serial seemed to take place; in the presence of any of the three
dilutions were made with PBS. latex fractions (Fe , Fb or Fa ) no real clotting, but a progres-
sive thickening of the blood was observed. The onset of the
thickening was around 1–2 min and progressed over a period
1.28 min with a range of 5–9 min. In the tubes containing
of about 12 min.
the acid–citrate–dextrose (anticoagulant), the blood sample
did not clot, as expected; this was a control used to ensure
the integrity of the blood samples. 3.3. Effect of whole Jatropha curcas and fractions
When the effect of different dilutions of the latex on the on prothrombin time (PT) and activated partial
blood coagulation time was investigated for 16 of the vol- thromboplastin time (APTT)
unteers, it was observed that the clotting time increased as
the dilution increased or as the latex volume decreased. At Undiluted whole latex, 1:2 diluted and 1:4 diluted latex
1 in 8 dilution (0.125 ml latex) and 1 in 10 dilution (0.1 ml all caused the plasma to coagulate instantly in the PT test.
latex), the blood either had a long clotting time or did not With further dilutions, the plasma coagulated with PTs that
clot at all, even after 24 h. Fig. 1 confirms that there is an ex- were slightly higher than control values (Table 2). In the
ponential inverse relationship between the volume of latex
added to the blood and the clotting time. Further dilutions
Table 2
(1 in 16, 1 in 32 and 1 in 64) of the latex prevented all the Effect of Jatropha curcas latex on PT and APTT
blood samples from clotting.
Latex (dilutions) PT (s) APTT (s)
3.2. Effect of latex fractions on blood clotting Control (PBS) 16.00 ± 0.82 a 47.50 ± 0.71 a
Undiluted n.d. n.d.
Another set of adult volunteers donated blood samples 1:2 n.d. n.d.
1:4 n.d. >240a
for this investigation. There was no significant difference in 1:8 20.17 ± 0.41 d >240a
clotting times between blood with PBS added, i.e control 1:16 18.17 ± 0.75 d >240a
(PBS) and the clotting time of blood in the absence of PBS. 1:32 17.67 ± 0.52 c >240a
Each of the latex fractions (Fe , Fb and Fa ) exhibited a bipha- 1:64 17.50 ± 0.84 b >240a
sic effect on the blood coagulation. At low concentrations 1:128 17.33 ± 1.03 b 76.00 ± 5.66 b
1:256 17.67 ± 1.50 b 56.00 ± 5.66 a
the ethyl acetate fraction (Fe ) significantly (P < 0.05) de-
creased the blood clotting time while the butanol (Fb ) frac- Values are mean±S.D. (n = 6). n.d.: not determinable because coagulation
was instantaneous. For PT, letters (b, c, d) are significantly different from
tion significantly increased (P < 0.05) the clotting time in a
letter (a) at P < 0.05, P < 0.01 and P < 0.001, respectively using
concentration-dependent manner. The aqueous fraction had Student’s t-test. For APTT, letter (b) is significantly different from letter
no significant effect on the clotting time (Table 1). At high (a) at P < 0.05.
a No statistical tests were performed with values greater than 240 s.
concentrations however, some processes other than clotting
104 O. Osoniyi, F. Onajobi / Journal of Ethnopharmacology 89 (2003) 101–105

Table 3 et al. (1999) have also observed a procoagulant activity in

Effect of Jatropha curcas latex fractions on PT and APTT the latex of Jatropha curcas. One would normally expect the
Latex fraction Amount PT (s) APTT (s) procoagulant activity of the latex to decrease with dilution
(␮g) and that the clotting time of blood would approach the value
Control (PBS) – 16.08 ± 1.08 a 42.43 ± 4.61 a obtained in the absence of the latex, or in the presence of
Fe 10 16.17 ± 0.75 a 55.50 ± 0.58 b PBS alone, as the dilution increased. The observation that
Fe 20 17.00 ± 0.50 a 110.00 ± 11.55 c the latex actually changed from being a procoagulant to an
Fe 40 17.00 ± 0.65 a 155.00 ± 23.45 c anticoagulant upon dilution raises a few possibilities. One
Fe 60 18.00 ± 0.55a b >240b possibility is that the two activities are exhibited by different
Fe 80 22.50 ± 0.58a b >240b
Fe 100 26.50 ± 3.50a b >240b
factors, each of which functions optimally at different con-
centrations. It is also possible that the same factor acts as a
Fb 10 16.00 ± 1.55 a 137.50 ± 53.03 c
procoagulant and as an anticoagulant under different condi-
Fb 20 18.50 ± 1.73 b >240b
Fb 40 27.50 ± 2.89 c >240b tions. For example, it has been shown that thrombin acts as
Fb 60 31.00 ± 1.15 c >240b a procoagulant when it cleaves fibrinogen and promotes the
Fb 80 41.00 ± 1.16a c >240b formation of a fibrin clot but functions as an anticoagulant
Fb 100 42.13 ± 9.75a c >240b when it activates protein C in the presence of the cofactor
Fa 10 15.25 ± 0.96 a 50.50 ± 0.71 a thrombomodulin (Cartwell and Di Cera, 2000).
Fa 20 16.00 ± 0.41 a 54.50 ± 0.71 b The results obtained on the effect of the whole latex on
Fa 40 16.00 ± 0.54 a 62.50 ± 3.54 c the PT and APTT tests further support the conclusion that
Fa 60 17.00 ± 0.58 a 105.00 ± 13.29 c
Fa 80 17.00 ± 0.65 a 120.00 ± 0.55 c
the whole latex contains both coagulant and anticoagulant
Fa 100 17.17 ± 0.98 a 127.00 ± 4.24 c activities, with the coagulant activity being evident at high
concentrations (lower dilutions) of the latex, while the anti-
Values are mean ± S.D. (n = 6). Letters (b, c) are significantly different
from letter (a) at P < 0.01 and P < 0.001, respectively using Student’s coagulant activity was evident at low concentrations (higher
t-test. dilutions) of the latex.
a Improper clotting. For the latex fractions Fe , Fb and Fa , the results of the PT
b No statistical tests were performed with values greater than 240 s.
and APTT tests failed to show the presence of a procoagu-
lant in any of the fractions, yet the whole blood clotting time
test indicates that the coagulant activity of Jatropha curcas
APTT test, undiluted and 1:2 diluted latex caused instant latex is associated with the ethyl acetate fraction (Fe ). The
coagulation of the plasma. On the other hand, further dilu- coagulant activity of Fe may have been weakened during
tions of the latex strongly inhibited coagulation of the plasma the process of solvent partitioning. The presence of antico-
(Table 2). agulant activity in all the fractions is however, shown by the
The ethyl acetate fractions of the latex (Fe ) generally APTT tests, this activity being highest in the butanol frac-
had no significant effect on the PT at low concentrations. tion (Fb ) and least in the aqueous fraction (Fa ). Thus, the
At high concentrations, there was improper clotting in solvent partitioning of the latex has not succeeded in sepa-
that no gel-like clot was formed, although there was some rating the anticoagulant activity into a single solvent fraction
kind of granulation. Fe , however, caused the APTT to in- but it has concentrated this activity into the butanol fraction
crease significantly in a concentration-dependent manner (Fb ). Therefore, a partial separation of the coagulant and an-
(Table 3). ticoagulant activities of the latex has been achieved by the
The butanol fraction of the latex (Fb ) elongated the PT solvent partitioning process, locating the procoagulant ac-
at levels of 10–100 ␮g in the assay mixture, with the PT tivity in the Fe and concentrating the anticoagulant activity
increasing with increasing concentration of Fb (Table 3). in the Fb .
However, the same kind of improper clots described above Blood clotting process is very complex, involving many
were observed at 80 and 100 ␮g levels. Fb increased APTT factors found in the plasma and tissues. It involves both
very strongly at all concentrations used (Table 3). the intrinsic and extrinsic pathways (Brown, 1988; Jandl,
Fa , the aqueous fraction of the latex, had no effect on the 1996). Inhibitors (anticoagulants) and activators (procoagu-
PT as shown in Table 3. This fraction, however, increased lant) of blood coagulation may affect any of the factors. The
the APTT in a concentration-dependent manner. PT test and the APTT test are used for distinguishing be-
tween the effects of test agents on the extrinsic and intrinsic
pathways (Brown, 1988). Substances that affect the PT are
4. Discussion thought to act on the extrinsic pathway factors: factors V,
VII, X, prothrombin and fibrinogen, while those that affect
The above results indicate that whole Jatropha curcas la- the APTT act on the components of the intrinsic pathway,
tex possesses both procoagulant and anticoagulant activities, that is, all coagulation factors except factors VII and XIII.
the former being evident at high concentrations of the latex, The results reported here show that while the Jatropha cur-
while the latter is exhibited at low concentrations. Odusote cas latex or its fractions show little or no anti-clotting effect
 O. Osoniyi, F. Onajobi / Journal of Ethnopharmacology 89 (2003) 101–105
when examined by the PT test, a very strong anticoagulant
effect is portrayed by the APTT test. This indicates that the
Jatropha curcas latex anticoagulant inhibits a factor or fac-
tors in the intrinsic pathway of blood coagulation. At this
stage, there is no indication of how the procoagulant in the
latex acts since it affected the PT and APTT in a similar
manner, causing instant coagulation in both cases.
These results, which demonstrate the procoagulant activ-
ity of Jatropha curcas latex, give a scientific explanation for
its folkloric use as a styptic. The results further show that
the diluted latex can be used as an anticoagulant. The in-
tegrity of the red cell membrane of blood to which latex has
been added, under the non-clotting condition, is presumably
intact as we have shown in our laboratory that ethanolic
extracts of Jatropha curcas latex stabilizes the erythrocyte
membrane. Even this crude non-purified anticoagulant in
the latex could be useful clinically, for in vitro assays when
blood has to be obtained and kept for some time under cir-
cumstances in which chemical anticoagulants are not readily
available, such as in villages and other remote places. Any
in vivo use of blood kept from clotting with diluted Jatropha
curcas latex cannot be recommended at present in view of
the reported presence of some toxic substances in the latex
(Abdu-Aguye et al., 1986; Joubert et al., 1984). The great
dilution required for the latex to act as an anticoagulant may
render the toxic substances ineffective but this still requires
some investigation.
Work is in progress to further investigate and purify the
coagulant and anticoagulant factors in the Jatropha curcas
latex.
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