BMB

reports

Anticoagulant activities of curcumin and its derivative

& Jong-Sup Bae *
1,# 2,# 3,
Dong-Chan Kim , Sae-Kwang Ku
1
Laboratory of Microvascular Circulation Research, NEUORNEX Inc. Daegu 711-823, 2Department of Anatomy and Histology, College of
Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, 3College of Pharmacy, Research Institute of Pharmaceutical Sciences,
Kyungpook National University, Daegu 702-701, Korea

Curcumin, a polyphenol responsible for the yellow color of alternate routes are used, each giving rise to a different form of
the curry spice turmeric, possesses antiinflammatory, anti- the prothrombin activator (1-5). In the extrinsic pathway, pro-
proliferative and antiangiogenic activities. However, anti- thrombin activator complex consists of activated factor X (FXa),
coagulant activities of curcumin have not been studied. Here, tissue factor (TF), activated factor VII (FVIIa) and the cofactor
the anticoagulant properties of curcumin and its derivative activated factor V (FVa) (1-5). This complex, specifically FXa,
(bisdemethoxycurcumin, BDMC) were determined by monitor- along with the cofactor FVa, then converts prothrombin to ac-
ing activated partial thromboplastin time (aPTT), prothrombin tive thrombin. Fibrin forms a mesh within the platelet aggregate
time (PT) as well as cell-based thrombin and activated factor X to stabilize clots (1-5). In contrast, in the intrinsic pathway, pro-
(FXa) generation activities. Data showed that curcumin and thrombin activator complex consists of FXa, FVa, activated fac-
BDMC prolonged aPTT and PT significantly and inhibited tor VIII (FVIIIa) and phopspholipid (PL) (1-5). The clotting time
thrombin and FXa activities. They inhibited the generation of assay measures the lag time of thrombin generation (6) and the
thrombin or FXa. In accordance with these anticoagulant activ- activated partial thromboplastin time (aPTT) is a performance
ities, curcumin and BDMC showed anticoagulant effect in indicator measuring the efficacy of both the contact activation
vivo. Surprisingly, these anticoagulant effects of curcumin pathway and the common coagulation pathways (6). Further,
were better than those of BDMC indicating that methoxy the prothrombin time (PT) is measure of the extrinsic pathway
group in curcumin positively regulated anticoagulant function of coagulation (7, 8).
of curcumin. Therefore, these results suggest that curcumin 　The rhizome of Curcuma longa has been used in indigenous
and BDMC possess antithrombotic activities and daily con- medicine for the treatment of inflammatory disorders and its me-
sumption of the curry spice turmeric might help maintain anti- dicinal activity has been known since ancient times. Turmeric
coagulant status. [BMB reports 2012; 45(4): 221-226] derived from the rhizome has been widely used by the people in
the Middle East for centuries as a food component (9, 10). The
use of turmeric extract or turmeric oil as a spice and household
INTRODUCTION remedy has been known to be safe for centuries. Bhide, et al. al-
so revealed the safety and tolerance of turmeric through human
The key of the blood clotting pathway is the production of clinical trials (11). In many previous studies, extracts prepared
thrombin which is required for the conversion of fibrinogen to from Curcuma longa have been used as antiinflammatory agents
fibrin (1, 2). Thrombin resides in the cell in an inactive form, to treat gas, colic, toothaches, chest pains, menstrual difficulties,
called prothrombin, and is activated by the coagulation casca- stomach and liver ailmetns (9, 12, 13). Polyphenolic phytochem-
devia formation of a complex called the prothrombin activator icals are common in the diet and have been suggested to have a
complex (1-5). The formation of the prothrombin activator com- wide range of beneficial health effects and the polyphenolic
plex occurs by two different pathways: the intrinsic pro- compounds in turmeric are responsible for a number of its bene-
thrombin activation pathway and the extrinsic prothrombin ac- ficial health effects (14, 15). Turmeric contains three major poly-
tivation pathway. Though the ultimate goal of both the path- phenolic analogues. The majority is curcumin and the com-
ways is the generation of the prothrombin activator complex, pounds in smaller amounts are demethoxycurcumin, and bisde-
methoxycurcumin (BDMC) (16, 17). Recent studies indicate that
*Corresponding author. Tel: +82-53-950-8570; Fax: +82-53-950- dietary administration of curcumin may have beneficial effects in
8557; E-mail: [email protected] conditions such as cancer (18), Alzheimer’s disease (19) and
#
First two authors contributed equally to this work. cystic fibrosis (20). With regard to mode of action, curcumin ex-
http://dx.doi.org/10.5483/BMBRep.2012.45.4.221 hibits a diverse array of metabolic, cellular and molecular activi-
ties. Although curcumin analogues exhibit activities very similar
Received 9 August 2011, Revised 28 September 2011, to curcumin, their potencies compared to curcumin have not
Accepted 11 October 2011
been clearly established. In most systems, curcumin is found to
Keywords: aPTT, Curcumin, HUVECs, PT
be most potent (21, 22) and in some systems, BDMC was found

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 Anticoagulant activities of curcumin
Dong-Chan Kim, et al.

to exhibit different (in some cases, more potent) activities (23-25). the anticoagulant function of curcumin.
There is an increasing demand for comparison study between
curcumin and BDMC, due to the discovery of their new bio- Effects of curcumin and BDMC on inactivation of thrombin or
logical activities (21, 26, 27). Identification of novel biological FXa
activities of curcumin and its analogues is of interest both pre- To elucidate the inhibitory mechanism of curcumin and
clinically and clinically. Additionally, anticoagulant activities of BDMC on coagulation time, their inhibitory effect on thrombin
curcumin have not been well studied. Herein, the anticoagulant and FXa activities was measured using chromogenic substrates
properties of curcumin and its derivative, BDMC on the gen- in the absence or presence of antithrombin III (AT III). In the
eration of FXa and thrombin as well as the regulation of clotting absence of AT III, the amidolytic activity of thrombin was in-
time (PT and aPTT) were determined. hibited by curcumin and BDMC in a dose-dependent manner,
showing that the anticoagulant directly inhibited thrombin
RESULTS activity. However, in the presence of AT III, thrombin activity
was essentially unchanged (Fig. 1A, B). Thus, AT III was un-
Effects of curcumin and BDMC on aPTT and PT able to potentiate the activity of curcumin or BDMC. Further,
The anticoagulant properties of curcumin and BDMC were the effects of curcumin and BDMC on FXa activity in the ab-
tested in aPTT and PT assays using human plasma and are sence or presence of AT III were also investigated. The anti-
summarized in Table 1 and 2. Although the anticoagulant ac- coagulant showed direct inhibitory effects on FXa activities at
tivities of curcumin and BDMC were weaker than those of high concentrations, and the inhibitory effect of AT III was not
heparin, aPTT and PT were significantly prolonged by curcu- changed by curcumin or BDMC (Fig. 1C, D). These results
min or BDMC at concentrations at or greater than 5 μM. were consistent with the antithrombin assay. Therefore, these
Prolongation of aPTT suggests inhibition of the intrinsic and/or results suggested that the antithrombotic mechanism of curcu-
the common pathway while prolonged PT indicates that curcu- min and BDMC appears to be due to inhibition of fibrin poly-
min and BDMC could also inhibit the extrinsic pathway of merization and/or the intrinsic/extrinsic pathway without po-
coagulation. To confirm these in vitro data, in vivo tail bleed- tentiation by AT III. Furthermore, the methoxy group in curcu-
ing time was determined. As shown in Table 1 and 2, tail min positively regulates the anticoagulant effects on the in-
bleeding time was significantly prolonged by curcumin or hibition of thrombin or FXa activity because the anticoagulant
BDMC with respect to the control. Surprisingly, effects of cur- effects of curcumin were better than those of BDMC.
cumin on the clotting time were better than that of BDMC sug-
gesting that methoxy group in curcumin positively regulates

Table 1. Anticoagulant activity of curcumina Table 2. Anticoagulant activity of BDMCa

In vitro coagulant assay In vitro coagulant assay

Sample Dose aPTT (s) PT (s) Sample Dose aPTT (s) PT (s)

Control Saline 36.2 ± 1.2 17.5 ± 0.4 Control Saline 35.8 ± 1.3 17.5 ± 0.4
Curcumin 0.1 μM 37.2 ± 1.3 17.4 ± 0.3 BDMC 0.1 μM 38.9 ± 0.8 17.5 ± 0.5
b b
0.5 μM 48.5 ± 1.4 18.2 ± 0.7 0.5 μM 41.6 ± 1.5 17.9 ± 0.6
c b b b
1 μM 52.6 ± 1.8 19.8 ± 0.5 1 μM 48.5 ± 2.14 18.2 ± 0.5
5 μM 65.3 ± 1.5c 21.6 ± 0.6b 5 μM 68.5 ± 1.2c 19.2 ± 0.7c
c c c c
10 μM 77.5 ± 2.1 27.5 ± 0.5 10 μM 70.5 ± 1.8 20.9 ± 0.4
c c c c
20 μM 91.8 ± 1.5 31.8 ± 0.4 20 μM 87.6 ± 1.5 25.4 ± 0.3
50 μM 119.8 ± 0.9c 35.2 ± 0.4c 50 μM 98.6 ± 1.4c 29.8 ± 0.5c
Heparin 1.5 (μg/ml) 15 (μg/ml) Heparin 1.5 (μg/ml) 15 (μg/ml)
c c c c
＞300 61.5 ± 0.5 ＞300 61.5 ± 0.5

In vivo bleeding time In vivo bleeding time

Sample Dose Tail Bleeding time (s) n Sample Dose Tail Bleeding time (s) n

Control Saline 54.2 ± 8 3 Control Saline 54.2 ± 8 3

Curcumin 100 mg/kg 102 ± 2c 3 BDMC 100 mg/kg 82 ± 2b 3
c c
Heparin 50 mg/kg 158.6 ± 4 3 Heparin 50 mg/kg 158.6 ± 4 3
a
Each value represents the means ± SD (n = 5). bP ＜ 0.05 as com- a
Each value represents the means ± SD (n = 5). bP ＜ 0.05 as
c c
pared to control. P ＜ 0.01 as compared to control compared to control. P ＜ 0.01 as compared to control

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 Anticoagulant activities of curcumin
Dong-Chan Kim, et al.

Fig. 1. Effect of curcumin and BDMC on the inactivation of

thrombin and factor Xa. Inhibition of thrombin (Th) in the ab- Fig. 2. Inhibition of thrombin and FXa generation by curcumin
sence of antithrombin III (○) or in the presence of antithrombin and BDMC in HUVECs. (A) FVa (100 pM) and FXa (1 nM) were
III (●) by curcumin (A) or DBMC (B) was monitored by a chro- preincubated on HUVECs monolayers for 10 min with indicated
mogenic assay as described in “Materials and Methods”. Inhibition concentrations of curcumin (□) or BCMC (■). Prothrombin was
of factor Xa (FXa) in the absence of antithrombin III (○) or in added to a final concentration of 1 μM and the amounts of pro-
the presence of antithrombin III (●) by curcumin (C) or DBMC thrombin activated were determined at 30 min as described in
(D) was monitored by chromogenic assay as described in “Materials and Methods”. (B) HUVECs were preincubated with in-
“Materials and Methods”. *P ＜ 0.01 as compared to 0. dicated concentrations of curcumin (□) or BCMC (■) for 10 min.
TNF-α (10 ng/ml for 6 h) stimulated HUVECs were incubated with
FVIIa (10 nM) and FX (175 nM) in the absence or presence of
anti-TF IgG (25 μg/ml) and FXa generation was then determined
Effects of curcumin and BDMC on the generation of thrombin as described in “Materials and Methods”. (C) HUVECs were cul-
and FXa tured with curcumin (□) or BCMC (■) in the absence or pres-
Sugo et al. reported that endothelial cells are able to support ence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentration in the
prothrombin activation by FXa (28). Preincubation of FVa and culture mediums was examined as described in “Materials and
Methods”. (D) the same as C except that cells were preincubated
FXa in the presence of CaCl2 with HUVECs before addition of with curcumin (□) or BCMC (■) and cultured with SP600125 (2
prothrombin resulted in thrombin generation (Fig. 2A). The ef- μM), emodin (2 μg/ml), and PD98059 (10 μM) in presence of
fect of curcumin and BDMC on thrombin generation showed TNF-α (10 ng/ml) for 18 h. *P ＜ 0.05 as compared to 0 (A) or
that curcumin and BDMC inhibited thrombin activation of pro- TNF-α alone (B, C) or to (+) control (D).
thrombin dose- dependently (Fig. 2A). Rao et al. showed that
the endothelium provides the functional equivalent of procoa-
gulant phospholipids and supports FX activation (29) and that induced PAI-1 secretion from HUVECs. To define the molec-
in TNF-α stimulated HUVECs, FVIIa could activate FX, which ular targets of curcumin in the signal transduction pathways
was completely dependent on TF expression (30). Thus, it is leading to TNF-α induced PAI-1 expression, we investigated
likely that the endothelium can provide support for FVIIa acti- the effects of three signal transduction inhibitors, emodin (a
vation of FX. If so, it would be of interest to investigate the ef- NF-κB inhibitor), PD98059 (an extracellular signal regulated
fect of curcumin and BDMC on FVIIa activation of FX. kinase, ERK, inhibitor), and SP600125 (a c-Jun N-terminal kin-
HUVECs were stimulated with TNF-α to induce TF expression. ase, JNK, inhibitor) on TNF-α induced PAI-1 expression in the
As shown in Fig. 2B, the rate of FX activation by FVIIa was presence or absence of curcumin. Experiments performed
100-fold higher in stimulated HUVECs (53.3 ± 5 nM) com- showed that neither SP600125 nor emodin showed any addi-
pared with non-stimulated HUVECs (0.54 ± 0.2 nM), which tional inhibitory effects in the presence of curcumin (Fig. 2D).
was completely attenuated by anti-TF IgG (4.8 ± 0.4 nM). However, the inhibitory effects of PD98059 were essentially
Moreover, preincubation with curcumin or BDMC dose-de- additive with those of curcumin (Fig. 2D).
pendently inhibited FVIIa activation of FX (Fig. 2B). Therefore,
these results suggested that curcumin could inhibit the gen- DISCUSSION
eration of thrombin or FXa and the methoxy group in curcu-
min positively regulated these functions of curcumin. The vascular endothelium provides a number of important
　Because plasminogen activator inhibitor type 1 (PAI-1) de- functions in order to maintain adequate blood supply to vital
termines fibrinolytic activity (1), the effect of TNF-α and curcu- organs. These functions include prevention of coagulation,
min on PAI-1 secretion from HUVECs were investigated. As regulation of vascular tonus, orchestration of the migration of
shown in Fig. 2C, curcumin dose dependently inhibited TNF-α blood cells by the expression of adhesion molecules and regu-

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 Anticoagulant activities of curcumin
Dong-Chan Kim, et al.

lation of vasopermeability (31). Among these, regulation of he- of curcumin could be mainly caused by interaction of the target
mostatic activity was regulated through a balance of pro- and molecules with the ortho-methoxy group.
anticoagulant properties (1). Impaired endothelial function 　The significant progress made in understanding the role of
causes thrombus-related complications including myocardial FXa and thrombin in various thrombotic disease states has
infarction, stroke and thromboembolism (32). In this study, we clearly demonstrated potential therapeutic benefits of blocking
presented curcumin as a potent anticoagulant by inhibitiing these key enzymes in the blood coagulation cascade (39). A
thrombin or FXa. The anticoagulant activity of curcumin was potent and selective small molecule FXa or thrombin inhibitor
evidenced by the prolongation of the clotting time in plas- has the potential to offer substantial therapeutic benefits (39).
ma-based PT and APTT assays. Additionally, the inhibitory ef- Curcumin exhibits the potency and selectivity required for such
fects by curcumin on FXa generation and further thrombin a candidate and is currently undergoing additional evaluations.
generation support the anticoagulant activities of curcumin. 　In conclusion, this study showed that curcumin inhibited the
　It is well known that FXa has no effect on platelet activation, extrinsic and intrinsic pathways of blood coagulation by in-
however, once it is assembled into the prothrombinase com- hibiting FXa and thrombin generation in HUVECs. These re-
plex, it triggers enormous amounts of thrombin (2, 5). Throm- sults adds to previous work and may be helpful for the rational
bin is the final enzyme in the blood clotting cascade respon- design of pharmacological strategies for treating or preventing
sible for clot formation and platelet activation (2, 5). Based on vascular diseases via regulation of thrombin generation.
the results that curcumin could inhibit generation of FXa and
thrombin, the anticoagulant activity of curcumin was initiated MATERIALS AND METHODS
from the inhibition of the penultimate and final enzyme in the
blood clotting cascade. Reagents
　TNF-α has been known to activate JNK, NF-κB, and ERK in Curcumin (product catalog #: C2302) and bisdemethoxycurcu-
human endothelial cells (33-35). Here, we used a JNK min (product catalog #: B3347) were purchased from TCI
(SP600125), NF-κB (emodin), and ERK (PD98059) inhibitors to Korea (Tokyo Chemical Industry Co., Ltd. Seoul, South Korea).
define the molecular target of curcumin. We observed that TNF-α, JNK inhibitor (SP600125), NF-κB inhibitor (emodin),
PD98059, but not emodin or SP600125, was additive to the in- and ERK inhibitor (PD98059) were purchased from R&D
hibitory effects of curcumin on TNF-α induced PAI-1 secretion. Systems (Minneapolis, MN). Anti-tissue factor antibody was
These results suggest that the NF-κB and JNK pathway are in- purchased from Santa Cruz Biologics (Santa Cruz, CA). Factor
volved in curcumin mediated inhibition of TNF-α induced V, Vll, Vlla, FX, FXa, antithrombin III (AT III), prothrombin and
PAI-1 expression in HUVECs. Thus, these results seem to in- thrombin were obtained from Haematologic Technologies
dicate that curcumin decreases PAI-1 levels via inhibition of the (Essex Junction, VT, USA). aPTT assay reagent and PT reagents
NF-κB and JNK pathways. were purchased from Fisher Diagnostics (Middletown, Virgi-
　Noting that the effects of curcumin on the anticoagulant ac- nia, USA). Chromogenic substrates S-2222, and S-2238 were
tivity was better than BDMC, it suggests that the ortho-methoxy from Chromogenix AB (Sweden).
group in curcumin positively regulates anticoagulant functions
of curcumin. In a previous report, curcumin and BDMC had Anticoagulation assay
different redox properties due to the presence of the ortho-me- Determination of aPTT and PT were performed according to
thoxy group in position 3 of the phenyl moiety in curcumin. the manufacture’s specifications using Thrombotimer (Behnk
(36) While curcumin has two symmetric ortho-methoxy phe- Elektronik, Germany). In brief, citrated normal human plasma
nols linked through the a,b-unsaturated b-diketone moiety, (90 μl) was mixed with 10 μl of curcumin or BDMC and in-
BDMC, which is also symmetric, is deficient in the two or- cubated for 1 min at 37°C. Then, aPTT assay reagent (100 μl)
tho-methoxy substitutions. Although curcumin and bisdeme- was added to the mixture and incubated for 1 min at 37°C.
thoxycurcumin differ in their chemical structures only with re- Thereafter, 20 mM CaCl2 (100 μl) was added and the clotting
gard to the ortho-methoxy substitution, they exhibit signifi- time was recorded. For the PT assay, citrated normal human
cantly different antioxidant, antitumor, and antiinflammatory plasma (90 μl) was mixed with 10 μl of a curcumin or BDMC
o
activities. The hydrogen bonding interaction between the phe- stock and incubated for 1 min at 37 C. Then, PT assay reagent
o
nolic OH and the ortho-methoxy groups in curcumin markedly (200 μl), preincubated for 10 min at 37 C, was added and the
influences the O-H bond energy and H-atom abstraction by clotting time was recorded.
free radicals, thus making it a better free radical scavenger than
BDMC (37). In another investigation, the ortho-methoxy- defi- Cell culture
cient BDMC was a more potent ROS inducer and the ortho-me- Primary HUVECs were obtained from Cambrex Bio Science
thoxy substituted curcumin was a more potent suppressor of (Charles City, IA) and maintained as described before (40).
o
NF-kB activation (38). According to our results, the ortho-me- Briefly, cells were cultured to confluency at 37 C at 5% CO2
thoxy group in curcumin is important for the anticoagulant in EBM-2 basal media supplemented with growth supplements
effect. Thus, we can postulate that the anticoagulant activities (Cambrex Bio Science).

224 BMB reports http://bmbreports.org

 Anticoagulant activities of curcumin
Dong-Chan Kim, et al.

Factor Xa generation on the surface of HUVECs transected at 2 mm from the tip. Bleeding time was measured
HUVECs were preincubated with indicated concentrations of as time elapsed until bleeding stopped. When bleeding time
curcumin or BDMC for 10 min. TNF-α (10 ng/ml for 6 h in lasted longer than 15 min, measurement was stopped and
serum-free medium) stimulated confluent monolayers of bleeding time was recorded as 15 min for statistical analyses.
HUVECs in a 96-well culture plate were incubated with FVIIa
(10 nM) in buffer B for 5 min at 37°C in presence or absence Statistical analysis
of anti-TF IgG (25 μg/ml). FX (175 nM) was then added to the Data are expressed as the means ± standard deviation of at
cells (final reaction mixture volume, 100 μl) and incubated for least three independent experiments. Statistical significance
15 min. The reaction was stopped by adding buffer A contain- between two groups was determined by a Student’s t-test. The
ing 10 mM EDTA and the amount of FXa generated in the re- significance level was set at P ＜ 0.05.
action period was measured by using a chromogenic substrate,
and the change in absorbance at 405 nm was monitored in a Acknowledgements
microplate reader for 2 min. The initial rate of color develop- This work was supported by the National Research Foundation
ment was converted into FXa concentrations from a standard of Korea (NRF) grant funded by the Korea government [MEST]
curve prepared with known dilutions of purified human FXa. (No. 2011-003410, 2011-0026695, 2011-0030124).

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