Pro-coagulant Activity of Phenolic Acids Isolated from Blumea Riparia

Abstract

The effects of extracts of the aerial part of Blumea riparia DC. and their phenolic acids on hemostasis were evaluated. The EtOAc fraction showed significantly reduced blood clotting time (CT) and tail bleeding time of transection (BT) of mice in vivo. This fraction contained vanillic acid (1), syringic acid (2), p-coumaric acid (3), caffeic acid (4), and protocatechuic acid (5). Compound 1 reduced prothrombin time (PT), and strengthened mice uterine contractions. Compound 3 reduced CT and the activated partial thromboplastin time (APTT). Compound 5 reduced CT and increased the frequency of mice uterine contraction in a dose-dependent manner. Compound 2 reduced APTT. Compound 4 remarkably strengthened uterine contraction. Taken together, these data suggest that compounds 1, 3, and 5 possess procoagulant activity which jointly synergize blood coagulation via different mechanisms.

Full text

Pro-coagulant Activity of Phenolic Acids Isolated from
Blumea riparia
Li Huanga,c, Cuiwu Lina ,*, Aiyuan Lib, Baoyao Weic, Jianwen Tengc and Lue Lia
aDepartment of Chemistry and Chemical Engineering, Guangxi University, Nanning,
Daxue Road, 88, 530004, People’s Republic of China
bGuang Xi Traditional Chinese Medical University, Nanning, Mingxiu East Road, 88, 530021,
People’s Republic of China
cInstitute of Light Industry and Food Engineering, Guangxi University, Daxue Road, 88,
530004, People’s Republic of China
lincuiwu@126.com
Received: April 15th, 2010; Accepted: June 9th, 2010
The effects of extracts of the aerial part of Blumea riparia DC. and their phenolic acids on hemostasis were evaluated. The
EtOAc fraction showed significantly reduced blood clotting time (CT) and tail bleeding time of transection (BT) of mice in
vivo. This fraction contained vanillic acid (1), syringic acid (2), p-coumaric acid (3), caffeic acid (4), and protocatechuic acid
(5). Compound 1 reduced prothrombin time (PT), and strengthened mice uterine contractions. Compound 3 reduced CT and the
activated partial thromboplastin time (APTT). Compound 5 reduced CT and increased the frequency of mice uterine
contraction in a dose-dependent manner. Compound 2 reduced APTT. Compound 4 remarkably strengthened uterine
contraction. Taken together, these data suggest that compounds 1, 3, and 5 possess procoagulant activity which jointly
synergize blood coagulation via different mechanisms.
Keywords: Blumea riparia, procoagulant, blood, hemostasis, phenolic acids, coagulant.
Menorrhagia and postpartum hemorrhage are common
illnesses that affect many women [1a, 1b]. Oxytocin,
ergot alkaloids, prostaglandins, tranexamic acid,
ethamsylate, and recombinant activated factor VII are
used for treatment but produce adverse reactions [1c].
Some compounds isolated from plants display
hemostatic activity. For example, an extract from
Blumea riparia (BL.) DC. (Compositae) is used in
traditional Chinese medicine for reducing uterus
blood, preventing gynecological inflammation, and
accelerating body recovery postpartum. In addition, an
herbal medicine produced from B. riparia DC. is
clinically used for the treatment of postabortal
metrorrhagia, and other gynecological problems [2a,2b].
Previous chemical studies of this plant characterized
flavones, organic acids, acetylenes, and xanthene
sesquiterpenes [3a-3d], while the present study, found
several additional constituents and evaluated their
procoagulant activity and structure-activity relationship.
Our findings provide a molecular basis for the clinical
application of B.riparia in the treatment of menorrhagia
and postpartum hemorrhage.
To analyze the procoagulant activity of B. riparia, the
effects of orally administered fractions Fa, Fb, and Fc
on blood clotting time (CT) and tail transection bleeding
time (BT) in rats were examined. As shown in Table 1,
a dose of 1.08 and 2.16 g /kg Fb significantly reduced
CT and BT (P<0.05), in comparison with the control
group. Fa, Fb, and Fc exhibited a biphasic effect on the
blood coagulation, by which the CT and BT increased at
a dose of 4.32 g /kg. Similarly, polysaccharides from
green seaweeds also showed a dual hemostatic effect,
that is, inhibition or activation of coagulation pathways
at different doses [4a]. These results give a scientific
explanation for the folkloric use of B. riparia as a
hemostatic in gynecology, as well as an inhibitor of
thrombus formation at larger doses.
As Fb exhibited better hemostatic activity than the other
fractions, it was fractionated to give the five phenolic
acids, vanillic (1), syringic (2), p-coumaric (3), caffeic
(4), and protocatechuic (5). The hemostatic activities of
these compounds were examined by CT, prothrombin
time (PT) and activated partial thromboplastin time
(APTT), and uterine contractility, which are commonly
used for evaluating hemostatic agents. As large amounts
NPC Natural Product Communications 2010
Vol. 5
No. 8
1263 - 1266

1264 Natural Product Communications Vol. 5 (8) 2010 Huang et al.
Table 1: Effect of the different fractions on procoagulant activity in mice
in vivo.
Groups Dose (g/kg) CT(min) BT(min)
control - 7.83±2.00 3.89±1.32
Ethamsylate 0.25 3.73±0.94** 1.28±0.51**
4.32 6.19±2.25 2.72±1.48
2.16 5.45±2.01 2.34±0.94
Fa
1.08 5.50±1.36 1.98±1.13
4.32 6.36±1.93 3.40±1.89
2.16 4.57±1.35** 1.65±0.68*
Fb
1.08 4.78±1.56* 1.73±1.48*
4.32 8.52±1.54 2.64±0.78
2.16 9.06±1.05 2.52±1.18
Fc
1.08 5.58±1.56 3.00±1.62
Negative control, saline solution; positive control, ethamsylate. Values
are mean±SD. (n = 8).Statistical comparison was performed using
ANOVA followed by Tukey’s test. * Significant vs. the control group (p
<0.05); ** Significant vs. the control group (p <0.01)
of test samples were required for the week- long daily
oral administration, sufficient quantities of compounds
1–5 had to be purchased. Interestingly, at a dose of
3 mmol/kg, only compound 3 (Figure 1) significantly
decreased CT (P <0.05), suggesting that, while
compound 3 may be effective, other procoagulant
components may be present to account for the high dose
of compound 3 needed.
Blood coagulation results from a series of proteolytic
reactions involving the step-wise activation of
coagulation factors I-XII. Subsets of these factors can
be activated by two distinct pathways, the extrinsic and
the intrinsic pathway.[4b] The APTT is commonly used
for determining the overall efficiency of the intrinsic
coagulation pathway, while PT is the screening test for
the extrinsic coagulation pathway [4c]. The APTT was
reduced to12.5, 13.3, and 15.6% in mice treated with
compounds 2, 3, and 5 when compared to the control
group. However, the PT values remained unchanged in
the presence or absence of these compounds (Table 2).
These data suggest that the intrinsic coagulation
pathway may be the target of these compounds.
Protocatechuic acid (5) can enhance the activity of
anticoagulant factors such as antithrombin-III and
protein C [4d].. This observation may be explained if it
has dual functions in the coagulation system. It may act
as a procoagulant to promote thrombus when bleeding
occurs and also act as an anticoagulant when combined
with the cofactor thrombomodulin in the protein case
complex [5a, 5b].
Vanillic acid (1) significantly lowered PT, but had no
effect on APTT. The lessening of PT suggested that 1
could activate the extrinsic coagulation pathway. A
similar observation showed that 1 had weak
antithrombotic effects when evaluated for thrombosis in
the mice model [6a]. It also decreased the recalcification
time in a dose-dependent manner and inhibited
5’nucleotidase activity specifically, which mediated the
Figure 1: Effect of phenolic acids on CT in mice in vivo.
Negative control, saline solution; positive control, ethamsylate. The
treatment with compound 1–5 were used in a dose of 0.2mmol/kg or
3mmol/kg, respectively. Values are mean±SD. (n = 8). Statistical
comparison was performed using ANOVA followed by Tukey’s test.
* Significant vs. the control group (p <0.05).
Table 2: Effect of phenolic acids on PT and APTT in vivo.
Groups Dose( mmol/kg) PT (s) APTT(s)
control - 9.61±1.72 19.69±1.49
Positive control 1(g·kg-1) 7.23±0.80** 15.38±0.81**
1 0.75 7.38±1.29* 18.31±2.37
2 0.75 7.77±1.62 17.22±1.53*
3 0.75 7.88±0.80 17.08±1.93*
4 0.75 8.16±1.30 18.28±2.36
5 0.75 7.94±0.75 16.60±1.43**
Negative control, saline solution; positive control, Yunnan white power.
PT values are mean±SD. (n = 8) and APTT values are mean±SD. (n =
10). Statistical comparison was performed using ANOVA followed by
Tukey’s test. * Significant vs. the control group (p <0.05); ** Significant
vs. the control group (p <0.01).
anticoagulant effect of Naja naja venom [6b] Therefore,
B. riparia DC. may affect both intrinsic and extrinsic
pathways to regulate heavy menstrual bleeding or
postpartum hemorrhage.
The effects of different phenolic acids on spontaneous
contractile activity of uterine smooth muscle in estrous
mice were compared. As summarized in Table 3, there
was no obvious difference in the tension of uterine
contraction before and after the addition of phenolic
acids to the bath, except for compounds 2 and 4.
Surprisingly, compounds 4 and 5 significantly increased
the frequency of uterine contraction in a dose-dependent
manner (P < 0.01). Compound 1 also accelerated the
frequency of uterine contraction at 1.43×10-4 mmol/ml
(P < 0.05). These results indicate that phenolic acids can
strengthen the frequency of mice uterine contractions,
while only affecting tension slightly. Alteration of
uterine contractions by drugs is important in obstetrics
practice. Inadequate uterine contractions could impede
labor and increase the incidence of emergency caesarian
sections, whereas promotion of uterine contractions
could prevent or treat excessive hemorrhage in the
immediate postpartum period [7]. Based on the above it
may be assumed that 4 and 5 strengthen contraction
of uterine and blood vessel to promote the repair and

Pro-coagulant activity of phenolic acids Natural Product Communications Vol. 5 (8) 2010 1265
Table 3: Effect of phenolic acids on the uterine contraction of mice in vitro.
Frequency of uterine
contractioncycle/5min Tension of uterine contraction
n ( g
groups
dose×10-
4mmol/
mL Before After Before After
control - 6.26±2.14 6.21±2.20 1.80±0.54 1.77±0.49
positive - 5.25±1.39 15.25±3.81** 1.45±0.46 3.43±0.92**
0.71 11.88±5.03 14.75±5.12 1.96±0.73 2.03±0.75
1.43 10.13±3.27 12.25±4.27* 1.59±0.58 1.55±0.55 1
2.14 11.13±4.49 11.75±3.24 1.86±0.70 1.70±0.48
0.36 12.63±4.87 13.75±5.06 2.15±0.72 2.24±0.74
0.71 10.38±3.20 11.00±4.90 1.72±0.67 1.93±0.66* 2
1.43 8.75±4.92 9.50±4.41 1.65±0.55 2.19±0.74
0.71 9.50±2.45 9.75±4.27 1.85±0.64 1.87±0.69
1.07 8.75±4.37 8.50±4.21 1.37±0.10 1.70±0.49
3
1.43 7.88±2.47 8.50±4.69 1.70±0.49 1.96±0.66
0.71 7.88±3.68 13.00±5.95* 3.08±1.06 3.16±1.09
1.43 9.13±4.16 18.50±6.19* 2.06±0.72 2.80±1.00
4
2.86 5.25±1.67 23.13±10.62** 1.73±0.48 2.61±0.83*
0.89 10.13±3.48 14.25±4.65* 2.30±0.59 2.25±0.69
1.07 6.75±2.60 15.38±3.78** 2.11±0.59 2.41±0.56
5
1.25 7.13±2.80 16.13±6.96** 2.45±0.76 2.56±0.84
Negative control, saline solution; positive control, oxytocin and dose is 1×10-
3unit/mL.Values are mean±S.D. (n = 8). The uterine contractions were
recorded for 10 min immediately before and after addition of phenolic acids to
the bath. *Contraction after addition of phenolic acids is significantly different
from the values before its addition to the organ bath at p <0.05 using paired t-
test with a two-tailed p-value. **Contraction after addition of phenolic acids is
significantly different from the values before its addition to the organ bath at p
<0.01 using paired sample t-test with a two-tailed p-value.
regeneration of tissue, thus preventing hemorrhage. B.
riparia DC. can therefore not only be used for
postpartum care, but also for treating irregular, painful,
or excessive menstruation, uterine pain and dysfunction.
Experimental
General: The ethanol extract of B. riparia DC. was
provided by Guixi Pharmacy Co., Ltd. of Naning,
Guangxi, China. Caffeic acid (4) was purchased from
Shanghai Junchuang Biological Tech. co., (Shanghai,
China). p-Coumaric acid (3), vanillic acid (1), syringic
acid (2) and protocatechuic acid (5) were purchased
from Wuhan Hezhong Bio-Chemical Manufacture Co.,
(Wuhan China). APTT assay kit and PT assay kit were
purchased from Beijing Bo Lai Bio. Co. (Beijing,
China). Column chromatography was performed on
Silica gel (200–300 mesh), gel H (Qingdao Mar. Chem.
Ind. Co.Ltd), and Sephadex LH-20 (Pharmacia).
Extraction and sample preparations: The ethanol
extract of (4.5 kg) B. riparia. was evaporated under
reduced pressure to obtain a residue (3.2 kg), which was
diluted with water (6.4 kg) and extracted several times
with petroleum ether. The petroleum ether extracts were
combined to form fraction (Fa). The aqueous layer was
further extracted with EtOAc to give fraction (Fb) and
the residual aqueous layer gave fraction (Fc). All these
fractions were evaporated under reduced pressure to
give dried products, Fa (142.7 g), Fb (286.7 g), and
Fc (2520 g).
Among the extracts and fractions tested for their
procoagulant activities (Table 1), Fb showed significant
inhibition activities. Fb (100 g) was repeatedly
chromatographed over a silica-gel to give vanillic acid
(1) (14 mg) and syringic acid (2) (18 mg), p-coumaric
acid (3) (22 mg), caffeic acid (4) (54 mg) and
protocatechuic acid (5) (36 mg). These phenolic acids
were identified by comparison of physical and
spectroscopic data with literature data. [8a-8d]
Animals: Mice (Kingming strain) weighing 18–22 g
were used to assay CT and BT in vivo. Female mice
(Kingming strain) weighing 25–30 g were used to assay
uterine contractility. New Zealand rabbits weighing
1.8–2.2 kg were used to assay PT and APTT in vivo.
The animal experiments were conducted in accordance
with international guidelines.
Assay of blood clotting time in vivo (CT): The blood
clotting time was evaluated by the glass slide method
[9a]. The fractions (Fa, Fb, and Fc) and phenolic acids
were dissolved in water. Experimental mice were
divided into 25 groups of 8 and administrated with the
fractions or reagents at 0.1ml/10g by an oral gavage
with a syringe. The control groups were treated with
saline solution. The positive groups were injected with
ethamsylate (Beijing Pharmaceutical Factory, Beijing,
China) at 0.25g/kg. After three days, each mouse’s
inner canthus was punctured with a glass capillary and a
drop of blood from the supraorbital vein was collected
on a glass slide. The bleeding time was recorded
between blood collection and blood streak formation.
Tail transection bleeding time in vivo (BT):
Experimental mice were divided into 11 groups each
consisting of 8 mice. The preparations of the fractions,
control group, positive groups and the manner of
administration are similar to that described above for
the CT assay. After three days, the mice were
anesthetized with sodium pentobarbital. The bleeding
time was recorded according to the method by Dejana
et al. [9b].
Assay for prothrombin time (PT) and activated
partial thromboplastin time (APTT) in vivo:
Experimental rabbits were divided into 8 groups of 3
and 10 ml/kg of the reagent administered with a syringe
by an oral gavage for once a day for three days at the
same time. The positive groups were given 1g/kg
Yunnan white power (Yunnan Baiyao Group Co., Ltd.,
Yunnan, China). Rabbit blood was collected with
plastic disposable syringes 1h after the rabbits were
administered in the third day. Fresh blood was mixed
with 1:9 volume of 3.2% sodium citrate. Platelet poor
plasma was obtained by centrifuging citrated blood for
15 min at 3000 × g. PT and APTT were determined
immediately using a kit according to the cited method

1266 Natural Product Communications Vol. 5 (8) 2010 Huang et al.
[9c] All experiments were carried out in at least
duplicates.
Measurement of uterine contractility in vitro: Female
mice were injected subcutaneously with 0.1mg/kg
estradiol (Mingxin Pharmaceutical Co., Ltd.,
Guangzhou, China). After two days, the mice were
sacrificed by decapitation. Both uterine horns were
isolated, cut in half, and placed in isolated organ baths
incubated with De Jalon’s solution bubbled with
carbogen mixture (95% O2 and 5% CO2) at 37±0.5ºC.
Each uterine horn was subjected to a resting tension of
0.5-1g and equilibrated for at least 30-45 min before the
reagents were added to the organ baths. The positive
groups were treated with oxytocin 1×10-3 unit/mL. The
mice uterus contractions were recorded by polygraph
system (HW-400S) (Chengdu, China.) [9d].
Statistical analysis: Data are expressed as
mean±SD(standard deviations), and statistical
significance was determined by one-way analysis of
variance (ANOVA) and Tukey’s test. In the
measurement of uterine contractility, the differences
between before and after addition of samples were
tested for paired t-test with a two-tailed p-value. A
value of p<0.05 was considered significant.
Acknowledgments - We are grateful to Guixi
Pharmacy Co., Ltd., Guangxi, China for providing raw
materials for experimentation. This work was supported
by a major grant from the National Natural Science
Foundation of China (20962002 and 20662001), a
major grant from the Key Science Project of Guangxi
Province (04080005), and an innovative pilot scheme
for the College Students of China (200714).
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