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Anticoagulant and Antioxidant Activities of

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American Journal of Biomedical Research, 2013, Vol. 1, No. 4, 86-92
Available online at http://pubs.sciepub.com/ajbr/1/4/4
© Science and Education Publishing
DOI:10.12691/ajbr-1-4-4

Anticoagulant and Antioxidant Activities of Dracaena

arborea Leaves (Wild.) Link
Nwaehujor Chinaka O.1,*, Ode Julius O.2, Nwinyi Florence C.2, Madubuike Stella A.3
1
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Nigeria
2
Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Nigeria
3
Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
*Corresponding author: [email protected]

Received October 04, 2013; Revised October 16, 2013; Accepted October 20, 2013
Abstract The crude methanol extract of Dracaena arborea leaves induced significant (p<0.01) increase in the
clotting times of 21 ± 0.54 sec and 25 ± 1.1 sec at 5% and 10% concentrations of the extract respectively compared
to the baseline clotting time of 7 ± 0.63 sec for the blood sample. The extract also exhibited potent in vivo and in
vitro anticoagulant activities. Increased doses (100 and 200 mg/kg) of the extract, heparin (0.75 and 1.5 mg/kg) and
aspirin (1.0 and 2.0 mg/kg) were found to have significantly (p < 0.01) prolonged the mean bleeding times with
respect to the baseline in rabbits. However, in thrombin-induced clotting assay, the extract demonstrated a reduced
potency compared to heparin. DPPH (1, 1-Diphenyl-2-picrylhydrazyl) and FRAP (Ferric reducing/antioxidant
power) spetrophotometric assays revealed that the crude leaf extract possesses appreciable high antioxidant
potentials. Dracaena arborea leaves (Wild.) Link could be a source of novel anticoagulant and antioxidant
compounds for the management of various hematological disorders.
Keywords: anticoagulant, Dracaena arborea, antioxidant, clotting time, stroke, thrombin
Cite This Article: Nwaehujor Chinaka O., Ode Julius O., Nwinyi Florence C., and Madubuike Stella A.,
“Anticoagulant and Antioxidant Activities of Dracaena arborea Leaves (Wild.) Link.” American Journal of
Biomedical Research 1, no. 4 (2013): 86-92. doi: 10.12691/ajbr-1-4-4.

recently produced as alternatives to oral vitamin K

antagonists for stroke prevention in atrial fibrillation. But
1. Introduction each of these drugs has distinct pharmacological
properties that could influence optimal use in clinical
Heart attacks and strokes resulting from thrombosis and practice [6]. There is critical need to explore for effective
intravascular blood clots are increasingly becoming the and alternative anticoagulants from natural products with
most common cause of debilities and untimely deaths minimal side effects.
worldwide. Prevention of these diseases has been a major Fortunately, plants offer prospect as sources of various
health challenge over decades because of inefficacies medicaments and potent chemotherapeutic agents.
associated with most of the readily available drugs. Dracaena arborea (Asparagaceae) commonly called tree
Antithrombotic agents include anticoagulants, antiplatelets Dracaena is a popular plant in Nigerian tradomedical
and thrombolytics that decrease clots in the body by practice for its use in the treatment of various diseases. It
dissolving already formed clot or prevent clot formation is locally known as ope kannakanna, peregun, akuku in
[1]. Anticoagulants are indicated for strokes, transient Yoruba dialects of western Nigeria and ogirisi in Igede
ischaemic attacks, deep vein thrombosis and pulmonary (Middle belt, Nigeria) and in Ibo (Eastern Nigeria). The
embolism [2]. Oral anticoagulants have been used in the roots, leaves and fruits are used for effective treatment of
management of atherothrombotic stroke treatment [3] skin infections, stomach ache and dental caries in western
which accounts for 61% of all strokes and have been Nigeria [7]. The plant leaves are also employed locally by
relied upon for prevention and treatment for several the Ibo people in Nsukka community to relieve
decades. These drugs however, produce a highly variable hypertension, stroke and body pains. The leaves boiled in
anticoagulant effect in patients, requiring their effect to be palm nut soup with the fern, Nephrolepis biserrata and
measured by special blood tests and their dose adjusted Dissotis multiflora, is used to strengthen pregnant women
according to the results [4]. Frequent development of and those delivered of their babies in Ghana [8]. In a bid
immune thrombocytopenia, haemorrhages and to establish the antihypertensive effects and other folkloric
idiosyncratic adverse reactions during treatment of uses, the anticoagulant and antioxidant activities of the
patients with the drugs [5] are some of the limitations plant leaves were investigated. Increased oxidative stress
encountered in anticoagulant therapy. The oral direct generates reactive oxygen species (ROS) which are
thrombin inhibitors (e.g., dabigatran) and oral direct implicated in a variety of pathologies including heart
inhibitors of factor Xa (e.g., rivaroxaban, apixaban) were attacks, strokes, renal failure, diabetes, neurodegenerative
 American Journal of Biomedical Research 87

diseases, etc. [9]. Presently, there is paucity of information before been pulverized into fine particles using a
on the in vitro antioxidative activities of D. arborea leaves. laboratory hammer mill. The plant material was
In a previous study, the ethyl acetate and aqueous exhaustively extracted by cold maceration in 80%
fractions of the leaf extract demonstrated insecticidal methanol with intermittent shaking at 2 h intervals for 48
activity against Sitophilus zeamais (Motsch.) and h. The extract was filtered and concentrated in vacuo
Callosobruchus maculatus (Fab.) [10] and offered using a vacuum rotary evaporator. The concentration and
protection to stored grains. percentage yield of the extract were determined. The
The present study focused on evaluation of the concentrated D. arborea extract, subsequently denoted as
anticoagulant and antioxidant effects of the methanol DAE was stored in a refrigerator at 40C until further use.
extract of D. arborea (Willd.) Link leaves using standard
models. 2.5. Acute Oral Toxicity Studies
Acute toxicity studies were conducted using the method
2. Materials and Methods described by Lorke [12]. Thirty (30) matured Wistar rats
of both sexes were marked with 10% picric acid, weighed
and randomly separated into 6 groups (A-F) of 5 rats each.
2.1. Experimental Animals Groups A–E were given varying oral doses (150; 300; 600;
Matured inbred albino rats of both sexes weighing 1200 and 1500 mg/kg) of DAE respectively, while group
between 80-190 g and locally bred albino rabbits (2.0-2.2 F (6th group) received an equivalent volume (10 ml/kg) of
kg) were obtained from the Laboratory Animal Unit of the distilled water. All treatments were given orally by gastric
Department of Biochemistry, Faculty of Basic Medical intubation. The rats were observed for signs suggestive of
Sciences, University of Calabar, Calabar, Nigeria. The toxicity within 72 h. The animals that survived were
animals were kept in different cages in the same room further monitored for two weeks for toxic effects. The test
with a temperature varying between 28 and 30°C; lighting was terminated after two weeks and all the animals were
period was between 15 and 17 h daily. The rats were kept humanely sacrificed and postmortem examinations carried
in stainless steel wire mesh cages which separated them out on them.
from their faeces to prevent coprophagy. They were
supplied clean drinking water and fed standard feed 2.6. Blood Clotting Time
(Grower mash pellets, Vital feeds®, Nigeria). Rabbits were
given fresh forages ad libitum. The animals were allowed The ability of D. arborea to inhibit coagulation of
two weeks to acclimatize prior to commencement of the blood in vitro was quantitatively assayed following the
experiments. The laboratory animals were used in procedure adopted by Koffuor and Amoateng [13]. A
accordance with laboratory practice regulation and the fixed volume (1.0 ml) of whole blood drawn from the
principle of laboratory animal care as documented by marginal ear vein of rabbits was added to 0.2 ml of D.
Zimmerman [11]. arborea (5 and 10% w/v) in test tubes placed in a water
bath at 37°C. The time taken for the blood samples to clot
was recorded. Five determinants were made. A similar
2.2. Chemicals, Reagents, Drugs and determination was carried out using heparin (1.5 µL of 5%
Equipment w/v) as a reference anti-coagulant. Controls were set up
Freshly prepared solutions, analytical grade chemicals using distilled water (0.2 ml) and blood, and blood alone
and reagents were used in the experiments. Methanol, 1, (baseline clotting time).
1-Diphenyl-2-picrylhydrazyl (DPPH), Ferric
reducing/antioxidant power (FRAP) reagents, Ferric 2.7. Rabbit Bleeding Time
sulphate (FeSO4 x 7H2O), Ascorbic acid (vitamin C), The in vivo anticoagulant activity of D. arborea at 200
bovine thrombin purchased from Sigma Aldrich, USA; and 400 mg/kg was investigated as described by Elg et al.
heparin (Mayne Pharma, Espana), soluble aspirin tablets [14]. Rabbits were pre-treated orally with DAE for 30 min.
75 mg (Bristol laboratories Ltd, UK), Sodium phosphate Pricking a small vein in the margin of the ear induced
(BDH, Poole, England), Spectrophotometer (Spectrumlab bleeding. The bleeding vein was gently blotted with filter
752 S., B. Bran Scientific and instrument company, paper every 5 s till cessation of bleeding (when no more
England) were used for the tests. bleeding was observed for 60 s). The observation time was
limited to 10 min. Care was taken that no pressure was
2.3. Plant Collection and Identification exerted on the ear tips that could affect homeostasis. The
Fresh leaves of D. arborea were collected from Obukpa above procedure was repeated using aspirin (1-2 mg/kg),
village, in Nsukka Local Government Area of Enugu State, distilled water and after intravenous administration of
Nigeria in April, 2013. The plant roots were authenticated heparin (0.75-1.5 mg/kg). A baseline bleeding time was
by Mr A.O. Ozioko, a taxonomist with Bioresources determined before any drug administration.
Development and Conservation Programme (BDCP), Aku
road, Nsukka, Enugu State, Nigeria. 2.8. Thrombin-induced Clotting Time Assay
This assay measures the prolongation of thrombin
2.4. Preparation and Extraction of Plant generation. When human plasma is incubated with a
Material compound which inhibits blood coagulation, the time
The plant leaves were dried under mild sunlight, and taken for the clot formation will be prolonged as
then reduced to coarse particles with mortar and pestle compared to the control (no inhibitor added). In the assay,
100 µl of human plasma (pre-incubated at 37°C for 5 min
88 American Journal of Biomedical Research

before use) was incubated with different concentrations of 5% level of significance. Dunnet’s test was used to detect
the extract for 5 min at 37°C. Heparin was used as a the difference among the treatment groups.
positive control while buffer served as the negative control.
One hundred µl of bovine thrombin (2.5 U/ml, Sigma)
was then added to initiate the reaction. The test was 4. Results
carried out in triplicates. The time for clot formation was
recorded and compared with the reference (heparin) 4.1. Description of the Extract
values [15].
The methanol extract of D. arborea leaves was light
brownish in colour. The extraction process gave a yield of
2.9. Antioxidant Capacity of D. Arborea Leaf 10.23 % w/w.
Extract Using the 1, 1- diphenyl-2-
picrylhydrazyl Radical (DPPH) 4.2. Acute Toxicity Study of the Extract in
Spectrophotometric Assay Rats
The method of Mensor et al. [16] was adopted. Two (2) DAE did not cause death in the experimental rats by the
ml of the test extract at concentrations ranging from10 oral route even at the highest test dose of 1500 mg per kg
µg/ml to 400 µg/ml was each mixed with 1 ml of 0.5 mM body mass. There were also no changes in faecal
DPPH (in methanol). Absorbance at 517 nm was taken consistency of the animals within the period. At post
after 30 min incubation in the dark at room temperature. mortem, there was no observable gross lesion in the liver,
The concentrations were prepared in triplicates. The gastro-intestinal tract, spleen, heart and kidneys of the
percentage antioxidant activity was calculated as follows: experimental rats.
 absorbance of sample 
 − absorbance of blank  4.3. Clotting Time
100 − ( 
%[ AA] =  ∗100)
DAE at concentrations of 5% and 10% significantly (p
Absorbance of control
< 0.01) produced increased clotting times of 21 ± 0.54 and
1ml of methanol plus 2 ml of the extract was used as 25 ± 1.1 s respectively compared to the baseline clotting
blank while 1ml of 0.5 mM DPPH solution plus 2 ml of time of 7 ± 0.63 s for the blood sample (Table 1).
methanol was used as control. Ascorbic acid was used as However, the blood added to heparin failed to clot.
the reference standard.
Table 1. The in vitro effect of DAE (5 and 10%) on clotting time of
blood taken from the marginal ear vein of rabbits in a clotting time
2.10. Ferric Reducing/Antioxidant Power test
(FRAP) Assay Sample Volume Mean clotting time (s)
The total antioxidant potential of the extract was Blood 1.0 ml 7 ± 0.63a
determined using a ferric reducing ability of plasma
(FRAP) assay of Benzie and Strain [17] as a measure of Blood + vehicle 1.2 ml 11± 0.63a
“antioxidant power”. FRAP assay measures the change in Blood + 5% DAE 1.2 ml 21 ± 0.54b**
absorbance at 593 nm owing to the formation of a blue
colored Fe11-tripyridyltriazine compound from colorless Blood + 10% DAE 1.2 ml 25 ± 1.1b**
oxidized Fe111 form by the action of electron donating Values are mean ± SEM (n=5). b ** Significant (p<0.01) difference
between the samples and the control (blood alone).
antioxidants. Standard curve was prepared using different
concentrations (100-1000 µmol/L) of FeSO4 x 7H2O. All
solutions were used on the day of preparation. In the 4.4. Rabbit Bleeding Time
FRAP assay the antioxidant efficiency of the extract under DAE was observed to have significantly (p<0.01)
the test was calculated with reference to the reaction prolonged the bleeding time with respect to the baseline.
signal given by an Fe2+ solution of known concentration, At a dose of 100 mg/kg, the extract induced a bleeding
this representing a one-electron exchange reaction. The time of 61.8 ± 1.4 s but when the dose was doubled (200
results were corrected for dilution and expressed in µmol mg/kg), the bleeding time became elevated to 125.1± 1.0 s
Fe11/L. Vitamin C was measured within 1 h after which was proportionately a more than a double increase.
preparation. The sample to be analyzed was first Heparin (0.75 and 1.5 mg/kg) and aspirin (1.0 and 2.0
adequately diluted to fit within the linearity range. All mg/kg) also significantly (p<0.01) induced increased
determinations were performed in triplicate. Calculations bleeding times compared to the baseline. The bleeding
were made by a calibration curve. time of 125.1± 1.0 s at the highest test dose (200 mg/kg)
FRAP value of sample (mM ) of the extract was seen to be a significant (p<0.01)
increase relative to 61.0 ± 1.7 s with heparin (1.5 mg/kg)
chang in abs from 0 − 4 min
× FRAP value of std and 70.02 ± 0.0 s with aspirin (2.0 mg/kg) (Figure 1).
chang in abs of std 0 − 4 min
4.5. Thrombin-induced Clotting Time Assay
3. Statistical Analysis A comparison of the in vitro activity of crude extract of
D. arborea leaves with heparin in thrombin-induced
All data were expressed as Mean ± SEM. Data were clotting time is presented in Figure 2. DAE produced a
analyzed using One way analysis of variance (ANOVA) at significant (p<0.01) reduction in thrombin-induced
 American Journal of Biomedical Research 89

clotting times compared to heparin. At 100 µg/ml, DAE 170.5 ± 0.5 s but the same concentration of heparin
produced clotting at 50.3 ± 0.6 s relative to 330.8 ± 2 s for prolonged the effect to occur at 460.1 ± 0.8 s.
heparin. Similarly, DAE (1000 µg/ml) induced clotting at

Figure 1. The effect of DAE (100-200 mg/kg), heparin (0.75-1.5 mg/kg) and aspirin (1-2 mg/kg) on the bleeding time in rabbits. Values are Means ±
SEM (n=5). **p<0.01, *p<0.05 compared to vehicle treated group. ++p<0.01, +p<0.05 significant differences between dose levels

Figure 2. A comparison of the in vitro activity of DAE and heparin in thrombin-induced clotting time

Figure 3. The antioxidant activity of DAE with DPPH spectrophotometric assay

90 American Journal of Biomedical Research

4.7. Ferric Reducing/Antioxidant Power

4.6. DPPH Spectrophotometric Assay Assay (FRAP)
The crude methanol extract of D. arborea leaves The ferric reducing ability of plasma, a measure of the
demonstrated appreciable but reduced antioxidant activity antioxidant ability, showed that the methanol extract of D.
relative to the reference values of ascorbic with DPPH arborea produced a dose dependent antioxidant effect. At
radical scavenging method. At the lowest concentration 10 µg/ml, the mean antioxidant power (FRAP value) was
(10 µg/ml), the extract exhibited a mean antioxidant 0.12 mM; this increased to 0.43 mM at 100 µg/ml and
activity of 15.7 percent while ascorbic acid produced 73.6 then 1.16 mM at 400 µg/ml. DAE had antioxidant power
percent (Figure 3). However, at the highest concentration of 1.35 mM at 1000 µg/ml compared to ascorbic acid with
(400 µg/ml), the extract had 50.3 percent antioxidant a standard FRAP value of 2 mM at the same maximum
activity compared to 79.2 percent with ascorbic acid at the concentration (Figure 4).
same concentration.

Figure 4. The antioxidant activity of D. arborea leaf methanol extract determined by the FRAP assay

Heparin can inhibit both the generation of thrombin and

also the formed thrombin. The synthesis of clotting factors
5. Discussion II, VII, IX and X in the liver depends on adequate
amounts of vitamin K. Coumarin and inadedione group of
The extract was tolerated by the experimental rats at the oral anticoagulant drugs antagonizes the synthesis of non
highest oral test dose of 1500 mg/kg; hence there were no functional forms of coagulant proteins and thereby
observable mortalities. The methanol leaf extract of D. prevents blood clot formation [20].
arborea significantly increased the in vitro time of blood Blood coagulation is an important part of homeostasis
clotting (Table 1) but along with heparin, and aspirin, the in which platelets; a non nucleated cell derived from
in vivo effect shows prolongation in the bleeding times megakaryocyte plays a prominent role to prevent
(Figure 1). The high doses (100-200 mg/kg) of the extract excessive bleeding [21]. Platelets change shape and adhere
exerted the most profound bleeding effect compared to to damaged endothelium or aggregate to themselves
heparin or aspirin. The extract however displayed a resulting in plug formation which is strengthened by fibrin
comparatively lower potency than heparin in thrombin- containing clot to stop bleeding and for repair of damaged
induced clotting time. A given concentration (1000 µg/ml) vessel to begin. Disorders of coagulation are known to
of the extract produced clotting at 170.5 ± 0.5 s while cause haemorrhage or thrombosis [22]. Fibrin is the more
heparin at the same concentration prolonged the clotting important component of clots that form in veins but
time to 460.1 ± 0.8 s. This is logical since heparin is a platelets are the major component of clots that form in
pure compound but the extract is a crude and mixture of arteries where they can cause heart attacks and strokes by
many compounds. The mechanism by which the extract blocking the flow of blood in the heart and brain,
exhibited anticoagulant activity is not understood but respectively [4].
chelating agents, heparin and vitamin K antagonists are The crude extract produced a significant (p < 0.05)
known to interfere with blood clotting processes. concentration dependent antioxidant activity with a
Chelating agents including trisodium citrate, sodium maximal effect of 50.3 percent against 79.2 percent with
oxalate and ethylene diaminetetraacetic acid (EDTA) bind ascorbic acid at 400 µg/ml in DPPH photometric assay.
with calcium ions and render them unavailable to facilitate DPPH assay reveals the ability of the extract to scavenge
coagulation reactions [18]. Heparin, contained in mast free radicals. DPPH is a stable free radical and accepts an
cells is released into circulation when mast cells are electron or hydrogen radical to become a stable
disrupted during inflammation. Heparin can inhibit diamagnetic molecule [23]. The reduction capability of
clotting factor IXa, XIa and thrombin but its action on DPPH is determined by the decrease in the absorbance at
factor Xa accounts for its potency as an anticoagulant [19]. 517 nm induced by antioxidants. This system is valid for
 American Journal of Biomedical Research 91

the primary characterization of the scavenging potential [2] Holbrook, A.M., Pereira, J.A., Labiris, R., McDonald, H.,
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