De ce mber 2012, Volume 3, No.

6
Inte rnational Journal of Chemical and Environmental Engineering

Preliminary Assessment of In vitro Anticoagulant

Activity vs. Heparin 1,000I.U. and Cytotoxicity of
Selected Philippine Medicinal Plants
J.H. Evangelistaa,b, M.J. De Veraa,b, R.S. Garciaa , M.G. Joven a , M.J.A. Nerosa a , J.N. Soliduma*
aSchool of PharmacyEmilio Aguinaldo College
San Marcelino St. Ermita, Manila, Philippines
bProfessor III Emilio Aguinaldo College
San Marcelino St. Ermita, Manila, Philippines
*Corresponding author E-mail: graloheus@yahoo.com

Abstract:
One main cause of mortality in developing countries is thromboembolic disorders such as pulmonary emboli, deep vein thrombosis
and heart attacks. Several agents and interventions were available; however, there are still side effects that are acquired t hrough these
therapies. Herbal plants are popularly used nowadays in drug discovery due to their ancient medicinal use. Philippines, a tropical
country, have a variety of herbal plants. The aim of the study was to assess the anticoagulant activity and cytotoxicity of b oth flesh
and peels/seeds of selected plants available in the Philippines. The plants Allium sativum, Cucurma longa, Ananas comosus and
Lycopersicum esculentum were used in the in vitro method using Heparin and water as controls. Percentage clot lysis of the plants
were 18.30%, 21.77%, 21.85%, 35.91%, 15.67% and 24.52%, respectively. Only C. longa peel extracts and L. esculentum showed
clot lysis beyond the negative control. C. longa peels showed a higher percentage clot lysis as compared with Heparin. Using one-way
ANOVA, statistics showed the p value of 0.674574 from the clot lysis activity of all plant extracts. Cytotoxicity of the herbal plants
was also determined using brine shrimp lethality assay (BSLA). The LC50 values of the herbal extracts ranges from 6.72 to
31.2g/mL. A. sativum and C.longa flesh have the lowest values (most potent among the extracts). Descriptively, C. longa peels
extract exhibited promising clot lysis activity, however further studies are still needed to strengthen the effectiveness of these
Philippine plants as anticoagulant. Furthermore, it was also seen in this study that fruit and vegetable peels have prospective
therapeutic application. It will not only benefit the industrial waste products reduction, but it can also serve as an alternative source of
pharmacologic agents.
Keywords: Alliu m sativum, Curcu ma longa, Ananas comosus, Lycopersicm esculentum, thro mbolytic activity, fruit and

vegetable waste and by products, brine shrimp lethality assay

1. Introduction
1.1. Background of the study
In vitro hemolytic activ ities are beco ming a new area of
drug research to discover new interventions for diseases
such as thromboembolic d isorders. Interest in studying
plants is emerging for their immense potential to be used
in various traditional systems. Efforts have been shifted
towards discovery and development of natural products
FR fro m various plant and animal sources which have
anticoagulant, antithrombotic and thrombolytic activity
[1-3]. Fruit and vegetable waste products (e.g. peels and
seeds) are also being studied for their potential usage.
They may also contain a variety of bioactive components, which
can have a beneficial effect on health. The therapeutic use of
agro waste is gradually gaining popularity and its recovery will

be economically pro mising [4]. Fruit and vegetable peels

and seeds can serve as natural, eco-friendly and economic
sources of agents which can be used in the prevention of

diseases and the use of such can also reduce waste

pollution. Screening and fractionation of hysiologically
active plant extracts can be perfo med using brine shrimp
lethality test (BST), in which lethality in brine shrimps
(Artemia salina Leach) is monitored [5].This general
bioassay determines a wide range of biological activ ities.
A basic premise of this test is that toxicology is simply
pharmacology at a higher dose, thus if a to xic co mpound
is found, a lower non-toxic dose might elicit a useful and
pharmacological effect on a physiologic system [6].
The use of med icinal plants to meet the drug requirements
of a developing country like the Philippines is highly
significant [7]. Four Ph ilippine medicinal plants of
interest the authors included were Allium sativum flesh
and peels, Curcuma longa flesh and peels, Ananas
comosus flesh and peels, and Lycopersicum esculentum
flesh and seeds.

Pre liminary Assessment of In vitro Anticoagulant Activity vs. Heparin 1,000I.U. and Cytotoxicity of Selected Philippine Medic inal Plants

Allium sativum L. or locally known as bawang

belongs to Lilliaceae family and is described as a
perennial, erect, bu lbous herb up to 70cm tall, with strong
smelling odor when crushed [8]. The main b iological
active constituents are allicin (diallylthiosulfate) and
thiosulfonates that greatly participates in its thro mbolytic
activity [9]. Studies have proven that garlic has a great
potential in inhibit ing plate aggregation and enhancing
fibrinolytic activ ity [10]. Th is significantly found
property of garlic led to further develop ments using garlic
in drug treatment of thro mboembolic disorder. Studies of
garlic skins (peels) extract showed strong antioxidant
activity, and some responsible constituents were isolated
and identified. It has been used as herbal med icine, but
there was no report on the health benefits of the skin or
peel [11, 12].
Curcuma longa L. also known as turmeric or luyang
dilaw, is a member of the Zingiberaceae family. Its
active ingredient is curcumin wh ich was identified to be
responsible for most of the biological effects of turmeric.
Turmeric p lant ext ract (curcu min and other derivatives)
also have wide range activities such as, anti-arthritic,
antioxidant, anti-microbial, anti-leishmanial, hepato
protective, anti-cancer, anti-ulcer and anti diabetic
activity [13]. Curcu min also shows anticoagulant activity
by inhibiting collagen and adrenaline-induced platelet
aggregation in vitro as well as in vivo in rat thoracic aorta
[14]. Studies have shown that C. longa peel wastes
possess antioxidant properties which could probably work
by counteracting and or quenching of reactive o xygen
species [15]. To the best of our knowledge, there has been
no reported study on the peels of C. longa, aside fro m the
one mentioned above.
Lycopersicum esculentum Mill. co mmonly known as
tomato or kamatis is a member of the Solanaceae
family. It contains important carotenoids e.g. lycopene,
lutein, and -carotene, v itamin C, -tocopherol,
flavonoids, phenols, sugars, inorganic ions and organic
acids [16]. Studies have found out that ingestion of
tomato co mponents with in vit ro antiplatelet activity
significantly affects ex vivo platelet function. Moreover,
its reported cardioprotective effects are potentially linked
to a modulation of platelet function [17]. The observed
cardiovascular benefits attributed to the tomato could be
lin ked to its antiplatelet activity and thus suppression of
platelet function in vivo. This type of natural antithrombotic agent could have an application in primary
prevention of cardiovascular diseases [18]. M oreover, it has
been investigated that daily intake of tomato extracts could offer
a convenient and effective way of preventing thrombotic
activity. Studies done on tomato seeds have shown that it also
contains lycopene and flavonoids such as quercetin, kaempferol,

and isorhamnetin derivatives. A significant cell

proliferation inhib ition, acetylcholinesterase inhibitory
activity, and antioxidant capacity were also observed
from tomato seed extracts using rat models [19-21].
Ananas comosus (L.) Merr., fro m the Bro meliaceae
family, is co mmonly known as pineapple or pinya. It
contains bromelain, a protease which was said to illicit

the pharmacological effects of p ineapple. Bro melain is

potent enzy me that naturally supports the body's ability to
break down blood clots caused by the blood-clotting
protein fibrin, as they develop and d iminish inflammation
[22]. Studies have also shown other beneficial effects of
bromelain to humans such as thinning of mucus,
prevention of platelet aggregation, prevention or
minimizat ion of the severity of angina pectoris and
transient ischemic attacks, and prevention and treatment
of thro mbosis and thrombophebitis. Pineapple peels are
also a well-known ingredient in ethnomedicine and also
possess antioxidant activity. A study has showed that
pineapple peel ext ract can help protect against alcoholinduced oxidative stress in brain tissues, an effect
attributed to its bromelain and phytochemical constituents
[23- 25]. Pineapple peel is rich in cellulose, hemicellulose
and other carbohydrates. It also produces methane which
can be used as a biogas. Anaerobic digestion takes place
and the digested slurry may find further application as
animal, poultry and fish feeds [26].
The aim of this paper was to investigate the potential
thrombolytic activ ity and cytotoxicity through brine
shrimp lethality assay, of both flesh and peels/seeds of
A.sativum, C. longa, L. esculentum and A. comosus.
1.2. Objectives
The aim of this paper was to investigate the potential clot
lysing activity (thrombolytic) of several palnt substances.
Specifically it aimed to determine the said act ivity against
controls (Heparin and distilled water). Fu rther it aimed to
determine the cytotoxicity through brine shrimp lethality
assay, of both flesh and peels/seeds of the samples. The
plants analyzed were A.sativum, C. longa, L. esculentum
and A. comosus.
1.3. Hypothesis
The following are the null and alternative hypotheses Ho :
There is no significant difference among percent clot lysis
among plant samples and controls.
Ha: There is a significant difference among percent clot
lysis among plant samples and controls.
1.4. Significance of the study
This study would be significant in the continuous quest
for alternative and less expensive sources of med icine e.g.
thrombolytics. The inclusion of fru it and vegetable by products e.g. peels and seeds in the research would also
be a potential solution in the reduction of industrial generated wastes. Screening of plant ext racts via BSLA is
of equal importance in determin ing its appropriate
fraction or dose required to exert pharmacological action.

2. Materials and Method

The methods used in the clot lysis experiment were
adapted fro m the study done by Khan, et al with slight
modifications [2].

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Pre liminary Assessment of In vitro Anticoagulant Activity vs. Heparin 1,000I.U. and Cytotoxicity of Selected Philippine Medic inal Plants

2.1. Plant Material Collection and Extraction

Allium sativum (garlic), Cucurma longa (luyang dilaw),
Ananas comosus (pineapple) and Lycopersicum
esculentum (to mato) were collected fro m selected
localities in the Ph ilippines. All of the collected samp les
were identified and certified by Dr. W ilfredo F. Vendivil
of the Philippine Nat ional Museum Botany Division.
After the plant samples were rinsed with running water to
remove the dirt, the peels/seeds were separated from the
flesh. The garlic flesh and peels, luyang dilaw flesh and
peels, pineapple peels and to mato seeds were air-dried for
5 days prior to trituration. The powdered, air - dried plant
samples were soaked in ethanol for 48 hours and were
filtered via vacuum filtrat ion. The pineapple and tomato
flesh were freshly prepared, wherein both of wh ich were
cut, juiced and filtered. Plant sample extracts were stored
in sample bottles in the refrigerator prior to their use.
2.2 Herbal Preparation
A weight of 100 mg of the plant ext ract was suspended
and shaken vigorously with 10 ml d istilled water. The
suspension was kept for 24 hours and decanted to remove
the soluble supernatant. From the aqueous herbal
preparation, 0.1ml was added to each of the sterilized
microtube containing the clots to check its thrombolytic
activity.
2.3 Preparation of controls
The commercially available Heparin 1000 I.U and
distilled water were used as the controls for the study. The
Heparin 1000 I.U was diluted with 5ml distilled water and
served as the positive control of the experiment whereas
0.1ml of distilled water served as the negative control.
2.4 Inclusion and exclusion criteria
Qualified participants for blood extraction were all
students of School of Pharmacy - Emilio Aguinaldo
College with age range of 20-30 years old. The
participants were oriented and provided with in formed
consent signed by them prior to blood sample collection
[5]. In the consent the participants were provided with a
checklist to verify if they were suitable to the blood
extraction activity. The exclusion criteria included the
history of oral contraceptive, anticoagulant therapy,
presence of bleeding disorders, and pregnancy [27].
2.5 Specimen Collection
Whole blood (5mL) was withdrawn fro m healthy
volunteers (n=5). Blood ext raction procedure was
performed by Charmaine Talento and Kimberly Cailao,
both are trained medical technology students under the
supervision of Professor Cesar Mendoza, RMT of the
Emilio Aguinaldo College School of Medical
Technology. Collected blood samples we re immed iately
transferred into sterilized microtubes.
2.6 Clot lysis
The withdrawn venous blood was transferred in different

pre-weighed sterile microtubes (0.5ml/tube) and was

incubated at 37C for 45 minutes. After clot formation,
the incubated microtubes were centrifuged for 2 minutes
and serum was removed co mpletely using a micropipette
(aspirated out without disturbing the clot formed). Each
microtubes with clotted blood were weighed again to
determine the initial clot weight (1), wherein Cwi is the
initial clot weight; Cwt is the weight of tube with clot; and
wt is the weight of tube alone.
Cwi = Cwt - wt
(1)
After the determination of the in itial clot weight, each
microtube containing the clot was labeled properly and
0.1 ml of each of the prepared p lant ext ract were added.
For the controls, 0.1ml of Heparin and 0.1ml of distilled
water were separately added to the control tubes and were
labeled accordingly. The microtubes containing the
treated clots were incubated again at 37C for 90 minutes
and clot lysis was observed. The incubated tubes were
centrifuged for 2 minutes and the fluid obtained was
removed. The microtubes were weighed again to
determine the weight after clot disruption (2), where Cw f
is the final weight of clot; Cw T is weight of tube with
treated clot; and wt is the weight of tube alone. The
difference obtained taken before and after clot lysis were
expresses as percentage of clot lysis (3). The procedure
was repeated 5 times.
Cwf = CwT - wt
% Clot lysis = (Cwi -Cwf)/Cwi x 100

(2)
(3)

2.7 Brine shrimp lethality assay

The procedure carried on was based on the writings of
Cantoria, Peteros and Uy with slight modifications [5, 7].
The plant samples were washed and air-dried for a week.
A water tank with a perforated divider was used to
contain a mixture of 2.5L water and 9.5g of sea salt
obtained fro m a pet store. Brine shrimp (Artemia salina)
eggs were p lace to one side of the div ided tank wh ich was
covered. A lamp was turned on to attract the nauplii
(larvae) through the perforated divider. The brine shrimp
were left to hatch and grow for 2 days, after wh ich,
weighed 20mg each of the flesh and peels/seeds of
Lycopersicum esculentum, Ananas comosus, Curcuma
longa and Allium sativum were added with 2ml ethanol
individually. Each carefully labeled sample served as a
stock solution fro m wh ich the volumes 500l, 50l and
5l were transferred to 3 vials labeled as 1,000, 100 and
10 g/ml respectively. Seawater (5mL) was added to each
vial wherein 10 brine shrimps were placed. The vials
were maintained under illu mination. Nu mber of survivors
was recorded after 24 hours and the percentage mortality
at each vial was determined by (4), where Nc is the
number of dead nauplii and Nt is the total number of the
sample.
% mortality = (Nc/Nt ) x 100
(4)
The lethal concentration (LC50 ) to brine shrimps was
determined using Finneys probit analysis (5) in which y

373

Pre liminary Assessment of In vitro Anticoagulant Activity vs. Heparin 1,000I.U. and Cytotoxicity of Selected Philippine Medic inal Plants

corresponds to the probit value of 5 (probit of 50%), b

and m are the values obtained from regression of Log 10

distilled water as the negative control showed 27.21% clot

lysis. The clots treated with 0.1ml of Allium sativum flesh
and peels, Curcuma longa flesh and peels, Ananas
comosus flesh and peels and Lycopersicum esculentum
flesh and seeds prepared extracts exhibited clot lysis:
18.30% and 21.77%; 21.85% and 35.91%; 15.67% and
24.52%; 28.34%, and 26.10% respectively. The p value
generated from one-way ANOVA (0.674574) was greater
than the set = 0.05. The percent clot lysis obtained after
the treatment with herbal ext racts and controls is shown in
Figure 1

Figure 1. Pe rce ntage of total clot lysis from Plant Extracts as

compared to the positive (Heparin 1,000I.U) and negative (distilled
water) controls.

Table 1. Results of Brine Shrimp Lethality Assay on crude e thanol

extracts from both flesh and peels/seeds
of four Philippine medicinal plants

PLANT SAMPLE

Figure 2. Clot lysis after deducting the value of distilled water: (1)
A.sativum flesh; (2) A sativum peels; (3) C.longa flesh; (4) C. longa
pe e ls; (5) A.comosus flesh; (6) A. comosus peels; (7) L.
esculentum flesh; (8) L. esculentum seeds; (9) Heparin 1,000 I.U; (10)
distilled water

concentration on probit value, X is the Log10 of the

concentration.
y = -b + mX
LC50 = -log X

(5)

Abbotts formula for controlled mo rtality was also used in

the calculations since there was no control group used in
the procedure, where CM is the percentage of corrected
mortality, M obs is the observed treatment mortality and
M ctrl is the control or percentage of organism wh ich died
due to factors other than the treatment (6), [28].
CM (%) = (Mobs Mctrl )/ (100-Mctrl) x100
(6)
2.8. Statistical analysis
The significance of the percentage clot lysis by herbal
extracts by means of weight difference was tested using
one-way ANOVA. The statistical software Microsoft
Excel 2010 was utilized in the data analysis of the
study.

3. Results and Discussion

3.1. Clot lysis
The addition of the positive control, 0.1ml Heparin (500
I.U.) to the clots after incubation for 90 minutes at 37o C
showed a 31.11% clot lysis. The clots treated with 0.1ml

% MORTALITY AT
DIFFERENT
CONCENTRATION

1000
ug/mL
A. sativum flesh
100
A. sativum peels
100
C. longa flesh
100
C. longa peels
100
A. comosus flesh
100
A. comosus peels
100
L. esculentum flesh
100
L. esculentum seeds
100

100
ug/mL
100
100
100
100
80
80
100
20

10
ug/mL
90
90
70
70
50
60
80
50

LC50, 24h
ug/mL
11.5
11.5
6.72
6.72
25
29.6
8.6
13.2

Only Curcu ma longa peels and Lycopersicum esculentum

flesh showed ability to clot lyse beyond that of distilled
water as shown in Figure 2. On ly the former showed the
ability to lyse clots beyond that of Heparin 1000IU. It can
be seen values that C. longa peels and L..esculentum flesh
showed percent clot lysis of 8. 68 and 1.08 respectively
The anticoagulant effect of C.longa peels was not proven
statistically, results still have shown that it has a strong
potential to d issolve blood clots. This finding may
reinforce the efforts being done by other researchers find
methods on how to maximize the use of fruit and
vegetable wastes and to address the potential therapeutic
application of these substances. Discovering med icinal
uses from peels like the potential of C. longa as an anti
coagulant is advantageous. Food security will not in any
way be affected by the use of the said peels. Further,
recycling of biodegradable wastes will help in the
improvement of environmental integrity.
3.2. Brine shrimp lethality assay
The results of the assay on ethanol extracts of the plant
samples wh ich include percent mortality at concentrations
of 10g/mL, 100 g/mL, and 1000 g/ mL and LC50
values, are shown in Table 1. A ll of the ext racts exhibited
100% mortality to the brine shrimps at the concentration
of 1000 g/mL. At 100 g/ mL concentration, A.sativum
flesh and peels, C.longa flesh and A. comosus peels have
shown 100% mortality. C. longa peels and A. comosus
flesh both showed 80% mortality whereas L. esculentum
flesh and seeds showed 20% and 50% mortality
respectively. At the concentration of 10g/mL, A. sativum

374

Pre liminary Assessment of In vitro Anticoagulant Activity vs. Heparin 1,000I.U. and Cytotoxicity of Selected Philippine Medic inal Plants

flesh gave the highest result which was 90% mortality.

Both A. comosus peels and L. esculentum seeds showed
80% mo rtality. A. sativum peels and C. longa flesh, A.
comosus flesh, C. longa peels and L. esculentum flesh

there is potential for thrombolytic products to be

ormu lated fro m it. Further studies in order to verify the
anticoagulant properties of the plant especially C. longa is
highly suggested. Isolation and purification of the active
ingredient/s should be done. BSLA have shown that A.
sativum peels and C. longa flesh are the most toxic among
the plant samples. This result may be used as a guide for
the choice of crude plant extract dose to be used in future
studies. In order to thoroughly understand the
effectiveness of these Philippine medicinal plants, further
studies using varied solvent in ext raction for an
exploratory research should be done. Streptokinase may
be used as a positive control in p lace of heparin.
Increasing the amount of blood from the volunteers and
concentration of the plant extracts to be used in the study
should also be employed.

Figure 3. Comparisons of the effect of different plant flesh and

pe e ls/ seeds extract concentrations on brine shrimp mortality.

have shown 70%, 60%, 50% respectively. LC50 values

ranged fro m 6.72 to 31.2g/ mL with A. sativum peels and
C.longa flesh having the lowest value (most potent) [7];
followed by A.comosus peels (8.62g/mL), A. sativu m
flesh (11.52g/ mL), L. esculentum flesh (13.22g/ mL),
C. longa peels (252g/mL), A. co mosus (29.62g/ mL)
and L. esculentum seeds (31.22g/mL).The brine shrimp
lethality assay (BSLA) has been used routinely in the
primary screening of the crude extracts to assess its
toxicity towards brine shrimp. Th is could also provide an
indication of possible cytotoxic propert ies of the plant
extracts. The variation in BSLA results may be due to the
difference in the amount and kind of cytotoxic substances
(e.g. tannins, flavonoids, triterpenoids, or coumarins)
present in the crude ext racts. Moreover, this significant
lethality of the crude plant extracts (LC50 values less than
100 pp m or g/ mL) to brine shrimp is indicative of the
presence of potent cytotoxic co mpound [7]. Abbotts
formula for control mortality was used to determine
whether any bioassay organisms died due to factor o ther
than exposure to our test chemical [28]. It was also used
in the calculations since there was no controlled group
used in the experiment. These results may be used as
guide for succeeding studies as a basis of which crude
plant extract fractions to be utilized.

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