Anticoagulant activity of Calotropis gigantea leaves

Abstract

Calotropia gigantea (Asclcpiadacae) is widely growing plant and has been reported to possess multiple therapeutic properties like anti-pyretic, analgesic, anticonvulsant, anxiolytic, sedative, wound healing and antidiabetic. Chemical investigation of Calotropis gigantea report isolation of different type of photochemicals such as flavonoid, glycoside, terpenoid, steroid etc. However its anti thrombotic property has not been yet elucidated. Hence, in the present study we investigated the anti-thrombotic activity of aqueous and alcoholic extract of leaves of Calotropis gigantea in comparison with standard and normal saline as negative control.

Full text

Research J. Pharm. and Tech. 9(9): September2016
1493
ISSN 0974-3618 (Print) www.rjptonline.org
0974-360X (Online)
RESEARCH ARTICLE
Anticoagulant activity of Calotropis gigantea leaves
Jyoti B. Wadekar1*, Ramesh L. Sawant2, Kunal R. Sanklecha2, Amol S. Ghodechor2,
Priyanka B. Sable2, Ganesh D. Barkade2
1Department of Pharmacognosy, Pad. Dr. Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Vilad Ghat,
Ahmednagar 414111, India.
2Department of Pharmaceutical Chemistry and P G Studies, Pad. Dr. Vithalrao Vikhe Patil Foundation’s
College of Pharmacy, Vilad Ghat, Ahmednagar, 414111, India.
*Corresponding Author E-mail: jyotsfriend21@yahoo.com
ABSTRACT:
Calotropia gigantea (Asclcpiadacae) is widely growing plant and has been reported to possess multiple
therapeutic properties like anti-pyretic, analgesic, anticonvulsant, anxiolytic, sedative, wound healing and anti-
diabetic. Chemical investigation of Calotropis gigantea report isolation of different type of photochemicals such
as flavonoid, glycoside, terpenoid, steroid etc. However its anti thrombotic property has not been yet elucidated.
Hence, in the present study we investigated the anti-thrombotic activity of aqueous and alcoholic extract of
leaves of Calotropis gigantea in comparison with standard and normal saline as negative control.
KEYWORDS: Calotropis gigentea, flavonoid, Quick‘s method.
Cardiovascular disease and stroke are the major causes
of morbidity and mortality in high-income countries.
Although many factors contribute to the development of
cardiovascular disease, thrombus formation is the main
trigger event in acute coronary syndrome and stroke.
Injury to the endothelial wall causes platelets to activate
and adhere to the exposed endothelium, leading to
thrombus formation [1].
Herbs and plants have been in use as a source of
therapeutic compounds in traditional medicinal system
since ancient time. Plant-produced compounds are of
interest as sources of safer or more effective substitutes
for synthetically produced antithrombotic agents. Herbal
medicines have been used from the earliest times to the
present day. [2]
Received on 20.01.2016 Modified on 25.01.2016
Accepted on 14.02.2016 © RJPT All right reserved
Research J. Pharm. and Tech 2016; 9(9):1493-1495.
DOI: 10.5958/0974-360X.2016.00290.0
Calotropis species, belonging to the family of
Asclepidaceae in plant kingdom, are the well known
plants throughout the tropical world and they are native
to the tropical and subtropical parts of Asia and Africa
[3]. C. gigantea is a wasteland weed better known as
milkweed, habitat of Asian countries that includes, India,
Indonesia, Malaysia, Philippines, Thailand, Sri Lanka
and China. Tribal people were using this plant parts to
cure several illnesses such as toothache, ear-ache, sprain,
anxiety, pain, epilepsy, diarrhoea and mental disorders
[4]. C. gigantea is scientifically reported for its anti-
Candida activity, cytotoxic activity, antipyretic activity
and wound healing activity.
Figure 1: Calotropis gigantea leaves

Research J. Pharm. and Tech. 9(9): September2016
1494
Phytochemicals present in the various parts of C.
gigantea especially in the leaves are usharin, gigantin,
alpha and beta-calotropeol, beta-amyrin, fatty acids (both
saturated and unsaturated), hydrocarbons, acetates and
the benzoates, a mixture of tetracyclic triterpene
compounds, sterols, giganteol and giganteol are also
found to be present. However cardenolide calotropin, α-
amyrin, β-amyrin, β-sitosterol, α-amyrin
methylbutazone, β-amyrin methylbutazone, α-amyrin
acetate, β-amyrin acetate, lupeol acetate B,
gigantursenyl acetate A, gigantursenyl acetate B,
flavonol glycoside, akundarol, uscharidin, calotropin,
frugoside, calotroposides A to G are responsible for
many of its activities [5]. Current study was focused to
investigate antithrombotic activity of aqueous and
alcoholic extract of C. gigantea leaves.
MATERIALS AND METHODS:
Plant material:
C. gigantea plant was collected from the natural
population growing in the Vilad ghat, Ahmednagar,
India during December 2014. The plant material was
identified at the field using standard keys and
descriptions.
Processing of the plant:
Plant leaves were collected washed and were shade dried
at room temperature. Dried leaves were uniformly
grinded using mechanical grinder. Ten gram of
powdered leaves was soaked in 100 ml of distilled water
and alcohol separately in a conical flask and loaded on
an orbit shaker at a speed of 120 rpm for 24 hours. The
mixture was filtered using Whatman filter paper number
1. The filtrate was concentrated using rotary vacuum
evaporator and dried. Dried aqueous and alcoholic
extracts were collected in an air tight container and
stored at 4°C. The aqueous and alcoholic extracts were
dissolved in sterilized distilled water separately to make
1000 μg/ml stock solution and used to perform
antithrombotic assay [1].
Determination of Prothrombin Time (PT) by Quick‘s
Method:
Pipette out 0.1 ml of plasma in small test tube. Add 0.1
ml of brain thromboplastin and mix. Wait for 2 min and
add pre warmed calcium chloride solution at 37°C, mix
Start the stop watch. Hold the tube in front a source of
light and keep tilting the test tube gently. At first
appearance of fibrin clot stop the watch immediately and
record the time as control reading. Pipette 0.1 ml of
plasma in small test tube; add 0.1 ml of 100 mg/ml test
compound solution. Incubate for 5 min and repeat the
procedure to record the elongation in mean prothrombin
time. [6]
RESULTS AND DISCUSSION:
The aqueous and ethanolic extracts of C. gigantea were
subjected to preliminary phytochemicals screening and
revealed the presence of Alkaloids, Phenolic
compounds/tannins and flavonoids and Sterols. Anti-
coagulant activities of aqueous and ethanolic extracts of
C. gigantea were carried out by Quick’s method and
results are shown in Table 1 and Table 2. The clot lysis
of C. gigantea was found to be increased with the
increase in concentration of the sample. Result shows
that the extracts exhibit moderate anti-coagulant activity.
Literature shows that tannins and flavonoids are
responsible for anti-coagulation [7-8]. Hence the
presence of tannin and flavonoid may contribute the said
activity. Further study is under progress to isolate the
pure component fraction.
Table no. 1: In-vitro anti-coagulant activity of ethanolic extract of
Calotropis gigantia leaves
Sr. No.
Concentration
(mg per dl)
Time
(In sec.)
Mean
1.
100
42
41
40
41.00
2.
250
75
72
77
74.66
3.
500
180
182
180
180.66
Table no. 2: In-vitro anti-coagulant activity of aqueous extract of
Calotropis gigantia leaves
Sr. No.
Concentration
(mg per dl)
Time
(In sec.)
Mean
1.
100
35
35
38
36.00
2.
250
70
70
70
70.33
3.
500
167
160
168
165.00
Table no. 3: In-vitro anti-coagulant activity of Warfarin
Sr. No.
Concentration
(mg per dl)
Time (In sec.)
Mean
1.
100
102
103
103
102.66
CONCLUSION:
From the present study it is evident that aqueous and
ethanolic extract of C. gigantea possess anticoagulant
property. However, in vivo activity of C.gigantea is yet
to be discovered and is in pipeline. By above result, it
can be suggested that the application of the C. gigantea
component may be accessible to the society for the
treatment of cardiovascular diseases.
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