Research Article ISSN 2250-0480 Vol 2/Issue 4/Oct-Dec 2012

IN VITRO ANTI PLATELET AGGREGATION ACTIVITY AND

THROMBOLYTIC ACTIVITY OF CHEENALINGA CHENDHURAM

E. SATHYAPRIYA*1, V. VELPANDIAN1, J. ANBU2 AND ASHWINI ANJANA2

1
Department of Pharmacology (Gunapadam), Govt. Siddha Medical College, Arumbakkam, Chennai-
600106, India
2
Department of Pharmacology, School of Pharmaceutical Sciences, (VISTAS) Vels University,
Pallavaram, Chennai-600117 India

ABSTRACT

Cellular metabolism plays a vital role in the homeostasis of the body. This metabolism is disturbed in
conditions like thrombus which inhibits the blood supply to the body tissues leading to ischemia and finally
necrosis of the cells. Hence there is an urgent need for research in the field of antiplatelet and thrombolytic
agents in order to prevent and cure thrombus which leads to the pathological conditions like Myocardial
Infarction (MI), Hemiplegia, etc. Siddha system of medicine boasts of a variety of medicines to prevent,
cure and to revive the body from various diseases which are coded based on the known 96 principles.
Though, herbal medicines have the above mentioned properties, it is essential to search for mineral
medicines of similar nature which are considered to be cost effective, with a speedy recovery and having a
longer shelf life. Cheenalinga chendhuram [CLC] is a mineral drug and as in literature is mentioned for its
use in cardiac ailments predominantly angina and MI in which thrombus is the pathological background. In
this study, CLC was evaluated for its in vitro antiplatelet aggregation and thrombolytic activity which was
showed effective at the dose of 300µg/ml and 75µg/ml respectively. We conclude therefore, that CLC is an
effective drug in the treatment of cardiovascular diseases and cerebrovascular accidents.

Keywords: Cheenalinga Chendhuram, Anti-platelet activity, thrombolytic, angina and MI.

1. INTRODUCTION

In this modernised world, we are facing major drugs, siddhars spiritually and holistically
threats against many stress related disease. emphasized various wonderful medicines to
Thrombus is a pathological condition that plays a prevent stroke, cheenalinga chendhurum is one
vital role in causing many diseases like Stroke, among them that posses thrombolytic activity
Deep vein thrombosis and Myocardial infarction. and anti platelet aggregation activity. There is
Major clinical manifestation of thrombus is many Antiplatelet aggregation and Thrombolytic
stroke. Stroke or Cerebro Vascular Accident medicine in siddha, yet there is no proper
(CVA) Causes 2,00,000 deaths each year in the scientifical evaluation or an attempt to establish
world and are a major cause of disability. The cheenalinga chendhuram as good anti-platelet
most common forms of Cerebro Vascular aggregation thrombolytic drug in siddha
Accident (CVA) are Cerebral Thrombosis (40% system.These beneficial effects result in the
of cases), Cerebral embolism (30% of cases) and improvement of blood fluidity. However, as
cerebral hemorrhage (20% of cases).Long before siddha drug significantly enhances fibrinolytic
inventions of various modern techniques and activity, it is theoretically possible that its over-

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activity could cause platelets to aggregate 2.3 PREPARATION OF THE DRUG

through the release of fibrinogen degradation Lingam : 35gm
products because it has been reported that Vediuppu : 70gm
excessive fibrinolysis is associated with the Padikaram : 70gm
release of FDP. These findings clearly showed Purified Vediuppu and Padikaram were
that siddha drug has anticoagulant properties. Its powdered, taken together in new mud vessel and
medicinal uses include treatment for including it was burnt on low flame. When it melts,
Stroke, Deep vein thrombosis and Myocardial purified lingam bar was added and fried. Then
infarction. The interaction between platelets and Lingam was taken from it and scrubbed. Then it
blood vessel walls are important in the was washed in distilled water and grinded in
development of thrombosis and cardiovascular stone mortar into fine powder. This powdered
diseases such as myocardial infarction, stroke medicine was kept in a closed air tight glass
and atherosclerosis. Among the family of platelet vessel for the further use.
activating factors (PAF), Arachidonic acid, and
ADP are three important platelet stimulants, 2.4 TOXICITY STUDY
which induce platelet aggregation via different Animals:
mechanism. Platelet plays a key role in the Healthy adult Wistar albino rats (200-250g) and
physiological hemostatic process and pathologic Swiss albino mice (28-35g); individually in
thrombosis. CheenaLinga Chendhuram possess polypropylene cages, maintained under standard
anti-platelet activity in a dose-dependent manner. conditions (12 h light and 12 h dark cycle; 25±
A physically active life style has an important 30oC; 35 - 60% humidity). The animals were fed
role in preventing thrombotic events and with standard rat pellet diet and water ad libitum.
decreasing the risk of cardiovascular disease. The Institutional Animal Ethical Committee
Platelets are involved in the pathogenesis and (IAEC/VELS/08/10-2011) approved the study
progression of cardiovascular diseases. These
beneficial effects result in the return of normal ACUTE TOXICITY STUDY:
blood flow. Acute toxicity studies were conducted to
determine the safe dose as per OECD-423
2. MATERIALS AND METHODS guidelines. Drugs were administered orally to
overnight fasted animals. After drug
administration the animals were observed
2.1 PREPARATION OF CHENDHURAM
continuously for 1 hour, frequently for the next
The preparation was collected from ANUBOGA
four hours, and then after 24 hours. After
VAITHIYA NAVANEETHAM – PART 4, P.no-54
administration, Irwin's test was conducted, where
has written by HAKKIM P.MOHAMMED
the animals were observed for gross behavioral
ABDULLAH SAIBU.
changes. The toxic dose was determined by
observing the mortality rate in the drug treated
2.2 COLLECTION AND AUTHENTICATION
groups. From this the therapeutic dose was
OF THE TEST DRUG
selected for the further study.
The raw drugs 500mg of lingam, 2kg of
vediuppu and 2kg of padikaram which are
2.5 PHARMACOLOGICAL STUDY
ingredients of CHEENALINGA CHENDHURAM
Drugs and Chemicals
collected from raw drug shop Tampcol at The drugs and reagents used in this study were as
Chennai.They were identified and confirmed by follows: Heparin sodium and adenosine
gunapadam experts, PG Department of diphosphate (ADP) (Sigma Chemical Co., St.
Gunapadam G.S.M. college at Chennai-106. Louis, MO, U.S.A). Other reagents used were of
These specimens were kept in PG Department of analytical grade. On the day of the assay the
Gunapadam, G.S.M. College, Chennai-106, for CLC was dissolved in 2% CMC in saline to yield
further reference. a final concentration of 0.5% (v/v) for InVitro
Anti-platelet Aggregation studies.

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ANTI-PLATELET AGGREGATION (20µg/ml) was used as reference standard. The

ACTIVITY maximal aggregation was recorded.
Platelet rich plasma (PRP) was prepared by The aggregation is expressed as % inhibition (X)
centrifugation (1000 rpm for 5min) of blood calculated by using the following equation:
collected from normal aspirin free blood bank X(%)=(A-B)/A×100. where A=maximal
donors. 1.5 ml of acid citrate dextrose was used aggregation of the control, and B=maximal
as anticoagulant for every 8.5 ml of blood. PRP aggregation of drug-treated PRP.
was taken into siliconized glass cuvettes. Platelet
poor plasma (PPP) collected by centrifugation ADP induced platelet aggregation
(3000 rpm for 5 min) was kept as reference. The The reaction mixture contains different
cuvettes were incubated at 37°C for 5 min. The concentration of drug CLC 0.5 ml (100, 200, 400
aggregation was initiated by `adding 20 µl of µg/ml) solution and platelet rich plasma 0.5 ml.
ADP (10 µM) to 1ml of PRP. The aggregation These reaction mixtures was maintained at 370C
was recorded for 5 min at 600 nm. The effect of and kept for 2 min with constant stirring, 0.5 ml
different concentrations (50–250µg) of of ADP solution was added and incubated for 4
Cheenalinga Chendhuram was studied by mins and absorbance was measured at 414 nm.
incubation with PRP at 37 °C for 5 min before ADP induced platelet aggregation was calculated
the addition of ADP. Commercial heparin by following formula:

Where, T1= ADP + Platelet, T2= ADP + CLC +Platelet and T3= ADP + CLC +Platelet.

Statistical analysis 0.2ml of fresh blood of a donor was added in

The data of In vitro anti-platelet aggregation seven sterile cuvettes were added serially and the
activity of Cheenalinga Chendhuram was test drug SLC at different concentration from 25-
analyzed statistically using One-Way ANOVA 125microgram was added after fifteen minutes of
followed by Dunnett’s t test by INSTAT-V3 blood collection. Then the contents were mixed
computer software programme. slowly by tilting the cuvettes and then a small
amount of thrombus was transferred carefully
2.7 THROMBOLYTIC ACTIVITY with the help of capillary tube to the plain glass
Method plate and observed for the cell distribution under
In the present study the thrombolytic activity was microscope.
analysed by the modified invitro method. The

3. RESULTS
Table 1. Modulatory effect of different concentrations of Cheenalinga Chendhuram on platelet deaggregation
activity.

S.No. Groups Inhibition of platelet aggregation (%)

1. Control ------------
2. Cheenalinga Chendhuram 100 (µg/ml) 23.6 ± 2.62**
3. Cheenalinga Chendhuram 200 (µg/ml) 38.19 ± 2.44**
4. Cheenalinga Chendhuram 300 (µg/ml) 49.60 ± 2.53**
5. Heparin (20 µg/ml) 78.99 ± 2.84
Values are expressed as mean ± SEM. P<0.01 compared to standard (n=6)

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The doses fixed for the Anti platelet aggregation coronary syndromes, and vessel wall injury leads
activity were 100, 200, and 300µg/ml to to the adherence of platelets and subsequent
ascertain the dose dependent activity. In vitro platelet activation. Platelet aggregation is
inhibitory activity of Cheenalinga Chendhuram absolutely essential to the formation of a
against ADP-induced platelet aggregation were hemostatic plug when normal blood vessels are
measured. Cheenalinga Chendhuram inhibited injured. However, the interactions between
platelet aggregation in vitro potently compared to platelets and collagen can also cause circulatory
heparin reference drug widely used as anti- disorders, such as thrombosis, atherosclerosis,
platelet agent in clinical practice. and myocardial infarction. Inhibition of the
Intravascular thrombosis is one of the generators platelet-collagen interaction might be a
of a variety of cardiovascular disease and platelet promising approach to the prevention of
aggregation is believed to play a crucial role in thrombosis. Cheenalinga Chendhuram may have
atherothrombotic processes. In vitro, Cheenalinga an anti-platelet function by elevating the cyclic
Chendhuram inhibits platelet aggregation in a adenosine monophosphate (cAMP) level, and
dose-dependent manner stimulated by a ADP, in then by decreasing the [Ca2+], an essential factor
human platelet-rich plasma. Tissue factor, also for platelet aggregation. Intravascular thrombosis
called platelet tissue factor, is necessary for the is one of the generators of a variety of
initiation of thrombin formation from the cardiovascular disease and platelet aggregation is
zymogen prothrombin. Taken together, all these believed to play a crucial role in
results suggest that Cheenalinga Chendhuram has atherothrombotic processes. It is reported that
an effective anti-platelet effect in vitro, and be a CLC have anti-platelet aggregation activity. In
potential therapeutic agent for arterial vitro, CLC (40–200 µM) inhibits platelet
thrombosis. aggregation in a dose-dependent manner
stimulated by an agonist ADP, in human platelet-
THROMBOLYTIC ACTIVITY rich plasma. Further investigation reveals that
The microscopical examination reveals that the those effects are due to the inhibition of
Cheenalinga chenduram at the concentration of phospholipase C activity, leading to reduced
75microgram added content showed RBC phosphoinositide breakdown, followed by the
redistribution at dose dependent manner. As a inhibition of thromboxane A2 formation, and
result of drug treatment at various concentrations then inhibition of [Ca2+] mobilization of platelet
it was identified that the CLC possess effective aggregation stimulated by agonists.
deaggregation compared to aspirin and indicates Tissue factor (TF), also called platelet tissue
the drug can be an alternative for aspirin in the factor, is necessary for the initiation of thrombin
treatment of cardiac diseases since the activity formation from the zymogen prothrombin.
was almost equivalent to that of the positive Besides the above mechanism, the antiplatelet
control aspirin. activity of CLC is related to the inhibition of the
release of platelet-derived TF by stimulating the
4. DISCUSSION synthesis and releases of cGRP (cyclic
g\Guanosine Releasing Peptide). Taken together,
Platelets play a pivotal role in health and all these results suggest that CLC has an
diseases, given their central involvement in effective anti-platelet effect both in vivo and in
homeostasis and thrombosis. Recently several vitro, and be a potential therapeutic agent for
natural antiplatelet agents from natural products arterial thrombosis.
including polyphenols and flavonoids have been
reported. Plant preparations containing 5. CONCLUSION
polyphenols/flavonoids have been used for
centuries as herbal remedies for a variety of In conclusion, many cardiovascular diseases can
diseases and found to have an impact on diabetes be attributed to excessive platelet aggregation,
and obesity related disorders. Thrombosis plays which has a critical role in thrombus formation.
an important role in the pathogenesis of acute It appears that Cheenalinga Chendhuram can

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inhibit platelet aggregation in vitro; therefore, cardiovascular disorder patients. However, this
they may be used to treat or prevent some drug should be used with caution by patients
cardiovascular diseases. The present study with bleeding or other haematological disorders
ensures the anti-platelet aggregation of as it may increase the risk of bleeding and
Cheenalinga Chendhuram that is beneficial for complications.

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