Research Proposal

Presented to:
Dr. Alamgeer
Presented by:
Khadija Arshad
Roll no. 04
 CONTENTS:
1- INTRODUCTION

2- AIMS AND OBJECTIVES

3- RESEARCH PROTOCOL
4- MATERIALS AND METHODS
4.1- MATERIALS
4.1.1 Animals to be used
4.1.2 Chemicals to be used
4.1.3 Drugs to be used in different models
4.1.4 Apparatus to be used

5- REFERENCES
 1-INTRODUCTION
• Anti-thrombotics include anticoagulants,
anti-platelets
thrombolytics

that decrease the rate of blood clotting in the body by dissolving

already formed ones or prevent clot fomation.[1,2]

• Platelets are blood cells that participate in human primary

homeostatic mechanism.[3,4]
 Platelets

maintainance regulation of
of CV integrity clot formation

uncontrolled
aggregation leads to
CV diseases e.g
HF [5,6]
 Endothelial injury

platelets activated

ADP Thrombin AA Collagen

platelet aggregation

Thrombus formation

Primary homeostasis[7,8,9] Atherothrombotic diseases

e.g, mayocardial infarction[10]
 EPIDEMIOLOGY:

 Pulmonary embolism (PE) is the leading cause of direct

maternal death in the United Kingdom.
 60,ooo cases of PE / years
 5,000 deaths /year from pulmonary embolism[11].

 2.5 million cases of deep vein thrombosis/year

 According to WHO, CVD due to thrombus formation will be the

leading cause of death in 2015. These CVDs are the cause of
death, now a days, in western countries. [12]
PATHOPHYSIOLOGY:
Thrombus formation include platelet
adhesion
activation
aggregation. [13,14]
PROBLEMS WITH CURRENT MEDICATIONS:

Most of the drugs used for antiplatelet activity are

synthetic drug. e.g, aspirin,clopidogrel and heparin
treat the cardiovascular diseases but also lead to
serious adverse reactions in certain patients which
include
• Bleeding
• GIT toxicity
• Neutropenia
• Thrombocytopenia
• Drug resistance.[15]
• Thrombolytic agents such as tissue plasminogen
activator, urokinase, streptokinase (SK), etc are used
to dissolve the formed clots in the blood
vessels[16,17].

• Drugs have certain limitations and cause serious and

sometimes fatal results including hemorrhage,
severe anaphylactic reaction, lacked specificity, etc.
[18,19].
Demand For New Drugs:

There is a great demand in identifying new drugs for

thrombosis. Although, a lots of drugs like aspirin , heparin etc.
are available in the market for the treatment of thrombosis
but still there is need of new drugs with

 less side effects

 less resistance
 better tolerability.

Agents from plant source are expected to be

 less antigenic
 Cheaper[20].
Various herbal plants have been used and
claimed to have antithrombotic activities such
as:
• Synclisia scabrida [21]
• Allium sativum [22]
• Zingiber officinale [23]
2-AIMS AND OBJECTIVES:

The aim of this study is :

 To confirm its traditional use in CVDs by evaluating its

antithrombotic effect.

 To evaluate the antithrombotic effects of selected plant by

administration of extract in different experimental subjects
3. RESEARCH PROTOCOL:

PLAN OF WORK
Collection of plant

Preparation of extract

Selection of animal

experimentation
 4 -MATERIAL AND METHODS
4.1-MATERIALS:
4.1.1- Plants to be used:
Grewia asciatica
Acacia leuciphloea

4.1.2-Animals to be used:
Human volunteers
Rabbit
Rats

4.1.3-Chemicals to be used:
Arachidonic acid Methanol
Sodium citrate solution Chloroform
Normal saline
4.1.3- Standard Drugs to be used in different models:

•Heparin
•Aspirin
•Xylane
•Streptokinase

4.1.4- Apparatus to be used:

•Centrifuge
•Aggregometer
•Microscope
•Blotted paper
4.2-METHODOLOGY:

4.2.1-Preparation of extract: [24]

Concentrate the
Collection of filterate to
plant dryness using
evaporator

Cleaning and
filteration
drying at RT

Pulverization to 500g powder will

coarse powder be soaked in 80%
using hammer of methanol for
mill 48 hrs
4.2.2-Preparation of platelets:

Blood will be taken via venipuncture from normal

volunteers who will reported to be free of medication for 7
days. Blood samples will be mixed with 3.8% (w/v) sodium
citrate solution and will be centrifuged at 1000 rpm for 15
min to obtain platelet rich plasma (PRP). All aggregation
studies will be carried out at 37°C with PRP.[25]
Acute toxicity testing:[26]
:
4.2.4- MODELS FOR ANTI-
THROMBOTIC ACTIVITY:
 Rabbit bleeding time
For antiplatelet
By aggregometer

Rabbit bleeding time

Models for anti- For anticoagulant
thrombotic
activity Prothrombin time

Clotting time

For thrombolytic

Percentage weight
loss of clot
 A- Rabbit Bleeding Time:
(Model for antiplatelet & anticoagulant activity)

Rabbits(free of medication , 2-2.2 kg, n=6)

group 1 group 2 group 3 group 4 group 5

200mg/kg 400mg/kg 1-2mg/kg 0.75-1.5mg/kg distilled

Of extract of extract of aspirin of heparin water

• Then ear marginal vein will be pricked.

• Bleeding time will be checked after every 5 seconds with blotted
paper.
• Baseline bleeding time will be measured before giving extract or
drugs.[27]
 B- Thrombolytic Activity By Percentage Weight Loss Of Clot
In Human Volunteers: [28]

(clot weight = weight of clot containing tube – weight of tube alone)

Add blood in 5 Serum will be 100 ,200,

2.5 ml microcentrifuge removed from 300 µl of
blood from tubes(0.5ml/tub clot and clot
e) and will
extract in
healthy weight will be
incubate for 45 pre-weighed
volunteer measured
minutes clot

100µl of
100µl of streptokinase
Fluid will be
Incubate at water will suspension will
removed and
37º for 90 be used as be used as
again weigh
minutes negative positive control
the clot
control (2.5mg vial in
PBS)
 C-Antiplatelet Activity By Light Transmission
By Aggregometer:

Aggregation will be measured by Dual-channel Lumi- aggregometer

using 0.45 ml aliquots of PRP.

Aggregation will be induced by using arachidonic acid (AA).

The anti aggregatory effects of extract will be studied by incubating PRP

with 0.5 ml of extract for 1 min followed by the addition of aggregating
agent (AA).

The resulting aggregation will be recorded for 5 min after the challenge,
by the change in light transmission as a function of time.[29]
 D- Prothrombin Time:
The prothrombin time test will be screening procedure for the extrinsic coagulation
mechanism. It will detect deficiencies in factor V, VII, and X. The prothrombin time test
will be used to follow oral anticoagulant therapy that inhibit factors VII, IX and X.
Treatment Amount of Amount of Calcium Time of
groups plasma extract chloride soln. coagulation
Control (0.9% 0.2 ml 0.1 ml (NS) 0.3 ml 70 Sec
saline water)
extract (200 0.2 ml 0.1 ml 0.3 ml 8:32 min
µg/ ml)
extract(400 µg/ 0.2 ml 0.1 ml 0.3 ml 22:56 min
ml)

All test tubes will be incubated at 37ºC. The coagulation time

will be recorded with stopwatch by tilting the test tubes every 5
seconds. This time is called the prothrombin time. [30]
 E- Blood Clotting Time :

The time taken for the blood samples to clot will be recorded
and compared in this model.[31]

1.0ml of blood from 1.0 ml of blood + 1.5µl 1.0 ml of blood + 0.2 1ml of blood for
marginal ear vein of of heparin as positive ml of distilled water baseline clotting time
rabbit + 2ml of extract control as negative control
• Incubate at 37º in • Incubate at 37º in • Incubate at 37º in • Incubate at 37º in
water bath water bath water bath water bath
 5-Impact of study:

 This study will provide the scientific evidence to the folkloric

claims regarding selected plants extracts possessing Anti-
platelet, Anti-coagulant and Thrombolytic effects.

 This study will also provide basis for the development of safer
and cheaper alternatives of already available synthetic drugs.
 5-REFRENCES
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2-Chinaka O. Nwaehujor, Rita I. Udegbunam, Julius O. Ode, Stella A.

Madubuike, Antithrombotic activities of Newbouldia laevis (P.
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3- Park M, Rhee Y, Lee H, Lee E, Kim K, Park M, Jeon B, Shim

B, Jung C, Choi S, Ahn K, Kim S.(2008). Antiplatelet and
antithrombotic activity of indole-3-carbinol in vitro and in
vivo. Phytother Res, 22,58-64.

4- Davies MJ, Thomas AC (1985). Plaque fissuring - the cause

of acute myocardial infarction, sudden ischaemic death, and
crescendo angina. Br. Heart J., 53: 363-373
5- Park M, Rhee Y, Lee H, Lee E, Kim K, Park M, Jeon B, Shim B,
Jung C, Choi S, Ahn K, Kim S.(2008). Antiplatelet and
antithrombotic activity of indole-3-carbinol in vitro and in vivo.
Phytother Res, 22,58-64.

6- Mirza Bojic, Zeljko Debeljak, Maja Tomicic, Marica Medic-Saric

and Sinisa Tomic . evaluation of antiaggregatory activity of flavonoid
aglycone series(2011).

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activation mechanisms in arterial thrombpsis and ischaemic stroke.
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platelet adhesion and activation. Arterioscler. Thromb. Vasc.
Boil.2010,30,2341-2349.

9- Offermanns, S. Activation of platelet function through G-Protein

couple receptors. Circ. Res.2006,99,1293-1304.
11-Confidential Enquiries into Maternal Deaths in the United Kingdom.The fifth report:
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Zingali, R.B. Metabólitos secundários de origem vegetal: Uma fonte potencial de fármacos
antitrombóticos. Quim Nova 2010, 33, 172–180.

13-Sanjay K Banerjee and Subir K Maulik , Effect of garlic on cardiovascular disorders. A

review, 19 november 2002.

14-Eduardo Fuentes, Marcelo Alarcón , Luis Astudillo , Claudio Valenzuela , Margarita

Gutiérrez and Iván Palomo, Protective Mechanisms of Guanosine from Solanum
lycopersicum on Agonist-Induced Platelet Activation: Role of sCD40L, 10 July 2013.

15- Shah, R.; Keough, L.A.; Belalcazar-Portacio, A.; Ramanathan, K.B. Ticagrelor as an
alternative in clopidogrel-associated neutropenia. Platelets 2014, 26, 80–82.

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Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Dhaka,Bangladesh, Thrombolytic activity of some spices and plants available in
Bangladesh, Sci. 36 (2012) 72-77.
17- M.W. Gillman, L.A. Cupples, D. Gagnon, B.M. Posner, R.C. Ellison, W.P.
Castelli, and P.A. Wolf. Protective effect of fruits and vegetables on development
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Mughal Qayum and Inamullah Khan, Anti-platelet activity of methanolic extract
of Grewia asiatica L. leaves and Terminalla chebula Retz. Fruits(6 January, 2012)

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Castelli, and P.A. Wolf. Protective effect of fruits and vegetables on development
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21- Afonne O.J., Orisakwe O.E., Obi E., Orish C. and Akumba D.D. Some Pharmacological
properties of Synclisia scabrida III. Indian J. Pharmacol. 2000; 32: 239-241.

22-Tattelman E. Health effects of garlic. Am. Fam. Physician. 2005; 72 (01): 103-106.

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platelet function. Methods Mol. Biol. 2010; 663: 22940

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Antithrombotic activities of Newbouldia laevis (P. Beauv) seem. ex Bureau leaves , Vol.
5 (05), pp. 075-079, May, 2015.

25-M. Zia-Ul-Haq1, Shakir Ahmad Shahid, Sagheer Ahmed, Shakeel Ahmad,

Mughal Qayum and Inamullah Khan, Anti-platelet activity of methanolic extract
of Grewia asiatica L. leaves and Terminalla chebula Retz. Fruits(6 January,
2012)

26- Nwaehujor Chinaka O., Ode Julius O., Nwinyi Florence C., Madubuike Stella A.
Anticoagulant and Antioxidant Activities of Dracaena arborea Leaves (Wild.) Link, Vol. 1,
No. 4, 86-92, 2013.
27-Chinaka O. Nwaehujor, Rita . Udegbunam, Julius O. Ode, Stella A. Madubuike,
Antithrombotic activities of Newbouldia laevis (P. Beauv) seem. ex Bureau leaves,
Vol. 5 (05), pp. 075-079, May, 2015.

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Londonkar, evaluation of in vitro anti-thrombolytic activity and cytotoxicity
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asiatica L. leaves and Terminalla chebula Retz. Fruits(6 January, 2012)

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4, 86-92, 2013