This document outlines a study to evaluate the anti-ischemic activity of indigenous medicinal plants using in-vivo and in-vitro models. The study will investigate the effects of extracts from Terminalia arjuna and Carthamus tinctorius on several animal models of ischemia, including isoproterenol-induced ischemia in rats and sympathetic hyperactivity-induced ischemia in rabbits. Cell culture models of ischemia in neonatal rat heart cells will also be used. The methodology describes preparation of plant extracts, fractionation, toxicity testing, and experimental procedures to assess anti-ischemic effects.

3. Anti ischemic activity of some
indeginous medicinal plants by
using In-vivo and In-vitro models.

4.  Introduction
 Aims & Objectives
 Plan of Work
 Methodology of Work
(a) Plant Material
(b)Animal Used
(c)Experimental Procedures
(d)Statistical Analysis
 Impact of Study
 References

5.  Cardiovascular diseases are an important
medical and public health issue through out the
world[1,2]
 CVS diseases the first killer for human being
,constitute the global economic burden .The
updated data from WHO addresses that the
mortality rank due to CVS diseases will increase
from No.5 in 2000 to Top 1 till 2020 .And 30 % of
overall mortality is due to CVS diseases [3].

6.  This is a bigger problem for developing
countries like Pakistan[4].
 The rise in prevalence of CVS diseases in
developing countries is mostly related to
demographic changes ,urbanization ,life style
modification and high risk factor levels [5].
 In Pakistan, cardiac ailments contribute to about
25% deaths in the country [6].

7.  IHD is a condition in which the vascular supply to the heart is impeded by
atheroma, thrombosis or spasm of coronary artries.This may impair the
supply of oxygenated blood to cardiac tissue sufficiently to cause Myocardial
ischemia.
Pathogenesis:
Myocardial ischemia occurs by 2 ways :
 When oxygen demand exceeds myocardial oxygen supply.The resultant
ischemic myocardium release adenosine,the main mediator of chest pain ,by
stimulating the A1 receptors located on the cardiac nerve endings.
 Myocardial Ischemia can also arise if oxygen demand is abnormally
increased,as may occur in patients with thyrotoxicosis or severe ventricular
hypertrophy due to hypertension.MI supply is dependent on the luminal cross
sectional area of the coronary artery and coronary arteriolar tone .

9.  IHD is the leading cause of death and disability
worldword. It kills over 6.5 million people worldwide
each year[7].
 From 1990 to 2020 the rise in mortality due to ischemic
heart disease (IHD) in developing countries (137% in
men and 120% in women) is predicted to be much
higher than that in the developed countries (48% in
men and 29% in women ) [8].
 Existing treatment for Myocardial
Ischemia…….Nitrates, Beta blockers , Calcium
channel blockers etc[9].

10.  But these synthetic drugs have ADRs like
Headache,constipation ,weakness,dry mouth etc and
are expensive. Development of more influential anti-
ischemic drugs with lesser ADRs is necessary. SO,the
use of herbal medicine has been steadily
increasing[10].
 Natural products are easily biodegradable, possess
least environmental hazards, represent minimum side
effects and are available at affordable prices [11].
 In different studies, Panax notoginseng, Tinospora
cordifolia , Zingiber officinale exhibited to have anti-
ischemic activity [12,13,14].

11. The aim of this study will be:
 To evaluate the anti-ischemic activity of plant
extract.
 To explore new novel medicinal plants which
may be of commercial value.
 To develop a herbal medicine and evaluate it
scientifically.

12. Selection of
experimental
animals
Selection of plants
Shade drying
Preparation of
crude extract
Fractionation of
crude extract
Preliminary
phytochemical
analysis
Acute toxicity test
Experimental
procedures on
animals

13. (a) Instruments:
Following intruments will be used during experimentation .
Spectrophotometer
Electrocardiogram
Techno positive pressure Respirator
Teflon coated stainless steel electrode

14. (b) Standard drugs & Chemicals:
The following drugs will be used in this investigation
 Ramipril
 Sublingual Nitrates
 Sodium pentobarbital
 Solvents include saline solution,Ethanol ,Kreb solution
 2,3,5 triphenyltetrazolium chloride (TTC) stain
All chemicals will be purchased from Merck (Germany) ,Sigma
Chemical Co (St. Louis, MO, USA).

15. (c) Animals:
Following animals will be used for various experiments in-vivo and in-
vitro .
 Sprague-Dawley rats
 Dogs
 Rabbit
All animals will be treated according to the standard procedures guided
by national research council [15].

16. (d) Plant material:
All plants for this study will be selected according to their
Folkloric claim to cure the conditions of Ischemia.
 Terminalia arjuna
 Carthamus tinctorius (Safflower)

18.  Preparation of crude extract:
 Dried flower petals will be extracted 3 times in 50% ethanol.
 The extracts will be combined,filtered and evaporated in
evaporator to obtain condensed solution.
 Macroporous resin chromatography will be used to purify the
condensed extract solution .
 Finally the concentrated solution will be stored at cool place
in capped bottles.
 Extracts will be dissolved in normal saline before
administration [16].

20.  Fractionation of the crude extract:
 Activity directed fractionation of the crude extract will be
carried out by using different organic solvents in order to separate
or concentrate the activities in any one of the corresponding fraction
[17].
 Preliminary Phytochemical analysis:
 The fraction with highest activity will be selected & analyzed for
the presence of different phytochemical groups such as,
flavonoids, flavones, terpenoids, tannins, alkaloids by
using phytochemical methods [18].

21.  Acute toxicity test:
 Acute toxicity test will be performed on rat as
per OECD guidelines 2002 . [19].
 The test will be done for observation of
mortality, behavioral changes and toxic
effects

22. Animals models
of Ischemia
In-vivo models
In-vitro models

23.  Left coronary artery ligation method
 Open chest method
 Closed chest method
 Exercise induced Myocardial Ischemia
 Wire induced Myocardial Ischemia
 Sympathetic overactivity induced myocardial
ischemia
 Isoproterinol Induced myocardial ischemia
 Cocaine induced myocardial ischemia

24.  Myocardial ischemia will be induced by subcutaneous injection of isoproterenol
hydrochloride (ISO, 85 mg kg−1 bw), dissolved in physiological saline solution,
for 2 consecutive days.
 The rats will be randomly divided into 6 groups with 8 rats each.
 Group 1 will serve as control (received 5% DMSO sc, for 7 days).
 Group 2 rats will be administered isoproterenol (ISO, 85 mg kg−1 bw, sc,
twice at an interval of 24 hours) on 8th and 9th days.
 Group 3 rats will be administered with plant extract(60 mg kg−1 bw, sc) for 7
days.
 Groups 4, 5, and 6 rats will receive 20, 40, and 60 mg kg−1 b.w., of plant
extract subcutaneously, for 7 days and will receive ISO (85 mg kg−1 bw, sc,
twice at an interval of 24 hours) on the 8th and 9th days, respectively.
 Plasma cardiac Troponin T and I (cTnT and cTnI) will be quantitatively
measured by means of a highly specific enzyme immunoassay using
commercially available kits .

25. In this experiment ,we will induce sympathetic
hyperactivity by intracerebroventricular
injection of excitatory amino acid L-
Glutamate(10u mol)( in pentobarbital
anaesthetized albino rabbits of either sex (2-3
kg),associated with inhibition of nitric oxide
synthesis with L-NAME(40mg/kg, iv).
Glutamate will trigger ventricular arrythmia
and persistent ST segment shifts in the
ECG,it will indicate myocardial ischemia.

26. 
Divide in 4
Groups
Control
group(1)=L-
NAME(40mg/kg
) adm.then
after 1 hour L-
Glutamate(10u
mol) adm.ECG
changes will
be recorded
Pretreated
group(2)=cloni
dine(10 ug/kg
iv.)Rilmenidine
(300ug/kg
iv.)adm 10
mint before L-
NAME.ECG
changes will
be observed
Post treated
group(3)=Cloni
dine,Rilmenidi
ne adm .10
min. before L-
Glutamate.EC
G chenges will
be noted.
4th group =
adm only L-
Glutamate
and will
investigate
effects of
sympathetic
hyperactivity
in absence of
L-NAME.

27. The IV adm. Of clonidine(10ug/kg) and Rilmenidine(300ug/kg) will decrease the
incidence of myocardial ischemia.

28. Open chest method:
This procedure will be performed in larger animals like
dog and pig etc ,in which major branch of coronary artery
will be ligated.
By the use of anaesthesia ,chest will be opened for a
long time,and by coronary artery ligation ischemia will be
induced and we will map the ischemic areas by means of
multi positioning of a surface electrode.Ischemic damage
will be measured by ST segment elevation
Some metabolic changes in ischemic myocardium will be
monitored by taking blood samples from the vein.

30. Closed chest method:
Myocardial ischemia in closed chest method will be brought about by 3 ways:
1. The production of thrombus will be done either by passing a current via an
intracoronary catheter or simply by leaving a helical occlusion of the lumen by a balloon
catheter.
2. Copper wire will be inserted in the lumen of the coronary artery.
3. Embolization will be done by radioopaque micro-spheres of different sizes or by
intracoronary injection of mercury.

32.  Cell culture model of seal induced Ischemia
 Ischemia(blockage of circulation ;total or low
flow).
 Ischemia(global or regional)

33. Neontal rat heart cells will be cultured in a sealed
flask for 24 to 72 hours(cells will be exposed to
hypoxia).PO2 and pH of medium will be decresed
during the procedure
This will trigger severe cell injury including
Morphological degeneration,ATP depletion,beating
impairment.Apoptosis will also be occured in some
cardiomyocytes after onset of ischemia.
This will be evident by using Hoechst 33258 .After
72 hr,DNA fragmentation will also be observed in
some of the myocytes.

34. This study will :
 be valuable for screening of various
plant extracts having Anti-ischemic
effect.
 give scientific evidence about its
tradtional use.

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