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Research Article
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Chronic Administration of Tribulus terrestris Linn. Extract Improves Cardiac Function and Attenuates Myocardial Infarction in Rats |
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Shreesh K. Ojha,
Mukesh Nandave,
Sachin Arora,
Rajeev Narang,
Amit K. Dinda
and
Dharamvir Singh Arya
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ABSTRACT
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The present study was undertaken to evaluate the cardioprotective
potential of hydro-alcoholic extract of Tribulus terrestris Linn.
(Family; Zygophyllaceae), a traditional medicine used in Indian and Chinese
systems of medicine. Wistar male albino rats weighing 150-200 g were randomly
divided into three main experimental groups; sham (saline treated only),
isoproterenol (ISP) control (saline and ISP) and Tribulus terrestris
treatment groups (T. terrestris and ISP). Saline or T. terrestris
extract 250 mg kg-1 once daily were orally administered for
30 days. Isoproterenol was administered in rats to induce myocardial infarction.
On days 29 and 30, the animals of ISP control and T. terrestris
treatment group were administered ISP (85 mg kg-1, subcutaneously)
at an interval of 24 h. On the day 31, 48 h after first dose of ISP, hemodynamic
parameters were recorded. After sacrificing the animals the hearts were
excised and subjected to biochemical, histopathological and ultrastructural
studies. ISP-administration produced a significant decrease in the activities
of endogenous antioxidant defence enzymes viz. superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GSHPx) and tissue antioxidant,
reduced glutathione (GSH) along with a concomitant increase in the lipid
peroxidation product malonaldehyde (MDA). In addition, a significant decrease
in the activities of myocardial injury markers i.e., creatine phosphokinase-MB
(CK-MB isoenzyme) and lactate dehydrogenase (LDH) was also observed in
the heart of ISP control group as compared to sham control. Cardiac dysfunction
was observed as a decrease in mean arterial pressure (MAP), heart rate
(HR), left ventricular rate of peak positive and negative pressure change
{(+) and (-) LV dP/dt} and elevated left ventricular end diastolic pressure
(LVEDP) following ISP administration. These functional alterations were
supported by severe modifications in histopathological and ultrastructural
assessment. Pretreatment with T. terrestris resulted in the increased
activities of SOD, CAT, GSHPx and prevention of depletion of tissue glutathione
along with inhibition of lipid peroxidation. In addition treatment with
T. terrestris decreased the leakage of CK-MB and LDH enzymes from
myocardium, there was a significant improvement in cardiac function as
evidenced by correction of MAP, HR, LVEDP and contractility and relaxation.
The possible underlying mechanism of the cardioprotective effect of T.
terrestris could be due to restoration of endogenous myocardial antioxidant
status or free radical scavenging activity along with correction of the
altered hemodynamic parameters and preservation of histoarchitectural
and ultrastructural alterations.
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How
to cite this article:
Shreesh K. Ojha, Mukesh Nandave, Sachin Arora, Rajeev Narang, Amit K. Dinda and Dharamvir Singh Arya, 2008. Chronic Administration of Tribulus terrestris Linn. Extract Improves Cardiac Function and Attenuates Myocardial Infarction in Rats. International Journal of Pharmacology, 4: 1-10. DOI: 10.3923/ijp.2008.1.10 URL: https://scialert.net/abstract/?doi=ijp.2008.1.10
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INTRODUCTION
Among cardiovascular diseases (CVDs), myocardial infarction (MI) accounts
for majority of death and disability and has high incidence rate in both
developed and developing countries as seen over the past quarter century
(Agrawal et al., 2006). Reduction of death incidences and prevention
of myocardial infarction are of utmost importance. In spite of development
in modern drugs, there is a growing interest in the use of herb based
drugs from alternative medicine for long-term prevention of myocardial
ischemia in high risk patients along with a realization that the use of
herbal products can influence the course of heart diseases and may provides
anintegrated approach of nutritional substances, which help in restoring
and maintaining, the balanced body systems (Hertog et al., 1993;
Dhar et al., 1968).
The plant Tribulus terrestris Linn, a deciduous tree of the Zygophyllaceae
family, is an important herb from Indian and Chinese traditional medicine
literature for the treatment of various diseases especially ischemic heart
diseases (Warrier, 1994). Modern pharmacological studies have reported
its hypoglycemic (Amin et al., 2006), nephroprotective (Kavitha
and Jagdeesan, 2006), diuretic (Anand et al., 1994), aphrodisiac
(Gauthaman et al., 2002) and immunomodulator (Bhattacharya et
al., 2000) activities. T. terrestris extract standardized to
contain saponins have been reported to dilate coronary arteries, increase
coronary blood flow and scavenge free radicals in patients with ischemic
heart diseases (Sharifi et al., 2003; Guo et al., 2005)
and indicated for angina pectoris, myocardial infarction, congestive heart
failure and stroke (Phillips et al., 2006; Guo et al., 2005).
In addition to the antianginal activity, their antihypertensive effect
has been demonstrated owing to its angiotensin converting enzyme (ACE)
inhibitor property (Sharifi et al., 2003). Experimental studies
also showed that ACE inhibitors administered chronically before acute
MI might limit myocardial infarct size, improve cardiac function and prevent
cardiac hypertrophy (Stauss et al., 1994). The extract of the plant
contains a large amount of flavonoids, saponins, alkaloids and tannins,
which possess significant antioxidant (Wang et al., 1997) and cardiac
(Achenbach et al., 1996) activity. In addition to antioxidant constituents,
it has been reported to consist of adaptogenic components which provide
resistance against stress (Gauthaman et al., 2002). Several herbal
antioxidants and adaptogens rich herbs have been shown to possess cardioprotective
activity. However, so far very few scientific studies has been undertaken
to evaluate the cardioprotective potential of T. terrestris. In
addition, the exact mechanism of its cardioprotective effects with respect
to the present knowledge of the pathophysiology of myocardial infarction
has not been investigated. In view of the above, the present study was
designed to evaluate the cardioprotective potential of lyophilized extract
T. terrestris in isoproterenol (ISP)-induced myocardial infarction
in rats.
We aimed to investigate the effects of chronic administration of the
hydro-alcoholic extract of T. terrestris on: (i) hemodynamics e.g.,
heart rate (HR), systolic, diastolic and mean arterial blood pressure
(SAP, DAP and MAP, respectively) (ii) left ventricular function e.g.,
left ventricular peak positive pressure change (rate of pressure development),
left ventricular rate of peak negative pressure change (rate of pressure
decline) {(+) and (-) LV dP/dt} indicating the inotropic and lusitropic
state of heart respectively and left ventricular end-diastolic pressure
(LVEDP), a surrogate marker of preload (iii) endogenous antioxidant status
e.g., reduced glutathione (GSH) and antioxidant enzymes; superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GSHPx) (iv) lipid peroxidation
marker e.g., malonaldialdehyde (MDA) (v) myocyte injury marker enzymes
e.g., creatine phosphokinase-MB isoenzyme (CK-MB) and lactate dehydrogenase
(LDH) (vi) histoarchitectural changes and (vii) ultramicroscopic changes
in rats subjected to acute ischemic injury of heart following ISP-administration.
MATERIALS AND METHODS
Plant extract: Standardized hydro-alcoholic lyophilized extract
of whole plant of Tribulus terrestris was procured from Sanat Products
Ltd., New Delhi, India. The identity of Tribulus terrestris was
authenticated on the basis of routine pharmacognistical studies including
organoleptic tests and macroscopic and microscopic observations. The voucher
specimen of lyophilized extract of T. terrestris (No. TTS 104)
has been deposited in Cardiovascular Laboratory, Department of Pharmacology,
All India Institute of Medical Sciences, New Delhi, India for further
reference. Phytochemical analysis of the extract was performed for saponin
content determination. The total saponin content was 43.77% w/w and the
extractive values of 1 g sample of Tribulus terrestris in water
and 50% v/v methanol were 81.36 and 90.31%, respectively. The dose of
T. terrestris (250 mg kg-1) in the present study was
selected on the basis of a pilot study assessing the antioxidant activity
of T. terrestris in rat hearts (Ojha et al., 2006). The
doses screened were 75, 150, 250 and 350 mg kg-1 day-1
and 250 mg kg-1 of T. terrestris exhibited maximum antioxidant
and anti-peroxidative activity in hearts. Therefore, this dose was selected
for further evaluation in the ISP-induced myocardial infarction incorporating
biochemical, hemodynamic, histological and ultra-structural assessments.
Chemicals: All chemicals used in the study were of analytical
grade and obtained from standard drug houses. Isoproterenol hydrochloride
was obtained from Sigma Chemicals Co. (St. Louis, MO), USA.
Animals: Laboratory bred Wistar male albino rats weighing 150-200
g were obtained from the Central Animal House Facility of All India Institute
of Medical Sciences, New Delhi, India. The study protocol was reviewed
and approved by the Institutional Animal Ethics Committee (227/IAEC/03)
and conducted in accordance with the Indian National Science Academy Guidelines
for the use and care of experimental animals in research. The animals
were acclimatized with the atmosphere of departmental animal house and
housed under standard laboratory conditions of temperature at 25±2°C,
relative humidity of 50±10% and light: dark cycle of 12 h photoperiod.
All experiments were performed between 9.00 and 16.00 h. They were group
housed in polypropylene cages (38x23x10 cm) with not more than four animals
per cage and had free access to food pellets and tap water.
Study design: A total of thirty-nine rats were used in the study
and equally divided into three main experimental groups as follows:
Control group: Rats of this group received oral saline once daily
for 30 days and on days 29 and 30 administered 0.5 mL saline s.c. at an
interval of 24 h.
ISP group: Rats of this group received oral saline once daily
for 30 days and on days 29 and 30 administered ISP (85 mg kg-1,
s.c.) at an interval of 24 h.
T. terrestris plus ISP group: Rats of this group received
T. terrestris extract 250 mg kg-1 day-1 for
30 days and on days 29 and 30 administered ISP (85 mg kg-1,
s.c.) at an interval of 24 h.
On day 31, 48 h after injection of ISP administration or saline, hemodynamic
parameters were recorded.
Induction of myocardial infarction and hemodynamic assessment: Animals
of all the experimental groups were anesthetized intraperitoneally with
pentobarbitone sodium (60 mg kg-1). Atropine (4 mg kg-1)
was administered along with the anesthetic agent to maintain the heart
rate especially during the surgery and to reduce tracheo-bronchial secretions.
The body temperature was monitored and maintained at 37°C during the
surgical period. The neck was opened with a ventral midline incision to
perform tracheostomy and rats were ventilated with room air from a positive
pressure ventilator (Inco, India) using compressed air at a rate of 90
strokes/min and a tidal volume of 10 mL kg-1. Ventilator setting
and PO2 were adjusted as needed to maintain the arterial blood
gas parameters within the physiological range. The left jugular vein was
cannulated with polyethylene tube for continuous infusion of 0.9% saline
solution. The right carotid artery was cannulated and the cannula was
filled with heparinized saline. The cannula was connected with Cardiosys
CO-101 (Experimentria, Hungary) using a pressure transducer and the signal
was amplified by means of an amplifier for the measurement of SAP, DAP,
MAP and HR. The left thoracotomy was preformed at the fifth intercostal
space on left side and the heart was exposed. A sterile metal cannula
(1.5 mm bore) was introduced into the cavity of the left ventricle from
the posterior apical region of the heart for measuring left ventricular
dynamics such as (+) LV dP/dt, (-) LV dP/dt and LVEDP. The canula was
connected to a pressure transducer (Gould Statham P23ID, USA) through
a pressure-recording catheter on the Polygraph (Grass 7D, USA). After
the stabilization time of 10 min the tracings has been recorded polygraph
paper following baseline measurements at different sensitivity and speed.
After recording hemodynamic parameters, the animals were sacrificed under
an overdose of anesthesia (pentobarbitone, 100 mg kg-1; i.v.)
and the heart was excised. The excised hearts were rinsed with ice cold
saline and snap frozen in liquid nitrogen for biochemical analysis. However,
the hearts subjected for histopathological studies were fixed in 10% buffered
formalin and for ultrastructural studies in Karnowsky reagent.
Biochemical studies: Hearts stored in liquid nitrogen were brought
to room temperature and weighed. A 10% homogenate of whole heart was prepared
in 50 mM phosphate buffer (pH 7.4) and an aliquot of 0.5 mL was used for
the assay of MDA (Ohkawa et al., 1979) and reduced GSH (Moron et
al., 1979). Remaining the homogenate was centrifuged at 7000 rpm for
15 min and the supernatant was used for estimation of the following biochemical
parameters: SOD (Misra and Fridovich, 1976), CAT (Aebi, 1974), GSHPx (Paglia
and Valentine, 1967) and protein (Lowry et al., 1951). Myocardial
injury markers, CK-MB isoenzyme (Lamprecht et al., 1974) and LDH
(Cabaud and Wroblewski, 1958) were estimated spectrophotometrically.
Histopathological studies (Light microscopy): For histopathological
studies, the hearts fixed in 10% buffered neutral formalin solution were
cut in to four segments from apex to bottom to visualize myocardial lesions
at different levels. The segments of the tissue fixed tissues were embedded
in paraffin wax and serial semi thin sections of 4 μm thickness were
cut. After hematoxylin and eosin staining, for histoarchitectural evaluation,
these sections were examined under light microscope (Nikon, Tokyo, Japan)
and photomicrographs were taken. Representative area images were captured
in an image analysis system. The Image Analyzer consisted of BX-50 Research
Microscope (Olympus, Japan), Coolsnap 10 bit Digital Camera (Media Cyberneticus,
USA) with an image analysis software Image Plus Pro (Media Cyberneticus,
USA). The slides were evaluated for myonecrosis, inflammatory cell infiltration
and edema.
Ultrastructural studies (Transmission electron microscopy): For
ultrastructural study, at the end of experiment small pieces of myocardial
tissue (approximately 1-2 mm in thickness) were immediately fixed in ice-cold
Karnovsky`s fixative. The tissues were then washed in phosphate buffer
(0.1 M, pH 7.4) and post fixed for 2 h in 1% osmium tetroxide in the same
buffer at 4°C. The specimens were then washed in phosphate buffer,
dehydrated with graded acetone and then embedded in araldite CY 212 to
make tissue blocks. The semi thin as well as ultra thin sections (80-100
nm) were cut by an ultramicrotome (Ultracut E, Reichert, Austria). The
sections were stained with uranyl acetate and lead acetate and examined
under transmission electron microscope (Morgagni 268D, Fei Co., The Netherlands)
operated at 60 KV.
Statistical analysis: The statistical analysis was carried out
by SPPSS statistical package version 10. In tables, figures and text the
data are expressed as mean±SD. Differences between groups were
examined for statistical significance using one-way analysis of variance
(ANOVA) followed by Bonferroni Multiple Range post-hoc analysis. p<0.05
was considered as statistical significance level.
RESULTS
There was no significant difference in body weight of the treated rats,
when compared with sham control, either at the beginning or at end of
the study period.
ISP induced myocardial necrosis produced a significant reduction in activities
of SOD, CAT and GSHPx (Table 1) as compared to sham
control. Treatment with T. terrestris significantly prevented fall
in enzyme activities and increased the activities of SOD, CAT and GSHPx
as compared to ISP control.
A significant decrease in myocardial GSH level and increase in lipid
peroxidation product MDA, was observed in the ISP control group in comparison
to sham control (Table 2). However, T. terrestris
extract significantly prevented lipid peroxidation and decline of GSH
level and as compared to ISP control.
We observed a significant decrease in the myocardial enzyme activities,
CK-MB isoenzyme and LDH (Table 3) in the rat myocardium
of ISP control group as compared to sham control. Treatment with T.
terrestris extract significantly prevented loss of CK-MB isoenzyme
and LDH enzymes and restored both in the myocardium.
A significant fall in SAP, DAP and MAP was observed in the ISP control
group as compared to sham control (Fig. 1). T. terrestris
treatment significantly
increased MAP with a slight but insignificant improvement in SAP and DAP
in comparison to sham control. Subsequent to ISP administration, a significant
fall in HR was observed as compared to sham control (Fig.
2). T. terrestris treatment significantly improved HR in comparison
with ISP control.
Table 1: |
Changes in activities of antioxidant enzymes in heart
homogenates |
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*: p<0.05 vs sham control; #: p<0.05
vs ISP control. Each value represents mean±SD of six readings
(n = 6) |
Table 2: |
Changes in lipid peroxidation and GSH level in heart
homogenates |
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*: p<0.05 vs sham control; #: p<0.05
vs ISP control. Each value represents mean±SD of six readings
(n = 6) |
Table 3: |
Changes in the activities of CK-MB and LDH in heart
homogenates |
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*: p<0.05 vs sham control; #: p<0.05
vs ISP control. Each value represents mean±SD of six readings
(n = 6) |
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Fig. 1: |
Changes in SAP, DAP and MAP in different experimental
groups, *: p<0.05 vs sham control; #: p<0.05 vs ISP
control. Each value represents mean±SD of six readings (n =
6) |
ISP administration resulted in left ventricular dysfunction, as indicated
by a significant fall in (+) and (-) LVdP/dt (Fig. 3)
and increase in LVEDP as compared to sham control (Fig.
4). Myocardial relaxation (lusitropic state) denoted by (-) LVdP/dt
was significantly restored by T. terrestris treatment as compared
with ISP control (Fig. 3). Additionally, T. terrestris
treatment markedly reduced LVEDP as compared to ISP control (Fig.
4).
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Fig. 2: |
Changes in heart rate in different experimental groups,
*: p<0.05 vs sham control; #: p<0.05 vs ISP control.
Each value represents mean±SD of six readings (n = 6) |
 |
Fig. 3: |
Changes in (+) and (-) LV dP/dt in different experimental
groups, *: p<0.05 vs sham control; #: p<0.05 vs ISP
control. Each value represents mean±SD of six readings (n =
6) |
 |
Fig. 4: |
Changes in left ventricular end-diastolic pressure in
experimental groups, *: p<0.05 vs sham control; #: p<0.05
vs ISP control. Each value represents mean±SD of six readings
(n = 6) |
On histopathological examination depicted in Fig. 5A-C,
we observed normal architecture of the heart in sham control (Fig.
5A). However, significant myocyte membrane damage with extensive myonecrosis,
fibroblastic proliferation and infiltration of inflammatory cells was
observed in ISP control hearts (Fig. 5B) in comparison
with sham control. T. terrestris treatment extract prevented myonecrosis
(Fig. 5C) as demonstrated by significant reduction in
the infiltration of inflammatory cells, vacuolar changes as well as edema
as compared to the ISP control group.
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Fig. 5: |
Photomicrograph of heart tissue (H and E X 100) (A)
Sham group showing normal architecture. Endocardium and pericardium
are seen within normal limits without infiltration of inflammatory
cells (B) ISP group showing focal myonecrosis with myophagocytosis
and lymphocytic infiltration. Vacuolar changes and prominent edema
along with chronic inflammatory cells were seen in subendocardium
(C) Tribulus terrestris treated group administered showing
lesser infiltration of inflammatory cells and myonecrosis |
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Fig. 6: |
Electron micrograph of heart tissue (A) Normal ultra
structure of heart tissue of sham group (1250X) (B) Extensive muscle
necrosis with significant disruption of myofibrils, intracytoplasmic
vacuoles and lipid droplets with irregular mitochondria in ISP control
group (4400X) (C) Lesser muscle necrosis with slight disruption
of myofibrils and mitochondria in Tribulus terrestris treated
group (2800X) |
Electron microscopic examination of the myocardium revealed normal appearance
of cell organelles in sham control rats showing sarcomere and abundant
mitochondria (Fig. 6A). In ISP control group, the myocardial
damage was marked by significant disruption of myofibrils (Fig.
6B). In addition, the myocardial tissue of ISP control group revealed
several intracytoplasmic vacuoles and lipid droplets. Other ultrastructural
changes include the appearance of small and irregular mitochondria with
loss of cristae (Fig. 6B). However, treatment of animals
with T. terrestris showed structural protection of the myocardium
with respect to ISP control group (Fig. 6C).
DISCUSSION
Tribulus terrestris has significant cardioprotective activity
as shown by its mitigating effects on ISP-induced hemodynamic, biochemical,
histopathological and ultrastructural perturbations. In the present study,
ISP, a β-adrenergic agonist was used to produce myocardial necrosis
and interstitial fibrosis at its supra-maximal dosages in a multi-step
manner. In addition, isoproterenol causes substrate loss, depletion of
energy reserves, hypoxia due to myocardial hyperactivity, increased inotropic
activity, coronary hypotension, a high-oxygen demand and production of
free radicals resulting from oxidative metabolism of catecholamine (Tappia
et al., 2001). Free radical mediated peroxidation of membrane phospholipids
and consequent changes in membrane permeability are the primary reasons
for cardiotoxicity induced by ISP (Rathore et al., 1998; Blasig
et al., 1984). ISP-induced oxidative stress also depresses the
sarcolemmal Ca2+ transport and results in the development of
intracellular Ca2+ overload and ventricular dysfunction (Tappia
et al., 2001). The oxidative stress may be exerted through quinine
metabolites of ISP which react with oxygen to produce superoxide anions
and others reactive oxygen species and interfere with SOD, glutathione
reductase and ATP pumps (Rathore et al., 1998; Remiao et al.,
2000). Experimental and clinical studies have demonstrated that therapeutic
interventions having antioxidant or free radical scavenging activity may
exert cardioprotective effects against oxidative stress associated with
various cardiovascular diseases, including ischemic heart disease (Bandyopadhyay
et al., 2004). Hence, medicinal herbs or natural products possessing
antioxidant constituents has recently gained a great deal of scientific
interest (Gupta et al., 2004; Mohanty et al., 2004; Karthikeyan
et al., 2003) as it is expected to offer better protection against
oxidative stress associated diseases through cellular adaptive mechanism
and may provide a lead for promotion of therapeutic agent especially of
natural origin.
In the present study, chronic administration of T. terrestris
extract maintained the antioxidant activity of the endogenous antioxidants
as evidenced by increase in the levels of reduced GSH and activities of
endogenous antioxidant enzymes such as SOD, CAT and GSHPx along with decrease
in the level of lipid peroxidation product, MDA. The depletion in GSH
levels along with increased plasma lipid peroxides (measured as TBARS
or thiobarbituric acid-reactive substances) has been observed during myocardial
ischemia and associated with the consumption of endogenous antioxidant
and enzymes such as SOD, CAT and GSHPx. These antioxidant enzymes are
known to remove the ISP-induced generation of toxic oxygen species by
their peroxidative and free radical scavenging activity. In addition,
an increase in MDA levels was observed in the heart tissue following ISP
administration. The myocardial necrosis observed in the animals receiving
ISP can also be attributed to per-oxidative damage as it has been previously
reported that ISP generates lipid peroxide (Blasig et al., 1984).
The fall in activity of GSHPx in the ISP group might be co-related with
decreased availability of its substrate, GSH. Moreover, due to impairment
in both enzymatic and non-enzymatic antioxidant defense mechanisms, it
is possible that the free radicals may not be effectively neutralized,
thereby rendering the myocardium susceptible to lipid peroxidation. The
increased GSH levels observed in the T. terrestris groups in the
present study may be due to its enhanced synthesis, as antioxidant compounds
have been shown to increase glutathione reductase activity, which, maintains
GSH in its reduced state (Mohanty et al., 2004). The observations
of our study are in conformity with earlier reports that have demonstrated
the modulation of synthesis of cellular antioxidants by treatment with
natural products (Gupta et al., 2004; Mohanty et al.,
2004; Karthikeyan et al., 2003).
In addition to the antioxidant parameters, the estimation of CK-MB isoenzyme
and LDH levels, both of which are useful parameters for assessing cardiomyocyte
damage (Jennings et al., 1990). These enzymes normally exist in
cellular compartment and leak out into the plasma during myocardial injury
due to disintegration of contractile elements and sarcoplasmic reticulum.
ISP challenge in the present study produced significant depletion of myocardial
CK-MB isoenzyme and LDH. T. terrestris treatment prevented the
leakage of these enzymes and restored their activities as compared to
the ISP control. The observation that T. terrestris treatment significantly
restored CK-MB isoenzyme and LDH levels is suggestive of its cardioprotective
effect.
As described earlier, hemodynamic parameters were also incorporated into
the experimental design for a better understanding of the co-relation
between biochemical and functional changes in the myocardium subjected
to ISP-induced damage. Previous studies have shown that ISP mediated oxidative
stress could progresses to myocardial necrosis which leads to cardiac
dysfunction characterized by increased end-diastolic volume and pressure
and left ventricular wall thickness (Grimm et al., 1998; Teerlink
et al., 1994) and these changes are significantly prevented by
antioxidants. In the present study, the favorable modulation of hemodynamic
and left ventricular function by T. terrestris treatment also supports
the previous observations it may modify the course of action and help
in retaining the function of heart to normal level.
In the ISP control group, myocardial dysfunction was clearly evident
by a significant fall in SAP, DAP and MAP, HR, LV (+) and (-) dP/dt along
with a rise in LVEDP, which might be ascribed to its ISP-induced myocardial
infarction. The left ventricular rate of negative pressure change was
more markedly depressed indicating a more diastolic dysfunction per se
which may result in the persistence of elevated LVEDP. It is speculated
that deteriorating myocardial contractile status following ISP-induced
necrosis might be responsible for the significant fall in MAP. Normally
a fall in MAP reflexly increases HR and myocardial contractility. However,
none of these effects were observed in the present study, suggesting impairment
in the conducting system of the heart following ISP-induced myocardial
necrosis. T. terrestris treatment in our study significantly increases
MAP and HR as compared to the isoproterenol control group. Moreover, T.
terrestris appeared to improve left ventricular function as evidenced
by significant preservation of both inotropic and lusitropic functions
of the heart along with correction of elevated LVEDP. Preservation of
cardiac reflexes resulting in increased heart rate and ventricular function
to maintain cardiac output may be on account of myocardial salvage, produced
by T. terrestris.
The histopathological examination of myocardium in ISP control animals
showed presence of focal myonecrosis with myophagocytosis and lymphocytic
infiltration in sub-endocardial region indicative of infarct like lesions
similar to previous studies (Grimm et al., 1998; Teerlink et
al., 1994). Treatment with T. terrestris extract preserved
the normal histoarchitecture and ultrastructure of the myocardium as evidenced
by reduced myonecrosis and lesser infiltration of inflammatory cells.
Taken together, the biochemical and histopathological results of the present
study demonstrate the cardioprotective effects of T. terrestris.
Additionally, ultrastructural perturbations in ISP administered rat hearts
further confirmed the injured state of myocardium. Restoration of myocardial
CK-MB activity, along with preservation of myofibrils and mitochondrial
morphology is indicative of cytoprotective activity of T. terrestris.
Recently, adaptogens are demonstrated to confer resistance against a
variety of stresses and aid in myocardial adaptation. Various experimental
studies have demonstrated the adaptogenecity i.e., increased non-specific
resistance to stress of medicinal herbs is considered as a function of
antioxidant components which may produce myocardial adaptation (Bhattacharya
and Muruganandam, 2003; Lei and Chiou, 1986). The major active constituents
of T. terrestris are flavonoids and steroidal saponins, lignanamides,
protodioscin and tribulosins (Wang et al., 1997; Achenbach et
al., 1996). It has been reported that protodioscin and tribulosins
are the adaptogenic components present in T. terrestris (Gauthaman
et al., 2002). The myocardial adaptation against ischemic stress
may be mediated through augmentation of cellular tissue antioxidant enzymes
such as SOD, CAT and GSHPx and stress proteins. Although, the exact mechanism
of such myocardial adaptation is not known. Further, flavonoids have been
shown to exhibit wide spectrum of biological activities including free
radical scavenging, antioxidant and anti-ischemic activities (Hertog et
al., 1993). Therefore, it could be possible that T. terrestris
may have exerted its cardioprotective effects partly due to its antioxidant
as well as adaptogenic constituents.
In summary, the present study demonstrated that multiple mechanisms may
be responsible for the cardioprotective effect of Tribulus terrestris.
Improved hemodynamics and left ventricular function along with improved
endogenous myocardial antioxidant status and myocardial adaptability assure
its cardioprotective potential in ISP-induced myocardial necrosis. The
histopathological and ultrastructural examination, further confirmed its
cardioprotective effects through preservation of cell organelle and myocyte
membrane integrity. In conclusions, the observations of present study
provide a scientific basis for cardioprotective effect of Tribulus
terrestris during myocardial ischemia and demonstrate its therapeutic
potential in the treatment of ischemic heart disease.
ACKNOWLEDGMENTS
The financial aid from UGC, New Delhi is gratefully acknowledged. The
authors also like to express their thanks to Mr. B.M. Sharma, Department
of Pharmacology, AllMS, New Delhi, India for his valuable technical assistance
in conducting the study.
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