Indian Journal of Experimental Biology

Vol. 47, January 2009, pp. 41-46

Cardioprotective effect of lipistat against doxorubicin induced myocardial

toxicity in albino rats
B C Koti*, A H M Vishwanathswamy, Jyoti Wagawade & A H M Thippeswamy
Department of Pharmacology, K L E Society’s College of Pharmacy, Hubli 580 031, India

Received 25 March 2008; revised 18 November 2008

Preventive role of lipistat against doxorubicin induced myocardial toxicity in rats has been reported.. Cardiotoxicity was
produced by doxorubicin administration (15 mg/kg for 2 weeks). Lipistat (350 mg/kg, orally) was administered as
pretreatment for 2 weeks and then for 2 weeks alternated with doxorubicin. The general observations, mortality,
histopathology, biomarker enzymes like lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), serum lipid
profiles like total cholesterol, triglycerides, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), antioxidant
enzymes such as glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were monitored after 3 weeks of last
dose. Pretreatment with the lipistat significantly protected myocardium from the toxic effects of doxorubicin by reducing the
elevated level of biomarker enzymes like LDH and CPK to the normal and serum lipids such as total cholesterol,
triglyceride and LDL back to normal. Lipistat increases the decreased level of GSH, SOD and CAT and decreases the
increased level of malondialdehyde in cardiac tissue. The repeated administration of doxorubicin causes cardiomyopathy
associated with an antioxidant deficit and increased level of lipid profiles by interfering with fatty acid metabolism. The
results support the lipid lowering and antioxidant properties of lipistat, which indicate the cardioprotective property against
doxorubicin induced cardiotoxicity.

Keywords: Antioxidant, Cardiotoxicity, Doxorubicin, Lipistat

Doxorubicin an anthracycline is well-established and
highly efficacious drug in the fight against many
kinds of cancers like solid tumors, leukemia, soft
tissue sarcoma, breast cancer, small cell carcinoma of
the lung and oesophageal carcinomas1,2. But, its
clinical usefulness is still restricted due to its specific
toxicities to cardiac tissue3. Congestive heart failure,
cardiomyopathy and electrocardiographic changes
were demonstrated after cumulative doxorubicin
administration4. The mechanisms proposed for
cardiotoxic effects of doxorubicin include free radical
induced myocardial injury, lipid peroxidation5,
mitochondria damage6, decreased activity of Na+
K+ATPase7, vasoactive amine release8, impairment in
myocardial adrenergic signaling/regulation, increase
in serum total cholesterol, triglyceride and low
density lipoproteins9. Generation of reactive oxygen
species like superoxide anion and hydrogen peroxide
by doxorubicin leads to causing impairment of cell
functioning and cytolysis10. Due to the presence of
__________
*Correspondent author
Telephone: +91-836-2373174
Fax: +91-836-2371694
E-mail: bc_koti@yahoo.com
less developed antioxidant defense mechanisms, heart
is particularly vulnerable to injury by anthracycline-
induced reactive oxygen species. Liberation of free
radicals is central to the mechanism of doxorubicin-
induced damage to the myocardium11. It also causes
the elevation of serum enzymes like lactate
dehydrogenase (LDH) and creatine phosphokinase
(CPK)12. Endogenous antioxidant deficits have been
suggested to play a major role in doxorubicin induced
cardiomyopathy and heart failure13.
Polyherbal formulations/antioxidant compounds
have shown protective effects in doxorubicin induced
cardiotoxicity without reducing their therapeutic
efficacy. Moreover, there is a growing interest in the
usage of natural antioxidants as a protective strategy
against the cardiovascular related problems in
experiments such as ischemia reperfusion14 and
doxorubicin induced cardiotoxicity15. Probucol a lipid
lowering agent and potent antioxidant, provides
complete protection against doxorubicin induced
cardiomyopathy, without interfering with the
antitumor properties of this antibiotic16. Lipistat
capsule contains 500 mg of Terminalia arjuna, Inula
racemosa and Commiphora mukul in equal
42 INDIAN J EXP BIOL, JANUARY 2009
proportions (Dabur India). Lipistat (1 capsule twice
daily) promotes hypolipidemia and improves
optimum cardiac function. Terminalia arjuna bark has
remarkable cardioprotective and heart muscle
strengthening properties17. It possesses cardiac
stimulant properties with variable effect on heart,
associated with increase in threshold for myocardial
ischemia and angina18. The bark powder of Arjuna
exerts hypocholesterolaemic and antioxidant effect19.
It is thought that saponin glycosides may be
responsible for the inotropic effect of T. arjuna, while
the flavonoids and oligomeric proanthocyanidins
provide free radical antioxidant activity20 and vascular
strengthening. Inula racemosa is another ingredient
with potent beneficial effects on cardiovascular
system. It has antianginal and beta blocking activity21.
Commiphora mukul has hyperlipidemic22, thrombosis
and fibrinolytic activity23,24. Efforts have been made
to use antioxidants, iron chelators, beta blockers and
hypolipidemic agents to prevent doxorubicin induced
cardiotoxicity.
The present study has been undertaken to
investigate the effect of lipistat against doxorubicin
induced myocardial toxicity in rats.
Materials and Methods
Animals — Albino rats of either sex weighing
150-200 g were procured from animal house of
K.L.E.S’s College of Pharmacy, Hubli, were used for
the study after the clearance from Institutional Animal
Ethical Committee. Animals were acclimatized for
one week to laboratory conditions before starting the
experiment; they had free access to water and
standard rat feed but 12 hr prior to an experiment, the
rats were deprived of food but not water.
Materials — Doxorubicin was a generous gift from
Get Well Pharmaceuticals, India. Lipistat and other
chemicals used were of analytical grade and procured
locally. Analyzing kits were obtained from ERBA
Diagnostics, Daman, India.
Dosage fixation — Lipisat (350 mg/kg) showed
protection against isoproterenal induced myocardial
necrosis25. The same dose was used in the present
study.
Experimental design — After one week of
acclimatization, the animals were randomly divided
into 4 groups of 6 animals in each. Group 1 served as
normal control and received normal saline 5 ml/kg
body weight (ip). Group 2 animals were treated with
doxorubicin (2.5 mg/kg body weight ip) in 6 equal
injections alternatively for 2 weeks to make a total
cumulative dose of 15 mg/kg body weight. Group 3
animals received lipistat (350 mg/kg body weight po)
for 2 weeks and then alternatively with vehicle for
next 2 weeks. Group 4 animals received lipistat
(350 mg/kg body weight po, for two weeks) as a
pretreatment followed by doxorubicin administration
as in group 2.
Enzyme assays — After 36 hr of the last treatment,
orbital blood samples were obtained under light ether
anesthesia using heparinized microcapillaries for the
estimation of cardiac biomarkers CPK26 and LDH 27.
Both control and treated animals were observed for as
long as 3 weeks after the last injection for the general
appearance, behaviour and mortality. At the end of 3
weeks post treatment period, animals were sacrificed
under ether anesthesia and a midline abdominal
incision was performed and heart tissue was quickly
dissected out and washed in ice cold saline, dried on
filter paper and weighed immediately. A portion of
each heart was taken from all the groups and a 30%
w/v homogenate was prepared in 0.9% buffered KCl
(pH 7.4) for the estimation of glutathione (GSH)28,
superoxide dismutase (SOD)29, catalase (CAT)30 and
malondialdehyde (MDA)31. Orbital blood samples
were collected before sacrificing the animals and used
for estimation of cholesterol32, triglycerides33, low
density lipoprotein (LDL)34 and high density
lipoprotein (HDL)35. The remaining portion of the
heart tissue was used for histopathological studies.
Statistical analysis — The results were expressed as
the mean ± SE. The results obtained were analyzed
using one-way ANOVA followed by Dunnett’s
multiple comparison tests. Data were computed for
statistical analysis by using Graph Pad Prism
Software.
Results
Chronic administration of doxorubicin induced
cardiac toxicity and effect of lipistat was established
by significant increase in cardiac biomarker enzymes
and endogenous antioxidants and heart tissue
histolopathology.
General observations — The general appearance of
all groups of animals was recorded throughout the
study. In doxorubicin treated group, the animal fur
became scruffy and developed a pink tinge. These rats
also had red exudates around the eyes and nose, soft
watery feces and enlargement of abdomen. These
observations were significantly decreased in lipistat
treated group.
 KOTI et.al: CARDIOPROTECTIVE EFFECT OF LIPISTAT IN RATS 43

Heart weight, body weight and ratio of heart weight the heart tissue from control and lipistat treated
to body weight — Effect of doxorubicin on heart animals showed normal morphological appearances,
weight, body weight, liver weight, ratio of heart whereas in group 2 disruption of loss of myofibrils
weight to body weight and liver weight to body and vacuolization of the cytoplasm were observed.
weight is shown in Table 1. The food and water intake The histology of heart tissues from group 4 showed
in doxorubicin treated group was significantly less loss of myofibrils and vacuolization of the
decreased as compared to group 1. The food and cytoplasm.
water intake in the group 4 was significantly
increased as compared to group 2. The heart weight, Table 2—Effect of lipistat on lipid profiles in doxorubicin
body weight, liver weight, ratio of heart weight to induced cardiotoxicity in rats
body weight and liver weight to body weight in group [Values expressed in mg/dl are mean ± SE from 6 rats]
2 treated rats were significantly increased compared Treatment Choles- Trigly- HDL LDL
with normal rats. The heart weight, body weight, liver terol cerides
weight, ratio of heart weight to body weight and liver
weight to body weight in group 4 was significantly Normal 59.602 ± 108.12 ± 27.34 ± 28.645 ±
decreased compared with group 2. 1.123 1.690 0.924 2.387
Doxorubicin 106.50 ± 332.17 ± 31.3 ± 71.543 ±
Serum lipid levels — Animals treated with
1.409a 3.176a 0.384a 3.539a
doxorubicin produced significant increase in the Lipistat 56.072 ± 101.87 ± 42.870 17.827 ±
levels of cholesterol, triglycerides and LDL compared 0.977ns 1.394ns ± 1.251b 3.033ns
to group 1 and there was very slight difference in Lipistat + 85.488 ± 256.22 ± 33.312 49.500 ±
HDL levels compared to group 1 (Table 2). Group 4 doxorubicin 2.120b 3.588b ± 1.487b 5.126b
produced significant decrease in the level of
cholesterol, triglycerides and LDL but significant P values; <0.01; compared with anormal, bdoxorubicin
ns = not significant
increase in the level of HDL as compared to group 2.
Serum enzyme biomarkers __ Animals treated with
doxorubicin produced significant increase in the
levels of CPK and LDH compared to group 1 Table 3—Effect of lipistat on CPK and LDH levels in
doxorubicin induced cardiotoxicity in rats
(Table 3). Group 4 produced significant decrease in [Values expressed in IU/L are mean ± SE from 6 rats]
the level of CPK and LDH as compared to group 2.
Antioxidant status — Effect of doxorubicin on Treatment CPK LDH
tissue lipid peroxidation, antioxidant and antioxidant
enzymes is shown in Table 4. The malondialdehyde Normal 149.93 ± 2.809 141.71 ± 9.159
level was increased; GSH, SOD and CAT level were Doxorubicin 289.18 ± 26.255a 208.31 ± 6.517a
significantly decreased in doxorubicin treated group Lipistat 144.27 ± 10.347ns 132.79 ± 3.093ns
Lipistat +
as compared to normal animals. Group 4 produced 230.46 ± 4.396b 175.01 ± 13.907b
doxorubicin
significant decrease in the level of MDA and increase
in the status of antioxidant and antioxidant enzymes. P values; <0.01; compared with anormal, bdoxorubicin
Histopathological observation — The histology of ns = not significant

Table 1—Effect of lipistat on heart weight, body weight, liver weight and ratio of heart weight to body weight in doxorubicin
induced cardiotoxicity in rats
[Values are mean ± SE from 6 rats]

Treatment Body weight (g) Heart weight (g) Liver weight (g) Heart wt/body wt Liver wt/body wt
ratio (× 10-3) ratio (× 10-3)

Normal 253.33 ± 11.667 0.768 ± 0.01475 4.393 ± 0.04998 30.356 173.63

Doxorubicin 158.33 ± 5.270a 0.942 ± 0.0200a 5.796 ± 0.093a 59.62 366.83
Lipistat 232.50 ± 14.930ns 0.758 ± 0.1260ns 4.415 ± .08279ns 32.672 190.30
Lipistat + doxorubicin 190.83 ± 3.962b 0.850 ± 0.1609b 4.991 ± 0.104b 44.737 262.68

P values; <0.01; compared with anormal, bdoxorubicin.

ns = not significant
44 INDIAN J EXP BIOL, JANUARY 2009
Discussion
The study entails the cardioprotective effect of
lipistat against doxorubicin-induced cardiotoxicity.
Lipistat, an Indian herbal formulation possesses
cardioprotective, cardiotonic and lipid lowering
properties. The present study is aimed to explore
the cardioprotective effects of oral administration
of lipistat against doxorubicin-induced cardiotoxicity
in rats.
The existing experimental evidence suggests that
doxorubicin oxidative stress is due to the generation
of free radicals in the heart tissue36. The generated
reactive oxygen species such as superoxide radicals
and hydroxyl radicals are potential to cause damage to
various intracellular components. Heart tissue is
particularly susceptible to free-radical injury, because
it contains low levels of free-radical detoxifying
enzymes/molecules like SOD, GSH and CAT.
Further, doxorubicin also has high affinity for the
phospholipid component of mitochondrial membrane
in cardiac myocyte, leading to accumulation of
doxorubicin in the heart tissue37. The doxorubicin
induced mitochondrial injury is critical to the heart
because it would presumably have extreme adverse
effects on the contractile functioning of the cardiac
myocytes by alterations in the energy metabolism38.
Hence a protocol that would initiate doxorubicin
induced oxidative stress followed by lipistat
intervention was used to explore the extent of control
of progressive tissue damage.
Pretreatment of lipistat was able to reduce the
doxorubicin-induced cardiotoxic manifestations in
multiple ways. Increase in the level of plasma
triglycerides, total cholesterol and low density
lipoproteins in the doxorubicin treated group indicate
doxorubicin may be interfering with metabolism or
biosynthesis of lipids. Pretreatment with lipistat
Table 4—Effect of lipistat on malondialdehyde, glutathione, catalase and superoxide dismutase in doxorubicin induced
cardiotoxicity in rats
[Values are mean ± SE from 6 rats]
Malondialdehyde
Treatment
(n mol MDA/g of wet tissue)
Normal 17.467 ± 0.6086
Doxorubicin 48.173 ± 0.7414a
Lipistat 17.840 ± 0.8242ns
Lipistat + doxorubicin 36.855 ± 1.948b
P values; <0.01; compared with anormal, bdoxorubicin.
ns = not significant
t - µmole of H2O2 consumed / min.
tt - one unit of activity was taken as the enzyme reaction, which gave 50% inhibition of NTB reduction in one min.
showed reduction in blood lipid profile levels with
concomitantly increase in HDL cholesterol was
observed. Decrease in the blood lipid profiles and
increase in HDL cholesterol in lipistat treated group
may be due to the presence of C. mukul. Lipid
lowering effect of lipistat is due to inhibition of
hepatic cholesterol biosynthesis, increased fecal bile
acid secretion and stimulation of receptor mediated
catabolism of LDL cholesterol and increase in the
uptake of LDL from blood by liver39. Heart tissue
injury induced by doxorubicin in rats was indicated
by elevated level of the marker enzymes such as
serum LDH and CPK40. The increase of LDH level in
serum and extracellular fluid suggests an increased
leakage of this enzyme from mitochondria as a result
of toxicity induced by treatment with doxorubicin.
This index has been recently used in other studies to
test for cardiotoxicity41.
Lipistat was found to inhibit the doxorubicin-
induced CPK and LDH release in the serum of rats. It
is widely reported that doxorubicin-induced free-
radical generation triggers membrane peroxidation
and disruption of cardiac myocytes, which can lead to
increased release of CPK in the serum. Lipistat
pretreatment led to inhibition of CPK and LDH
release which resulted in either complete reversal or
considerable recovery of the serum enzyme activities.
The present results are in good agreement with those
of Murat et.al42. Another effect of doxorubicin
induced cardiotoxicity is characterized by decreased
body weight and increase in the heart weight43. The
results of the present study confirmed the earlier
findings that doxorubicin administration caused
decrease in the body weight and increase in
heart weight.
Cardioprotective activity of lipistat was further
supported by increased myocardial antioxidant
Glutathione Catalase Superoxide dismutase
(n mol/g of wet tissue) (units mg of protein)t (units/mg of protein)t t
2.867 ± 0.1606 60.568 ± 2.124 36.710 ± 0.8072
1.100 ± 0.1758a 40.643 ± 2.029a 23.907 ± 1.31a
2.432 ± 0.1840ns 61.425 ± 0.9377ns 37.677 ± 0.8215ns
1.697 ± 0.1106b 53.30 ± 1.084b 29.312 ± 1.075b
 KOTI et.al: CARDIOPROTECTIVE EFFECT OF LIPISTAT IN RATS
enzyme activity and decreased extent of lipid
peroxidation. Lipid peroxidation is known to cause
cellular damage and is primarily responsible for
reactive oxygen species induced organ damage.
Increased level of MDA and decreased levels of GSH,
SOD and CAT were observed in heart tissue in
doxorubicin treated animals. Pretreatment with lipistat
efficiently counteracted the doxorubicin induced
cardiac tissue damage by significant decrease in MDA
and increase in GSH, SOD and CAT levels. The
observed increase in CAT activity in doxorubicin
treated animals supports the above hypothesis that this
increase is possibly required to overcome excessive
oxidative stress44.
Histopathological report suggest lipistat pretreated
group attenuates the doxorubicin induced loss of
myofibrils, vacuolization of the cytoplasm and
swelling of mitochondria. The histopathological
changes observed in the doxorubicin treated rats were
similar to those previously reported. Flavonoids and
oligomeric proanthocyanidin present in T. arjuna and
guggulsterones in C. mukul may be responsible for
reducing oxidative damage. Hence, the antioxidant
activity of lipistat may be attributable to T. arjuna or
C. mukul. The other possible protective effect
mediated through the adrenergic blocking property of
I. racemosa, one of the important constituent of
lipistat, may be contributing to protective action
against doxorubicin induced cardiotoxicity.
β-Blockers have been reported to be useful in the
treatment of doxorubicin induced cardiomyopathy45.
In conclusion, the present results suggest that
lipistat prevented the doxorubicin induced myocardial
toxicity by boosting the endogenous antioxidant
activity. The cardioprotective property of lipistat
could be due to lipid lowering and antioxidant
properties. Further studies are needed in elucidate the
exact mechanisms of action of lipistat and its clinical
application.
Acknowledgement
The authors thank Dr. B. M. Patil, Principal, K.L.E.
Society’s, College of Pharmacy, Hubli, India for
support.
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