Download research article evaluation of cardioprotective activity of polyherbal

RESEARCH ARTICLE
EVALUATION
OF
CARDIOPROTECTIVE
ACTIVITY
OF
POLYHERBAL FORMULATION ON ISOPROTERENOL INDUCED
MYOCARDIAL INFARCTION IN WISTAR RATS
*Neelam1, Ayaz Ahmed2, Rohit Selkeri3, Parveen Kumari4, Sonu Sharma5
1, 2, 3
4, 5
Department of Pharmacology, Nims Institute of Pharmacy, NIMS University, Jaipur
Department of Pharmacognosy, Nims Institute of Pharmacy, NIMS University, Jaipur
*Corresponding Author
Ms. Neelam
Department of Pharmacology
NIMS Institute of Pharmacy
NIMS University, Shobha Nagar
Jaipur, Rajasthan (303121)
Email: [email protected]
Telephone no. + 91-8233930050,
+ 91-8816992175
ABSTRACT
Myocardial infarction is one of the killer diseases in many parts of the world and the allopathic
medicines has many side effects, but the herbal drugs have no or minimal side effects and the
polyherbal formulation has increased pharmacological effect than individual herbal formulations.
The effect of Cardioprotective polyherbal formulation, with an antioxidant property, has been
studied on isoproterenol (ISO)-induced cardiotoxicity in wistar rats. The cardioprotective effect
of polyherbal formulation on serum and heart tissue lipids and heart membrane bound enzymes
in isoproterenol induced myocardial infarction was studied. Male Wistar rats were administered
polyherbal formulation (200 mg/kg/day, orally) for 30 days with concurrent ISO administration
(85 mg/kg, subcutaneously, 2 doses at 24 h interval) on 29th and 30th day. Biochemical analysis
of heart tissue homogenate presented decreased levels of antioxidant enzymes viz., superoxide
dismutase, catalase and increased levels of cardiac markers viz. lactate dehydrogenase, creatine
kinase-MB isoenzyme. A marked reduction in reduced glutathione levels was observed in rat
myocardium. Rats treated with ISO (85mg/kg S.C.) recorded significant decrease in heart
weight, and significant increment in plasma lipid profile, plasma marker enzymes and cardiac
lipid peroxidation and activity levels of Ca2+ ATPase. The results indicated that the polyherbal
formulation improved cardiac function and this cardioprotection might be attributed to reduce
oxidative stress. Histopathological observation revealed a marked protection by polyherbal
formulation in myocardial infarction.
KEY WORDS
Myocardial infarction, Isoproterenol, Polyherbal formulation, Antioxidant
INTRODUCTION
MI commonly known as heart attack, which arrests the cardiac function, and leads to morbidity
and mortality in not only developing countries like India1. But also in developed countries like
U.S 2. MI is invariably followed by several biochemical alterations such as hyperlipidemia, lipid
peroxidation (LPO), free radical damage and leading to qualitative and quantitative alterations of
myocardium. Oxygen free radicals are the mediators of heart tissue injury 3. Myocardial
infarction (MI) is one of the main causes of death from cardiovascular disease. An increased risk
of coronary heart disease (CHD) is associated with high levels of serum total cholesterol4 and
low density lipoprotein (LDL) 5 and decreased levels of high density lipoprotein (HDL) 6.
Isoproterenol (ISO), a synthetic adrenoceptor agonist, has been found to induce myocardial
injury in rat as a result of disturbance in physiological balance between production of free
radicals and antioxidative defense system7. It is the acute condition of myocardial necrosis which
caused cardiac dysfunctions, increased lipid peroxidation, altered activities of cardiac enzymes
and antioxidants8. The pathophysiological and morphological changes observed in ISO-treated
rats have been similar to those observed in human MI
9.
Medicinal plants and their active
principles have received greater attention as anti-peroxidative agent 10.
Andrographolide, the main active constituent of Adrographis paniculata (A. paniculata), has
excellent anti-inflammatory, anti-bacterial and anti-viral effects. A. paniculata is an Indian herb,
well known as ‘king of bitter’. This bitter herb generally has an affinity with heart and liver 11.
Chicory (Cichorium intybus L.) is one of the most promising novel candidates among the
carbohydrates with potential for both food and non-food utilization. It has been implemented in
folk medicine from North Africa to South Asia for several 100 years (Jamshidzadeha et al.,
2006). Fresh chicory typically contains 68% inulin, 14% sucrose, 5% cellulose, 6% protein, 4%
ash, and 3% other compounds, while dried chicory contains approximately 98% inulin and 2%
other compounds 12 .
Chicory has a potent hepatoprotective, antioxidant, hypoglycemic, diuretic, anti-testicular
toxicity and immunomodulatory effects 13.
To the best of our knowledge, ISO-induced myocardial infarction and oxidative stress-related
parameters of polyherbal formulation have not been scientifically explored till date. Therefore,
the present study was designed to investigate the effects of oral administration of polyherbal
formulation on ISO-induced myocardial necrosis in rats.
MATERIAL AND METHODS
Plant authentication
Fresh leaves of all the different plants (Chichorium intybus, Andographis paniculata, Gymnema
sylvestre) were collected from the local market of jaipur and shade dried. All the plants were
identified by a Botanist(Dr. H.B. Singh, NISCAIR)
Extraction
The polyherbal formulation is containing three plant constituents. All the plant parts (leaves of
Andrographis paniculata, leaves of Cichorium intybus and leaves of Gymnema sylvestre were
collected fresh from jaipur district, Rajasthan, India and shade dried. All the plant parts were
ground and powdered. The dried and coarsely powdered leaves of plants (A. paniculata C.
intybus and G. sylvestre) were packed separately in soxhlet apparatus and subjected to extraction
with absolute ethyl alcohol. The extracted materials were evaporated to dryness under reduced
pressure at 45 ◦C14,15 and the leaves of C. intybus were extracted with 70% ethanol through
maceration(48hrs× 3 times) 16. The dried ethanolic extract was suspended in ethanol and used for
oral treatment of rats. The herb composition of CardiPro for each 100 mg(1:1:1) is as follows:
Andrographis paniculata (33.33mg leaves extract), Cichorium intybus (33.34mg leaves extract)
and Gymnema sylvestre (33.33mg leaves extract)17.
Phytochemical Screening of Extracts
The ethanolic extracts ofAndrographis paniculata contains Proteins and amino acids, Glycosides, Alkaloids, Flavonoids,
Tannin & phenolic Compounds.
Cichorium intybus contains carbohydrates, Glycosides, Flavonoids, Tannin & phenolic
Compounds.
Gymnema sylvestre contains carbohydrates, Proteins and amino acids, Glycosides, Flavonoids,
Alkaloids, saponins.
All the three plants were found to contain flavanoids by phytochemical estimation, due to which,
the plants are showing antioxidant activity.
Drugs and chemicals
ISO was purchased from Sigma Chemical Company, MO, and USA. All other chemicals used
were of analytical grade.
Animals
Wistar rats of either sex (150-200 g) were obtained from the Central Animal House facility of
this Institute. Animals were kept in the departmental animal house under controlled conditions of
temperature at25±1ºC, relative humidity of 55-75% and light–dark cycle of 12:12 h ten days
prior to start of the experiment to adapt to the environmental conditions. The animals were
housed in polypropylene cage with sterilized paddy husk bedding and food and tap water were
made available ad libitum throughout the experimental period. All the experiments were carried
out with the prior approval of institutional animal ethics committee (1302/ac/09/CPCSEA) 18.
Experimental design
The rats were divided into 4 groups of 6 animals each as follows: Group I: normal untreated
rats. Group II: rats were subcutaneously injected with ISO (85 mg /kg once a day for 2 days) on
29th and 30th day. Group III: normal rats were orally treated with polyherbal formulation using an
intragastric tube (200 mg/ kg /day, for 30 days) and then subcutaneously injected with ISO (85
mg /kg once a day for 2 days)19. Group IV: rats were orally treated with simvastatin (1mg/kg for
21days)
20
using an intragastric tube and then subcutaneously injected with ISO (85 mg /kg
dissolved in saline once a day for 2 days). At the end of the experimental period i.e., 12 h after
the second dose of ISO injection, all the rats were killed. Blood was collected and serum
separated after centrifugation. Serum was used for various biochemical estimations (21). The heart
was dissected out, washed immediately in ice-chilled saline, blotted and weighed. A known
weight (200 mg) of the heart tissue was homogenized in 5 ml of 0.1 M Tris–HCl (pH-7.4) buffer
solution. The homogenate was centrifuged at 3000 rpm for 5 min. The supernatant was used for
the estimation of various biochemical parameters22.
Biochemical estimation
Processing of heart tissue
The hearts were taken out from formalin, weighed and a 10% homogenate was prepared in icechilled phosphate buffer (50mM, pH7.4). Aliquots of homogenate were stored to be used for the
estimation of thiobarbituric acid reactive substances (TBARS) and GSH. The remaining
homogenate was then centrifuged at 5000 rpm for 20 min at 41ºC and supernatant was used for
the estimation of GSH, lactate dehydrogenase (LDH), CK-MB isoenzyme, SOD, CAT, Ca++
ATPase, lipid peroxidation and total cholesterol.
Reduced glutathione estimation
Myocardial GSH was estimated by the method of Moronetal. (1979). Briefly, 100 ml tissue
homogenate was mixed with 100 ml of 10% trichloroacetic acid (TCA) and centrifuged at 5000
rpm for 20 min. Subsequently, 0.05 ml of supernatant was incubated with a reaction mixture
containing 3.0ml 0.3M phosphate buffer (pH8.4) and 0.5ml DTNB [5, 5-dithiobis- (2nitrobenzoicacid)]. After 10min of incubation the absorbance was measured at 412nm using
spectrophotometer. GSH content was determined from a standard curve obtained using
commercially available standard GSH (SigmaChemicals, USA). Levels of GSH are expressed as
mmol/g tissue 23.
Plasma total cholesterol
Plasma total cholesterol (TC) was analysed using commercially available kits (Sigma, mumbai).
Creatine kinase-MB isoenzyme estimation
CK-MB isoenzyme was estimated using a commercial kit from Logotech Kit (Delhi, India). The
absorbance of reaction mixture was measured at 340nm for 3min at 60s intervals. CK-MB levels
are expressed as IU/mg protein.
Lactate dehydrogenase estimation
The reaction mixture contained 2.90ml of 0.2M Tris-buffer, 10 ml tissue supernatant, 100 ml of
30mM sodium pyruvate and 100 ml of NADH. The rate of change in absorbance was measured
at 340nm for 2min at 30s intervals. The oxidation of NADH in the above-mentioned reaction
was proportional to LDH present in the sample. LDH was determined from a standard curve
obtained using commercially available LDH (SigmaChemicals, USA). LDH levels are expressed
as IU/mg protein 24.
Thiobarbituric acid reactive substances estimation
Lipid peroxidation in hearts was determined by measuring malondialdehyde (MDA) content 25.
Briefly, 100 ml tissue homogenate was further centrifuged with 100 ml of10% TCA at 5000 rpm,
for 20 min at 41ºC. Subsequently, 0.2ml homogenate was mixed with 0.2ml of 8.1% sodium
dodecylsulfate, 1.5ml of 30% acetic acid (pH3.5) and 1.5 ml of 0.8% thiobarbituric acid. The
reaction mixture was heated for 60 min at 95ºC and then cooled. After cooling, 1.0ml of
redistilled water and 5.0ml of n-butanol: pyridine (15:1v/v) were added and centrifuged at
5000rpm for 20min. The absorbance of the pink colored organic layer was measured at 532nm.
1, 1, 3, 3-tetraethoxypropane (SigmaChemicals, USA) was used as the standard. MDA contents
are expressed as nmol/g tissue.
Catalase estimation
Catalase activity was estimated by the method described by Aebi (1974)
26
. To 50ml tissue
supernatant, 1.0 ml of 50mM phosphate buffer (pH7) and 0.1ml of 30mM hydrogenperoxide
were added and adecreasein absorbance at 240nm was measured every 5s for 30s. Catalase
content is expressed as U/mg protein.
Superoxide dismutase estimation
SOD activity was determined by the method of Marklundand Marklund(1974) 27. To 100 ml of
tissue supernatant, 2.85ml of 0.1M phosphate buffer(pH8.4) and 50 ml of 7.5mM pyrogallol
were added and absorbance was measured at 420nm for 3min at 30s intervals. SOD levels are
expressed as U/mg protein.
Statistical analysis
Statistical analysis was performed using one way analysis of variance (ANOVA) followed by
Dunnett's Multiple Range Test. Results were expressed as mean ± S.E.M. from six rats in each
group. P<0.05 were considered to be significant.
RESULT
Effect of polyherbal formulation on cardiac markers, TC and Ca++ ATPases
In the present study, ISO-treated rats showed significant (P<0.05) increase in the activities of
CK and LDH in serum but there was a significant increase in the concentration of total
cholesterol(TC) and Ca++ ATPase in heart tissue of ISO-treated rats when compared to normal
untreated rats (Table 1). Pretreatment with the polyherbal formulation (200 mg kg−1 day−1 for
30 days) to ISO-treated rats significantly prevented the altered levels of cardiac marker enzymes,
lipids and ATPases.
Effect of polyherbal formulation on antioxidant enzymes
ISO group recorded significant decrement in cardiac GSH content (from 3.23±0.11 to 1.27±0.06
µmol/g tissue) and lowered activity levels of SOD (from 11.38±0.37 to 7.07±0.291U/mg protein)
and CAT (from 44.77±1.78 to 30.78±1.871U/mg protein) when compared to control group.
Pretreated group with polyherbal formulation recorded significant increment in GSH content
(2.29±0.11µmol/g tissue) and elevated activity levels of SOD (8.64±0.551U/mg protein) and
CAT (39.32±1.751U/mg protein) (Table 2).
TBARS products
The product of lipid peroxidation measured as TBARS in heart tissue was significantly higher in
ISO-treated group (34.69±2.64 nmoles/g of heart tissue) compared to control (24.43±1.75
nmoles/g) (Fig 1). Coadministration of polyherbal formulation with ISO produced a significant
decrease in TBARS (27.31±1.73 nmoles/g of heart tissue).
Heart weight and microscopic evaluation of cardiac tissue:ISO group recorded a decrease in mean heart weight compared to control. Pre-treatment with
polyherbal formulation recorded an increment in heart weight compared to ISO group (Fig. 2).
The histoarchitecture of cardiac tissue of control group appeared to be normal as there was no
visible necrotic damage to the myocytes (Fig.3 A). However, extensive myocyte membrane
damage, myonecrosis, fibroblastic proliferation and infiltration of inflammatory cells was
observed in ISO group (Fig3.B). In ISO+formulation group, there was much less extent of
infiltration of inflammatory cells, myonecrosis, vacuolar changes and oedema as compared to IP
group (Fig. 3C).
Discussion
Present study demonstrates the cardioprotective potential of the polyherbal formulation in ISOinduced model of myocardial necrosis, as evidenced by amelioration of cardiac dysfunction,
improvement in endogenous antioxidant defense system and decreased lipid peroxidation.
Further, in light microscopic and ultra structural studies, polyherbal formulation pretreated
groups showed normal architecture of cells when compared with their respective controls.
Our results confirmed that a cumulative dose of isoproterenol (85 mg/kg) induces cardiotoxicity
in mice as evidenced by increase in mortality, accumulation of ascitic fluid, decrease in body
weight, and loss of cardiomyocytes. When myocardial cells, containing LDH and CK are
damaged or destroyed due to deficient oxygen supply or glucose, the cell membrane becomes
permeable or may rapture, which results in the leakage of enzymes. This accounts for the
decreased activities of LDH and CK in heart tissue of rats treated with isoproterenol
28
.
Pretreatment with polyherbal formulation elevated the decreased activities of the enzymes
comparable to the control. We suggest that this could be due to protective effect of polyherbal
formulation on the myocardium, reducing the myocardial damage and thereby restricting the
leakage of CK-MB isoenzyme and LDH.ISO-treated rats also showed an increase in the levels of
PLs in serum. Reports have shown that enhanced peroxidation of membrane phospholipids
releases FFAs via phospholipase A2 and Ca2+ ions have been reported to be one of the inducers
of phospholipase A2
29
So, the observed increase in FFAs concentration might be due to the
indirect effect of calcium level, which was reported to be altered in ISO-induced rats
30
.
Pretreatment with this polyherbal formulation decreases the levels of serum PLs and FFAs in
ISO-treated rats.
Abnormalities in lipid profile are associated with increased risk of myocardial infarction. High
level of circulating cholesterol and its accumulation in the heart tissue is usually accompanied by
cardiovascular damage 31. In the present study, polyherbal formulation restored total cholesterol
level to near normal levels; thereby reducing the risk of cardiovascular disease .Membrane bound
enzymes play a significant role in maintaining normal ion levels within themyocytes. Any
alterations in the properties of these enzymes affect the function of the heart. Vajreswari and
Narayanareddy
32
have shown that the failure of the cell membrane to maintain normal
transmembrane ionic distribution through ion pumps is considered to be a major event in the
pathogenesis of ischemia and arrhythmia. Previous studies have shown that activation of
adenylate cyclase increased the activity of Ca2+ATPase in ISO-induced rats 33. Our study shows
that pretreatment with polyherbal formulation maintains the activities of ATPases to near normal
in ISO-treated rats.It has been well documented that ISO causes increased oxidative stress in rat
heart as evidenced by reduction in myocardial SOD and CAT activities and reduced GSH level
34, 35, 36
. In the present study, we found that the polyherbal formulation increased activities of
SOD, CAT and GSH level in ISO-injected rats. To further characterize the modulatory action of
polyherbal formulation on ISO induced myocardial necrosis, light microscopic study was
performed. In histopathological study, ISO-induced myocardium showed severe infracted area
with edema and inflammatory cells and separation of cardiac muscle fibers. Only mild edema
and few inflammatory cells were observed in the myocardium of polyherbal formulation 200
mg/kg/day pretreated groups. Also the cardiac fibers were normal in architecture. Myocardium
of polyherbal formulation alone treated group showed normal cardiac fibers without any
pathological changes. This indicates that the formulation does not possess any adverse effects
under normal conditions. Our results showed that oral administration of polyherbal formulation
seems to protect the myocardium against ISO-induced MI.
CONCLUSION
It is concluded that the polyherbal formulation can mitigate the cardiotoxic effect of ISO in rat
heart. The present study provides experimental evidence that polyherbal formulation augmented
the myocardial antioxidant enzymes level, preserved histoarchitecture and improved cardiac
performance following ISO administration. These findings suggest the beneficial effects of
polyherbal formulation on rat heart against experimental myocardial necrosis.
ACKNOWLEDGEMENT
We are thankful to Principal, HODs and Guide, NIMS institute of pharmacy, NIMS University,
Jaipur, Rajasthan, India for providing all possible facilities and their support and constant
encouragement.
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Table 1:- Effect of polyherbal formulation on total cholesterol (TC), Ca++ATPase and cardiac
specific injury markers (LDH and CK-MB) in ISO induced myocardial infarction in rats
Group
TC (Mg/gwet tissue)
Ca++ ATPase
LDH(1U/mg protein)
CKMB(1U/mgprotein)
Group 1
62.31± 2.8
1.45±0.06
84.74±1.43
163.2±2.12
Group 2
110.2± 2.6#
3.38±0.81#
148.8±1.65#
121.5±2.35#
Group 3
90.35± 2.2***
1.92±0.08***
120.7±2.13***
139.5±2.47**
Group 4
80.48±2.8***
1.83±0.07***
97.84±2.19***
150.8±2.09***
TC- Total cholesterol; LDH- Lactate dehydrogenase; CK-MB – Creatinine kinase.
All values are expressed as mean± S.E.M. (n =6/group).
# P < 0.001 when compared with group 1, ***P < 0.001, **P < 0.01, *P < 0.05 when compared
with group 2.
Table 2. Effect of polyherbal formulation on the activities of CAT, SOD and GSH content.
Group
GSH (U/mg protein)
SOD (nmol/g tissue)
CAT (U/mg protein)
Group I
3.23±0.11
11.38±0.37
44.77±1.78
Group II
1.27±0.06#
7.07±0.29#
30.78±1.87#
Group III
2.29±0.11***
8.63±0.55*
39.32±1.75**
Group IV
2.76±0.11***
8.92±0.46*
41.19±1.78**
CAT: Catalase; SOD: Superoxide dismutase; GSH: Reduced glutathione.
All values are expressed as mean± S.E.M. (n =6/group).
# P < 0.001 when compared with group1, ***P < 0.001, **P < 0.01, *P < 0.05 when compared
with group
Fig. 1 Effect of polyherbal formulation on the level of thiobarbituric acid reactive substances
(TBARS) in ISO-induced myocardial necrosis in rats. All values are expressed as mean± S.D.
for each group (n=6/group).
40
TBARS(nmol/gm tissue)
35
30
25
20
15
10
5
0
control
ISO
PF
groups
Drug
Fig. 2: ISO-isoproterenol, PF-polyherbal formulation. Effect of polyherbal formulation on body
weight.
250
Body wt(grams)
200
150
100
Series1
50
0
control
ISO
PF
Groups
Drug
A
B
C
D
Fig.3 (A) Light micrograph of control rat heart showing normal architecture of myocytes.
(B)Light micrograph of ISO- group heart showing focal confluent necrosis of muscle fiber with
inflammatory cell infiltration, edema with fibroblastic proliferation and phagocytosis along with
extravasation of red blood cells. (C)Light micrograph of polyherbal formulation (200mg/kg/day)
+ ISO showing less marked myocardial necrosis, edema with inflammatory cells infiltration. (D)
Light micrograph of simvastatin (1mg/kg/day) + ISO showing mild myocardial injury, less
edema and inflammatory cells.