critical role in several cardiovascular diseases including myocardial infarction, myocardial I/R (ischemia/reperfusion), atherosclerosis, endothelial dysfunction, restenosis, hypertension as well as cardiomyopathies and heart failure ^{[3, 4]}. The oxidative stress associated injury is a direct result of an imbalance between an increase in ROS production and a decrease in antioxidant reserve under various pathological processes Ischemic injury occurs when there is reduced blood supply or complete occlusion of an artery. The causes for ischemic insults vary from organ to organ, and rupture of atherosclerotic plagues with resultant formation of thrombi represents a major cause for acute ischemic injury in the heart, brain, lung, intestinal tract and other organs. Intermittent constriction or compression from the outside of vessels also causes a reduction or cessation of blood supply. Lung, heart and liver transplantation remains the only effective therapy for end stage lung, heart or liver diseases.

Ischemic insults occur as results of a variety of conditions, leading to an accumulation of reactive oxygen species (ROS) and an imbalanced redox status in the tissues ^[5,6]. The oxidant stress may activate signaling mechanisms provoking more toxic events, and eventually causes tissue damage. Reactive oxygen species (ROS) are largely generated from mitochondrial energy metabolism via oxidative phosphorylation in the respiratory chain of eukaryotes. Because of the existence of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, and antioxidants, such as the reduced form of glutathione (GSH), as well as vitamin C and E, the redox balance is well maintained. Upon injurious insults including, inflammation, drugs, alcohol intake, or environmental pollutants, there is increased production of superoxide anion (O⁻) or other ROS from various sources resulting in the disturbance of this delicate balance. The increase in ROS consumes endogenous antioxidant compounds, such as GSH, and induces expression of antioxidant enzymes in order to maintain the redox balance ^{[7, 8]}. Thus, oxidant stress represents one of the major causes of ischemic injury, and antioxidant therapy may ameliorate the injury when it is properly delivered during an optimal time window and at right doses. A variety of antioxidants, scavengers, or scavenger mimetics have been evaluated in various ischemic conditions. Therefore, treatments with antioxidants, free radical scavengers and their mimetics, as well as gene transfer approaches to over express antioxidant genes represent potential therapeutic options to correct the redox imbalance.

It is now well recognized that isoproterenol (ISO) a synthetic catecholamine in large doses produces myocardial infarction ^[9]. Amongst various mechanisms proposed to explain ISO induced cardiac damage, generation of highly cytotoxic free radicals through autoxidation of catecholamines has been implicated as one of the important causative factor. Free radical mediated peroxidation of membrane phospholipids and consequent changes in membrane permeability appears to be the primary target responsible for cardiotoxicity induced by ISO^[10]. Proven cardio-protective and membrane stabilizing activity of few antioxidants in animal models of myocardial necrosis further strengthen this theory ^[11].

MI is a complex phenomenon affecting the mechanical, electrical, and structural and biochemical properties of the heart ^[12]. Although modern drugs are effective in preventing cardiovascular disorders, their use is often limited because of their side effects ^[13]. Recently, several plants of Indian origin have been found to possess antioxidant properties and their beneficial effects in pathological conditions like atherosclerosis, ischemia, cancer, cataract, and liver dysfunction have been related to their antioxidant properties. Drugs with multiple mechanisms of protective action, including antioxidant properties, may be one step forward in human disease. Though many antioxidant drugs for the protection against ischemic stroke are in the pipeline yet only few have successfully completed clinical trial. So proper screening of plant source for finding potential antioxidant drugs will definitely fulfill the dearth of suitable drugs for the treatment and protection against ischemia.

O. sanctum (the holy basil) is reported to possess antioxidant properties ^[14]. Ocimum canum Sims. (Hairy Basil) is a traditional medicinal plant distributes throughout Odisha and it is commonly known as Kala Tulasi in Odia has an unusual mint-like flavor. The plant branches out from its base, with angle stems and open foliage. The plant shows a pungent, aromatic ﬂavor and is commonly cultivated for culinary purposes. O. canum is used specially for treating various types of diseases and lowering blood glucose and also treats cold, fever, parasitic infestations on the body and inﬂammation of joints and headaches ^[15]. Protective role of O. canum in alcohol-induced oxidative stress has been already reported. Ocimum canum possess antibacterial ^[16]and mosquito repellent properties ^[17]and also lowers blood glucose level by facilitating the release of insulin from isolated pancreatic β-cells ^[18]; however, very little is known about its antioxidant properties. It is used in ritual as an incense as well to protect the home and welcome newborns into the world. It is an unusual and very useful addition to the medicinal garden. The hairy leaves and decorative flowers are very aromatic and form a lush mound about 2 feet in height. This annual plant grows well in full sun, well-drained soil and plenty of heat. The plant branches from the base and has an angled stems and oval pubescent leaves. Its leaves are tiny and fuzzy and have violet or white flowers, having a sweet scent resembling that of the clove. The leaves of the Ocimum canum are opposite and toothed. It is irregular and occurs in crowded whorls. The Ocimum canum has a small corolla. These plants have intense floral-fruity aromas. The oil of the Ocimum canum is composed of Linalool. The seeds may provide fiber or reduce constipation. This study aims to determine the role of O. canum as a cardio-protective and antioxidant agent in isoproterenol induced myocardial infarction in albino rats. A hydro-alcoholic extract of O. canum is evaluated for its abilities to scavenge free radicals, protect cell viability and inhibit the formation of lipid peroxides.

MATERIALS AND METHODS:

Method:

The experimental protocols were conducted with the approval of the Animal Research Committee at Royal College of Pharmacy and Health Sciences, Brahmapur. Odisha. All animals were maintained in accordance with the recommendations of the CPCSEA

Drugs and Chemicals:

Isoproterenol HCl, dihydro-diphosphopyridine nucleotide (DPNH), phenazinemethosulphate (PMS), nitrobluetetrazolium (NBT), 5,5-dithiobis (2-nitro benzoic acid) (DTNB), bovine serum albumin (BSA), 1,1,3,3-tetra methoxy propane, reduced glutathione (GSH), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and triphenyltetrazolium chloride (TTC), 1,1-diphenyl, 2- picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) ABTS were purchased from Sigma Chemical Co., USA through Nobel Enterprises, Brahmapur, Odisha. Dinitrophenylhydrazine solution (DNPH), sodium pyrophosphate (SPP), sodium dodecyl sulphate (SDS), thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were obtained from Nobel Enterprises, Brahmapur, Odisha, India. All other chemicals were of analytical grade

Animals:

Adult rates of either sex (150-200gm) were obtained from the animal house of R.C.P.H.S. and were housed and divided into 5 groups containing 6 animals each. All the experimental procedures and protocols used in this study were reviewed and approved by Institutional Animal Ethical Committee.

Plant collection

Leaves of Ocimum canum were collected in the month of December 2011 from its natural habitat from nearby Mohuda village, Berhampur, Ganjam district of Odisha. The plant was authenticated from Department of Botany, Khalikote College, Berhampur, Odisha. The leaves were cleaned and dried under the shade to avoid degradation of volatile oil. The leaves were dried in hot air woven at 55°C for 3 days and at 40°C for the next 4 days

Preparation of Plant Extracts:

The dried leaves were coarsely powdered and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50°C. The solvent was completely removed and obtained dried crude extract which was used for investigation. Further the extracts were subjected for the antioxidant study as well as pharmacological screening.

In vitro Antioxidant Study [19]:

Ocimum canum hydro-alcoholic leaf extract was tested for its antioxidant activity using different in vitro models as follows at concentrations of 250, 500, 1000 and 2000µg/ml

a) DPPH radical scavenging assay

To the Methanol solution of DPPH (1 mM) an equal volume of the extract dissolved in alcohol was added at various concentrations from 250 to 2000 μg/ml in a final volume of 1.0 ml. An equal amount of alcohol was added to the control. After 20 min, absorbance was recorded at 517 nm. Experiment was performed in triplicate.

b) ABTS radical scavenging assay

To the reaction mixture containing 0.3 ml of ABTS radical, 1.7 ml phosphate buffer and 0.5 ml extract was added at various concentrations from 250 to 2000 μg/ml. Blank was carried out without drug. Absorbance was recorded at 734 nm. Experiment was performed in triplicate

Toxicity Study:

The acute toxicity of the extract was evaluated in 56 normal albino rats. They were grouped into seven and each group contains eight rats (four male and four female). Each group was kept for fasting for 24hrs, after which they were treated once orally with one of the increasing doses of extract: 100, 200, 200,300 or 400 g/Kg/b. w. The volume of each administrated dose did not exceed one ml. the rats were then observed for at least 48hrs and up to seven days, for death, lethargy, jerkiness, sensitiveness to noise and touch, stools quality and frequency

Treatment Protocol

The rats were divided into six groups of six animals each.

· Group I served as a control (saline treated)

· Group II rats were administered with isoproterenol (200 mg/kg body weight administered subcutaneously twice at an interval of 24 h) dissolved in normal saline, to induce Myocardial ischemia ^[20].

· Group III rats were pretreated with Ocimum canum leaf extract (400 mg/kg) for a period of 30 days.

· Groups IV, V and VI animals were pretreated with Ocimum canum (hydro-alcoholic leaf extract) dissolved in distilled water was given to each animal orally (100 mg/kg, 200 mg/kg and 400 mg/ kg, respectively) for a period of 30 days ^[21] and isoproterenol (200 mg/ kg body weight subcutaneously twice at an interval of 24 hours) at the end of the treatment period on the 29th and 30th days. The animals were sacrificed 24 h after the second dose of ISO, under chloroform anesthesia. Hearts were removed and processed immediately for morphological and histopathological studies. For performing biochemical estimations hearts were immediately stored in cold formalin solution till further analysis

Determination of myocardial necrosis by direct staining

Using triphenyltetrazolium chloride (TTC) dye described by Lie et al. ^[22]. Myocardium of rat was frozen immediately after removal. When the tissue was firm, the heart was sliced into 1 mm segments and incubated at 37°C for 20 min in 1% TTC. The formazan precipitate resulting from the reaction of lactate dehydrogenase in normal and ischemic regions delineated the area at risk from the infarcted tissue.

Biochemical estimations

Hearts removed from liquid nitrogen were weighed. Ten percent homogenate was prepared in 0.1M Tris-buffer, pH 7.4 and used for all the assays.

The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated by the method of Reitman S, Frankel S ^[23]. The levels of lactate dehydrogenase (LDH) by the method of King J ^[24]and creatine phosphokinase (CPK) by the method of Okinaka S et al ^[25]were determined, using commercially available kits. The level of thio-barbituric acid reactive substances (TNARS) and reduced glutathione (GSH) were estimated by the method of Beuge and Aust ^[26]and Moron et al. ^[27]respectively. Troponin- T was estimated by the method of Bhaskar and Rao ^[28]. The protein content was estimated by the method of Lowry’s et al. ^[29]. The endogenous antioxidant enzyme superoxide dismutase’s (SOD) ^[29], catalase (CAT) ^[30] were estimated and for the estimation of MDA, the extent of lipid peroxidation was estimated by using the thiobarbituric acid method ^[31]. The absorbance was measured using spectrophotometer at 532 nm.

Histopathological studies

Myocardial tissue after removal was immediately fixed in 10% buffered neutral formalin solution. After fixation was complete, tissues were embedded in paraffin and serial sections were cut. Each section was stained with hematoxylin and eosin. The sections were examined under light microscope and photomicrographs were taken.

Statistical analysis

Descriptive statistics such as mean and standard deviation has been calculated for each and every variable for each group. One-way analysis of variance (ANOVA) has been applied for statistical analysis with post-hoc analysis (Bonferroni Multiple Range Test) and a value of p < 0.05 has been considered as statistical significance level ^[32]

RESULTS:

DPPH radical scavenging assay

The proton radical scavenging action is known to be one of the various mechanisms for measuring antioxidant activity. The DPPH test provides information on their activity of the test compounds with a stable free radical. This assay determines the scavenging of stable radical species of DPPH by antioxidants. The degree of reduction in absorbance measurement by Ocimum canum is indicative of the radical scavenging (antioxidant) power of the plant. The study showed that the hydro-alcoholic extract have the proton-donating ability and can serve as free radical inhibitors or scavenger, acting possibly as primary antioxidant.

Table 1:- Study on DPPH scavenging activity in Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	O. canum (% scavenging activity)
0	0	0
250	90.2±0.004	46.8 ± 0.005
500	91 ±0.009	56.7 ± 0.003
1000	92.4 ±0.005	72 ± 0.005
2000	93±0.007	79.2 ± 0.004

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Figure 1:- Study on DPPH radical scavenging activity in Ocimum canum leaves at 517 nm

ABTS radical scavenging activity

ABTS, a protonated radical, has characteristic absorbance maxima at 734 nm which decreases with the scavenging of the proton radicals (Mathew and Abraham 2006). The scavenging of the ABTS+ radical by the O. canum was found to be much higher than that of DPPH radical.

Table 2:- Study on ABTS scavenging activity in Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	O. canum (% scavenging activity)
0	0	0
250	68±0.014	52.65 ± 0.012
500	73.4±0.016	70.8 ± 0.013
1000	75±0.015	76.5 ± 0.014
2000	79.2±0.017	86.5 ± 0.011

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Figure 2:- Study on ABTS scavenging activity in Ocimum canum leaves at 734 nm

Figure 3:- Level of Lipid peroxide and reduced glutathione in plasma of normal and experimental groups

Table 3:- Level of lipid peroxide (LPO) and reduced glutathione (GSH) in plasma and heart tissue of normal and experimental groups of rats

Groups	Group I	Group II	Group III	Group IV	Group V	Group VI
Plasma
Lipid peroxide	1.76 ± 0.15	4.26 ± 0.31^a	1.02 ± 0.17	3.52 ± 0.27	2.02 ± 0.19	1.47 ± 0.13
Reduced glutathione	12.32 ± 0.62	8.59 ± 0.65^a	14.09 ± 0.64	9.87 ± 0.63	11.87 ± 0.67	13.02 + 0.67
Heart tissue homogenate
Lipid peroxide	0.98 ± 0.09	1.97 ± 0.11^a	0.66 ± 0.05	1.02 ± 0.10	0.97 ± 0.08	0.78 + 0.08
Reduced glutathione	5.21 ± 2.17	2.75 ± 0.29^a	6.47 ± 2.01	3.87 ± 1.20	4.98 ± 2.05	5.80 + 2.43

Results are mean ± SEM for 6 animals. Values expressed: Plasma lipid peroxide-nmol/ml, Reduced glutathione- mg/dl; Heart lipid peroxide - nmol/mg protein, Reduced glutathione - µg/g wet tissue; a р < 0.001 significantly different compared with Group I control animals.

Figure 4:- Level of Lipid peroxide and reduced glutathione in heart tissue of normal and experimental groups

Figure 5:- Level of MDA, SOD and CATALASE in heart tissue of normal and experimental groups

Table 4:- Level of Malondialdehyde (MDA) and Superoxide dismutases (SOD) and Catalase in heart tissue of normal and experimental groups of rats

Groups	MDA	SOD	CATALASE
Groups I	109.50 ± 22.08	4.11 ± 0.08	1.07 ± 0.08
Groups II	230.60 ± 44.82	1.77 ± 0.06	0.55 ± 0.23
Groups III	90.56 ± 14.00	4.02 ± 0.45	1.15 ± 0.11
Groups IV	165.00 ± 10.49	2.87 ± 0.38	0.78± 0.13
Groups V	111.80 ± 17.52	3.74 ± 0.40	1.09± 0.14
Groups VI	98.83 ± 9.61	4.12 ± 0.43	1.25± 0.10

Results are expressed as mean ± SEM for six animals. A p- value < 0.05 was considered as statistically significant. Value expressed: MDA, nmol/dl. SOD, one unit of SOD is described as the amount of enzyme required to cause 50%of inhibition of pyrogallol auto oxidation. CATALASE, units/ml of H₂O₂, decomposed/min

Table 5:- Effect of O. canum pretreatment on isoproterenol-induced changes in the activities of plasma AST, ALT, CK, LDH and Troponin T

Groups	ALT	AST	CPK	LDH	Troponin T
Group I	31.77± 0.32	14.62 ± 0.36	274.75 ± 0.31	78.8 ± 0.31	0.55 ± 0.24
Group II	52.63 ± 0.38	25.68 ± 0.43	530.52 ± 0.41	145.43 ± 0.27	1.64 ± 0.22
Group III	32.33 ± 0.88	13.75 ± 0.76	272.43 ± 0.68	78.23 ± 0.60	0.52 ± 0.24
Group IV	36.77 ± 0.58	15.47 ± 0.60	280.45 ± 0.68	81.0 ± 0.42	1.14 ± 0.37
Group V	35.37 ± 0.78	15.12 ± 0.47	277.70 ± 0.72	80.95 ± 0.55	0.88 ± 0.35
Group VI	33.65 ± 0.65	14.07 ± 0.89	274.57 ± 0.48	80.23 ± 0.63	0.78± 0.48

Results are mean ± SEM for 6 animals. Values expressed: ALT, AST and LDH-µmol pyruvate liberated/h/liter; CPK- µmol creatine liberated/ hr/ liter. Troponin T - mg/ dl.р< 0.001 significantly different compared with Group I control animals

Figure 6:- Effect of O. canum pretreatment on ISP –induced changes in the activities of ALT, AST, CPK and LDH

Figure 7:- Effect of O. canum pretreatment on ISP –induced changes in the activities of Troponin T

Table 6:- Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) in heart of normal and experimental groups of rats

Groups	ALT	AST	LDH	CPK
Groups I	43.51± 0.60	25.28 ±0.15	117.22 ± 0.15	13.13 ± 0.20
Groups II	27.9 ± 0.73	16.45 ± 0.16	80. 43 ± 0.18	9.45 ± 0.23
Groups III	44.46 ± 0.64	26.18 ± 0.16	115.43 ± 0.20	14.55 ± 0.23
Groups IV	41.68 ± 0.80	25.35 ± 0.15	114.78 ± 0.21	11.96 ± 0.22
Groups V	44.78 ± 0.75	25.92 ± 0.17	115.38 ± 0.14	13.28 ± 0.32
Groups VI	43.45 ± 0.73	26.25 ± 0.20	116.85 ± 0.26	14.16 ± 0.27

Results are mean ± SEM for 6 animals. Values expressed: ALT, AST and LDH- µmol pyruvate liberated/ h/mg protein; CPK- µmol creatine liberated/ hr/ mg protein. р< 0.001 significantly different compared with Group I control animals.

Figure 8:- Photomicrograph of rat heart

a) Photomicrograph showing normal architecture of rat heart of sham group. Endocardium and pericardium are seen within normal limits with no infiltration of inflammatory cells (H and E X100).

b) Photomicrograph of rat heart subjected to isoproterenol-induced focal myonecrosis with myophagocytosis and lymphocytic infiltration. In subendocardium vacuolar changes and prominent oedema along with chronic inflammatory cells are clearly visible (H and E X100) and

c) Photomicrograph of rat heart of the Ocimum canum treated group administered 200 mg kg-1 showing decreased degree of myonecrosis and lesser infiltration of inflammatory cells (H and E X100)

Biochemical estimations revealed a significant fall in the levels of LDH and antioxidant parameters in all Isoprenaline treated groups as compared to control. Table 3 and 4 depicts the levels of lipid peroxides and reduced glutathione and antiperoxidative enzymes in the heart tissue of normal and experimental groups of rats. There was a significant (p< 0.001) increase in the level of lipid peroxidation along with a concomitant decline in the level of GSH noted in the heart tissue of Group III isoprenaline-administered rats as compared to controls.

Also a significant (p< 0.001) reduction in the activities of reduced glutathione (GSH) and antiperoxidative enzymes (SOD and CAT) was observed. The prior administration of Ocimum canum significantly reduced the isoprenaline induced adverse effects and maintained the level of evaluated parameters at near normalcy. In Group II rats the oral administration of O. canum hydro-alcoholic leaf extracts resulted in a significant (p< 0.01) elevation in the level of reduced glutathione.

Table 5 depicts the levels of diagnostic marker enzymes (AST, ALT, LDH, CPK and Troponin T) in the plasma of normal and experimental groups of rats. There was a significant (p<0.001) increase noticed in the levels of these marker enzymes in the plasma of Group III isoprenaline-induced myocardial infarcted rats as compared to that of Group I control rats. The oral pre-treatment with O. canum hydro-alcoholic leaf extracts significantly (p< 0.001) prevented the isoprenaline-induced release of these enzymes from the myocardium into the systemic circulation and maintained the rats at near normal status, indicating the cytoprotective action of Ocimum canum.

DISCUSSION:

Animals develop ‘infarct like’ lesions when injected with isoproterenol, a potent synthetic catecholamine. These lesions are morphologically similar to those of ‘coagulativemyocytolysis’ (COAM) or myofibrillar degeneration, one of the findings described in acute myocardial infarction (AMI) and sudden death in human ^[33].

Though the pathogenesis of AMI has not yet been completely understood, the studies on ISO induced cardiotoxicity clearly demonstrate the involvement of oxidative stress in this pathology ^{[10, 34]}. Therapeutic intervention that could improve impaired antioxidant defense mechanisms or diminish free radical production in the ischemic myocardium has been of great interest ^[10]. Recently there has been an increase in interest to explore the cardio-protective potential of natural products ^[11].

A number of investigations have suggested that catecholamines in large doses produce myocardial necrosis ^{[9, 10, 35]}. Various mechanisms have been proposed to explain this catecholamine-induced necrosis which includes an increase in cAMP levels ^{[36, 37]}, intracellular calcium overload, and exhaustion of high-energy phosphates ^[38]. Since catecholamines readily undergo oxidation, it has been suggested that the oxidation products of catecholamines, rather than catecholamine themselves, are responsible for myocardial changes observed following the administration of the parent compounds ^[39]. There is strong evidence that adrenochrome and other oxidation metabolites of catecholamines can cause cell necrosis and contractile failure in the rat heart ^{[40, 41]}. It is also proved that autoxidation of catecholamines results in the generation of highly cytotoxic free radicals ^[42]. Free radicals can initiate the formation of alkyl, alkoxy and hydro-peroxy radicals plus hydro-peroxides from polyunsaturated fatty acids. The localization of highly unsaturated fatty acids in membrane makes the cell membranes vulnerable to free radical induced lipid peroxidation ^[43]. These studies strongly suggests that free radicals play an important role in catecholamine-induced cardiotoxicity by causing peroxidation of membrane phospholipids, which can result in permeability changes in the membrane as well as intracellular calcium overload.

Free radical scavenging enzymes such as catalase, superoxide dismutase, glutathione peroxidase are the first line of cellular defense against oxidative injury, decomposing O₂ and H₂O₂before their interaction to form the more reactive hydroxyl radical (OH·). The equilibrium between these enzymes is an important process for the effective removal of oxidative stress in intracellular organelles. The second line of defense consists of the non- enzymatic scavenger’s like, ascorbic acid, α tocopherol, ceruloplasmin and sulphydryl containing compounds, which can scavenge residual free radicals escaping decomposition caused by the antioxidant enzymes.

Besides, antioxidant enzymes and physiological antioxidants, alteration in LDH has been considered as one of the most important marker of myocardial infarction. Wexler and Kittinger ^[44] in their study demonstrated that there was a dramatic rise and fall in serum CPK and LDH following ISO induced MI in rats, and the degree of rise and fall in serum enzyme activities were proportionate to the extent of the myocardium infarcted. Various studies have shown and increase in plasma LDH activity with the consecutive injections of ISO and appearance of degenerative changes in myocardial cell membranes ^[45]. In the present study, LDH was estimated in heart tissue and a significant (p < 0.001) fall in the levels was observed in ISO treated rats to those of control. This observation is in conformity with previous reports and can be attributed to the fact that LDH, being the myocardial enzyme, leaked out from the tissue to plasma on development of degenerative changes in myocardial cell membranes. Detection of myocardial necrosis by direct staining using TTC dye, which forms a red formazan precipitate with LDH of the viable myocardial tissue also confirmed that in all ISO administered groups there was a significant leakage of LDH as compared to control.

In this study, it was observed that 200 mg/kg dose of ISO, induced marked lesions in myocardium and significantly altered various biochemical parameters.The present study was designed to systematically evaluate the hydro-alcoholic extracts of OC leaves for its potential as a cardio-protective agent. For this, effect of OC on morphology, biochemistry and histopathology of heart was studied against ISO induced myocardial infarction.

In the present study pre-treatment of OC exhibited significant protection against ISO induced histopathological and biochemical changes. The cadioprotective mechanism(s) appear to be through modulation of various antioxidant parameters thereby improving the overall antioxidant defense of the myocardial tissue. Present data on GSH, SOD, and MDA demonstrated that antioxidant status of the myocardial cell in ISO treated group is significantly hampered. Significant fall in GSH levels and impaired SOD activity together with increased LP appears to be the initial insult to the tissue making it more susceptible to oxidative damage. Increased ·OH production in such a compromised situation may be responsible for the observed membrane damage as evidenced by the elevated LP in terms of TBARS.

Glutathione is implicated in the removal of free oxygen species such as H₂O₂superoxide radicals, alkoxy radicals, and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase (GPX) and glutathione-stransferase (GST). Enhanced levels of GSH could either be because of its increased synthesis or due to improved glutathione reductase activity in presence of OC. Experiments performed in the present study demonstrates inhibition of the LP, enhancement of SOD activity and improvement in GSH levels in OC administered groups, which further implies that the cardio-protective effect of OC may be by virtue of its antioxidant properties. Antioxidant properties of OC could be attributed to its constituents like eugenol, flavonols, flavones and anthocyanins etc. Pre-treatment of animals with OC (100 and 200 mg/kg) offered a significant at p < 0.001 protection against ISO induced MI. However OC at doses 400 mg/kg failed to exhibit any significant cardio-protection in the present study, which might be due to the pro-oxidant activity at higher doses.

The fall in SOD levels may be due to the involvement of superoxide free radical in myocardial cell damage. A decrease in activity of SOD can result in the decreased removal of superoxide ion, which can be harmful to the myocardium ^[46]. It is possible that in presence of OC either generation of free radical it is impaired or enhanced SOD activity could effectively scavenge the first free radical superoxide from the system.

Serum lactate dehydrogenase has been reported to elevate markedly in ISO induced MI due to its leakage from heart tissue as a consequence of ISO induced LP and membrane damage ^[45]. In the present study, the level of LDH falls significantly (p < 0.001) in heart tissue following membrane damage. A significant protection in heart LDH levels is indicative of the fact that OC have cardioprotective action and maintain membrane integrity of myocytes.

A serum marker that once held promise as cardiac specific marker for MI is the cardiac troponin. Troponin is a protein found in cardiac tissue and located in the thin filament of striated muscles consisting of the three subunits Troponin T, Troponin I and Troponin C. Out of the three troponins; Troponin T and I are being used as the biochemical markers for the diagnosis myocardial injury. When the myocardial damage occurs thecytosolic troponins reach the blood stream quickly resulting in a rapid peak of serum troponin observed during the first few hours.In this study, significant increased level of Troponin T in serum of ISO-treated rats. Increased level of Troponin T was due to the leakage from the damaged heart tissues into the blood stream as a result of necrosis induced by isoproterenol in rats. Pretreatment with Ocimum canum to ISO-treated rats restored the level of Troponin T in serum indicates the protective action of Ocimum canum against peroxidative damage.

On histopathological examination, ISO 200 mg/kg group, (Fig. 8) demonstrates focal myonecrosis and chronic infiltration of inflammatory cells. Marked vacuolar changes and edema were seen. Pre-treatment with OC (200 mg/kg) demonstrated reversal of myonecrosis and lymphocytic infiltration (myocarditis) seen with ISO treated group. Inflammatory cells were seen with reduced density in the OC treated groups as compared to ISO 400 mg/kg group confirming further the cardioprotective activity exerted by the hydro-alcoholic leaf extract of OC in the present study. Our data indicate that OC may provide potential therapeutic value in the treatment of MI. If the beneficial effects of Ocimum canum can be reproduced in human beings, these findings may represent a novel prophylactic therapy for MI.

CONCLUSION:

Ocimum canum hydro-alcoholic extract decreased the leakage of CK-MB and LDH enzymes from myocardium. Presence of antioxidant constituents (flavanoids) in the extract might be responsible for its cardioprotective effect

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Received on 26.05.2012

Modified on 30.05.2012

Accepted on 07.06.2012

Research J. Pharmacology and Pharmacodynamics. 4(4): July –August, 2012, 191-201