Pharmacodynamic interaction of Terminalia arjuna (Roxb) with Ocimum sanctum (Linn) in Isoproterenol induced cardiac necrosis

Chetan Savant¹*, Venkatrao Kulkarni¹, Prasanna Habbu², Preeti Kulkarni¹,

Muhammed Majeed³, Mahadeva Nayak³

¹Department of Pharmacology, SET’s College of Pharmacy, Dharwad-580002, Karnataka, India.

²Department of Pharmacognosy, SET’s College of Pharmacy, Dharwad- 580002, Karnataka, India.

³Sami Labs Limited, R&D Peenya Industrial Area, Bangalore- 560058, Karnataka, India.

*Corresponding Author E-mail: chetan.savant@yahoo.com

ABSTRACT:

Herbal drugs are used widely even when their biologically active compounds are unknown, probably because of their effectiveness, lesser side effects and affordability. In the present study we investigated the potential cardio-protective effect of combination of standardized extracts of Terminalia arjuna and Ocimum sanctum in isoproterenol induced myocardial infarction. Myocardial infarction was induced in rats by subcutaneous injection of Isoproterenol for two consecutive days at an interval of 24h. Rats were pre-treated with test drugs for the period of 21 days and Isoproterenol was administered on 20 and 21^st day. At the end of experiment i.e on 22^nd day hemodynamic, biochemical and histopathological study of heart tissue were evaluated from control and experimental groups. Isoproterenol treated animals showed significant increase in heart rate and cardiac biomarkers. Whereas, mean arterial blood pressure and antioxidant parameters were significantly decreased. Animals administered with different combinations of standardized extracts of Terminalia arjuna and Ocimum sanctum significantly maintained the levels of cardiac biomarkers, antioxidant parameters, mean arterial pressure and heart rate. Histopathology study showed marked myocardial necrosis in Isoproterenol treated animals and significant cardiac protection of animals treated with extract combinations. Hence, we may conclude that herb-herb combinations were found to be effective in prevention of myocardial injury induced by isoproterenol.

KEYWORDS: Cardiac necrosis, Isoproterenol, Ocimum sanctum, Terminalia arjuna and Myocardial infarction.

INTRODUCTION:

Ischemic heart diseases are among the most threatening diseases to human life due to high rates of attack and associated disability and mortality. Myocardial infarction (MI) is an acute condition of necrosis of the myocardium that occurs as a result of sudden or continual interruption of blood supply to the myocardium¹.

Isoproterenol (ISO) is a sympathomimetic drug that acts almost exclusively on beta-adrenergic receptors, used in the treatment of allergic emergencies, bronchial asthma, ventricular bradycardia, cardiac arrest, and glaucoma². ISO administration in experimental animals provided a rapid, simple, and non-invasive method to generate myocardial damage status similar to that seen in humans with acute MI. In addition, ISO produced a model that had low mortality, high reproducibility, and validity compared with other animal models, which make it more appropriate for the assessment of potential cardioprotective agents³. The main mechanism involved in ISO-induction of myocardial ischemia is the generation of free radicals, reactive oxygen species, lipid peroxidation, oxidative stress, and calcium overload, which lead to the alteration in membrane permeability, causing apoptosis and necrosis and finally slowing the conduction between myocardial cells, triggering alterations in heart electrical activity^4,5.

The plant Terminalia arjuna (Combretaceae) is found abundantly throughout Indian subcontinent, Sri Lanka, Burma and Mauritius. The plant is used to treat many ailments due to presence of various bioactive compounds like luteolin, arjungenin, terminic acid, arjunoside I, arjunoside II, arjunolic acid etc. The bark stem powder of this tree has been mentioned to be useful for “hritshool” (angina) and other related cardiac ailments by the ancient physicians. Recently there has been renewed interest in this plant because of its multimode cardioprotective activity⁶. Tulsi (Ocimum sanctum) in Hindi or Tulasi in Sanskrit (holy basil in English) is an exceptionally adored culinary and restorative fragrant herb from the family Lamiaceae that is indigenous to the Indian subcontinent and been utilized inside Ayurvedic medication over 3000 years. Nutritional compounds of basil leaves that show anti-inflammatory and antioxidant effects have a beneficial effect in reduction of cholesterol levels and prevention of oxidative stress-related injury. In general, polyphenolic compounds, especially flavonoids, are ubiquitous in dietary components from plant-derived foods. From medical view point, flavonoids have potentially been applied to decrease cholesterol level and oxidative stress⁷.

In earlier studies it was reported that both Terminalia arjuna and Ocimum sanctum have shown significant cardioprotective activity in isoproterenol induced cardiac necrosis^8,9. However, the data regarding combined effect of these drugs on isoproterenol induced cardiac necrosis is nil. Hence an attempt was made to evaluate the effect of Terminalia arjuna and Ocimum sanctum on ISO induced cardiac necrosis.

MATERIAL AND METHODS:

Materials:

Standardized extracts of Terminalia arjuna (Roxb), Ocimum sanctum (Linn) were obtained from Sami Labs Limited, 19/1, 19/2, I Main, II phase, Peenya Industrial Area, Bangalore, Karnataka 560058 as gift samples. Isoproterenol Hydrochloride was purchased from TCI Chemicals (India) Pvt. Ltd. Chennai, Tamil Nadu, India. Biochemical parameters were assayed by standard kits using semi auto-analyser.

Experimental Animals:

Experiments were carried out using male albino wistar rats weighing 150 to 200gm. They were housed in polypropylene cages (47cm x 34m x 20cm) lined with husk, renewed every 24 h under a 12:12 h light dark cycle at around 22ºC. The animals had free access to water and food, ad libitum. The animals were fed on a standard pellet diet. The experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals and approved by the Animal Ethical Committee of SET’s College of Pharmacy (Reg. No. 112/PO/ReS/1999/CPCSEA, letter dtd 7^th February 2017) S.R. Nagar, Dharwad, Karnataka, India.

Dose selection:

Based on the literature survey we have selected 250 mg/kg and 500mg/kg dose for Terminalia arjuna¹⁰ and 50mg/kg for Ocimum sanctum⁹. Cardio toxicity was induced by administration of ISO at the dose of 85 mg/kg subcutaneously (s.c.) on the 20^th and 21^st day at an interval of 24 h¹¹.

Experimental protocol:

The male albino wistar rats were divided into 7 groups of 6 animals each. Group I received distilled water (Vehicle control) at 1ml/kg p.o.; Group II ISO control (85mg/kg s.c.); Group III and IV received TAE at 250mg/kg and 500mg/kg p.o.; Group V received OSE at 50mg/kg p.o.; Group VI TAE 250mg/kg + OSE 50mg/kg p.o.; Group VII received TAE 500mg/kg + OSE 50mg/kg p.o. Weighed quantity of extracts were taken in mortar and triturated it to fine powder, add little quantity of suspending agent again triturate, and add 10ml of distilled water with triturating till the formation of suspension. All the animals were treated orally per day using oral feeding needle for 21 days.

Isoproterenol induced myocardial necrosis in rats:¹¹

After treatment, animals from Group II to VII were administered with ISO at 85mg/kg s.c. for two consecutive days on 20^th and 21^st day. After 24 h the final subcutaneous injection of ISO and blood pressure (BP) were measured using MRBP rat tail cuff method blood pressure system by IITC Life Science, USA.

Biochemical estimations:

After 24 hour of final subcutaneous injection of ISO blood was collected by retro orbital puncture under ether anesthesia and allowed to clot for 30 min at room temperature. The serum was separated by centrifugation at 2500rpm at 30⁰C for 15 min and used for the estimation of marker enzymes viz., creatine kinase (CK-MB), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and creatine phosphokinase (CPK). Animals were sacrificed and hearts were dissected out immediately, washed with ice-cold saline and one half was used to prepare 10% (w/v) homogenates in phosphate buffer (50 mM, pH 7.4). The homogenates were centrifuged at 7000rpm for 10 min at 4⁰C and the supernatants were used for the assays of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH). The remaining half was fixed in 10% (w/v) buffered formalin and used for histological studies.

SOD estimation:

SOD was assayed by the method of Sun and Zigman (1978) in which the activity of SOD was inversely proportional to the concentration of its oxidation product adrenochrome, which was measured spectrophotometrically at 320nm. One unit of SOD activity is defined as enzyme concentration required to inhibit the rate of autooxidation of epinephrine by 50% in 1 min at pH 10.¹²

Catalase estimation:

CAT was estimated by the method of Clairborne (1991), which is a quantitative spectroscopic method developed for following the breakdown of H₂O₂ at 240nm in unit time for routine studies of CAT kinetics.¹³

Glutathione estimation:

GSH was estimated in the heart homogenate using DTNB by the method of Ellman (1959). The absorbance was read at 412 nm and the results were expressed as mmol of GSH/g of wet tissue.¹⁴

Histological studies:

The hearts stored in 10% buffered formalin, were embedded in paraffin, sections cut at 5mm and stained with hematoxylin and eosin. These sections were then examined under a light microscope for histoarchitectural changes.

Statistical analysis:

All the experimental results were expressed as mean ± SEM. One-way ANOVA followed by Tukey’s test using GraphPad InStat, version 5.0. The intergroup difference was considered significant when p<0.05.

RESULTS:

Effect of Terminalia arjuna and Ocimum sanctum combination on Mean arterial blood pressure (MAB) and Heart rate:

The MAB was significantly (P< 0.001) decreased in the ISO treated group to 48.12 mmHg compared to normal control group. MAB was increased significantly in animals treated with TAE (500mg/kg) + OSE (50 mg/kg) (P< 0.001). Whereas animals treated with TAE (250 mg/kg) + OSE (50mg/kg) and TAE (500mg/kg) showed significant (P< 0.01) increase in MAB compared to ISO treated group. Animals treated with TAE (250mg/kg) and OSE (50mg/kg) showed significant (P< 0.05) increase in MAB. Further, a significant (p < 0.001) increase in heart rate was observed in ISO treated control group compared to normal control group. Animals treated with drug combinations of TAE (500mg/kg) + OSE (50mg/kg) showed significant (P< 0.001) decrease in heart rate compared to ISO treated control group (Table 1).

Table 1: Effect of drug combinations on MAB, heart rate

Treatment	MAB (mmHg)	Heart rate
Distilled water (1ml/kg)	90.45±1.326	332.6±2.142
Isoproterenol	48.12±1.618^###	421.1±2.857^###
TAE (250 mg/kg)	66.24±1.231*	390.4±2.471*
TAE (500 mg/kg)	69.23±1.629**	378.2±1.374**
OSE (50 mg/kg)	65.39±3.648*	392.1±1.244*
TAE (250 mg/kg) + OSE (50 mg/kg)	74.67±2.124**	361.6±2.740**
TAE (500 mg/kg) + OSE (50 mg/kg)	79.63±2.132***	353.4±2.621***

The values are expressed as mean ± SEM (n=6) ^*P< 0.05, **P< 0.01, ^***P< 0.001 as compared to ISO treated group. ^#P<0.05,^##P<0.01, ^###P<0.001 values compared to control groups.

Effects of drug combinations on cardiac biomarker enzymes such as CKMB, CPK, LDH, AST and ALT are shown in Table 2. Levels of these enzymes were significantly (P< 0.001) increased in the animals treated with ISO compared to normal control group. Pre-treatment with drug combination TAE (500mg/kg) + OSE (50mg/kg) reduced significantly (P< 0.001) the activities of CKMB, CPK, LDH, AST and ALT. The administration of TAE (250mg/kg) + OSE (50mg/kg) combination and TAE (500mg/kg) alone also showed significant (P< 0.01) reduction in activities of CKMB, CPK, LDH, AST and ALT when compared to ISO control group.

Effect of different extract combinations on antioxidant parameters like SOD, CAT and GSH were depicted in the Table 3. ISO alone treated animals shown significant (P< 0.001) reduction in the antioxidants compared to normal control animals. Pre-treatment with TAE (500 mg/kg) + OSE (50 mg/kg) has significantly (P< 0.001) restored the antioxidants. Whereas, animals treated with TAE (250 mg/kg) + OSE (50 mg/kg) combination and TAE (500 mg/kg) alone showed significant (P< 0.01) increase in antioxidants. While animals treated with TAE (250 mg/kg) and OSE (50 mg/kg) showed significant (P< 0.05) increase in antioxidants when compared to ISO control group.

Table 2: Effect of drug combinations on cardiac biomarker enzymes

Treatment	CKMB (IU/L)	CPK (IU/L)	LDH (IU/L)	AST/SGOT (IU/L)	ALT/SGPT (IU/L)
Distilled water (1ml/kg)	405.8± 11.25	181.6±1.626	381.2±2.754	41.85±1.201	85.24±2.532
Isoproterenol	852.3± 8.65^###	475.4±3.777^###	714.5±4.247^###	82.11±2.147^###	191.4±1.845^###
TAE (250 mg/kg)	741±3.52*	401.6±2.951*	611.3±4.658*	65.21±3.247*	151.2±3.525*
TAE (500 mg/kg)	715±4.27**	375.3±1.243**	571.2±2.124**	58.63±3.657**	139.4±4.255**
OSE (50 mg/kg)	743.2 ±8.53 *	403.8±3.473*	619.6±7.504*	63.00±2.414*	148.7±2.003*
TAE (250 mg/kg) + OSE (50 g/kg)	705.1±3.41**	360.7±3.241**	567.1±3.352**	56.22±2.032**	131.2±2.417**
TAE (500 mg/kg) +OSE (50 mg/kg)	685.7±9.64***	306.4±2.114***	512.4±5.470***	51.01±1.200***	115.4±1.785***

The values are expressed as mean ± SEM (n=6) ^*P< 0.05, **P< 0.01, ^***P< 0.001 as compared to ISO treated group. ^#P<0.05,^##P<0.01, ^###P<0.001 values compared to control groups.

Histopathological examination

Histopathological results of heart tissue are shown in [Figure 1]. The heart tissue from normal control group animals [1A] showed normal uniform cardiac fibre arrangement with clear strips and no apparent degeneration or necrosis was observed. ISO treated rats [1B] showed extensive hypertrophy, necrosis of subendocardial region and fibroblastic hyperplasia along with increased edematous intramuscular gap. Animals administered with TAE (250mg/kg), TAE (500mg/kg), OSE (50mg/kg) and TAE (250mg/kg) + OSE (50 mg/kg) [1C, 1D, 1E, 1F] showed mild myocytic necrosis with mild lymphocytic infiltration along the endocardium. Whereas animals administered with TAE (500mg/kg) + OSE (50mg/kg) [1G] combination showed significant protection against ISO induced myocardial damage.

DISCUSSION:

Present study screened the effect of different herb-herb combinations on cardiac biomarkers in ISO-induced-cardiac toxicity in rats. Subcutaneous treatment of ISO (85 mg/kg) leads to an obvious toxicity as result of marked increase in CKMB, CPK LDH, AST and ALT in sera and also increase in heart rate and decrease in mean arterial blood pressure occurs in the ISO group. Isoproterenol is a β-adrenergic agonist that has acute positive inotropic and chronotropic effects on the heart which can induce necrosis, hypertrophy, inflammatory cell infiltration, and fibrosis when administered at toxic dose¹⁵. Literature study revealed that significant myocardial injury followed by ISO induction (85mg/kg) with decrease in antioxidant enzymes like SOD, CAT and GSH^16,17,18. When heart cells are damaged due to hypoxia, the permeability of cardiac membrane changes or may rupture entirely, result in the leakage of enzymes. Furthermore, the level of the enzymes in serum is reported to be proportional to the extent of necrosis¹⁹.

This study showed that pre-treatment with TAE (500 mg/kg) + OSE (50mg/kg) and TAE (250mg/kg) + OSE (50mg/kg) combinations to rats: before, simultaneously and after ISO administration have remarkably lower levels of these biomarkers due to ISO toxicity as reflected by significant decreases in serum level of all the biomarkers analyzed in this study. These results corroborate an earlier report that TAE and OSE pre-treatment attenuated ISO-induced oxidative myocardial necrosis in rats^10,20. The significant increased levels of myocardial cytosolic enzymes could be due to increased synthesis of free radicals during ISO metabolism. These free radicals may be elevated by calcium mediated proteases activities that could damage the cellular proteins and liberates cytosolic enzymes into the blood serum²¹. The antioxidant activity of TAE and OSE has been proven to ameliorate the cardiac toxicity through reducing the free radical mediated lipid peroxidation and release of cytosolic enzymes from heart tissue^22,23.

The activity of LDH, CPK and ALT enzymes are not only present in the myocardium, but are also present in the liver, skeletal muscle, and kidney, can shows the extent of a tissue damage²⁴. ALT is found in profound quantities in the liver; an increased release from the liver following hepatic ischemia secondary to cardiac abnormality²⁵. Due to the hepatic cellular injury and inflammatory changes results in increased serum concentrations of ALT in the hepatic tissues²⁶. In the present study, all these enzymes were elevated in the ISO treated animals than the animals treated with TAE, OSE and their combinations.

The histopathology study indicates substantial myocardial necrosis in the ISO treated animals while animals treated with TAE, OSE and their combinations appeared fairly normal. The control animal showed normal cardiac morphology. The results of this study further corroborate the enhanced cardioprotective potential of drug combination than extract used alone.

CONCLUSION:

From the present study it may be concluded that the herb-herb combinations i.e standardized extracts of Terminalia arjuna (Roxb) and Ocimum sanctum (Linn) have shown increased cardioprotective activity than they were used alone. However, further dose adjustment and molecular mechanism study need to perform for better understanding.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to the President, Soniya Education Trust and Principal, SET’s College of Pharmacy, Dharwad for encouragement, support and providing the necessary facilities to carry out the research work. We are also thankful to Sami Labs Limited, Bangalore, Karnataka for providing standardised plant extracts as gift samples.

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Received on 28.05.2021 Modified on 30.10.2021

Asian J. Pharm. Res. 2022; 12(1):19-23.

DOI: 10.52711/2231-5691.2022.00004

Treatment	SOD (unit/mg protein)	CAT (unit/mg protein)	GSH (μmol/g wet tissue)
Distilled water (1ml/kg)	11.47±0.0234	21.32±0.0461	6.123±0.0427
Isoproterenol	4.967±0.0476^###	7.741±0.0236^###	2.118±0.0326^###
TAE (250 mg/kg)	5.325±0.0236*	11.241±0.0452*	3.345±0.0154*
TAE (500 mg/kg)	6.962±0.0532**	13.524±0.0246**	3.785±0.0476**
OSE (50 mg/kg)	5.532±0.02741*	11.147±0.0764*	3.239±0.0232*
TAE (250 mg/kg) + OSE (50 mg/kg)	7.475±0.07536**	13.789±0.0325**	3.964±0.0644**
TAE (500 mg/kg) + OSE (50 mg/kg)	8.248±0.0414***	15.25±0.0414***	4.848±0.0833***