Cardioprotective activity of Limonia acidissima against Isoproterenol Induced Myocardial Infarction in Rats

Hasitha Diana Manohar¹, Srinivasan D¹, Sengottuvelu S^2*

¹Pharmacology Department, Karpaga Vinayaga Institute of Medical Sciences and Research Centre, Madurantagam, Kancheepuram District, Tamilnadu, India

²Department of Pharmacology, Nandha College of Pharmacy and Research Institute, Erode, Tamilnadu, India

*Corresponding Author E-mail: sengt@rediffmail.com

Received on 01.03.2016 Modified on 15.03.2016

Research J. Pharm. and Tech. 2016; 9(5): 541-544.

DOI: 10.5958/0974-360X.2016.00102.5

ABSTRACT:

The objective of the current study is evaluate the cardioprotective activity of ethanolic fruit extract of Limonia acidissima against isoproterenol induced myocardial infarction in rats. The protective activity of Limonia acidissima was assessed by estimating the cardiac marker enzymes (Creatinine Phosphokinase and Lactate Dehydrogenase) and antioxidants enzymes (superoxide dismutase and Catalase). Thirty male Sprague – Dawley rats (180 - 220 gm) were randomly allocated in 5 groups of 6 each. Group I served as control, II to V animals were Isoproterenol (85mg/kg) induced myocardiaol infracted animals, pre treated with the reference control Lipistat (350mg/kg) and ethanolic fruit extract of Limonia acidissima (200 and 400 mg/kg). All the test drugs were administered orally for 15 days. On 16th day the blood was collected and subjected to the estimation of cardiac marker enzymes. The animals were sacrificed; hearts were homogenized and used for the estimation of antioxidant enzymes. The result shows that, Limonia acidissima significantly decreased the cardiac marker enzyme and increased the antioxidant enzymes. Limonia acidissima produced a dose dependent cardioprotective effect against the acute cardiac damage induced by Isoproterenol in rats. From the result it was concluded that, Limonia acidissima produced cardioprotective activity against isoproterenol induced cardiac damage in rats which was mediated by its antioxidant property.

KEYWORDS: Isoproterenol, Myocardial Infarction, Limonia acidissima, Creatinine Phosphokinase, Lactate Dehydrogenase, superoxide dismutase and Catalase.

INTRODUCTION:

Myocardial infarction (MI) is the acute condition of necrosis of the myocardium that occurs as a result of imbalance between coronary blood supply and myocardial demand¹. MI remains the major cause of death in the developed world and is a major pathological issue Worldwide despite rapid advancements made in the treatment of coronary artery diseases². According to the World Health Organization, MI is predicted to be the major cause of death in the world by the year of 2020³.

It occurs as a result of increased myocardial metabolic demand and decreased supply of oxygen and nutrients via the coronary circulation to the myocardium, leading to cell injury; it is one of the most lethal manifestations of cardiovascular diseases⁴. Some of the synthetic drugs available in the market for protecting the heart but they have some side effects. Recent attention has been taken on herbal drug research because of its minimal side effects. Therefore, there is a need in the search for safe and more effective cardioprotective agents on herbal based drug. Limonia acidissima L. Swingle Syn. Feronia elephantum Correa, Schinus Limonia L. (Rutaceae), is a tropical plant species, indigenous to India and locally known as elephant apple. Limonia acidissima is native to India and also cultivated in Bangladesh, Pakistan and Srilanka. The wood-apple is native and common in dry plains. It prefers a monsoon climate with a distinct dry season. The tree grows up to an elevation of 450 m in the western Himalayas. It is apparently drought tolerant and best adapted to light soils⁵. All the parts of Limonia are prescribed in indigenous system of medicine for the treatment of various ailments. Fruits are refrigerant, stomachic, stimulant, astringent, aphrodisiac, diuretic, cardiotonic, tonic to liver and lungs, cures cough, hiccup and good for asthma, consumption, tumours, opthalmia and leucorrhoea⁶. Unripe fruit is astringent while seeds are used in heart diseases. The fruits are used as a substitute for bael (Eagle marmelos) in diarrhea and dysentery ⁷. The bark and leaves are used for vitiated conditions of vata and pitta⁸. Leaves are astringent and carminative, good for vomiting, indigestions, hiccup and dysentery. The leaves have hepatoprotective activity⁹. The unripe fruits contain stigmasterol. Fruit pulp contains large quantity of citric acid and other fruit acids, mucilage and minerals. Alkaloids, coumarins, fatty acids and sterols have been detected in the pericarp. It also contains umbelliferone, dictamnine, xanthotoxol, scoparone, xanthotoxin, isopimpinellin, isoimperatorin and marmin¹⁰. Wood apple fruit can be eaten plain or mixed into a variety of beverages and desserts, or preserved as jam. The scooped-out sticky pulp, is eaten raw with or without sugar, or is blended with coconut milk and palm-sugar syrup and frozen as an ice cream. In Indonesia, wood apple is mixed with honey and eaten in breakfast¹¹. Some of the ethnobotonical claims like antidiabetic¹², Anticancer¹³, antioxidant¹⁴, diuretic¹⁵, antihistamine¹⁶, Antihyperlipidemic¹⁷ activities of Limonia acidissima plant were documented. So far no evidence is available for the cardio protective activity of Limonia acidissima fruit. The present study was conducted to evaluate the cardioptotective activity of ethanolic fruit extract of Limonia acidissima against Isoproterenol induced myocardial infarction in rats.

MATERIALS AND METHODS:

Plant Material

The plant and fruit of Limonia acidissima, Linn. were collected from the outskirts of Salem, in the month of February. The plant samples were identified and authenticated by the Scientist D, Botanical Survey of India, Southern Regional Center, Agricultural University, Coimbatore, India. The voucher specimen (A/12786) has been deposited in Herbarium for further reference.

Preparation of Extract

The fruits were washed with water and chopped in to small pieces. The pieces were dried in sunlight for one hour and then it was dried under shade. By the help of grinder the dried fruits were powered to get coarse. Dried course powders of the fruits were extracted with 70% ethanol by cold maceration for 7 days. The extracts were then concentrated, dried and stored in desiccators.

Animals and Ethical Considerations

Healthy male Sprague–Dawley (SD) rats weighing between 180 - 220 gm were used for this study. The animals were obtained from animal house, Nandha College of Pharmacy, Erode. The animals were placed at random and allocated to treatment groups in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of 24±2^oC and relative humidity of 30 – 70 %. A 12:12 light: day cycle was followed. All animals were allowed to free access to water and fed with standard commercial pelleted rat chaw (M/s. Hindustan Lever Ltd, Mumbai). All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee (688/02/c/CPCSEA) and were in accordance with the Institutional ethical guidelines.

Pharmacological Activity

Cardioprotective Activity¹⁸

The rats were divided into 5 groups of 6 animals each. Group I served as normal control, administered with 1ml/kg of 0.1% CMC solution for 15 days. Group II served as induced control, administered with isoproterenol (85mg/kg, bw) intraperitoneally twice at an interval of 24 hours on 14^th and 15^th day. Group III served as reference control, administered orally with Lipistat (350 mg/kg, bw), once daily for 15 days. Group IV and V served as test controls, administered orally with 200 and 400 mg/kg, bw of ethanolic fruit extract of Limonia acidissima respectively. Group III, IV and V along with test drugs, the animals administered with isoproterenol (85mg/kg, bw) as mentioned earlier. All the test drugs were administered orally by suspending in 0.1% CMC solution.

On 16^th day blood was collected in heparinized tube by retro orbital sinus puncture, under thiopentone sodium anaesthesia. The collected blood samples were centrifuged for 10 minutes at 2000 r.p.m. and plasma was separated. The separated plasma was subjected to various biochemical tests like estimation of cardiac biomarkers Creatinine Phosphokinase (CPK)¹⁹ and Lactate Dehydrogenase (LDH)²⁰.

After blood collection, the animals were sacrificed by excess thiopentone sodium and heart tissue was quickly dissected out and washed in ice cold saline. A weighed quantity 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 superoxide dismutase (SOD)²¹ and catalase (CAT)²²

Table.No: 1. Effect of ethanolic fruit extract of Limonia acidissima against Isoproterenol induced myocardial infarction in rats

Groups	Drug Treatment	Plasma		Cardiac Tissue Homogenate
Groups	Drug Treatment	CPK (IU/L)	LDH (IU/L)	SOD (units/mg of protein)	CAT (units/mg of protein)
I	Vehicle Control 0.1% CMC	138.62±5.40	142.88±6.62	34.76±2.39	59.19±2.42
II	Isoproterenol (85 mg /kg)	265.86±9.43	211.53±8.55	18.24±1.03	32.86±1.11
III	Lipistat (350mg/kg) + Isoproterenol (85 mg /kg)	176.55±7.66***	163.52±9.20**	29.31±1.52**	50.45±2.35**
IV	Limonia acidissima (200mg/kg) + Isoproterenol (85 mg /kg)	201.39±8.07**	186.39±5.34*	24.57±2.05*	44.72±1.71**
V	Limonia acidissima (400mg/kg) + Isoproterenol (85 mg /kg)	182.97±9.61***	171.21±6.93*	27.32±1.15**	48.09±2.65**

Values are in mean ± SEM (n=6),

*P<0.05 , **P<0.01, ***P<0.001 Vs Isoproterenol Control

Statistical Analysis

Results were expressed as mean ± SEM. The data were analyzed by using one way analysis of variance (ANOVA) followed by Dunnet’s t test. P values < 0.05 were considered as significant.

RESULT AND DISCUSSION:

Cardioprotective activity of ethanolic fruit extract of Limonia acidissima against Isoproterenol induced myocardial infarction in SD rats was performed. Isoproterenol is a ß – adrenergic, synthetic catechlomine which induces severe stress in the cardiac muscle leading to Myocardial Infarction. Studies suggest that free radical plays a crucial role in the pathogenesis of Isoproterenol induced Myocardial Infarction. Myocardium contains an abundant concentration of diagnostic marker enzymes like CPK, LDH etc,. Once the muscle metabolically damaged, these enzymes released its content into the extra cellular fluid²⁰. In the present study, the cardioprotective effect of Lismonia acidissima, was assessed by determining the marker enzymes CPK, LDH and the levels of SOD and catalase. In Isoproterenol alone treated animals, the cardiac enzyme levels (CPK and LDH) were increased and the free radical enzymes (SOD and CAT) were decreased, when compared to the levels of normal control animals, which confirms the damage of cardiac tissues. Pretreatment of Lipistat, the reference control, significantly reduced the cardiac marker enzymes CPK (P<0.001) and LDH (P<0.01) and increased the levels of SOD and CAT ((P<0.01). The animals pretreated with 200 mg/kg of Limonia acidissima significantly decreased the cardiac marker enzyme CPK (P<0.01) and LDH (P<0.05) and increased the levels of SOD (P<0.05) and CAT (P<0.01). The animals pretreated with 400 mg/kg of Limonia acidissima significantly decreased the cardiac marker enzyme CPK (P<0.001) and LDH (P<0.05) and increased the antioxidant enzymes SOD and CAT (P<0.01). Limonia acidissima produced a dose dependent cardioprotective effect against the acute cardiac damage induced by Isoproterenol in rats.

CONCLUSION:

From the result, it was concluded that, the ethanolic fruit extract of Limonia acidissima exhibited cardioprotective activity against, Isoproterenol induced Myocardial infarction in rats. The probable mechanism of action of Limonia acidissima may be due to its antioxidant property. Further study is require in this plant to identify the active principle responsible for its cardioprotective activity.

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