Evaluation of Cardio Protective Activity of Musa Acuminata in Ischemia-Reperfusion Induced Myocardial Infarction in Rats

INTRODUCTION

Heart ailments are the significant reasons for a large portion of the mortalities worldwide. World Health Organization (WHO) depicted coronary illness as the best scourge. In India, heart illness has turned into the third most prominent executioner after diabetics and malignant growth.¹

As per the World Health Report, cardiovascular diseases (CVD) will be the biggest cause of death in India by 2020. 17 million deaths were estimated worldwide in 2017 due to CVD, it incorporates congestive cardiovascular breakdown, heart failure and so forth, caused because of the demise (ischemia) of cardiovascular tissue.²

Myocardial damage that has created through a time of ischemia-reperfusion may have numerous causes. Before, most research has focused on the components causing cell damage during ischemia and on defensive techniques intended to decrease the advancement of ischemic damage. Potential reasons for damage that create during reperfusion have been hard to break down, as these must be separated from ischemic causes. Ischemia typically advances from hypoxia (decreased oxygen supply) and results in a condition in which the heart can’t keep up its pace of cell oxidation promoting metabolic irregular characteristics. Following aggregate or incomplete impediment of the coronary course, metabolic and practical changes are started inside a second. These progressions are at first of a reversible sort; be that as it may, if oxygen is denied for an all-inclusive timeframe, these progressions logically become increasingly extreme, prompting tissue harm and in the end irreversible damage. On the other hand, if the stock of blood is immediately reestablished, reaccessibility of oxygen advances the re-foundation of typical high-impact digestion. Moreover, the seriousness and movement of ischemia aren’t exclusively controlled by the degree of oxygen diminished, yet by numerous different components including the overall amassing of poisonous metabolites. The decrease in blood supply during ischemia likewise restricts the evacuation of these metabolites further adding to the extreme metabolic damage.^3,4

Myocardial ischemia and the resultant metabolic changes progressions are connected to different phases of cell harm including the oxidation of proteins, loss of chemicals, cell layer disturbance, mitochondrial and myofibril ultrastructural changes, discouraged contractile capacity and eventually cell passing.⁵

Remedial intercessions for patients with the ischemic coronary illness include the reviving of blocked coronary supply routes. Impediment might be counteracted pharmacologically with hostile to hypertensive or against cholesterol operators or if impediment doesnot occur it is dealt with pharmacologically with enemies of thrombolytics, vasodilators, inotropic specialists, and in increasingly extreme cases with obtrusive strategies. Notably, the rescue of ischemic tissue is best when intercessions are started at the earliest opportunity after vessel impediment, bringing about the reclamation of the bloodstream (reperfusion) to the influenced myocardium causing the myocardium damage. Reperfusion damage is phone harm and brokenness that happens during the re-foundation of the bloodstream, and may perhaps prompt deadly arrhythmias and the demise of cells that were debilitated yet possibly work before reflow.⁶

MATERIAL AND METHODS

Plant Materials

Collection and authentication of plant:

Musa acuminata L. was gathered from Botanical nursery, Bengaluru and was confirmed by Botanist Prof R Nijagunaiah, Department of Botany, Bengaluru University.

Extraction of plant:

Musa acuminata L. strips were isolated from the natural product, and shade dried. At that point the strips were coarsely powdered with a processor. Powdered strips of Musa acuminata L. were exposed to soxhlet extraction. 100 g of strip powder was broken down in 300 ml of dissolvable (70% ethanol). The concentrate acquired after 4-5 h of extraction was kept for vanishing in porcelain plate.⁷

Phytochemical screening:

The ethanol unrefined concentrate (1 g) was totally broken up in 100 ml of its own mom dissolvable (ethanol) to set up the stock arrangement, and stock arrangement was utilized for the phytochemical screening by adhering to the technique referenced in standard pharmacognosy book.⁸

Total Flavonoid Content

Dried concentrates (10 mg) was broken down in 10 ml of 70% methanol. Concentrate arrangement (1000 µl) was blended with 1 ml of 2% aluminum chloride and 6 ml of 5% potassium acetic acid derivation. At that point the blends were hatching at room temperature for 40 m. The absorbance of the response blend was estimated at 415 nm versus the readied clear utilizing spectrophotometer. All out flavonoids were communicated in mg of quercitin proportionate per gram of the dry plant extricate (mg QE/g).⁹

Experimental animals:

The examination was done by rules of Committee with the end goal of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and convention was affirmed by the Institutional Animal Ethical Committee (IAEC) of Acharya and B. M. Reddy College of Pharmacy with Ref.: IAEC/ABMRCP/2016-2017/13.

Wistar rats weighing 150-200 g were chosen for screening cardio defensive action. The rats were housed in a polypropylene confine, kept up at temperature 25 ± 2 °C under 12 h light and dull conditions and provided with standard eating regimen and water not indispensable, from multi week prior and till the consummation of the examination.

Induction of myocardial infarction:

Rodents were anesthetized with thiopental sodium (40 mg/kg, i.p.) at that point the trachea was opened with a ventral midline cut and intubated through a tracheotomy and ventilate with room air by a techno positive weight respirator. The body temperature was screen and keep up at 37 °C all through the trial convention. A left thoracotomy and pericardiotomy were performed, trailed by distinguishing the left front plummeting coronary corridor. A silk string was passed behind corridor and was impeded by a bunch for 30 m. The silk string was expelled after 30 m with the assistance of two bunch releasers to permit reperfusion of heart for 4h. Where, as the trick control gathering exposed to whole surgery and string go underneath coronary course, however the coronary supply route was not ligated.

In the typical control gathering and I/R gathering, saline was directed orally for 21 days before ischemic reperfusion and for treatment bunches creatures were pretreated with Musa acuminata L., strip remove at the dosages of 200 and 400 mg/ kg orally for 21 days before ischemic reperfusion. Past research demonstrated no critical sex contrasts in infarct size in charge or treated rodents; hence, male and female qualities were joined in the present examination.¹⁰

Experimental design:

Rats were randomly divided into four groups each consisting of EIGHT animals.

Group 1: Sham group-Control group (without I/ R)

Group 2: Rats were received 0.2 ml of saline and served as a control subjected to ischemia reperfusion (with I/ R).

Group 3: Rats were received Musa acuminata L. peel extract (200 mg/kg) for 21 days before ischemic reperfusion.

Group 4: Rats were received Musa acuminata L. peel extract (400 mg/kg) for 21 days before ischemic reperfusion.

Animals of all groups were used to carry out Cardio protective activity by collection of blood, sacrification and isolation of the heart tissue, which were further used to carry out bio-chemical estimations and histopathological studies.

Preparation of Tissue Homogenate

The heart was isolated, washed promptly in ice-chilled physiological saline, smudged and weight was recorded. 0.5 g of the heart tissue was homogenized in 0.1 M Tris-HCl cushion (pH 7.4) arrangement. The homogenate was centrifuged at 3000 rpm for 5 m and the supernatant was gathered. Further serum was isolated from blood tests by centrifugation at 3000 rpm for 10 m and the supernatant was gathered.

To quantify infarct size:

In every one of the gatherings, in the wake of relinquishing the creature, the heart was extracted from the thorax and the more noteworthy vessels were expelled. The left ventricle was isolated from the heart and was gauged. It was cut parallel to the atrioventricular furrow to 0.1 cm thick areas and the cuts were hatched in 1% TTC arrangement arranged in phosphate support pH 7.4 for 30 m at 37 °C. In practical myocardium TTC is changed over by dehydrogenase catalysts to a red formazan color that stains tissue dim. The infarcted myocardium that did not take TTC recolor where the dehydrogenase compounds were depleted off stays pale in shading. The pale necrotic tissue was isolated from the recolored parcels and burdened an electronic equalization. Myocardial infarct size was communicated quantitatively as far as percent left ventricle rot (PLVN).¹⁰

Serum parameters:

Aspartate Aminotransferase (AST/GOT)

Serum AST/ GOT was estimated with the help of clinical semi-auto analyzer and the diagnostic kit. Working reagent was set up in the extent of 4:1 proportion of reagent and reagent B. 100 μl of serum test was added to 1000 μl of working reagents and blended. After 1 min, absorbance was recorded.¹¹

Alanine Aminotransferase (ALT/GPT)

Working reagent was set up in the extent of 4:1 proportion of reagent and reagent B. 100 μl of serum test was added to 1000 μl of working reagents and blended. After 1 min, absorbance was recorded.¹¹

Assay of creatinine kinase (CK).

This procedure involves measurement of Creatinine Kinase (CK) activity in the presence of an antibody to Ck-M monomer. This antibody completely inhibits the activity of CK-MM and half of the activity of CKMB while not affecting the B subunit activity of CKMB and CK-BB.¹²

Assay of lactate dehydrogenase (LDH).

1000 µl was mixed to 25 µl of sample in effendroff’s tube. This mixture was aspirated and absorbance was directly recorded at 405 nm by using Auto Analyzers BTS-350.¹³

Antioxidant profile

Estimation of superoxide dismutase (SOD):

Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. The principle of this method is the inhibition of reduction of nitrobluetetrazolium to blue colouredtetrazolium in presence of phenazinemethosulphate and NADH by superoxide dismutase enzyme. The colour intensity was measured at 560 nm.¹⁴

Estimation of Catalase (CAT):

Catalase measurement was carried out by the ability of CAT to oxidize hydrogen peroxide (H₂ O₂ ). Decomposition of H₂ O₂ gives water and oxygen. The UV light absorption of hydrogen peroxide solution can be easily measured between 230 to 250 nm. On decomposition of hydrogen peroxide by catalase, the absorption decreases its time. The enzyme activity could be arrived at this decrease. But this method is applicable only with enzyme solution which do not absorb strongly at 230-250 nm.¹⁵

Estimation of reduced Glutathione

Glutathione (GSH) is a tripeptide. It helps protect cells from reactive oxygen species such as free radicals and peroxides. The thiol group present in GSH will react with 5,5`- dithio bis-2-nitrobenzoic acid (DTNB) to form mercapto benzoic acid which gives yellow colour to the solution. The colour intensity was measured at 412 nm.¹⁶

Estimation of Glutathione Peroxidase

Glutathione peroxidase is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. The biological function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water.¹⁷

Estimation of Malonaldehyde (MDA) in tissue

The generated oxygen derived free radicals react with membrane lipids to form lipid Peroxidise which were heated with TBA in the presence of acetic acid. The resulting Chromogen is extracted with a mixture of n-butanol and pyridine and the absorbance of organic phase is determined at a wavelength of 532 nm.¹⁸

Estimation of MPO

Myeloperoxidase (MPO) was estimated by the strategy for Mullane et al., Heart tissue was homogenized with homogenizer in volume of 1/10 of the heart weight with super cold phosphate cradle containing 0.5% hexadecyl trimethyl ammonium bromide. The homogenate was centrifuged at 8000 rpm for 30 m at 4 °C. 40 µl supernatant was added to 960 µl of phosphate cradle containing o-dianisidine dihydrochloride (0.167 mg/ml) and hydrogen peroxide (0.0005%) arrangement and shaken overwhelmingly. The adjustment in the absorbance was estimated at 460 nm for 3 m at an interim of 60 s. One unit of movement was characterized as the measure of MPO that causes an adjustment in absorbance estimated at 460 nm for 3 m. MPO movement information are introduced as units/ ml.¹⁹

Statistical analysis

Results were communicated as mean ± SD. Contrasts in cardiovascular markers (CK, LDH, AST, and ALT) and tissue lipid peroxide levels, SOD, CAT, GSH, GPx, MPO and MDA were controlled by One-Way Analysis of Variance (ANOVA). Individual gatherings were looked at utilizing Tukey’s test. Contrasts were viewed as factually critical p <0.05.

RESULT

Phytochemical Screening: The ethanol extract of Musa acuminata L.peel. shows the presence of Flavonoids, Tannins, Alkaloids, Saponins, Phytosterols, Phenols and Glycosides. The total falvonoid content in the extract was found to be 0.3594 mg QE/g.

Effect of ethanol extract of Musa acuminate L. peel on serum CK and LDH levels in normal and I/R induced myocardial infarction in rats.

Experimental results showed that compared with sham group, serum CK-MB and LDH levels were significantly increased in I/R control group, (P<0.05). Compared with control group, serum CK-MB and LDH levels were all decreased in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg (P<0.05).

All values are expressed as Mean ± SD (n=6), where “***” represents p<0.001 compared I/R group, where “#” represents p<0.05 compared to test 1, where “**” represents p<0.01 compared to I/R group using one-way ANOVA followed by TUKEY’S post hoc test.

Effect of ethanol extract of Musa acuminate L. peel on serum SGOT (AST) and SGPT (ALT) levels in normal and I/R induced myocardial infarction in rats.

Experimental results showed that compared with sham group, tissue SGOT and SGPT levels were significantly increased in I/R control group (P<0.05) and significantly decreased in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg(P<0.05), compared to control group.

All values are expressed as Mean ± SD (n=6), where “***” represents p<0.001 compared I/R group, using one-way ANOVA followed by TUKEY’S post hoc test.

Effect of ethanol extract of Musa acuminate L. peel on tissue antioxidant enzymes in I/R induced myocardial infarction in rats.

Experimental results showed that compared with sham group, tissue SOD, CAT, GSH levels were significantly decreased in I/R control group (P<0.05) and significantly increased in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg(P<0.05), compared to control group.

Experimental results showed that compared with sham group, tissue and serum MDA and MPO levels were significantly increased in I/R control group (P<0.05). Compared with control group, tissue and serum MDA and MPO levels were all decreased in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg(P<0.05), in compared to control group.

Experimental results also showed that compared with sham group, tissue GPx levels were significantly decreased in control group (P<0.05) and significantly increased in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg(P<0.05), compared to control group.

All values are expressed as Mean ± SD (n=6), where “***” represents p<0.001 compared I/R group, where “**” represents p<0.01 compared to I/R group, where “*” represents p<0.05 compared to I/R groupusing one-way ANOVA followed by TUKEY’S post hoc test.

Myocardial infarct size:

The myocardial infarct size of I/R control group was significantly higher than that in the sham group (P<0.05). Compared with the I/R control group, the myocardial infarct size in Musa acuminata L. peel extract treated group with 200 and 400 mg/kg(P<0.05), were significantly decreased (P <0.05).

All values are expressed as Mean ± SD (n=6), where “*** “represents p<0.001 compared I/R group, using one-way ANOVA followed by TUKEY’S post hoc test.

All values are expressed as Mean ± SD (n=6), where “***” represents p<0.001 compared I/R group, using one-way ANOVA followed by TUKEY’S post hoc test.

Histopathological studies:

The histopathological observations revealed that ethanol extract of Musa acuminate L. peel at higher dose (400 mg/kg) showed better cardioprotective response against I/R induced myocardial infarction in rats.

Discussion

In an acute model of cardiac ischemia-reperfusion injury, we performed transient ligation of the left coronary artery in rat model in order to obtain results closer to the clinical situation in which almost all patients with myocardial infarction are reperfused. Pre treatment for 21 days with ethanol extract of Musa acuminate L. peel before ischemiareperfusion has shown significant reduction in infarct size. Studies suggest that there is a direct relationship between mortality and the extent of infarct size, because the myocardium does not have the ability to regenerate its tissue.²⁰

Flavonoids and phenolic compounds derived from different plant species are found to be potential therapeutic agents in the prevention and management of cardiovascular diseases due to their antioxidant nature. Cardioprotective effects of ethanol extract of Musa acuminate L. may be attributed to its anti-oxidant, ROS scavenging, anti-apoptotic, and anti-inflammatory activities. Reperfusion of the ischemic myocardium results in cardiomyocyte apoptosis and heart dysfunction.Reperfusion of the ischemic myocardium results in cardiomyocyte apoptosis and heart dysfunction.^21,22

In the present investigation, we found that there is a critical height in serum lactate dehydrogenase (LDH) and creatine kinase (CK) affirming the intense myocardial dead tissue in rodents. The myocardial cells contain numerous cardiovascular compounds like creatinine kinase, lactate dehydrogenase, asparate transaminase and so forth, upon left plunging coronary conduit ligation, the oxygen request of the heart expanded with increment in inotropic impact in the heart, bringing about delayed ischemia. The cells are harmed with expanded muscle contractility, which results in expanded cell layer penetrability enabling cardiovascular chemicals to hole out into the bloodstream.2³ Creatine kinase is a compound prepared to do reversibly moving a phosphate bunch from the vitality stockpiling type of creatine phosphate, to an atom of ADP, delivering ATP. CK is confined prevalently in the heart and this makes it an important symptomatic instrument for MI since harm explicit to the myocardium would bring about height of CK levels.^24,25 LDH has been utilized generally as a vague analytic instrument for myocardial localized necrosis. An ascent in the extent of LDH in the serum can be indicative guide of myocardial localized necrosis. Upon treatment with ethanol concentrate of Musa acuminata L. strip there is a critical diminished degree of CK-MB and LDH.

Bioflavonoids have been reported to have antioxidant activity and also to possess cardioprotective potential.²⁶ Because oxidative damage is a potential culprit in the injury of viable myocardium, it would be beneficial to limit oxidative damage to a minimum and reinforce the antioxidant defense mechanism by supplementing antioxidants. Indeed, several clinical studies and experimental studies reported that the administration of antioxidants resulted in beneficial outcomes in I/R injury. The increased generation of reactive oxygen species or depletion of the antioxidants in the defense system may contribute to oxidative stress and affect the pathogenesis of myocardial infarction. Free radical scavenging enzymes such as superoxide dismutase, glutathione peroxidase and catalase are the first line cellular defense against oxidative stress, eliminating reactive oxygen radical such as superoxide (•O2−) and hydrogen peroxide (H2O2) and preventing the formation of more reactive radical of hydroxyl radical (•OH).27 Treatment with ethanol extract of Musa acuminate L. peel increased the endogenous antioxidant enzyme SOD, CAT, GPx, GSH levels in I/R injured rats. Epidemiological studies, suggest an inverse correlation between the severity of oxidative stress-induced diseases and the levels of anti-oxidants. Therefore, one of the mechanisms of the cardio protection of Ethanol extract of Musa acuminata L. peel is associated with antioxidant effect.

Lipid peroxidation is an important pathogenic event in myocardial infarction, and the accumulation of lipid peroxides reflects the various stages of this disease and its complications. Because MDA is the endproduct of lipid peroxidation, the results clearly indicate the cytotoxic free radical activity on the loss of membrane integrity with disintegration of polyunsaturated fatty acids in the membrane bilayer and exert unfavourable effects on the heart structure and function.28 Treatment with ethanol extract of Musa acuminate L. peel significantly reduced the elevated serum and tissue lipid peroxides levels in rats exposed to I/R injury.

Histopathological examination revealed that treatment with ethanol extract of Musa acuminate L. peel (200, 400 mg/kg) shown strong protection of myocardial cells from cellular tissue injury produced with ischemia-reperfusion.in compared to control group.

The therapeutic efficacy of ethanol extract of Musa acuminate L. peel may be due to its antioxidant, anti-lipidperoxidative, anti-apoptotic and anti-inflammatory property that could have prevented ischemia-reperfusion induced injury. Thus, on hypothesis suggested that ethanol extract of Musa acuminate L. peel shown significant cardio protective action.

CONCLUSION

The ethanolic extract of Musa acuminata L. peel showed significant cardio protective effect against ischemia reperfusion induced myocardial infarction in rats.

Acknowledgement:

We are thankful to Management, Principal, Acharya and BM Reddy College of Pharmacy, Bangalore for necessary support provided to carry out this research activity.

Conflict of interest: Nil