Evaluation of Cardioprotective Potential of Hydroalcoholic Peel Extract of Citrullus Colocynthis


 Background and objective: Myocardial infarction is the number one cause of death both in males and females in many developed countries. Citrullus colocynthis is a medicinal plant used for the treatment for asthma, constipation, cough. The current study evaluates the cardioprotective potential of hydroalcoholic peel extract of Citrullus colocynthis against adrenaline-induced myocardial infarction in rabbits.Materials and Methods: Animals were divided into four major groups each group consisting of six rabbits. Group - 1 was given adrenaline 2mg/kg subcutaneously for two successive days. Considerably (p<0.001) accelerated the ALP, AST, ALT, Troponin, CK-MB, LDH, and CRP (p<0.05). Group 2, 3, 4 rabbits pre-treated with the peel extract of C. colocynthis 100mg/kg, 200mg/kg, 300mg/kg respectively orally for 14 successive days and on 14th and 15th-day adrenaline was inoculated considerably improve the impact of adrenaline through lowering the levels of CK-MB, LDH, Troponin, AST, CRP, ALT, and ALP.Results: The myocardial tissues of adrenaline induced group have more necrotic lesions. A slight deteriorating modifications of myocardial tissues usually in cardiac cells structure were observed in the group treated with extract.Conclusion: The out-turn showed the cardioprotective potential of hydroalcoholic peel extract of C. colocynthis in a dose-dependent manner.


Introduction
Myocardial infarction is the number one cause of death both in males and females in many developed countries. Because of the changing lifestyle patterns in many developing countries such as Pakistan, India, Bangladesh, and Afghanistan are rapidly catching up with this epidemic at an alarming rate (1). Though synthetic medicines are very useful in the treatment of cardiovascular ailments their use is restricted due to their harmful effects (1). Myocardial infarction is a serious ischemic situation in which extreme necrosis of myocardial tissue takes place. It happens because of the imbalance between coronary blood supply and myocardial oxygen requirement (2).
Adrenaline is a catecholamine synthesized through the adrenal medulla. Adrenaline is a nonselective agonist of all adrenergic receptors consisting of alpha-1, alpha-2, beta-1, beta-2, and beta-3, and various others. It causes myocardial infarction in a higher dose (3). The myocardial infarction is because of lipid peroxidation leading to depletion of cellular antioxidants. It causes the overproduction of nitrosative derivatives (4). Overproduction of ROS results in oxidative stress (3). This method is applied to induce the myocardial infarction in experimental animal.
The importance of herbal medicines in the treatment of various ailments is invaluable Globally. Herbal medicines continue to provide important therapeutic agents both in traditional and modern medicines and also because they are less toxic than synthetic medicines. Citrullus colocynthis is a medicinal plant that is from the family Cucurbitaceae. In Arab, it was used to deal with the skin eruption in camels and for insect bites (5). It has been stated that traditionally, it was used to treat asthma, cough, oedema, constipation (6). Its fruit is used as hepatoprotective (7), and its pulp and seeds as an anti-inflammatory (8), diabetes (9). Phytochemical screening of the plant displayed that it comprises phenolics, flavonoids, glycosides, alkaloids, and its leaves showed antioxidant activity (10). Till now there is no recorded experimental foundation for its utilization as cardiotprotection. The current study was performed to evaluate the cardioprotective potential of hydroalcoholic peel extract of C. colocynthis.

Plant material and extract preparation
Plants of C. colocynthis had been collected fresh from the nearby area of Multan. It was authenticated by the cooperation of an expert taxonomist at the Department of Botany, Bahauddin Zakariya University, Multan. The voucher specimen (R.R. Stewart F.W. Pak.702/10) has been placed for further reference. The fresh plant was subjected to shade dry. All the foreign adulterants and vegetative waste were eliminated through manual picking before grinding the peel part of the plant into a coarse powder with the assist of a special herbal grinder. The crushed plant powder is store in airtight jars waiting for extraction. The powder attained from only one batch was used in the experiment. 250g C. colocynthis peel powder was soaking in a hydroalcoholic solvent (70:30 v/v) for 9 days in 2.5L ambered colored air-tight jars. Then it was filtered and the filtrate was evaporated at 37 degrees centigrade under reduced pressure on a rotatory evaporator to obtain a thick paste-like consistency (11). The obtained extract of the peel of C. colocynthis was utilized for evaluating the adrenaline-induced cardioprotective activity.

Animals used in the Experiment
Male rabbits weighing 1.5 kg had been obtained from the nearby market of Multan. They had been nourished with standard commercially available food and tap water add libitum. The rabbits had been kept at the Department of Biochemistry, Muhammad Institute of Medical and Allied Sciences, Multan. The temperature was maintained at 25 degrees centigrade. The experiments were performed according to the rules of the National research council (12) and approved by the Ethical Committee of Muhammad Institute of Medical and Allied science Multan, Pakistan (03/pharma/times/NOV/19).

Chemicals
Adrenaline, Ethanol, Distilled water was purchased from M/s. Sigma Chemical Company, St. Louis. MO. All the other chemicals used in the experiment were of analytical grade. AST, ALP, ALT, LDH, CRP Kits obtained from Javed Pharmacy Multan.

Acute Oral toxicity Dose Test
The acute oral toxicity of the extract was evaluated in 12 rabbits. They were divided into 3 groups and each group contained 4 rabbits and they were kept on fasting for 24 hours and dosed in the following manner 350mg/kg, 550mg/kg, 2000mg/kg body weight. After the dosing, the rabbits had been noticed for 14 days for lethargy, Jerkiness, and death. The LD50 was calculated through graph pad prism version 7.3.

Induction of Myocardial Infarction
Adrenaline (2mg/kg) was administrated subcutaneously once daily for two successive days in rabbits (3)

Experimental design
The animals had been divided into 4 major groups each group contained 6 rabbits. Group, I rabbit administrated adrenaline 2mg/kg, subcutaneously at 24 hours of a gap for two successive days. Group II rabbits were pre-treated with 100mg/kg of extract for a time of fourteen successive days, and besides administrated adrenaline 2mg/kg, on the 14 th and 15 th day at a gap of 24 hrs. The rabbits of group 3 were pre-treated with 200mg/kg of extract for fourteen days consecutively and on the 14 th and 15 th -day adrenaline 2mg/kg was administrated subcutaneously at a gap of the 24 hrs. Group 4 rabbits were pre-treated with extract in an amount of 300 mg/kg for fourteen days successively and on the 14 th and 15 th -day adrenaline 2mg/kg was inoculated subcutaneously at an interval of 24 hours (1).
24 hours later, after the final dose of adrenaline rabbits had been anesthetized and the blood samples of rabbits had been taken from the marginal ear vein of rabbits to assess the biochemical parameters including CK-MB, LDH, Troponin. CRP, ALT, AST, ALP levels in serum were measured by commercially available Kits. Rabbits had been Killed under intense anesthesia and the heart was isolated and for histological examination, then the ventricular portion of the heart swiftly transferred to a 10 % formalin solution. After that tissue had been submerged in the paraffin. A 5μm thick segment had been cut and stained with a hematoxylin-eosin dye then mounted in the diphenyl xyline. The histopathology of heart tissue had been determined beneath a compound microscope and micro-images had been taken (2).

Statistical Analysis
The experimental statistics had been demonstrated as mean ± SD. Statistical analysis had been accomplished through one-way ANOVA. The statistical value with p˂0.05 was taken as significant.

Results
The acute oral toxicity test showed that any dose of hydroalcoholic peel extract of C. colocynthis up to 2,000 mg/kg did not yield any mortality.
Biochemical estimations showed a significant rise in the level of AST, ALP, ALT, CKMB, CRP, Troponin, LDH in adrenaline treated group in comparison with extract-treated Group 2, Group 3, Group 4. Table 1 shows the levels of different biochemical parameters in the heart tissues of adrenaline-induced and extract-treated groups. There was a highly significant (p<0.001) increased noticed in the levels of AST, ALT, ALP, CKMB, LDH, Troponin and a significant (p<0.05) increase in the level of CRP in adrenaline treated group 1. Group 2 rabbits pretreated with the extract 100mg/kg for 14 successive days showed a mild decrease in the levels of biochemical parameters. Group 3 rabbits pretreated with the extract 200mg/kg for 14 successive days showed a mild to moderate decrease in the biochemical parameters. Group 3 rabbits pretreated with extract 300mg/kg for 14 successive days significantly (p<0.001) prevented the effect of adrenaline and maintained the biochemical parameters at a normal level.

Histopathological observation of Heart tissues
Microscopic observation of the ventricular portion of the heart tissues from different groups were carried to assess the adrenaline effect injection on the cardiac cells structure as well as its variation by the drug test inoculation. Analysis of segments of heart tissues in Adrenaline induced group showed a remarkable change in cardiac cell structure. In adrenaline administrated group histological variations for example mononucleate cellular infiltration, mottled staining, interstitial edema, fragmentation and breakdown of muscle fiber, vacuolar disintegration, dilatation of capillaries, hemorrhage, and congestion of the myocardium were observed. Severe necrotic lesions were noticed in all the rabbits treated with adrenaline alone. Pant extract treated groups observed decreased histopathological variations in dose dependent fashion.

Discussions
Myocardial infarction is a serious ischemic situation in which extreme necrosis of myocardial tissue takes place. It happens because of the imbalance between coronary blood supply and myocardial oxygen requirement (2). Adrenaline is a catecholamine synthesized through the adrenal medulla. Adrenaline is a non-selective agonist of all the adrenergic receptors consisting of alpha-1, alpha-2, beta-1, beta-, beta-3, and various others. It causes myocardial infarction in a higher dose (3). The myocardial infarction is because of lipid peroxidation leading to depletion of cellular antioxidants. It causes the overproduction of nitrosative derivatives (4). Overproduction of ROS results in oxidative stress (3). It is applied as an approach to induce myocardial infarction in the experimental animals. The importance of herbal medicines in the treatment of various ailments is invaluable Globally. Herbal medicines continue to provide important therapeutic agents both in traditional and modern medicines and also because they are less toxic than synthetic medicines. C. colocyntis is a medicinal plant that is from the family Cucurbitaceae. In Arab, it was used to deal with the skin eruption in camels and for insect bites (5). It has been stated that traditionally, it was used to treat asthma, cough, oedema, constipation (6). Its fruit is used as hepatoprotective (7), and its pulp and seeds an anti-inflammatory (8), diabetes (13). Phytochemical screening of the plant displayed that it comprises phenolics, flavonoids, glycosides, alkaloids, and its leaves showed antioxidant activity because of the presence of phenolics (10). Till now, there is no recorded experimental foundation for its utilization as cardioprotection, the current study has been performed to evaluate the cardioprotective potential of hydroalcoholic peel extract of C. colocynthis. The heart comprises a rich concentration of diagnostic marker enzymes like LDH, CKMB, Troponin, and CRP and transaminases such as AST, ALT, ALP and once the heart is metabolically injured, it releases its content into Extra Cellular Fluid (ECF). In the current study, it was noted that in adrenaline myocardial infarcted rabbits, the enhanced activities of the serum marker enzymes accompanied by their associated reduction in the heart homogenate approve the beginning of myocardial necrosis. So, the whole concentration of the marker enzymes was found to be an increase in heart tissue of adrenaline-infracted rabbits as compared to Group 2, Group 3, and Group 4, which may be the reflection of consequences of cellular injury due to lipid peroxidation. Adrenaline is well known to generate free radicals and to stimulate lipid peroxidation, which may be a causative issue for the irreversible damage to the myocardium. In the current study AST, ALT, ALP, LDH, CKMB, CRP, and Troponin were increased significantly after the administration of adrenaline. Our result is in agreement with other investigators findings. Previously (14,15) found significantly elevated biochemical parameters after the inoculation of adrenaline. Our results are also according to (16) that showed a highly significant elevation in the cardiac biomarkers. Histological variation resembled with the rise in serum enzyme level. In this study, all the variations support the fact that adrenaline produced intense necrosis of the myocardium in the doses used in the current experiment. In the current study pretreatment with the extract 300mg/kg in adrenaline-treated rabbits caused a significant decrease in the myocardium. This result is similar to other investigators. The result of this stud indicates that adrenaline administration induced myocardial damages evidenced by increase levels of as well as histomorphological changes in the myocardium which was associated with free radical production as shown in myocardial tissue. C. colocynthis afforded cardioprotection by reducing oxidative stress-induced in experimental myocardial infarction through prevention of free radical-mediated injury of catecholamine assault.

Conclusion
Hydroalcoholic peel extract of C. colocynthis showed cardioprotective potential in experimentally induced myocardial infarction in rabbits that is shown by the amelioration of histological variations as well as by the estimation of different biochemical and inflammatory markers of the cardiac tissue injury.

Ethics approval and consent to participate
The experiments were performed according to the rules of the National research council and approved by the Ethical Committee of Muhammad Institute of Medical and Allied science Multan, Pakistan (03/pharma/times/NOV/19).