CARDIOPROTECTIVE INTERACTION OF CUCUMIS MELO WITH ENALAPRIL IN ISOPROTERENOL INDUCED MYOCARDIAL DAMAGE IN RATS

a) BRIEF RESUME OF THE INTENDED WORK:

6.1 Need of study:

The use of complementary and alternative medicines is burgeoning globally, especially in developed countries including US1. It is interesting to note that herbs are often administered in combination with therapeutic drugs, raising the potential of herb-drug interactions2. Certain herbal supplements can cause potentially dangerous side effects when taken with prescription drugs and the number of cases reported for the emerging herb-drug interactions are already on the rise 3. Hence it is widely accepted that in-depth and appropriate studies on drug-herb interactions should be carried out to confirm the efficacy of combined drug-herb treatments.

Myocardial infarction is the commonest single cause of death in many parts of the world. Its therapy includes administration of thrombolytic agents, anticoagulants, -blockers etc., or surgical angioplasty or coronary bypass surgery4. It has been shown that cardiac renin angiotensin system (RAS) rather than the circulatory system may play an important role in myocardial infarction. Recent experimental and limited clinical trials strongly indicate a role for ACE inhibitors in limiting myocardial ischemia-reperfusion induced injury 5-8. Coronary vasodilation due to interference with kininase II or complex changes in systemic haemodynamics that reduce oxygen demand or inhibition of cardiac angiotensin II formation may account for cardioprotection5. In addition, altered prostaglandin production or oxygen free radical scavenging properties of ACE inhibitors have been postulated to reduce myocardial infarction5. One of the most commonly used ACE inhibitor is enalapril. Enalapril is used in the treatment of hypertension and some types of chronic heart failure. Enalapril was the first member of the group of ACE inhibitors known as the dicarboxylate-containing ACE inhibitors. Enalapril is a prodrug that is converted by deesterification to converting enzyme inhibitor, enalaprilat, with effects similar to those of captopril. Enalaprilat itself is available only for intravenous use, primarily for hypertensive emergencies. The consensus study of enalapril therapy in heart failure demonstrated that an ACE inhibitor could prolong survival in patients with advanced heart failure9. Further, reduction of reperfusion arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme inhibitors was also demonstrated 10.

Muskmelon (Cucumis melo) is a species of melonthat has been developed into many cultivated varieties. It is a native to central asia. It is grown in the tropicalregions for a very long time. Musk melon is widely known as 'Cantaloupe'. Musk melon being an annual plant is cultivated from the seeds. The plants have to be watered lightly. It is a monoecious plant where the male and the female flowers are distinct. The melons are easily grown in sandy soil. Dry river beds are the most suitable for melon cultivation. Unripe melons should be stored at room temperature until they ripen. The two principal varieties of muskmelon are those with netted skinsand those with smooth skins.The creamy flesh can be consumed chilled or as fruit juice. With scientific advancements the melon growers have introduced many hybrids which are much tastier. Melons make a good combination for custards and fruit salads. Musk Melon juice is beneficial to be consumed duringconditions like lack of appetite, weight loss, urinary tract infections, constipation, acidity, ulcer. Musk melon reduces heat in the body to a great extent, relieves tiredness, enhances appetite and is an effective laxative. It is a good source of Vitamins A, B, and C. Musk Melons are rich in potassium, a nutrient that may help control blood pressure, regulate heart beat, and possibly prevent strokes11. Even though muskmelon enjoys the great benefit of regulating heart beat and could be the possible candidate for cardioprotection, there is no scientific report available confirming its efficacy and also many dietary supplements and home remedies are used in conjunction with conventional drugs, hence our interest is to explore the possible cardioprotective potential of Cucumis melo and also to evaluate the pharmacodynamic interaction of Cucumis melo with conventional cardioprotective drug, enalapril in isoproterenol induced acute and chronic myocardial damage in rats.

6.2 Review of literature

One of the study was conducted to evaluate in vitro and in vivo the antioxidant and anti-inflammatory properties of a cantaloupe melon (Cucumis melo LC., Cucurbitaceae) extract (CME) selected for its high superoxide dismutase activity. Peritoneal macrophages were pre-activated in vitro with 300IU of interferon-γ (IFN-γ) and were then challenged in culture with IgGl/anti-IgG1 immune complexes (IgG1IC) in presence of various CME extracts. The subsequent production of free radicals (superoxide anion, nitric oxide, and peroxynitrite) and of pro-(TNF-α) and anti-(IL-10) inflammatory cytokines was evaluated. These data did not only indicate that the SOD activity is important for the antioxidant and anti-inflammatory properties of the CME extract, but also demonstrated that when the SOD activity is preserved during the digestive process by its combination with wheat gliadin it is possible to elicit in vivo the pharmacological effects of this antioxidant enzyme12.

Oil extracted from the seeds of Cucumis melovar. agrestis,collected from Ghibaish (sandy soil) and Gezira (heavy clay soil) provinces in Sudan, was studied in terms of the profile of fatty acids, tocopherols and sterols as well as phenolic compounds and oxidative stability by Rancimat (Metrohm AG, Herisau, Switzerland). The predominant fatty acid was 18:2n-6, representing 61.3 and 61.4% for Ghibaish and Gezira samples, respectively. There was little variability according to location between the two samples. Two samples from dried seeds of Cucumis melovar. agrestiswere collected. Seeds were crushed and ground using a grinding mill; the oil was extracted from the ground seeds by extraction with petroleum ether in a Soxhlet apparatus, and the physicochemical properties of C. melovar. agrestisoil were determined. The fatty acid and sterol composition were investigated using gas chromatography; the different tocopherol homologs were identified by high-performance liquid chromatography (HPLC), and the oil oxidative stability was studied by Rancimat (Metrohm AG, Herisau, Switzerland) apparatus. The phenolic compounds were extracted from the obtained oil and then identified by HPLC with diode-array detection13.

Muskmelons, both cantaloupe (Cucumis melo Reticulatus Group) and orange-fleshed honeydew (C. melo Inodorus Group), a cross between orange-fleshed cantaloupe and green-fleshed honeydew, are excellent sources of β-carotene. Although β-carotene from melon is an important dietary antioxidant and precursor of vitamin A, its bioaccessibility/bioavailability is unknown. Orange-fleshed honeydew melon fruit had higher amounts of β-carotene than cantaloupe. The bioaccessibility/bioavailability of β-carotene from orange-fleshed melons was comparable to that from carrot (Daucus carota) 14.

Among the major cucurbit vegetables, melon (Cucumis melo) has one of the greatest polymorphic fruit types and botanical varieties. Some melon fruits have excellent aroma, variety of flesh colors, deeper flavor, and more juice compared to other cucurbits. Despite numerous available melon cultivars, some of them are exceedingly susceptible to several diseases. The genetic background carrying the genes for tolerance and/or resistance for those diseases is found in wild melon landraces. Unfortunately, the commercial melon varieties are not able to produce viable hybrids when crossed with their wild melon counterparts. Plant tissue culture techniques are needed to surpass those genetic barriers. In vitro melon embryo rescue has played a main role to obtain viable hybrids originated from commercial versus wild melon crosses. In this chapter, an efficient and simple embryo rescue melon protocol is thoroughly described15.

6.3 Objective of study

The objective of the present research is to carry out cardioprotective interaction of Cucumis melowith enalapril in isoproterenol induced myocardial damage in rats.

SPECIFIC OBJECTIVES:

  • To collect and authenticate the cloves of Cucumis melo.
  • To prepare and carryout phytochemical evaluation of Cucumis melo homogenate.
  • To study the biochemical and antioxidant profile in serum and heart tissue homogenate upon chronic administration of Cucumis melo homogenate with or without enalapril.
  • To explore the role of enalapril in presence/absence of Cucumis melo during isoproterenol induced acute myocardial derangement in rats.
  • To study the benefits of adding Cucumis melo to cardioprotective therapy of enalapril in isoproterenol mediated chronic myocardial damage in rats.

b)MATERIALS AND METHODS:

7.1 Source of Data:

  • Data will be obtained from laboratory based studies by using Sprague dawley rats of either sex weighing between 150-200 gms maintained at room temperature having free access to food (std pellet diet), tap water ad libitum. These studies will be carried out in intact animal that will be supported by biochemical data and histopathological studies.

7.2 Method of Collection of Data:

Chemicals and reagents will be procured from standard companies. Isoproterenol induced myocardial damage will be used as model to evaluate cardioprotective efficacy. Suitable biochemical and histological investigation will be carried out in animal model. The data collected will be based on animal experimentation as per the parameters studied under each animal model.

Preparation of Cucumis melo homogenate

Cucumis melo bulbs will be purchased from the local fruit market. The fruit will be peeled, sliced and ground into a paste and suspended in distilled water. Two different concentrations of the Cucumis melo will be selected based on OECD guidelines16 and will be administered within 30 min of preparation.

Experimental protocol

The cardioprotective role of combined therapy of enalapril with Cucumis melo will be determined in isoproterenol induced ischemia-reperfusion rats. The Sprague dawley rats of either sex will be divided into following groups consisting of six animals each:

  • Group-I- animals kept as control without pretreatment
  • Group II- ISO control
  • Group-III- Cucumis melo (30 days oral treatment)
  • Group-IV- Cucumis melo (30 days oral treatment)
  • Group-V- Enalapril (10 mg/kg, orally for ten days)17
  • Group-VI- Cucumis melo (30 days oral treatment) + Enalapril (10 mg/kg, orally for ten days)
  • Group-VII- Cucumis melo (30 days oral treatment) + Enalapril (10 mg/kg, orally for ten days)

Isoproterenol induced chronic myocardial damage in rats:

During 30 days of Cucumis melo, ten days of enalapril and incorporation of enalapril in the last ten days of 30 days of Cucumis melo treatment in their respective groups, animals in all groups except group I will receive 3 mg/kg/day subcutaneously18. During treatment, haemodynamic parameters such as body weight, physiological changes and electrocardiographic observations will be noted. At the end of treatment, blood samples will be collected and serum will be separated. Heart tissue homogenate will be prepared in 0.25 M sucrose (10%) and both serum and heart tissue homogenate will be studied for CK-MB, LDH, SOD, Catalase, TBARS etc. Microscopic slides will be prepared to study histopathological and electron microscopic changes.

Isoproterenol (ISO) induced acute myocardial necrosis in rats19

At the end of treatment of animals as mentioned in experimental protocol, blood samples will be collected and serum will be separated. Influence of chronic therapy of Cucumis melo, in presence and absence of enalapril will be determined by estimating LDH, CK-MB and TBARS in serum. Subsequently, ISO (150 mg/kg, s.c) will be administered for two consecutive days. Symptoms and mortality in each group will be recorded and compared with those of the rats given ISO alone. After anesthetizing the rat with a combination of ketamine hydrochloride (75 mg/kg, i.p) and xylazine (8.0 mg/kg, i.p), leads will be attached to the dermal layer of both the front paws and hind legs and recording will be made on polygraph with the help of electrodes ECG system (subcutaneous lead II method). Blood samples will be collected and separated serum will be evaluated again for estimating LDH, CK-MB and TBARS20. Forty-eight hours after the first ISO administration, the rats will be sacrificed and autopsied. Three excised hearts will be homogenized to prepare heart tissue homogenate (HTH) using sucrose (0.25 M). The endogenous biological markers such as LDH, CK-MB and antioxidants (Superoxide dismutase and catalase) 21, 22 will be determined in heart tissue homogenate. Microscopic slides of remaining three hearts will be prepared for studying histopathological and electron microscopic studies.The myocardial damage based on histopathological and electron microscopic studies will be determined with the help of histopathologist in diagnostic centre by giving scores depending on the intensity as follows 23; no changes – score 00; mild – score 01 (focal myocytes damage or small multifocal degeneration with slight degree of inflammatory process); moderate – score 02 (extensive myofibrillar degeneration and/or diffuse inflammatory process); marked – score 03 (necrosis with diffuse inflammatory process).

Statistical analysis

The statistical significance will be assessed using one-way analysis of variance (ANOVA) followed by Dunnet comparison test. The values will be expressed as

mean ± SEM and p < 0.05 will be considered significant.

7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

YES, Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animals.

7.4 Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed.

c) List of References:

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