Download cardioprotective interaction of cucumis melo with enalapril in

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 infarction 5.
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 melon that has been developed into many cultivated
varieties. It is a native to central asia. It is grown in the tropical regions 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 skins and 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 during conditions 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 antiinflammatory properties of a cantaloupe melon (Cucumis melo LC., Cucurbitaceae) extract
(CME) selected for its high superoxide dismutase activity. Peritoneal macrophages were preactivated in vitro with 300 IU 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 melo var. 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 melo var.
agrestis were 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. melo var. agrestis oil 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 melo
with 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:
o Group-I- animals kept as control without pretreatment
o Group II- ISO control
o Group-III- Cucumis melo (30 days oral treatment)
o Group-IV- Cucumis melo (30 days oral treatment)
o Group-V- Enalapril (10 mg/kg, orally for ten days)17
o Group-VI- Cucumis melo (30 days oral treatment) + Enalapril (10 mg/kg, orally for ten
days)
o 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:
1.
Eisenberg DM, Davis RB, Ettner SL, Apple S, Wilkey S, Van Rompay M. Trends in
alternative medicine use in the United States JAMA 1990-1997;1998; 280:1569-75.
2.
Bruguera M, Barrera JM, Ampurdanes S, Forns X, Sanchez TJM. Use of
complementary and alternative medicine in patients with chronic hepatitis C. Med.
Clin. (Barc) 2004;122:334-35.
3.
Gohil K, Patel JA. Herb-drug interactions: A review and study based on assessment
of clinical case reports in literature. Ind. J. Pharmacol 2007;39:129-139.
4.
Chaudhuri SK. Quintessence of medical pharmacology. Ist ed. Calcutta; New Central
Book Agency;1997:329.
5.
Chopra K, Reddy DS, Singh M. Cardioprotective actions of ACE inhibitors on
ischemic myocardium: An overview. Indian Drugs 1996;32:464-70.
6.
Juggi JS, Koenig BE ,Van Gilst WH. Cardioprotection by ACE inhibitors. Can J
Cardiol 1993;9:336-52.
7.
Heuseh G, Rose J, Ehring T. Cardioprotection by ACE inhibitors in myocardial
ischemia – reperfusion. The importance of bradykinin. Drugs 1997;54:31-41.
8.
Remme WJ. Bradykinin - mediated cardiovascular protective actions of ACE
inhibitors. A new dimension in antiischemic therapy. Drugs 1997;54:59-70.
9.
The consensus Trial Study Group Effects of enalapril on
mortality in severe
congestive heart failure. Results of the Cooperative North Scandinavian Enalapril
Survival Study (consensus). N Engl J Med 1987;316:1429-35.
10.
Van Gilst WH, de Graeff PA, Wesseling H, de Langen CDJ. Reduction of reperfusion
arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme
inhibitors: a comparison of captopril, enalapril and HOE 498. J. cardiovasc. Pharmac
1986;8:722-28.
11.
Ioannis V, Dominique L, Caroline K, Philippe C, Alphonse C, Dominique M, et al.
Antioxidant and anti-inflammatory properties of a Cucumis melo LC. extract rich in
superoxide dismutase activity. Journal of Ethnopharmacology 2004;94:67-75.
12.
Abdalbasit M, Bertrand M, Bertrand M. Fatty acids, tocopherols, sterols, phenolic
profiles and oxidative stability of cucumis melo var. Agrestis oil. Journal of Food
Lipids 2008;15:56-67.
13.
Fleshman MK, Lester GE, Riedl KM, Kopec RE, Narayanasamy S, Curley RW, et al.
Carotene and Novel Apocarotenoid Concentrations in Orange-Fleshed Cucumis melo
Melons: Determinations of β-Carotene Bioaccessibility and Bioavailability. J Agric
Food Chem. 2011;59(9):4448-54.
14.
Nuñez-Palenius HG, Ramírez-Malagón R, Ochoa-Alejo N. Muskmelon embryo
rescue techniques using in vitro embryo culture. Methods Mol Biol 2011;710:107-15.
15.
http://www.healthtips.in/muskmelon.asp retrieved on 27/05/2011 at 10:50 am
16.
http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL420.pdf retreived
on 27/05/2011 at 11:00 am
17.
Yuejin Y, Pei Z, Laifeng S, Yingmao R, Xinlin X, Yongli L, et al. Comparison of
three doses of enalapril in preventing left ventricular remodeling after acute
myocardial infarction in the rat. Chin Med J 2002;115:347-51.
18.
Zhang G, Kimura S, Nishiyama A, Shokoji T, Rahman M, Yao L, et al. Cardiac
oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc Res
2005;65:230-8.
19.
Asdaq SM, Inamdar MN. Pharmacodynamic interaction of captopril with garlic in
isoproterenol induced myocardial damage in rat. Phytotherap Res 2010;24:720-5.
20.
Niehaus WG, Samuelsson B. Formation of malonaldehyde from phospholipid
arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-30.
21.
Erich F, Elastner M. Inhibition of nitrite formation from hydroxyl ammonium
chloride. A simple assay of super oxide dismutase. Anal. Chem 1976;70:616-20.
22.
Eva ML. Mechanism of pH dependent hydrogen peroxide cytotoxicity in vitro. Arch.
Biochem. Biophys 1998;365:362-72.
23.
Karthikeyan K, SaralaBai BR, Devaraj N. Cardioprotective effect of grape seed
proanthocyanidins on isoproterenol induced myocardial injury in rats. Int J Cardiol
2007;115:326-33.