Abstract: Cardiotonic effect mediated by Desmodium gangeticum (DG) chloroform root extract was determined in frog and assessed in isolated rat heart. Ischemia reperfusion injury was experimentally induced in rat by using Langendroff apparatus and was perfused with Kreb Hanseleit buffer through aorta. Ischemia-reperfusion injury was induced by 30 min of global ischemia and 45 min of reperfusion in isolated rat hearts. Heart rate, coronary flow and Left Ventricular Pressure (LVP) were recorded and the activity of lactate dehydrogenase in coronary effluent and creatine kinase and lactate dehydrogenase contents in myocardial tissues were measured. Administration of DG root chloroform extract (20 mg kg-1 b.wt.) before global ischemia caused a significant improvement of cardiac function and a decrease in the release of lactate dehydrogenase in coronary effluent. Kymogram result in isolated frog heart showed that DG root extract possess positive inotropic effect. GSMS and atomic absorption analysis of DG root extract confirm the presence of bio-molecules that can stimulate the release of calcium in heart. From the above observations of present study, we can conclude that DG chloroform root extract can protect the myocardium against the damages induced by ischemia reperfusion in rats and the effect of the extract may be related to calcium releasing property.
INTRODUCTION
Myocardium calcium overload is one of the patho-physiological consequences of cardiac injury mediated by reperfusion of ischemic myocardium. In cardiac tissue, the most important regulator of Ca2+ homeostasis is Sarcoplasmic Reticulum (SR), which serves as a sink for Ca2+ ions during relaxation and as a Ca2+ source during contraction (Lamb, 2009). It is well known that SR Ca2+-ATPase transports Ca2+ from the cytosol to the lumen of the SR at the cost of ATP and was depressed during ischemia reperfusion (Solaro and Arteaga, 2007).
Arrhythmogenesis, one of the clinical presentation of ischemia reperfusion injury (IRI), may be due to intracellular Na+ and Ca2+ loading (Huang et al., 2006). In fact, reperfusion of the ischemic myocardium results in an increase in intracellular Na+ and Ca2+ (Baczko et al., 2008). The exact mechanism for Ca2+ influx into the myocardial cells remains unsolved; it is, however, generally thought that both Ca2+ slow channels and Na+-Ca2+ exchangers play important roles in the Ca2+ overload during the myocardial ischemia and reperfusion (Satoh et al., 2000). Even the inhibitors of Na+-Ca2+ exchange system such as nickel was also found to be beneficial for the functional recovery of myocardium from the insult of ischemia reperfusion (Han et al., 2002). Indeed, divalent and trivalent cations have been shown to inhibit Ca2+ transport by Na+/Ca2+ exchanger in a concentration-dependent manner (Iwamoto and Shigekawa, 1998). Moreover, Mg2+ inhibits Na+ dependent Ca2+ uptake by competing with Ca2+ for the external exchanger site in cardiomyocytes and smooth muscle cells (Smith et al., 2001).
Despite of having ample experimental proof to reduce calcium overload in the myocardium, none of the agents were found to be effective in ameliorating IRI. Thus the search for new therapeutic agents continues.
A very few comparable studies investigated the possible role of herbal extract that modulate the Na+- and Ca2+-loading in ischemia induced reperfusion. We, therefore, decided to expand the above observations, using isolated rat model, to examine the possible role of herbal extract that can induce favorable cardiotonic effect and thereby act as possible agent against IRI. Desmodium gangeticum (DG) (Leguminosae) is absolutely found in India and is one of the important plants used in indigenous system of Indian medicine. Our laboratory previously reported the cardio protective effect of Desmodium gangeticum root methanol extract (muscarnic receptor agonist) in isolated rat heart as post conditioning agent (Kurian et al., 2008). But the negative inotropic response of the extract limited its use as a precondition mimetic drug. However our preliminary study with chloroform extract of DG root suggested using the extract as preconditioning mimetic drug. In this study, we explore the cardiotonic effect of DG root chloroform extract and its subsequent use as therapeutic agent against myocardial ischemia reperfusion.
MATERIALS AND METHODS
Preparation of Chloroform Root Extracts of Desmodium gangeticum
The plant, after collection from the herbal garden (September 2008)
was washed, cleaned and maintained in the Biochemistry department at SASTRA
University, Tamil Nadu, India. The voucher specimen A/C No. 3908 was retained
in our laboratory for future reference. The dried roots were then milled to
fine powder (10 kg) and extracted with chloroform in Soxhlet's apparatus for
24 h and the extract was evaporated to dryness under vacuum and dried in vacuum
desiccators (815.8 g). The residue thus obtained was used for the experiments.
Chemicals
DL isocitrate and N-Phenyl-P-Phenylenediamine were purchased from Acros
organics, New Jersy USA. Cytochrome C and ATP were purchased from sigma chemical
Co., St. Louis, MO USA. All other chemicals used were of analytical grade.
Animals
Adult male albino rats of the Wistar strain, weighing approximately 250-280
g were obtained from King Institute of Preventive Medicine, Chennai, India.
They were acclimatized to animal - house conditions and were fed commercial
pelleted rat chow (Hindustan Lever Ltd., Bangalore, India) and had free access
to water (ethically approved by Ministry of Social Justices and Empowerment
Government of India). The experimental protocol was approved by the institutional
animal ethical committee.
Animals for Experiment I and II
Male Wistar albino rats (150 to 200 g) and frogs of Rana hexadactyla
species were housed in cages and were maintained in controlled temperature at
23±2°C with 12 h light/dark cycle. The animals were fed with food
and water ad libitum. The animals were maintained as per the norms of
CPCSEA and the experiments were cleared by CPCSEA and the local ethics committee.
Protocol: Experiment I
Frog Heart in situ Preparation
Frog hearts were isolated from specimens of Rana hexadactyla (weighing
22.015±1.2 g (Mean±SE)) and connected to a perfusion apparatus
as previously described. Experiments were done at room temperature (18-21°C).
The hearts were perfused with frog-Ringer solution containing NaCl 6.5 g, KCl
0.14 g, CaCl 0.12 g and NaHCO2 0.2 g, NaH2PO4
0.01 g, Glucose 2.0 g in g L-1. The force of contraction was recorded
and the rate of contraction was counted and tabulated.
Protocol: Experiment II
The rats were divided into four groups (n = 6 in each group): group I, control;
group 2, ischemic control; group 3, reperfusion and group 4, drug.
Group 1: Normal Control
In normal control group hearts were perfused for 90 min with KH buffer and
used for the biochemical analysis.
Group 2: Reperfusion
In reperfusion control group, after 20 min of equilibration, rat hearts
were subjected to 30 min global ischemia followed by 45 min reperfusion.
Group 3: Pre Treatment of Nac Before Global Ischemia
Rat hearts (n = 6) in this group, after the equilibration were pretreated
with DG before global ischemia at a dose of 20 mg kg-1 body weight
for around 15 min. Hearts were then subjected to 30 min of global ischemia,
followed by 45 min of reperfusion.
Group 4: Pre Treatment of DG Chloroform Root Extract Before Global Ischemia
Rat hearts (n = 6) in this group, after the equilibration were pretreated
with DG before global ischemia at a dose of 100 mg kg-1 b.wt. for
around 15 min. Hearts were then subjected to 30 min of global ischemia, followed
by 45 min of reperfusion.
Heart Preparation
Wistar male rats weighing 250-280 g were anesthetized with 40 mg kg-1
sodium thiopentenone. After an intravenous injection of 300 U heparin, the heart
was rapidly excised via a mid-sternal thoracotomy and arrested in the ice cold
Krebs-Henseleit buffer (KH) containing (mM L-1) NaCl 118, KCl 4.7,
MgSO41.2, KH2PO4 1.2, CaCl2 1.8,
NaHCO3 25 and C6H12O6 11. The heart
was attached to a Lagendorff apparatus via an aorta for retrograde perfusion
with KH buffer maintained at 37EC and pH = 7.4 and saturated with a gas mixture
of 95% O2, 5% CO2. The coronary perfusion pressure was
maintained at 80 mm Hg the left ventricular pressure developed with the ventricle
filled with Krebs solution. The left ventricular pressure developed with ventricle
filled with Kreb solution was recorded with a with a pressure transducer, which
in turn was connected to a device amplifier and chart recorder. This left ventricular
pressure gave an indication of the mechanical performance of the heart. Coronary
flow was measured simply by collecting the perfusate draining from the heart
in a graduated cylinder for a defined time. The heart rate was measured by counting
the number of contractions (obtained from the left ventricular pressure record)
per minute.
Tissue Preparation
The heart was excised, rinsed in ice cold isotonic saline, blotted with
filter paper, weighed, homogenized in 0.1 M Tris-HCl (pH 7.4) buffer solution.
The homogenate was centrifuged at 3000 rpm for 5 min. The supernatant was used
for the estimation of various biochemical parameters.
Biochemical Assays
Mitochondria and microsomal fractions from the myocardium was isolated by
the method of Johnson and Lardy and Schenkman and Ciniti, respectively. Assay
of isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), succinate dehydrogenase
(SDH), α-ketoglutarate dehydrogenase (α-KGDH), NADH dehydrogenase
(NADH dH) and cytochrome c oxides were carried out in a UV-1601 Shimadzu spectrophotometer.
Protein concentration was measured with Folin phenol reagent, following the
procedure described by Lowry. Assay of creatine kinase, lactate dehydrogenase
and aspartate transaminase were also estimated.
Phytochemical Screening
Phytochemical screening for secondary metabolites in the chloroform extract
was carried out by Mayer = s and Dragendorff = s tests (alkaloids), Shinoda
= s test (flavonoids), ethanolic KOH test (coumarins), Libermann-Burchard test
(terpenoid/steroids) and froth formation test (saponins). A small quantity of
the chloroform extract of DG was dissolved in chloroform and applied as a band
(6 mm width) onto the HPTLC plate (silica gel 60 F 254, E. Merck, Germany, 10x10
cm) with an automatic Linomat V applicator (Camag, Switzerland). The HPTLC plate
was developed to a height of 80 mm in hexane-chloroform-methanol (1.5 : 7.5
: 1) with pre-saturation for 15 min in a Camag twin trough glass tank. After
development, the plate was derivatized using anisaldehyde-sulfuric acid reagent
and dried. The spots were scanned at 580 nm (visible, tungston lamp) using Camag
TLC Scanner 3 equipped with Wincat software at slit width 5x0.45 mm.
GS-MS Analysis
All analysis was conducted with a Perkin Elmer Clarus 500 GC equipped with
mass spectrometry. The chromatographic conditions were as follows: Column: Elite-1
(100% dimethyl poly siloxane). Helium was used as the carrier gas with a flow
rate of 1 mL min-1. The 1 μL chloroform root extract of DG was
injected into the GSBMS in split less mode at 250°C. The column oven temperature
was held at 110°C for 2 min, then programmed at 75°C min-1
to 200°C for 1 min, 5°C min-1 to 280°C and held for 9
min.
Statistical Analysis
All data were reported as Mean±SD. Results were statistically analyzed
by a one-way Analysis of Variance (ANOVA) by SPSS software 12.00, followed by
Duncan’s Multiple range Test (DMRT). p<0.05 was considered to be significant.
RESULTS AND DISCUSSION
The present study indicates inotropic action of Desmodium gangeticum chloroform root extract on isolated frog heart. The resultant effect of DG chloroform root extract was reassessed in vivo by subjecting it as anti ischemic reperfusion agent in isolated rat heart. The positive inotropic effect of the extract appeared to be linked to the release of calcium to myocardium. The calcium releasing effect of DG was utilized for cardio protection against ischemia reperfusion injury.
Phytochemical Screening
The chloroform extract of DG was tested positive for terpenoids and alkaloids
in preliminary phytochemical tests. The HPTLC fingerprint of the chloroform
extract is shown in Fig. 1. In the HPTLC fingerprinting of
chloroform extract, ten spots at the following Rf values: 0.05 (20.37%), 0.14
(12.72%), 0.25 (16.03%), 0.29 (10.96%), 0.33 (10.70%), 0.40 (7.54%), 0.51 (6.06%),
0.57 (5.36%), 0.64 (7.38%), 0.71 (2.88%) were observed.
Oleic acid, N hexadecanoic acid, 9,9 Dimethoxybicyclo (3,3,1) nona 2,4dione, 9 Dodecanoic acid methyl ester, Didodecyl phthalate. It represents around 89% (Fig. 2). Minor compounds such as 4 dodecanol, 10 undecenal, 1, 2 benzenedicarboxylic acid bis (2 methylpropyl) ester, 1, 14 Tetradecanediol and 2 methyl pentanal were identified (Table 1) through GSMS analysis. In fact olive oil reported to be an effective agent against oxidative stress associated diseases. Moreover, olive oil contains minor components with antioxidant properties.
| Fig. 1: | HPTLC profile of the chloroform extract of DG in 1.5 : 7.5 : 1 hexane-chloroform- methanol, derivatized in anisaldehyde-sulfuric acid and scanned at 580 nm |
| Fig. 2: | GSMS of chloroform extract of Desmodium gangeticum root extract |
The presence of iron, magnesium, sodium, potassium, calcium and nickel (Table 2) were confirmed in the extract through atomic absorption spectrophotometer. An early report confers protection against the incidence of diabetes, metabolic syndrome, hypertension and cardiovascular disease by increased dietary magnesium intake. It ameliorates insulin resistance, serum lipid profiles, lowers inflammation, endothelial dysfunction, oxidative stress and platelet aggregability (Bo and Pisu, 2008). Few other studies explain the efficacy of dietary sodium supplementation to cardiac patients (Mervaala et al., 1997).
Cardiotonic Assay
In situ frog heart study indicated no change in the amplitude and
force of contraction of frog myocardium when DG extract was given up to a dose
of 2 mg (Table 3). However, the positive inotropic effect
mediated by DG extract, initiates after a slight recession in the amplitude
and force of contraction, when calcium channel blocker was administered along
with it. On the other hand, 0.5 mg of calcium chloride along with calcium channel
blocker did not show any delay in the increased amplitude and force of contraction
(Fig. 3A, B). This suggested a delayed release of calcium
into the myocardium by alternate calcium release mechanism. This may be due
to sodium pump inhibition and elevated sodium (Sheu and
Fozzard, 1982). Indeed, slow calcium releases are known to show better cardiotonic
activity in hypodynamic hearts than in normal hearts (Garaliene
et al., 1998).
| Table 1: | Effect of DG on flow rate, heart rate and force of contraction in frog heart |
| Table 2: | Hemodynamic characteristic |
| Values are Mean±SD for 6 rats in each group. n,no. of hearts in each group; LVDP, left ventricular developed pressure; CF: Coronary flow; HR: Heart rate, RPP: Rate pressure product, MAP: Mean arterial pressure. *p<0.05, compared with control | |
| Table 3: | Level of creatine kinase and lactate dehydrogenase (LDH) in the myocardium of isolated rat heart and the activity of LDH in the myocardial perfusate |
| Results are Mean±SD (n = 6). Activity is expressed as μmoles of phosphorus liberated per sec per gram protein for Na+ K+ ATPase, Ca2+ ATPase and Mg2+ ATPase; mmoles of phosphorus released per mg protein per hour for 5'-nucleotidase. Values not sharing a common superscript differ significantly at p<0.05) when compared between the groups | |
| Table 4: | Metal composition of Desmodium gangeticum root chloroform extract by atomic absorption |
| Fig. 3: | The positive inotropic effect of Desmodium gangeticum root chloroform extract on isolated frog heart preparation. (A) with different concentrations of the drug and (B) insensitive to calcium channel blocker |
The positive inotropic effect of DG chloroform root extract was reassessed in isolated rat heart. The improved hemodynamic parameters in isolated rat heart are associated with DG root chloroform extract infusion (Table 4). Further, increased heart rate and force of contraction may be due to calcium influx, as predicted by above kymogram study. However, in isolated frog heart study, effect of DG root extract was observed to be insensitive to calcium channel blocker (Fig. 3), suggest that active ingredient of DG root extract may be acting indirectly through different sym or anti port system to release calcium. Further research is needed to establish the exact mechanism of action. But elevated calcium flux that may increased coronary flow rate observed in the present study suggest voltage independent calcium flux. Interestingly, our element analysis of DG root extract showed a significant level of nickel (Table 2) in the extract triggering a Ca2+ influx sensitive to nickel, an inhibitor of voltage-dependent calcium channels (Komai and Rusy, 1993).
Myocardial Ischemia Reperfusion Study
Cardiac biomarkers are usually measured to assess myocardial damage which
leads to the disintegration of heart cell membrane (Shell
et al., 1971; Irvine et al., 1980).
Hence the presence of cardiac enzymes like CK, LDH and Troponin in the coronary
perfusate of the present study indicates the cardiac damage which was observed
significantly in control animal.
| Table 5: | Chemical composition of Desmodium gangeticum root chloroform extract by GS-MS analysis |
However, administration of the DG root extract reduces the level of these enzymes in perfusate suggesting myocardial protection (Table 5). The myocardial depression is caused by the combined effect of a reduction in (1) trans sarcolemmal L type Ca2+ current, (2) sarcoplasmic reticulum Ca2+ content and (3) sensitivity of myofilaments to calcium (Pask et al., 1981). A high conductance Ca2+ activated potassium (KCa) channel, located in the inner membrane of the mitochondrion, has been shown to mediate cardio protection against ischemia/reperfusion (Xu et al., 2002), as does the mitochondrial ATP-sensitive potassium (mitoKATP) channel (Garlid et al., 1997). Thus a decline in cardiac biomarkers in the perfusate predicts the intact cardiac membrane architecture.
Metal content of DG extract analyzed by atomicd absorption spectroscopy also substantiate calcium like action of DG root extract (Table 2). Macro and microelements play a vital role in the medicinal value of plant therapy in health and diseases (Simon et al., 1990). It was previously reported that plants containing rich amount of calcium may support the medicinal uses of plants (Siddhuraju and Beckar, 2001). The high concentrations of Ca are very significant because Ca is known to enhance the qualities of bones and teeth and also of neuromuscular systemic and cardiac functions (Martin, 1984). Sodium and potassium content of the extract attribute to diuretic action (Sica et al., 2008) that can indirectly influence the cardiac function.
CONCLUSIONS
From the above observation we can infer that chloroform root extract of Desmodium gangeticum posses positive inotropic effect that mediate cardio protection against injury due to ischemia/reperfusion. This effect may be related to the delayed increase of Ca2+ in the myocardium due to the inhibition of sodium-potassium pump. The increased influx of Ca2+ could also be associated to the inhibition of voltage dependent calcium channels by Ni2+ as significant quantity is present in the extract. Ca2+-activated potassium (KCa) channel present in the mitochondrion could have been activated either due to the presence of trace elements like Mg2+ or by some principle component present in the drug for which further studies can help in confirming the mechanism involved in this drug mediated cardio protection.