Evaluation of cardioprotective effect of aqueous extract of Garcinia indica Linn. fruit rinds on isoprenaline-induced myocardial injury in Wistar albino rats.

In the present study, cardioprotective effect of aqueous extract of Garcinia indica Linn. fruit rinds in isoprenaline-induced myocardial infarction in Wistar albino rats was evaluated. In vitro total phenolic, total flavonoid content and 2, 2'-diphenyl-1-picrylhydrazyl hydrate radical scavenging activity was measured. In vivo effect of aqueous extract of G. indica was evaluated in Wistar albino rats by isoprenaline-induced myocardial injury model. Thirty six rats were randomly divided in 6 groups. Rats were treated with G. indica 250 mg/kg and 500 mg/kg doses for 21 days and myocardial injury was produced by subcutaneous injection of isoprenaline 85 mg/kg on day 20 and 21. Carvedilol 1 mg/kg for 21 days served as active control. Electrocardiogram parameters, cardiac injury markers (serum troponin-I, uric acid, lactate dehydrogenase, creatinine kinase-MB, aspartate aminotransferase and alanine aminotransferase), oxidative stress markers (superoxide dismutase, catalase and malondialdehyde level) and histopathological changes were evaluated in each group and compared using appropriate statistical tests. In vitro evaluation of aqueous extract showed significant antioxidant property. Isoprenaline produced significant myocardial ischemia as compared to normal control group (P<0.05). Administration of G. indica in both the doses did not significantly recover the altered electrocardiogram, cardiac injury markers, oxidative stress markers and histopathological myocardial damage as compared to disease control group (P>0.05). The aqueous extract of G. indica was not found to be cardioprotective against myocardial injury. Further study with more sample size and higher dose range may be required to evaluate its cardioprotective effect.

INTRODUCTION

Ischemic heart disease is a major non communicable disease and it has become an important problem worldwide. According to WHO, it will be a leading cause of mortality worldwide by 2020 (1). Among ischemic heart disease, acute myocardial infarction (MI) is the most alarming one and it occurs due to imbalance between coronary blood supply and myocardial demand. Free radical mediated myocardial damage is an important etiological mechanism that is associated with increased level of reactive oxygen species and/or inadequate antioxidant defense system (2).

Isoprenaline (ISO), a nonselective β agonist produces infarct like necrosis of myocardium (3). ISO-induced MI is the most widely used model to evaluate cardioprotective effect of various drugs (4). Administration of ISO in high doses produces myocardial lesions similar to MI in human.

Modern medicines are effective in preventing heart diseases but their use is limited due to various adverse effects (5). Many dietary plants and food antioxidants are increasingly being recognized as health promoter in heart diseases (6). Garcinia indica choisy belongs to the family Clusiaceae/Guttiferae. It is an evergreen tree that is commonly found along the west coast of India (7). The dried outer rind of the fruit of G. indica is known as ‘kokum’. In Charak Samhita, it has been mentioned as cardiotonic agent (8). Many other therapeutic uses of kokum for scalds and chaffed skin, sunstroke, dysentery, allergic skin rashes, burns, mucous diarrhoea, piles, anorexia, liver tonic and to allay thirst have been mentioned in traditional Indian medicine (9).

The fruit rind contains polyisoprenylated benzophenones garcinol, its isomer isogarcinol, xanthochymol, isoxanthochymol and hydroxycitric acid (10). Hydroxycitric acid and garcinol are the main ingredients in kokum with antioxidant effect (11). Other compounds with potential antioxidant properties in kokum are citric acid, malic acid, polyphenols, carbohydrates, anthocyanin pigments and ascorbic acid (12). Various plants have been found to have a cardioprotective effect due to their antioxidant properties (1314). So, this study was planned to evaluate cardioprotective effect of aqueous extract of G. indica on isoprenaline induced MI in Wistar albino rats.

MATERIALS AND METHODS

All experiments were performed after prior permission from Institutional Animal Ethics Committee (IAEC), Government Medical College, Bhavnagar, Gujarat, India. (IAEC No.28/2012, dated: 27/10/2012). Care of experimental animals was taken as per the guidelines given by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests (Animal Welfare Division), Government of India, New Delhi, India.

Experimental animals

Wistar albino rats (250 ± 50 g) of both sexes and female mice (25-30 g) were procured from central animal house of the institution.

They were housed in standard transparent polypropylene cages with wheat husk bedding, renewed every 24 h. They were kept under controlled room temperature and humidity (18 to 29 °C; 30 to 70%) in a 12 h light-dark cycle. Animals were acclimatized for one week to laboratory conditions before starting the experiment. The rats were given standard laboratory diet and water ad libitum.

Drugs and chemicals

Aqueous extract of G. indica was a generous gift from Leopard Investments Ltd, Mumbai. ISO, Gallic acid, quercetin, urethane and 2, 2’-diphenyl-1-picrylhydrazyl hydrate (DPPH) were obtained from the Sigma Chemical Company, St. Louis, MO, USA. Carvedilol was purchased from Selleck chemicals, Munich, Germany. ISO solution was freshly prepared in normal saline at the time of injection. G. indica was dissolved in distilled water.

Acute toxicity study

Acute toxicity study of G. indica extract was conducted using female swiss albino mice (25-30 g) in accordance with Organization for Economic Co-operation and Development guideline No. 423.

Determination of total phenols and flavonoids

Total phenolic content was determined according to Folin-Ciocalteu method (15). Flavonoid content in the extract was determined by a colorimetric method (16).

2, 2’-diphenyl-1-picrylhydrazyl hydrate radical scavenging assay

In this assay, free radical scavenging activity of G. indica was determined by measuring the bleaching of purple-colored methanol solution of DPPH (17).

Experimental groups

Total 36 Wistar albino rats were allocated randomly into 6 groups using rando software (6 animals per group). MI was induced in rats by giving ISO (85 mg/kg) subcutaneously (s.c.) for two subsequent days, on day 20 and 21 at the interval of 24 h. Distribution of study groups were as follow:

Group 1 (Normal control), Rats were given distilled water orally for 21 days and normal saline s.c.

Group 2 (ISO control), Rats were given distilled water orally for 21 days and ISO (85 mg/kg) s.c.

Group 3 (GI control), Rats were given G. indica extract 500 mg/kg orally for 21 days and normal saline s.c.

Group 4 (GI low dose), Rats were given G. indica extract 250 mg/kg orally for 21 days and ISO (85 mg/kg) s.c.

Group 5 (GI high dose), Rats were given G. indica extract 500 mg/kg orally for 21 days and ISO (85 mg/kg) s.c.

Group 6 (Active control), Rats were given carvedilol (1 mg/kg) orally for 21 days and ISO (85 mg/kg) s.c.

Outcome measures

After dosing and administration of ISO, the following parameters were recorded to evaluate the cardioprotective effect.

Electrocardiography recording

At the end of 21 days, 48 h after the first dose of ISO, all the animals were anaesthetized with intraperitoneal injection of urethane (125 mg/100 g, i.p) and was given stabilization period of 30 min before recording of electrocardiography (ECG). ECG was recorded with the help of student's physiograph and ECG coupler [Inco Ambala Co, Haryana, India; paper speed-50 mm/sec, sensitivity- 200 μV/cm, gain-maximum]. The electrodes constructed from 26 gauge hypodermic needle were attached to both front and hind paw. One precordial lead was used and was placed in a position corresponding to V4 in human. Heart rate, RR interval, QT interval, corrected QT interval and QRS inteval were calculated and compared between the groups.

Biochemical estimations

After recording of ECG, blood was collected through retro-orbital plexus using capillary tube. The serum was separated by centrifugation and used for the estimation of lactate dehydrogenase (LDH, measured by UV kinetic method), creatinine kinase-MB (CK-MB, measured by Immuno inhibition method), troponin I (measured by Immuno-fluorescence method), aspartate aminotransferase (AST, measured by UV kinetic method), alanine aminotransferase (ALT, measured by UV kinetic method) and uric acid (measured by Uricase pap method). The manufacturer for the kit used for Troponin I is Toshos Corporation, Tokyo, Japan. All other biochemical parameters were measured by kits of Instrumentation Laboratory, Lexington, USA.

After blood collection, rats were sacrificed and heart was dissected out with mid abdominal incision. It was blotted in filter paper, weighed and further proceeded for histopathology or antioxidant analyses. From all groups, 3 hearts were used for histopathological analysis and 3 hearts were used for antioxidant estimation.

Antioxidant estimation in heart tissue homogenate preparation

Hearts were washed in ice cold saline followed by in 0.25 M sucrose solution and were finely sliced. Homogenate was prepared in chilled tris-HCl buffer solution [10% w/v, 0.1 M, pH 7.4]. The homogenate was then centrifuged at 5000 rpm at 4 °C using compact high speed refrigerated centrifuge (Kubota 6500, Japan). The clear supernatant obtained was used for the estimation of superoxide dismutase (SOD) by the method of Misra and Fridovich (18), catalase by the method of Hugo e. Aebi (19) and marker of lipid peroxidation malondialdehyde (MDA) by the method of Slater and Sawyer (20).

Heart weight to body weight ratio

In each group, heart weight to body weight ratio was calculated. Body weight was the weight of animal on the day of sacrification. Heart weight was measured after washing it in heart in ice cold saline after removal from the body, squeezing out the blood and blotted on the filter paper.

Histopathological analysis

Three hearts from each group were preserved in 10% formalin, processed and embedded in paraffin wax. 5-6 μm thick sections were cut and stained with Hematoxyline and Eosin (H & E) stain and observed with light microscope to evaluate myocardial injury. All slides were coded and analyzed blindly by the trained pathologist from our institute without knowledge of treatment plan. Histological observations for oedema, infiltration and necrosis were categorised in to 5 different grades ranging from grade 0 to 4. Grade 0 indicates no change and +1, +2, +3, +4 indicates slight, mild, moderate and severe histopathological changes, respectively.

Statistical analysis

ll the values were presented as Mean ± SEM. Data were checked for normal distribution using the Kolmogorov–Smirnov test. Parametric data were analyzed by one way ANOVA followed by Tukey- Kramer multiple comparison test and Non parametric data were analyzed by Kruskal Wallis followed by Dunn's multiple comparison test using GraphPad InStat (version 3.00, GraphPad Software, California USA). A P value <0.05 was considered as statistically significant.

RESULTS

In toxicity study, no mortality was observed in mice treated up to 5000 mg/kg of aqueous extract of G. indica. Accordingly, 1/10 (500 mg/kg, orally) and 1/20 (250 mg/kg, orally) doses were selected for screening of cardioprotective activity.

During in vitro evaluation of antioxidant property of aqueous extract of G. indica, we found that extract had total phenolic content 252.87 mg/g GAE (Gallic Acid Equivalent), total flavonoid content 34 mg/g QUE (Quercetin Equivalents) and IC50 value for DPPH scavenging activity 231.85 ± 21.56 μg/ml.

As shown in Table 1, rats in ISO group showed significant increase in cardiac injury markers like troponin-I, LDH, CK-MB, ALT, AST and uric acid as compared to normal control group (P<0.05). In test groups, administration of G. indica extract could not significantly restore the elevated parameters due to ISO administration (P>0.05), whereas, carvedilol in active control group significantly restored the cardiac injury markers (P<0.05) (Table 1).

Table 1

Comparison of cardiac injury markers: serum Troponin-I, LDH, CK-MB, ALT, AST and Uric acid level between experimental groups.

Subcutaneous ISO administration caused marked increase in heart rate and decrease in RR interval as compared to normal control group (P<0.05; Table 2). Heart rate and RR interval was significantly restored in carvedilol group (P<0.05), whereas they were not restored in test groups (P>0.05).

Table 2

Comparison of electrocardiography parameters between experimental groups.

The rats in ISO control group showed marked increase in heart weight and heart weight/body weight ratio but ratio was decreased in active control group (Table 3). SOD and catalase level were decreased and thiobarbituric acid reactive substance (TBARS) level was increased significantly in ISO control group as compared to normal control group (P<0.05). In G. indica treated groups, the levels of antioxidant enzymes was not restored (P>0.05) (Table 4).

Table 3

Comparison of body weight, heart weight, heart weight/body weight ratio between experimental groups.

Table 4

Comparison of biomarkers of oxidative stress between experimental groups.

Average grading of hisopathological findings are shown in Table 5. Fig. 1 shows histopathological texture of myocardium in study groups. Administration of ISO significantly caused necrosis, oedema and neutrophilic infiltration in myocardium as compared to normal control group. Myocardium of animals treated with G. indica extract and carvedilol showed improvement as compared to ISO control group. However, it could not achieve the statistical significance. The G. indica control group was comparable to normal control group in relation to all observed parameters.

Table 5

Comparison of histopathological score between experimental groups.

Fig. 1

Histopathological comparisons between the various study groups. A; Normal control group, B; Isoprenaline control group, C; Garcinia indica control group, D; Garcinia indica low dose, E; Garcinia indica high dose, F; Active control group. Arrows indicates marked infiltration of neutrophils and mononuclear inflammatory cells and necrosis of the myocardium.

DISCUSSION

In vitro evaluation showed a good antioxidant property of aqueous extract of G. indica fruit rind. So, we evaluated cardio protective effect of G. indica extract in vivo by ISO-induced myocardial infarction model in Wistar albino rats.

ISO is a synthetic catecholamine and nonselective β agonist. It is proven that catecholamine, if given in high doses, produces necrosis of heart muscle (21). Exact mechanism of ISO induced MI is not proven yet. Intracellular calcium overload due to massive influx (22), hypoxemia due to increased cardiac work and O2 demand, free oxygen radical generation by auto oxidation of catecholamines, alteration of myocardial cell membrane permeability due to lipid peroxidation, interruption of mitochondrial oxidative phosphorylation by free fatty acid and changes in electrolyte contents are various proposed mechanisms. Among them, oxidative stress and free radical mediated injury appears to be the most important mechanism for ISO-induced MI (23).

In the present study, ISO caused significant myocardial damage as indicated by cardiac injury markers, ECG abnormalities, oxidative stress markers, heart weight and histopathological analysis. In the ISO control group significant increase in cardiac injury markers indicates necrosis of myocardium and leakiness of plasma membrane (24). The level of uric acid was also significantly increased. This could be due to proteolysis and excessive degradation of purine nucleotides (25). After injection of ISO, enormous amount of reactive oxygen species are generated due to auto-oxidation which reacts with polyunsaturated fatty acids present within cell membrane initiating chain of lipid peroxidation (26). The level of malondialdehyde, a marker of lipid peroxidation was increased and level of antioxidant enzymes like SOD and catalase was decreased in ISO group which indicates oxidative stress.

The plant phenolic compounds and flavonoids provide defense against oxidative stress (27). Flavonoids act as good antioxidants because of their free radical scavenging activity and protect tissue against free radical mediated lipid peroxidation and also chelate metal ions (28). Garcinol is a potent antioxidant present in G. indica which has structural similarity to curcumin as it contains both phenolic hydroxyl groups as well as a β-diketone moiety (29). It is a free radical scavenger (30). In our study, administration of aqueous extract of G. indica in a dose of 250 and 500 mg/kg for 21 days could not restore the altered parameters produced by ISO. This suggests the lack of cardio protective effect of aqueous extract of G. indica at given doses despite of its antioxidant property.

A recently published study by Kumar and coworkers (31) showed cardio protective effect of ethanolic extract of G. indica in doses of 250 and 500 mg/kg. This is in contrast to the present study. ISO causes dose dependant myocardial damage. Correlation between injected dose and severity of necrosis produced by ISO has been reported (32). Subcutaneous injection of ISO in various doses (21.2, 42.5, 85, 170, 340, 680 mg/kg) has shown differences in severity of myocardial necrosis. Doses above 85 mg/kg, cause more mortality in experimental animals. 85 mg/kg dose produces massive necrosis in apex and/or papillary muscles and adjacent sub-endocardial parts of left ventricles with an incidence of mortality around 10%. Doses less than 85 mg/kg produce cardiac necrosis with 100% incidence but extent and severity decreases with decreasing dose of ISO. In majority studies, 85 mg/kg ISO is used to induce MI. Hence, we administered 85 mg/kg ISO s.c. to produce MI. Whereas in study conducted by Kumar and colleagues, 25 mg/kg ISO intraperitoneally was used. This may be the reason for contradicting results in the present study. It may also be possible due to the different standards of extracts used in both the studies.

In vitro evaluation showed that aqueous extract of G. indica has dose dependent antioxidant potential (33). Various studies on G. indica for hepato protective evaluation have been conducted in a dose of 400 mg/kg and 800 mg/kg (3435). Hepatoprotective effect was found better with dose 800 mg/kg (3435).

In the present study, doses of 250 and 500 mg/kg of GI were not effective in restoring the myocardial damage caused by ISO may be because of lower dose range selection.

CONCLUSION

Aqueous extract of G. indica in doses of 250 mg/kg and 500 mg/kg for 21 days did not show cardioprotective effect in Wistar rats. Further evaluation can be planned with higher doses of aqueous extract of G. indica.