Cardioprotective efficacy of the active petroleum ether fraction of Gardenia gummifera  Linn.f. on isoproterenol induced myocardial infarction in rats

 

 

Abstract               Background: Gardenia gummifera  Linn.f. (Rubiaceae), a medicinal shrub used in Indian traditional medicine for the treatment of obesity, cardiac debility and lipolytic disorders. Oleanolic aldehyde and vernolic acid are the active components identified from the petroleum ether fraction (PEF) of Gardenia gummifera  Linn.f. The present work aim to study the cardioprotective effect of PEF of Gardenia gummifera   root on Isoproterenol (ISO) induced myocardial infarction (MI) in rats. Methodology: Myocardial infarction was induced by the subcutaneous injection of ISO (6mg/100g body weight) at an interval of 24h for 2 days. PEF (20 mg/kg, p.o.) was given to rats once daily for 15 days prior to the ISO challenge. The myocardial damage was assessed by quantifying the serum levels of cardiac marker enzymes (LDH, AST, ALT, CK-MB) and Cholesterol and triglyceride. The heart tissue antioxidants such as Catalase (CAT), Glutathione peroxidase (GPx), Glutathione reductase (GR), Glutathione-S-transferase (GST) and Reduced Glutathione (GSH) were altered in MI rats. The level of lipid peroxidation was measured as malondialdehyde (MDA) and quantification of histopathological changes also supported protective effects of MEGG. Results: PEF significantly (p ≤ 0.05) protected the above mentioned parameters to fall from the normal levels. Histopathological evaluation also supported the protective effects of PEF. Conclusion: Our results suggest that PEF of Gardenia gummifera  Linn.f. affords protection against isoproterenol-induced myocardial infarction.

Key Words: Cardioprotection; Gardenia gummifera; Isoproterenol; Myocardial infarction; ALT; AST.

 

 

INTRODUCTION

Ischemic heart diseases (IHD) remain the principal cause of death in both developed and developing countries, accounting for about 20% of death per year worldwide. Experimental and clinical studies have shown that during ischemic injury, oxidative stress produced by the generation of reactive oxygen species (ROS) plays a key role in the development of MI ¹. In ischemic tissues, the free radicals and ROS have been implicated in oxidative chain reactions which damage the cell membrane and subsequently, structural and metabolic alterations, leading to cardiac dysfunction and ultimately cell death ². The use of natural antioxidants is increasing as defensive agents against number of cardiovascular abnormalities. The bioactive agents from herbal sources have gained primary importance in modern system of medicines, reducing the risks of cardiac ailments by scavenging the free radicals formation ³. Cardioprotective potential of medicinal plants can be evaluated in chemically induced myocardial infarction in animal models.

 

MATERIALS AND METHODS

Chemicals

Isoproterenol hydrochloride was purchased from Sigma chemicals (St. Louis, MO, USA). Assay kits for serum lactate dehydrogenase (LDH), serum transaminases (ALT, AST), creatine kinase isoenzyme (CK-MB), triglycerides and cholesterol were purchased from Agappe Diagnostic Ltd., India.

Preparation of the active Petroleum Ether Fraction

Gardenia gummifera Linn. f. were collected from its natural habitat (Idukki Dist., Kerala, India) and identified. A voucher specimen (SBSBRL.05) is maintained in School of Biosciences, M.G University, Kottayam. The shade dried roots of G. gummifera were powdered and soxhlet extracted with methanol (50 g in 400 mL) and were concentrated under reduced pressure using a rotary evaporator. The percentage yield of methanolic extract in our study was approximately 10.3% (w/w). The methanolic extract thus obtained was then taken in a round bottom flask of simple condenser and further fractionated using solvents of increasing polarity, viz. petroleum ether, chloroform, ethyl acetate and methanol. For animal experiment the active sub-fraction (PEF) was prepared as a suspension in 5% Tween 80.

Animals and diets

Adult male wistar rats weighing 150 ± 7.6 gm (Mean ± S.D, n = 30) were used in this study. The rats were fed with standard laboratory chow (Hindustan Lever Foods, Bangalore, India) and provided with water ad libitum. The animals were maintained at a controlled condition of temperature of 26-28⁰C with a 12h dark cycle. Animal studies were followed according to Institute Animal Ethics Committee (IAEC) regulations approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Reg. No. B2442009/3) and conducted humanely.

Experimental Protocol

The animals were divided in to 5 groups (Six rats/group).

• Group I             :         Vehicle control: (5% Tween 80 and normal saline instead of PEF and ISO respectively).
• Group II           :         Toxic control: Animals were given subcutaneous injection of ISO (6mg/100g b.w., s.c.) dissolved in 0.1ml normal saline at an interval of 24hrs for 2 days
• Group III          :         Animals were given PEF orally for 15 days (10 mg/kg body weight) and received ISO (6 mg /100g b.w., s.c) on the 16^th and 17^th days, respectively.
• Group IV          :         Rats pretreated with PEF orally for 15 days (20 mg/kg body weight) and received ISO (6 mg /100g b.w., s.c.) on the 16^th and 17^th days, respectively.
• Group V           :         Animals were given PEF orally for 15 days (30mg/kg body weight) and received ISO (6 mg /100g bw, s.c) on the 16^th and 17^th days, respectively.

Twenty four hours after the second dose of ISO injection, the animals were sacrificed and the blood was collected and serum was separated by centrifugation. The heart tissue was excised immediately, washed with chilled isotonic saline, and used for antioxidant and histopathological studies.

Biochemical analysis.

Serum analysis

Cardiotoxicity was assessed by quantifying the serum levels of AST (EC 2.6.1.1), ALT (EC 2.6.1.2), LDH (EC 1.1.1.27) CK- MB (EC 2.7.3.2), TC and TG by using the kit of Agappe Diagnostic Ltd., India. Activities of these serum enzymes were measured using semi autoanalyzer (RMS, India).

Tissue analysis

Heart was excised, washed thoroughly in ice-cold saline to remove the blood. 10% of homogenate was prepared in 0.1M Tris HCl buffer (pH – 7.4). The homogenate was centrifuged at 3000 rpm for 20 min at 4^oC and the supernatant was used for the estimation of catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), lipid peroxidation product (Thiobarbituric Acid Reactive Substances – TBARS) and total protein.

Histopathological studies

Small pieces of heart fixed in 10% buffered formalin were processed for embedding in paraffin. Sections of (5-6 µm) were cut and stained with haematoxylin and eosin and examined for histopathological changes under the microscope (Motic AE 21, Germany). The microphotographs were taken using Moticam 1000 camera at original magnification of 100x.

Statistical Analysis

Results were expressed as mean + S.D. and all statistical comparisons were made by means of one way ANOVA test followed by Tukey’s post hoc analysis and p – values less than or equal to 0.05 were considered significant.

RESULTS

Biochemical analysis

Table 1and 2 shows the activities of serum marker enzymes such as AST, ALT, CK-MB, LDH and serum levels of total cholesterol (TC) and triglycerides (TG) in control and experimental groups of rats. Treatment with PEF showed a dose dependent decrease (P≤0.05) of AST, ALT, LDH and CK-MB.

 

Table 1: Levels of serum AST, ALT, LDH, CK-MB, Total cholesterol (TC) and triglycerides (TG) in control and experimental rats treated with PEF

 

Table 2: Levels of serum total cholesterol (TC) and triglycerides (TG) in control and experimental rats treated with PEF

Values are mean ± S.D., n = 6.Statistical significance: ^ap≤0.05 – ISO group differs significantly from normal control group.^bp≤0.05 –PEF (10mg/kg) + ISO (6mg/100g), 20mg/kg PEF+ISO (6mg.100g) and 30mg/kg PEF+ISO (6mg/100g) groups differs significantly from ISO control group.

Table 3 shows activities of GSH, GPx, CAT and levels of MDA in heart tissue of ISO control and PEF treated experimental groups. PEF pre-treated groups showed a significant (P≤0.05) increase in the activities of GSH, GPx and CAT when compared to ISO control groups. MDA level was decreased significantly (P≤0.05) in PEF pre-treated groups than the ISO control groups.

 

Table 3: Activities of GSH, GPx, CAT and levels of MDA in heart tissue of control and experimental rats treated with PEF

Treatment Groups	GSH(nmol/mg protein)	GPx(nmol of GSH oxidized/min/mg protein)	CAT(U/mg protein)	MDA (nmol/g tissue)
Normal	11.64 ± 1.13	9.25 ± 0.49	14.28 ± 0.97	1.72 ± 0.38
ISO Control (6mg/100g; s.c.)	4.13 ± 0.67 a	2.46 ± 0.39a	4.14 ± 0.68a	6.19 ± 1.40a
PEF (10mg/kg)+ISO	5.43 ± 0.29 b	5.16± 0.42b	6.13± 0.76b	3.57 ± 0.36b
PEF(20mg/kg)+ISO	9.12 ± 0.61 b	8.20 ± 0.46b	10.48 ± 0.85b	2.96 ± 0.43b
PEF (30mg/kg)+ISO	6.82 ± 0.63 b	5.96 ± 0.80 b	8.23± 0.49 b	3.84 ± 0.92 b

Values are mean ± S.D., n = 6.Statistical significance: ^ap≤ 0.05 – ISO group differs significantly from normal control group. ^bp≤0.05 –PEF (10mg/kg) + ISO (6mg/100g), 20mg/kg PEF+ISO (6mg.100g) and 30mg/kg PEF+ISO (6mg/100g) groups differs significantly from ISO control group.

Effect of PEF treatment on histopathological changes of rat myocardium.

The microscopic observations of myocardial histoarchitecture were qualitatively graded on the basis of myonecrosis, inflammatory cells and edema. The myocardium of control group showed a normal histoarchitecture (Fig.1A). Myocardium of ISO control rats showed massive necrosis of myofibers with cell infiltration, edema and increased connective tissue among myocardial fibers along with extra vasation of red blood cells (Fig. 1B). Pre-treatment with PEF at a dose of 10mg/kg and 30mg/kg in ISO- treated rats showed mild edema and inflammatory cells as compared to ISO control group (Fig.1 C and E). PEF pre-treated at a dose of 20mg/kg. b.w. showed almost the absence of myonecrosis, edema and inflammation (Fig.1D).

 

Figure 1: Histopathological changes occurred in groups treated with ISO and prevention by the treatment with PEF (hematoxylin and eosin, 100×)

A) Normal control; (B) ISO control, (6 mg/100g s.c.); (C) PEF (10 mg/kg) + ISO; (D) PEF (20 mg/kg) + ISO; (E) PEF(30 mg/kg) + ISO.