Orally administered aqueous bark extract of

Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478

Research Article ISSN: 0974-6943

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Orally administered aqueous bark extract of Terminalia arjuna protects against adrenaline-induced myocardial injury in rat heart through antioxidant mechanisms: an in vivo and an in vitro study Sanatan Mishra 1,2 , Shamreen Naaz2 , Arnab K. Ghosh2 , Sudeshna Paul1,2 , Nirajan Ghosal2 , Mousumi Dutta1,2, Debasish Bandyopadhyay2 , Aindrila Chattopadhyay1* 1 Department of Physiology, Vidyasagar College, Kolkata 700 006, India, 2Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, University College of Science and Technology, 92, APC Road, Kolkata 700 009, India. Received on:11-04-2016; Revised on: 19-05-2016; Accepted on: 27-06-2016 ABSTRACT Objective: The present study is aimed to evaluate the cardioprotective effects of the most effective dose of aqueous bark extract of Terminalia arjuna (TA) on adrenaline- bitartrate induced myocardial damages in male albino rats. Methods: After sacrifice of rats, the left ventricular portion of heart tissues were used for determination of biomarkers of oxidative stress, activities of antioxidant enzymes, Kreb’s cycle enzymes and respiratory chain enzymes by using standard methods. Results: Treatment of rats with adrenaline bitartrate induced alterations in the activities of serum lactate deh ydrogenase total (LDH -T), lactate deh ydrogenase-1(LDH -1), serum glutamate oxaloacetate transaminase (SGOT), tissue and serum nitric oxide (NO) concentration. Moreover, it caused elevation in the level of lipid peroxidation and protein carbon ylation, a decrease in glutathione content as well as altered the activities of antioxidant enzymes and the enzymes of Kreb’s cycle and respiratory chain. Tissue histomorphological studies also showed considerable damage following adrenaline treatmen t. Pre-treatment of rats with aqueous bark extract of TA significantly protected against these myocardial damages. Conclusion: The present studies suggest that the effective dose of aqueous bark extract of TA may be beneficial in ameliorating adrenaline-induced oxidative stress mediated myocardial injury. Keywords: Adrenaline, antioxidant, cardiac damages, oxidative stress, rats, Terminalia arjuna.

INTRODUCTION Adrenaline, a catecholamine synthesized by adrenal medulla, is generally considered as a hormone involved in “fight or flight” mechanism1. However, its role in the genesis of oxidative stress in humans is being increasingly recognized and is considered more dangerous in bringing about myocardial ischemia as well as myocardial infarction. In early phase of myocardial infarction systemic circulatory catecholamine level is vigorously increased2 and is released from ischemic region of myocardium3. In addition, auto-oxidation of catecholamine results in generation of cytotoxic free radicals4. It has been reported that during myocardial infarction, the components of the natural antioxidant defense system (i.e. GSH, superoxide dismutase and catalase) are depleted and subsequently the myocardial tissue becomes vulnerable to oxidative stress as a *Corresponding author. Dr. Aindrila Chattopadhyay Department of Physiology, Vidyasagar College, Kolkata 700 006, India

result of which cardiac-myopathy occurs. Adrenaline acts by binding to a variety of adrenergic receptors present in human system. Epinephrine is a nonselective agonist of all adrenergic receptors, including the major sub-type 1, 2, β1, β2 and β35. The binding of epinephrine to these receptors triggers a number of metabolic changes. Endogenous plasma adrenaline concentrations in resting adults have been reported normally to be less than 10 ng/L, but may increase 10-fold during exercise and 50-fold or more during time of stress6.Therefore, adrenaline is an endogenous stress inducer. Aerobic metabolism leads to production of reactive oxygen species (ROS), which is continuously removed by antioxidant defense system of an organism. A balance between production and removal of ROS is necessary to maintain normal physiology. Any impairment in antioxidant defense system or over production of ROS results in oxidative stress. ROS are known to cause numerous cellular anomalies including protein damage, deactivation of enzymes, and alteration of DNA and lipid peroxidation of membranes.

Journal of Pharmacy Research Vol.10 Issue 6 June 2016

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Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478 In modern medicine, many plants occupy a very significant berth as raw materials for many important drug preparations7.8,9. In this context, traditional Indian medicinal plants act as antiradicals and DNA cleavage protectors10. These plants have also been considered to protect health, longevity, intelligence, immunosurveillance, and body resistance against different infections and diseases. Terminalia arjuna (TA) is also an important medicinal plant widely used in the preparation of ayurvedic formulations for over three centuries, primarily as a cardiac tonic in India11,12,13 .The chronic oral a dministration of crude bark of Terminalia arjuna augments endogenous antioxidants of rat heart and also prevents oxidative stress associated with in vitro ischemic-reperfusion (IR) injury of the heart14,15,16,17. Clinical evaluation of this plant indicates that it can be of benefit in the treatment of coronary artery diseases, heart failure, and possibly, hypercholesterolemia18,19,20. It has also been found to be antibacterial and antimutagenic21,22. However, most of the beneficial works on this plant have been carried out on hepatic or renal disorders23. Herein, we provide evidence that oral treatment of aqueous bark extract of Terminalia arjuna ( 20 mg/kg bw) has potential to provide protection against adrenaline-induced oxidative stress mediated damages in rat cardiac tissue and this protection may be exerted through antioxidant mechanism(s). MATERIALS AND METHODS Animal Male albino rats of Wistar strain, weighing 150-200 g were handled as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of environment and forests, Government of India. All the experimental protocols had the approval of the Institutional Animal Ethics Committee (IAEC) of the Department of Physiology, University of Calcutta (approval no IAEC/proposal-Ph.D. /DB-01, 2013 dated 23.03.2013). Prof. P.K. Samanta, M.Sc.(Vet.), Ph.D, CPCSEA nominee to IAEC, Department of Physiology ,University of Calcutta, monitored care and experiments on animals. Chemicals and reagents Sodium pyruvate, isocitrate, succinate, a-ketoglutarate and bovine serum albumin (BSA) were purchased from SRL Chemicals, Mumbai, India. Adrenaline bitartrate was procured from Vulcan laboratories, India. Thiobarbituric acid (TBA) was procured from Spectro Chem Powder of bark of Terminalia arjuna (TA) was purchased from Herby House, Kolkata, India. All the other chemicals used including the solvents, were of analytical grade obtained from Sisco Research

Laboratories (SRL), Mumbai, India, Qualigens (India/Germany), SD fine chemicals (India), Merck Limited, Delhi, India. Preparation of aqueous extract of bark of Terminalia arjuna Five gm of TA bark powder was dissolved in 25ml of double distilled water. After proper mixing it was kept with cotton plugging for overnight (approximately 16 hours). Then it was centrifuged twice at 1300g for 10 minutes. Then the supernatant was collected and lyophilized. The yield of the aqueous extract of Terminalia arjuna from 5gm of TA bark powder was 10%. Induction of cardiac injury with adrenaline bitartrate - a doseresponse study Male Wistar rats (food and water ad libitum) weighing 150-200g were divided into four groups; each group comprised of 6 rats. The rats of the first group constituted the vehicle-treated control. The rats of the second, third and fourth groups were injected subcutaneously (s.c.) with different doses of adrenaline bitartrate (0.15, 0.3, 0.6 mg/kg body weight) daily for a period of 17 days. The animals were kept in polypropylene cages (Tarson cages) at light, temperature and humidity controlled animal house of Department of Physiology, University of Calcutta. After the last adrenaline bitartrate injection, the animals were fasted overnight and sacrificed early next morning by cervical dislocation following mild ether anaesthesia. Prior to sacrifice, the blood was collected by cardiac puncture after carefully opening the thoracic cavity for the preparation of serum. Thereafter, the hearts were surgically extirpated, thoroughly washed in cold saline and stored at -200C for further biochemical analyses. Protection against adrenaline induced cardiac injury - a dose dependent study using aqueous bark extract of Terminalia arjuna In a separate experiment, myocardial damages were induced in male Wistar rats by sub-cutaneous (s.c.) injection of adrenaline-bitartrate at the dose of 0.3mg/kg body weight. Briefly, rats were divided into eight groups. Each group of animals comprised of 6 rats. The rats of the first group constituted the vehicle-treated control. The rats of the second group were injected with sub-cutaneous (s.c.) injection of adrenaline at the dose of 0.3mg/ kg body weight. The rats of the third, fourth and the fifth group were orally fed, respectively, with different doses of aqueous bark extract of TA (10, 20, 40 mg/kg body weight) for 17 consecutive days where water was used as the vehicle. The rats of sixth, seventh and eighth groups were orally fed different doses of aqueous bark extract of TA(10,20,40 mg/kg body weight)for 17 consecutive days and injected with sub-cutaneous (s.c.) injection of adrenaline at the dose of 0.3mg/ kg body weight. After the completion of treatment, the animals were sacrificed by cervical dislocation following mild ether anesthesia. The cardiac tissues were surgically extirpated after carefully opening the thoracic cavity and were washed thoroughly in cold saline. The saline was soaked properly with a piece of blotting paper from the tissues and


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Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478 the tissues were stored at -20°C for further biochemical analyses. activity was obtained by measuring the oxidation of NADH (0.1 Prior to sacrifice, the blood was collected by cardiac puncture for the mM) to NAD+ at 340 nm using 1.0 mM sodium pyruvate as substrate, after incubating the serum samples at 65 °C which destroys all preparation of the serum. isoforms except LDH-1 for 30 min according to the method of Varcoe Protection of adrenaline induced cardiac injury by minimum ef- et al27 . fective dose of aqueous bark extract of Terminalia arjuna In another set of experiment, myocardial damages were induced in Measurement of nitric oxide (NO) concentration in the rat cardiac male Wistar rats by sub-cutaneous (s.c.) injection of adrenaline- tissues bitartrate at the dose of 0.3mg/kg body weight. Briefly, male rats Nitric oxide concentration in the cardiac tissues were measured were divided into four groups. Each group of animals comprised of 6 spectrophotometrically at 548nm according to the method of rats. The rats of the first group constituted the vehicle-treated Fiddler28 by using Griess reagent29 . control. The rats of the second group were injected with subcutaneous (s.c.) injection of adrenaline at the dose of 0.3mg/ kg Measurement of cardiac tissue lipid peroxidation (LPO) level and body weight. The rats of the third (positive control) and fourth group the contents of reduced glutathione (GSH), total glutathione (TSH) were orally fed, respectively, with aqueous bark extract of TA ( 20 as well as protein carbonyl (PCO) content mg/kg body weight; minimum effective dose) for 17 consecutive The lipid peroxides in the cardiac tissue homogenates were deterdays, and in fourth group, rats were injected with sub-cutaneous mined separately as thiobarbituric acid reactive substances (TBARS) (s.c.) injection of adrenaline at the dose of 0.3mg/ kg body weight. according to the method of Buege and Aust 30 with some After the completion of treatment the animals were sacrificed by modifications as adopted by Bandyopadhyay et al31. The reduced cervical dislocation following mild ether anesthesia. The cardiac glutathione (GSH) content (as acid soluble sulfhydryl) and total tissues were surgically extirpated after carefully opening the glutathione content of the cardiac tissue homogenates were thoracic cavity and were washed thoroughly in cold saline. The estimated separately by its reaction with DTNB (Ellman’s reagent) saline was soaked properly with a piece of blotting paper from the following the method of Sedlak et al32 with some modifications by tissues and the tissues were stored at -20°C for further biochemical Dutta et al33. Protein carbonyl content was estimated by DNPH34 analyses. Prior to sacrifice, the blood was collected by cardiac assay . The values were expressed as nmoles /mg protein. puncture for the preparation of the serum. Measurement of the activities of Cu-Zn superoxide dismutase (CuZn SOD), catalase, Mn superoxide dismutase (Mn SOD), gluPreparation of tissue homogenate A 10% tissue homogenate of heart was prepared in cold in ice cold tathione reductase (GR), glutathione peroxidase (GPx) and glu0.1M phosphate buffer (pH 7.4) using a Potter Elvenjem glass tathione-S-transferase (GST) of rat cardiac tissue homogenizer (Belco Glass Inc., Vineland, NJ, USA) for 30 sec. The Copper–zinc superoxide dismutase (Cu-Zn-SOD) activity was meahomogenates was kept in cold and processed for biochemical sured by Hematoxylin auto-oxidation method of Martin et al35 with analyses within 30 minutes of preparation. some modifications36. The enzyme activity was expressed as units / mg protein. Catalase activity was assayed by the method of Beers et Measurement of serum glutamate oxaloacetate transaminase al37 with some modifications38. The enzyme activity was expressed as (SGOT), total lactate dehydrogenase (LDH T), lactate dehydroge- μmoles of H O consumed/ mg protein. Manganese superoxide 2 2 nase 1 (LDH 1) activities dismutase (Mn-SOD) activity was measured by pyrogallol Serum GOT activity was measured by standard methods. Non- autooxidation method39 modified by Rudra et al40. The glutathione hemolyzed serum was mixed with glutamate pyruvate transaminase reductase activity was measured according to the method of Krohnesubstrate and incubated for 30 min at 37 0 C. Then, 2, 4-dinitrophenyl Ehrich et al41. The specific activity of the enzyme was calculated as hydrazine (DNPH) solution was added, mixed and kept for 20 min at units/ mg protein. The glutathione peroxidase activity was measured room temperature. Thereafter, 0.4(N) NaOH was added, mixed and according to the method of Paglia and Valentine42 with some kept at room temperature for 10 min. The intensity of the developed modifications38. The glutathione-S-transferase activity of the rat colour was noted at 540 nm after setting the UV/VIS spectrophotomcardiac tissue was measured sphectrophotometrically according to eter to zero with water (Bio-Rad, Hercules, CA, USA)25 . Habig et al43. GSSG was measured by the method of Sedlak and Lindsay 32 with some modification of Bandyopadhyay et al 31. The total serum lactate dehydrogenase (LDH T) activity was obtained by measuring the oxidation of NADH (0.1 mM) to NAD+ at Tissues were homogenized (10%) in 2 mM ice-cold ethylene diamine 340 nm using 1.0 mM sodium pyruvate as substrate, according to the tetra acetic acid (EDTA). The reaction mixture contained 0.1 mM method of Strittmatter26 with some modifications 27 .The cardiac sodium phosphate buffer, EDTA, NADPH and 0.14 units per ml specific Type 1 isoform of serum lactate dehydrogenase 1 (LDH-1) glutathione reductase. The absorbance was measured at 340 nm Journal of Pharmacy Research Vol.10 Issue 6 June 2016

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Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478 using a UV-VIS spectrophotometer to determine the GSSG content. The values were expressed as nmoles GSSG/mg protein. Determination of hydroxyl (OH.) radical scavenging activity in cardiac tissues The hydroxyl radical (OH.) generated in the cardiac tissues was measured by the method of Babbs and Steiner24 using DMSO as an OH. scavenger. DMSO forms methane sulfinic acid [MSA] on reaction with OH.. Accumulation of MSA was measured to estimate the OH. generated after forming a coloured complex with fast blue BB salt. Four groups of rat containing five animals in each group, was used for the experiment. The first group served as control. These were kept at room temperature without any stress after administration of DMSO. The second group was administered aqueous bark extract of TA (20 mg/kg body weight, orally) 30 minutes before DMSO injection. The third group was injected adrenaline- bitartrate ( 0.3 mg/kg body weight, s.c.) with 0.4 mL of 25% DMSO in saline per 100 g BW, 30 minutes before the oral administration of aqueous bark extract of Terminalia arjuna ( 20 mg/kg body weight). Animals of fourth group, was pre-treated with aqueous bark extract of Terminalia arjuna (20 mg/kg body weight) 30 minutes before DMSO injection and subsequent adrenaline-bi-tartrate (0.3. mg/kg BW.) administration. The cardiac muscle of each group was processed for MSA, which was allowed to react with fast blue BB salt to yield a yellow product. This was measured spectrophotometrically at 425 nm using benzenesulfinic acid as standard. Values obtained from the non- stressed DMSO treated group, DMSO treated stressed group and DMSO plus aqueous bark extract of Terminalia arjuna treated stressed group were expressed as nmol of OH. generated per g of cardiac tissues. Indirect assessment of the generation of superoxide anion free radical (O2•-) by determining the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) Xanthine oxidase activity of rat cardiac tissue was assayed by measuring the conversion of xanthine to uric acid following the method of Greenlee et al44. Xanthine dehydrogenase (XDH) activity was measured by following the reduction of NAD+ to NADH according to the method of Strittmatter26 with some modifications45. The enzyme activity was expressed as milli units/ mg protein. Isolation of mitochondria from rat heart tissues The mitochondria from heart tissues were isolated according to the procedure of Dutta et al46. A portion of the heart tissues were cleaned and cut into small pieces. Five hundred mg of both the tissues were placed separately in 10ml of sucrose buffer [0.25(M) sucrose, 0.001(M) EDTA, 0.05(M) Tris-HCl (pH 7.8)] at 25 0 C for 5min. The tissues were then homogenized separately in cold for 1 minute at low speed by using a Potter Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA). The homogenates were centrifuged at 1500rpm for 10 minutes at 4°C. The supernatant was poured through several

layers of cheesecloth and kept in ice. This filtered supernatant was centrifuged at 4000rpm for 5minutes at 4°C. The supernatant, thus obtained, was further centrifuged at 14000rpm for 20 minutes at 4°C. The final supernatant was discarded and the pellet was re-suspended in sucrose buffer and stored at -20°C for further biochemical and histochemical analyses. However, most of the enzymatic assays were carried out with freshly prepared mitochondria. Measurement of the activities of pyruvate dehydrogenase and some of the Kreb’s cycle enzymes Pyruvate dehydrogenase activity of rat cardiac tissue was measured spectrophotometrically according to the method of Chretien et al47 with some modifications as adopted by Mishra et al36. Isocitrate dehydrogenase activity of rat cardiac tissue was measured according to the method of Duncan et al48 by measuring the reduction of NAD+ to NADH at 340 nm with the help of a UV–VIS spectrophotometer. Alpha-Ketoglutarate dehydrogenase activity of rat cardiac tissue were measured spectrophotometrically according to the method of Duncan et al48. Likewise, succinate dehydrogenase activity of rat cardiac tissue was measured spectrophotometrically by following the reduction of potassium ferricyanide [K3Fe (CN) 6] at 420 nm according to the method of Veeger et al49 with some modifications50. Measurement of the activities of some of the mitochondrial respiratory chain enzymes NADH-Cytochrome c oxidoreductase activity was measured spectrophotometrically by following the reduction of oxidized cytochrome c at 565 nm according to the method of Goyal et al51. Cytochrome c oxidase activity was determined spectrophotometrically by following the oxidation of reduced cytochrome c at 550 nm according to the method of Goyal et al51. In vitro assessment of antioxidant capacity of aqueous bark extract of Terminalia arjuna (a) Assessment of hydroxyl radical (•OH) scavenging activity by using deoxyribose as a probe: Hydroxyl radical scavenging activity was performed according to the method of Halliwell and Gutteridge 52 as modified by Chattopadhyay et al53. (b) Assessment of hydroxyl radical (•OH) scavenging activity by using DMSO as a probe: Dimethyl sulfoxide forms a stable product [methane sulfinic acid (MSA)] on reaction with •OH during incubation which was measured by the method of Babbs and Steiner 24 as modified by Bandyopadhyay et al54. (c) Assessment of superoxide anion (O2.-) free radical scavenging activity: Superoxide anion (O2.-) free radical scavenging activity of aqueous bark extract of Terminalia arjuna was studied by the method of Misra and Fridovich et al55. (d) Assessment of DPPH free radical scavenging activity: The DPPH scavenging activity of Terminalia arjuna aqueous bark extract was determined by the method of Chen and Yeh et al56.


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Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478 (e) Assessment of reducing power: Potassium ferricyanide reducing activity of Terminalia arjuna aqueous bark extract was measured by the method of Oyaizu et al57. (f) Determination of hydrogen peroxide (H2O2) scavenging activity and metal chelating activity: The hydrogen peroxide (H2O2) scavenging activity was measured by studying the breakdown of H2O2 at 240 nm by using a UV / VIS spectrophotometer and metal chelating property of aqueous bark extract of Terminalia arjuna was estimated by the method of Ningappa et al58. Estimation of proteins Proteins of the different samples were determined by the method of Lowry et al59. Tissue morphological and histochemical studies (a) Staining of cardiac tissue sections using hematoxylin-eosin and periodic acid Schiff (PAS) stains: A portion of the extirpated rat heart was fixed immediately in 10% formalin and embedded in paraffin following routine procedure as used earlier by Dutta et al33.Sections of heart tissues (5 μm thick) were prepared. The cardiac tissue sections were stained with hematoxylin-eosin stain and periodic acid Schiff (PAS) stain. The stained tissue sections were examined under Leica microscope and the images were captured with a digital camera attached to it. (b) Quantification of fibrosis by confocal microscopy: A portion of the extirpated rat cardiac (left ventricular portion) tissue were fixed immediately in 10% formalin and embedded in paraffin following routine procedure. Additionally, the left ventricular tissue sections (5 μm thick) were stained with Sirius red (Direct Red 40; Sigma Chemical Co) and imaged with laser scanning confocal system (Zeiss LSM 510 META, Carl Zeiss Micro Imaging GmbH, Jena, Germany), and the stacked images through multiple slices were captured 60. (c) Scanning electron microscopy (SEM): Small pieces of cardiac tissues were fixed overnight with 2.5% glutaraldehyde. After washing three times with PBS, the pieces were dehydrated for 10 min at each concentration with a graded ethanol series (50, 70, 80, 90, 95 and 100%). The dehydrated pieces were immersed in pure tert-butyl alcohol and were then placed into a 4°C refrigerator until the tert-butyl alcohol solidified. The frozen tissue pieces were dried by placing them into a vacuum bottle. The cardiac tissue surface morphology was evaluated by scanning electron microscopy (SEM; Zeiss Evo 18 model EDS 8100). (d) Determination of nuclear DNA damage with agarose gel electrophoresis: The nuclear fraction from the cardiac tissue homogenates were prepared and dissolved in TE

buffer, and processed for DNA isolation from this nuclear fraction. The DNA, thus obtained, gave an average 260/ 280 absorbance ratio of 2-2.5. The obtained DNA samples were then mixed with 6X loading dye and resolved in 0.8% agarose gel. The gel was stained with ethidium bromide and DNA bands detected in a Gel-Doc apparatus (Biorad, Hercules CA). (e) Scanning electron microscopy (SEM) of mitochondria isolated from of the rat cardiac tissues: The incubated mitochondrial suspension was centrifuged, and the supernatant was removed. The pellet was fixed overnight with 2.5% glutaraldehyde. After washing three times with PBS, the pellet was dehydrated for 10 min at each concentration with a graded ethanol series (50, 70, 80, 90, 95 and 100%). The pellet was immersed in pure tert-butyl alcohol and was then placed into a 4°C refrigerator until the tert-butyl alcohol solidified. The frozen samples were dried by placing them into a vacuum bottle. Mitochondrial morphology was evaluated by scanning electron microscope (SEM; Zeiss Evo 18 model 8100). (f) Determination of rat cardiac mitochondrial intactness by using Janus green B stain: Following incubation, the cardiac mitochondria were spread on a slide. After that a few drops of Janus green B stain were put on the slide and was left for 5 min for staining in moist chamber. The mitochondria were rinsed once with distilled water so that the stain was not removed and a diluted stain remained. Then, the mitochondria were mounted in a drop of distilled water with a cover slip and imaged with a confocal system (BD Pathway 855, USA). Besides, 100μl of mitochondrial suspension following incubation, were mixed with 20μl of Janus Green B stain and incubated at room temperature, in dark. The mitochondria, stained with Janus Green B were analyzed by using flow cytometry (BDFACS Versa, USA). Statistical evaluation Each experiment was repeated at least three times with different rats. Data are presented as mean ± S.E.M. Significance of mean values of different parameters between the treated groups were analyzed using one way analysis of variances (ANOVA) after ascertaining the homogeneity of variances between the treatments. Pairwise comparisons were done by calculating the least significance. Statistical tests were performed using Microcal Origin version RESULTS Figure 1 depicts a dose-dependent significant increase in the serum level activities of SGOT [Fig. 1A] and LDH-1 [Fig. 1B], the biomarkers of cardiac injury as compared to control following treatment of rats


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with different doses of adrenaline bitartrate indicating damage to cardiac tissues. Moreover, a dose dependent elevation in the level of LPO [Fig.2A] and PCO [Fig. 2B] and reduction in the level of GSH [Fig. 2C] as compared to control suggests involvement of oxidative stress in this cardiac tissue damage. Futhermore, figure 3 shows a dose-dependent increase in the activities of antioxidant enzymes such as Cu-Zn SOD [Fig. 3A] and Mn-SOD [Fig. 3B] as compared to control and also a dose-dependent decrease in the activities of catalase [Fig. 3C], glutathione peroxidase [Fig. 3D] and glutathione reductase [Fig. 3E] as compared to control, following treatment of rats with different doses of adrenaline bitartrate, suggesting involvement of oxidative stress.

Lipid peroxidation level (nmoles TBARS/mg protein)

Sanatan Mishra et al. / Journal of Pharmacy Research 2016,10(6),454-478 3

A *

2 .5 2 1 .5 1 0 .5 0 CO N

A D R 0 .1 5

A DR 0 .3

B

A

Serum glutamate oxaloacetate transaminase(IU/L)

14

*

12 10 8 6 4

Reduced Glutathione Content (nmoles of GSH/mg protein)

35

16

30 25

*

20 15 10 5 0

2

CON

0 ADR0.15

ADR0.3

B

0.35

*

0.3 0.25 0.2 0.15 0.1 0.05 0 ADR 0.15

ADR0.3

ADR0.3

ADR0.6

C 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

*

CON CON

ADR 0.15

ADR0.6

Protein Carbonyl Content (nmoles /mg protein)

CON

Serum lactate dehydrogenase-I activity (IU/L)

A DR 0 .6

ADR0.15

ADR0.3

ADR0.6

ADR0.6

The values are expressed as mean ± S.E ; * P