PUBLICATIONS

Santosh Kumar Shukla, Suman Bala Sharma, Usha Rani Singh, Sayeed

Ahamad, Shridhar Dwivedi. Terminalia arjuna augments cardioprotection via

antioxidant and antiapoptotic cascade in isoproterenol induced cardiotoxicity in

rats. Asian pacific journal of Tropical Biomedicine 2012; 1-7 (In Press).

Santosh Kumar Shukla, Suman Bala Sharma, Usha Rani Singh, Shridhar

Dwivedi, Ankur Maheshwari, Manmohan Misro. Eugenia jambolana: A

Safeguard for Isoprotenol Induced Myocardial Infarction and Apoptosis in

Rats. International Journal of Cardiology 2012 (With editor).

Shukla S.K, Dwivedi S, Singh U.R, Sharma S.B. Terminalia arjuna as a

therapeutic and preventive modulator in experimentally induced myocardial

infarction. Diabetes and Vascular Disease Research 2011; 8: 49. DOI:

10.1177/1479164110390839

Santosh Kumar Shukla, Shridhar Dwivedi, Usha Rani Singh, Suman Bala

Sharma. Cardioprotective activity of Terminalia arjuna in isoproterenol-

induced cardiotoxicity in rats. Proceedings of Indian Society for

Atherosclerosis Research 2010; 86-91.

Santosh K. Shukla, Shipra Gupta, Shreesh K. Ojha and Suman B. Sharma.

Cardiovascular friendly natural products: A promising approach in the

management of CVD. Natural Product Research 2010; 24 (9):873-898.

Suman Bala Sharma, Shipra Gupta,Rini, A.C, Usha Rani singh, Reenu Singh

Tanwar, Santosh Kumar Shukla.Antidiabetogenic action of Morus rubra L.

leaf extract in streptozotocin induced diabetic rats. Journal of Pharmacy and

Pharmacology 2010; 67: 247-255.

Suman Bala Sharma, Reenu Singh Tanwar, Rini AC, Usha Rani singh, Shipra

Gupta & Santosh Kumar Shukla. Protective effect of Morus rubra L. leaf

extract on diet induced atherosclerosis in diabetic rats. Indian Journal of

Biochemistry and Biophysics 2010; 47 (1): 26-31.

Santosh Kumar Shukla, Shridhar Dwivedi, Usha Rani Singh, Suman Bala

Sharma. Studies on antioxidant activity of water and hydroalcoholic extract of

Terminalia arjuna in normal male albino rats. Journal of Advance Researches

in Biological Sciences 2009; 1(2): 6-11.

Chapter published in Book

Suman Bala Sharma, Seema Garg, Shipra Gupta, Reenu Singh Tanwar,

Santosh Kumar Shukla, Richa Gupta, and Shreesh Ojha. Pharmacotherap-

eutic Potential of Medicinal Plants against Cardiometabolic Diseases.

Traditional and Folk Herbal Medicine: Recent Researches 2012, Vol. 1 p. 155-

173 Daya Publishing House, New Delhi.

ABSTRACT PRESENTED IN CONFERNCES/ SYMPOSIUM

International Conferences

Poster presentation

S.K. Shukla, S.B. Sharma, U.R. Singh, S. Dwivedi Eugenia jambolana

augments cardioprotection via antioxidant cascade in isoproterenol-induced

myocardial ischemia in rats. Selected for poster discussion in “World Diabets

Congress, 4-8 Dec. 2011, Dubai ,UAE”

Santosh Kumar Shukla, Shridhar Dwivedi,Usha Rani Singh,Suman Bala

Sharma. Terminalia arjuna as a therapeutic and preventive modulator in

experimentally induced myocardial infarction.Poster presented at International

conference “Controversies and Advances in the treatment of cardiovascular

disease”-The tenth in series on 6th ,7th and 8th October 2010 at Montage Beverly

hills Los Angeles ,California, USA .

S.K.Shukla, S.Dwivedi, U.R. Singh, S.B. Sharma, Cardioprotective effect of

Terminalia arjuna on isoproterenol induced myocardial infarction in rats.

Indian Journal of Clinical Biochemistry supplement2009; 24:326.

Santosh Kumar Shukla, Shridhar Dwivedi, Suman Bala Sharma. Comparison

of antioxidant activity of water and hydroethanolic extract of Terminalia

arjuna in normal male albino rats. Poster presented at International conference

on Advances in free Radical Research: Natural products, antioxidants and

radioprotectors & 8th Annual meeting of the Society of Free Radical Research-

India, held at C.S.M medical University, Lucknow 19-21march 2009

S Gupta, Ac Rini, SK Shukla, SB Sharma, Antidiabetogenic activity of Morus

rubra leaf extract in streptozotocin-induced diabetic rats: A herbal remedy

Clinical Biochemistry reviews 2008, S 128.

Shukla SK, Dwivedi S, Singh UR, Sharma SB, Cardioprotective effect of

Terminalia arjuna on isoproternol induced myocardial infarction in rats:

Towards Drug Therapy, Poster presented at 8th International Congress on

Coronary Artery Disease From Prevention To Intervention ·(Abstract: A-150-

0010-00844)2008,Prague Crezh Republic.

Awards/achievements

Awarded Balaji Endowment Gold Medal for Basic Research in

Atherosclerosis during 23rd Annual conference of Indian Society For

Atherosclerosis Research (ISAR) & International Symposium on

Atherosclerosis From Bench to Bedside on 13th-14th November 2010 at

University College of Medical Sciences & GTB Hospital Delhi.

Awarded Young scientist Travel award to attend International conference

“Controversies and Advances in the treatment of cardiovascular disease”-The

tenth in series on 6th, 7th and 8th October 2010 at Montage Beverly hills Los

Angeles, California, USA by Department of Science & Technology (DST)

Govt. of India, New Delhi.

Awarded by ACBI Travel Fellowship Award in 36th Annual conference of

Association of Clinical Biochemists of India, 5th, 6th, 7th November 2009,

Amrita Institute of Medical Sciences, Kochi, Kerala.

1

Document heading

Terminalia arjuna augments cardioprotection via antioxidant and antiapoptotic cascade in isoproterenol induced cardiotoxicity in ratsSantosh Kumar Shukla1, Suman Bala Sharma1*, Usha Rani Singh2, Sayeed Ahamad3, Shridhar Dwivedi41Department of Biochemistry, University College of Medical Sciences (University of Delhi), Delhi 110095, India2Department of Pathology, University College of Medical Sciences (University of Delhi), Delhi 110095, India3Bioactive Natural Product Laboratory, Department of Phrmacognosy & Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi - 110062, India4Department of Medicine/Preventive Cardiology, University College of Medical Sciences (University of Delhi), Delhi-110095, India

Asian Pacific Journal of Tropical Biomedicine (2012)1-7

Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

*Corresponding author: Prof. Suman Bala Sharma, Department of Biochemistry, University College of Medical Sciences (University of Delhi), Delhi-110095, India. Tel: +91 22582972-74 Ex5204 (O) Fax: +91 11 22590495 E-mail: drsbs08@hotmail.com Foundation Project: Supported by Central Council for Research in Ayurveda and siddha (F. No. Z.31014/ 03/2007/EMR/CCRAS).

1. Introduction According to world health organization ischemic heart disease (IHD) is the leading cause of mortality worldwide and account for well over 20% of all deaths[1]. Myocardial infarction (MI) is a pathological condition which is associated with morphological changes and involves an interface of multiple pathophysiology of MI[2]. Isoproterenol (ISP) is a synthetic beta adrenoceptor agonist, which has been found to induce MI in rats[2,3]. Myocardial necrosis induced by ISP is responsible for increase in cardiac marker enzymes, pro-inflammatory cytokines, accumulation of lipid peroxides and damaged cardiac function[4-7]. The alterations in pathophysiological environment during MI in rats are comparable with those taking place in human myocardial ischemia[8-10]. There is an urgent need for the drug, which could limit the myocardial injury. It is well established that reactive oxygen species (ROS) have been

implicated in the pathophysiology of MI[2]. Therefore, therapeutic intervention with antioxidant may be useful in preventing these deleterious changes. Terminalia arjuna (T. arjuna), a deciduous tree of Combretaceae family, is traditionally used for the treatment of cardiovascular ailments since ancient time. The bark of T. arjuna has been mentioned in ancient Indian medicinal literature to have beneficial effects in heart diseases[11]. The crude bark of T. arjuna has strong antioxidant property and it augments endogenous antioxidant compound in rats[12]. The stem bark of T. arjuna has been reported to contain different groups of chemical constituents viz., hydrolysable tannins, triterpene, flavonoids, phenolics, and phytosterols[11]. Important triterpenes are arjunetin, arjunic acid, arjunolic acid, and arjungenin[11]. Arjunolic acid (2, 3, 23-trihydroxyolean-12-en-28-oic acid) possesses cardioprotective and hypotensive effect due to its strong antioxidant activity and it has beneficial effect on ISP-induced myocardial necrosis[13]. Despite the large number of studies on T. arjuna no study has yet addressed cardioprotective/antiapoptotic effect of hydroalcoholic extract of bark of T. arjuna in experimental myocardial ischemia. In this study, we aimed to investigate the molecular modulation of ISP related myocardial damage, biochemical and histopathological alterations with the

ARTICLE INFO ABSTRACT

Article history:Received 15 April 2011Received in revised form 27 April 2011Accepted 28 June 2011Available online 28 June 2011

Keywords:Terminalia arjunaIsoproterenolMyocardial infarctionOxidative stressApoptosis

Objective: To investigate protective effects of hydroalcoholic extract of bark of Terminalia arjuna (T. arjuna) on biochemical and apoptotic changes during cardiotoxicity induced by isoproterenol (ISP) in rats. Methods: Hydroalcoholic extract of bark of T. arjuna was evaluated for its antioxidant, anti-apoptotic and cardioprotective activity at a dose of 100, 200 and 400 mg/kg bodyweight, administered orally for 30 days with concurrent administration of ISP (85 mg/kg bodyweight) on days 29 and 30 at an interval of 24 h. To compare the antioxidant activity of the extract, alpha-tocopherol was used as a standard drug. Results: ISP caused deleterious changes in the myocardium viz., increased myonecrosis, inflammation, edema which finally leads to apoptotic cell death. However hydroalcoholic extract of bark of T. arjuna and alpha-tocopherol showed significant anti-oxidant and anti-apoptotic effect in isoproterenol induced oxidative stress. Pre-treatment with the extract and alpha-tocopherol, reduced the elevated level of cardiac marker enzymes. Histopathological findings revealed that architecture of the myocardium was restored towards normal after the extract and alpha-tocopherol pre-treatment. Conclusions: Hydroalcoholic extract of bark of T. arjuna attenuates oxidative stress, apoptosis and improves cardiac performance in ISP-induced cardiotoxicity in rats and hence attributes cardioprotection.

Contents lists available at ScienceDirect

Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-72

concomitant use of hydroalcoholic bark extract of T. arjuna.

2.Materials and methods

2.1.Animals

Forty eight of 10 to 12 week-old male wistar albino rats weighing 180-230 g [(210依20.58) g] were used in the study. The study protocol was reviewed and approved by the Institutional Animal Ethical Committee of University College of Medical Sciences, Delhi, India (IAEC/UCMS/DB-11/2008) and confirms to the Indian National Science Academy guidelines for the use and care of experimental animals in research. Animals were obtained from the central animal house facility of University College of Medical Sciences, Delhi, India. The rats were housed in polyacrylic cages (38x 23x 10 cm3) with not more than four animals per cage. They were housed under standard laboratory conditions with natural light and dark cycles (approximately 12 h light/12 h dark) and maintained at humidity of (55依5)% and an ambient temperature of (22依2) 曟. The animals were allowed free excess to standard pellet diet (Durga Brothers Pvt. Ltd.) and tap water ad libitum.

2.2. Drugs and reagents

ISP was obtained from Sigma Chemical Company, St. Louis, USA, and all other chemicals used were of analytical grade. CK-MB, SGOT and LDH assay kits were procured from Spinreact SA, Spain, and Troponin I ELISA kit from Calbiotech, USA. Immunohistostaining, kit based on HRP polymer detection system was purchased from Thermo Fisher Scientific, USA and primary antibodies (Bax mouse monoclonal IgG2b and Bcl-2 mouse monoclonal IgG1) from Santa Cruz Biotechnology, USA. TUNEL assay kit was obtained from R&D Systems Minneapolis, alpha-tocopherol was purchased from Merck, USA.

2.3. Plant material

T. arjuna was obtained from the campus of Institute of Human Behaviour and Allied Seciences (IHBAS), Delhi, India and authenticated by Dr. Sayeed Ahmad, Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi,India. The voucher specimen (BNPL/JH/078/2008) was kept for future reference.

2.4. Preparation of hydroalcoholic bark extract of T. arjuna

The bark was crushed in a mixer to coarse powder (sieve #60) and then extracted with 50% v/v ethanol thrice by macerating the material in 1:20 drug: solvent ratio for 24 h with occasional shaking at room temperature and sonicating the mixture for 30 min before filtration using 5-6 layers of muslin cloth. The filtrate was first centrifuged and then supernatant was lyophilized to store it for a longer duration. It was kept in a tightly closed bottle, protected from light in the refrigerator at 2 to 8 曟 to be used throughout the experiment. The yield of the hydroalcoholic extract was 18.5% w/w of the dried powder. The HPTLC fingerprint

analysis and estimation of arjunolic acid was done for quality control/standardization of extract.

2.5. Quantative analysis of the hydroalcoholic extract of T. arjuna by HPTLC

The sample was prepared by taking 750 mg of hydroalcoholic extract of T. arjuna in 25 mL of aqueous acidic solution (HCl 5.0 % v/v in water) and refluxed for one hour on water bath. The extract was filtered and taken into a separating funnel. It was then extracted with chloroform by taking same quantity and the process was repeated three times for complete extraction. The chloroform extracts were pooled and evaporated to dryness. The residue obtained was dissolved in 10 mL of HPLC grade methanol. The stock solution of standard arjunolic acid (purity 98%) was prepared in HPLC grade methanol to get 1.0 mg/mL solution. The estimation was carried out as per the method described by Singh et al[14] . The samples were spotted in the form of bands of width 4 mm using microlitre syringe on pre-coated silica aluminum sheet 60F254 (5.0 cm暳10.0 cm, 0.2 μm thickness) using Camag Linomat V sample applicator (Switzerland). The plates were pre-washed with methanol and activated at 60 曟 for 20 min prior to chromatography. A constant application rate of 0.12 μL per second was employed and space between two bands was 15 mm. The slit dimension was kept at 4.0 mm暳0.3 mm and 20 mm per second scanning speed were employed. The mobile phase consisted of choloroform: toluene: ethanol (4:4:1, v/v/v) and 10 mL of mobile phase was used for per chromatography. Linear ascending development was carried out in 10 cm×10 cm twin trough glass chamber, which was previously saturated with mobile phase for 15 min. The length of the chromatogram run was 80 mm. After the development, TLC plates were dried in a current of air with the help of an air dryer and sprayed with anisaldehyde sulphuric acid reagent and again air dried then kept in oven for 10 min at 110 曟. The densitometric scanning was performed on Camag TLC scanner III operated by winCats software using wavelength 600 nm.

2.6. Induction of myocardial ischemia

ISP was freshly prepared in normal saline and injected subcutaneously (s.c.) at a dose of 85 mg/kg bodyweight to the rats on 28th and 29th day at an interval of 24 h[15].

2.7. Experimental protocol

Animals were divided into six groups, each consisting of eight rats (n=8). Group 1 (Healthy control)-Orally administered normal saline; Group 2 (Ischemic control)-Normal saline+ISP (85 mg/kg bodyweight ) ; Group 3-hydroalcoholic extract of T. arjuna (HETA) 100 mg/kg bodyweight+ISP (85 mg/kg bodyweight); Group 4-HETA 200 mg/kg bodyweight+ISP (85 mg/kg bodyweight); Group 5- HETA 400 mg/kg bodyweight+ISP (85 mg/kg bodyweight); Group 6-Vit E (alpha-tocopherol) 100 mg/kg bodyweight+ISP (85 mg/kg bodyweight)[16]. The treatment was given for 30 consecutive days once in a day using standard orogastric intubation. On 29th and 30th days, rats were subjected to ISP (85 mg/kg bodyweight s.c.) with 24 h interval. The healthy control group was not

Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-7 3

subjected to ISP insult. Alpha-tocopherol was dissolved in 5% gum acacia.

2.8. Sample collection

Fasting blood samples were collected from retro orbital plexus of the animals at 0th, 21st and 30th days to perform biochemical studies. After blood sampling on 30th day, the rats were anesthetized with sodium pentobarbital (30 mg⁄kg, i.p.). To carry out immunohistochemical and histological studies, the heart was immediately dissected out, washed in ice-cold saline and stored in 10% buffered neutral formalin solution.

2.9. Measurement of oxidative stress parameters

Serum lipid peroxide levels were estimated using the method previously described by Satoh[17]. Activity of SOD in erythrocytes was assayed as described by Marklund &Marklund[18] and modified by Nandi&Chatterjee[19]. Erythrocyte’s glutathione (GSH) content was estimated by the method of Beutler et al[20].

2.10. Studies on cardiac marker enzymes

The myocardial injury markers such as CK-MB, SGOT and LDH in serum were estimated spectrophotometrically by using commercially available kits and serum troponin I was measured by using ELISA kit.

2.11. Apoptotic studies

Myocardial tissue samples preserved in 10% buffered formalin were carefully embedded in molten paraffin with the help of metallic blocks, covered with flexible plastic moulds and kept under freezing plates to allow the paraffin to solidify. Cross sections (5 μm thick) of the fixed myocardial tissues were cut from paraffin-embedded blocks on a microtome and mounted onto poly-lysine coated microscope slides and dried completely to proceed for immunohistostaining and TUNEL assay[21].

2.12. Immunostaining for the localization of Bax and bcl-2 proteins

A mouse monoclonal anti-bcl-2 and Bax proteins were used as the primary antibody for bcl-2/ Bax immunohistochemical staining. The Ultravision ONE HRP polymer detection system locates primary antibody by a universal secondary antibody polymer formulation. The amino acid polymer is conjugated to horseradish peroxidase (HRP) and the Fab fragments of secondary antibody. The polymer complex is visualized with an appropriate chromogen/substrate. Briefly, formalin-fixed paraffin-embedded myocardial sections were subjected to the immunohistochemical procedure for the localization of Bax and Bcl-2 proteins using specific mouse monoclonal primary antibodies. Sections are first blocked and then incubated in primary antibody followed by Ultra Vision One HRP polymer. The target protein (Bax/Bcl-2) was visualized by incubation in peroxidase substrate (H2O2) using 3, 3’ diaminobenzidine (DAB) as the chromogen.

2.13. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay)

Myocardial apoptosis was quantified by detection of DNA fragmentation using the TUNEL technique[21]. Briefly, the enzyme terminal deoxynucleotidyl tranferase was used to incorporate residues of digoxigenin nucleotide into 3’ OH ends of DNA fragments. The free end of cellular DNA was labeled by incubating the specimens in streptavidin conjugated to horse radish peroxidase enzyme and peroxidase substrate. TUNEL assay was used to identify apoptotic cells using secondary reaction with antibodies and DAB chromogen. The slides were counterstained in methyl green and total cell counts and TUNEL positive cells in the specimens were determined by means of a light microscope. The cells with clear nuclear labeling were defined as TUNEL positive cells.

2.14. Histopathological studies

Myocardial tissue fixed in buffered formalin was processed for paraffin embedding, sectioned at 5 μm and mounted onto microscope slides. These sections were stained with haematoxylin and eosin (H&E), and visualized under light microscope to study the histoarchitectural changes of the myocardium.

2.15. Data analysis

The results were expressed as mean依SEM. and statistical differences between mean values were determined by repeated measure analysis of variance (ANOVA) followed by the Tukey’s test for multiple comparisons. A value of P<0.05 was considered statistically significant.

3. Results

3.1. Characterization of hydroalcoholic extract of T. arjuna Characterization of hydroalcoholic extract of T. arjuna was done by HPTLC. The analysis of the extract showed the presence of 0.394 % w/w arjunolic acid (Figure 1).

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Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-74

ARJUNOLIC ACID

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3.2. Effect of hydroalcoholic extract of bark of T. arjuna on oxidative stress and cardiac function markers

Table 1 demonstrates the effect of hydroalcoholic extract of bark of T. arjuna on oxidative stress parameters. ISP control rats showed significant rise in serum MDA levels and significant reduction in SOD activity and GSH content in erythrocytes compared to healthy control group. T. arjuna extract at a dose of 100, 200, 400 mg/kg of produced dose dependent response on antioxidant markers (SOD and GSH). No significant effect was observed on erythrocyte’s SOD and GSH at a dose of 100 mg/kg of the extract when compared to ISP control group. However, extract at 200 and 400 mg/kg doses produced significant improvement in the activity of SOD (P<0.05 and P<0.001 respectively) and GSH levels (P<0.05 and P<0.01 respectively). MDA levels were found to be significantly reduced (P<0.001) in all the groups treated with extract compared to ISP control group. The rats treated with alpha-tocopherol also showed significant decrease (P<0.001) in MDA levels and significant increase in SOD (P<0.001) and GSH (P<0.05) levels.Table 1.Biochemical parameters of oxidative stress in rat serum.Group MDA (nmol/mL) GSH (mg/dL) SOD (U/gHb)

Normal 1.59依0.17 35.85依5.56 1250.00依76.07ISP control 3.59依0.42### 26.80依1.55## 940.17依60.29###

HETA+ISP100 2.10依0.28 33.21依3.66 1110.17依64.32HETA+ISP200 2.08依0.12*** 34.62依1.60* 1132.67依55.26*

HETA+ISP400 1.43依0.13*** 38.54依1.97** 1316.57依62.12***

Vit E+ISP 1.84依0.29*** 40.96依1.30** 1212.00依17.16***

MDA: malondialdehyde; GSH: reduced glutathione; SOD: superoxide dismutase; HETA: hydroalcoholic extract of T. arjuna; ISP: isoproterenol. Values are expressed as means依SEM (n=8); ## P≤0.01; ### P≤0.001 vs. normal rats; *P≤0.05, **P≤ 0.01, ***P≤0.001 vs. ISP control.

Table 2 shows the effect of T. arjuna extract on cardiac function markers. ISP control rats exhibited significantly (P<0.001) higher levels of serum myocardial injury markers

such as SGOT, CK-MB, troponin I and LDH as compared to normal rats. The pre-treatment with HETA at 100, 200 and 400 mg/kg doses produced significant reduction (P<0.001) in CK-MB, SGOT and LDH levels compared to ISP control group. The levels of troponin I was also significantly decreased with all the tested doses of the extract, however statistical difference with 100 mg/kg dose (P<0.01) was less significant than that of 200 and 400 mg/kg doses (P<0.001). Alpha-tocopherol treated group showed significant reduction (P<0.001) in all the oxidative stress and cardiac markers. There was no significant change in antioxidant status as well as cardiac function markers on 21st day of pre-treatment in all the groups, therefore they were excluded.Table 2.Biochemical parameters of myocardial injury in rat serum.

SGOT (U/L) CPK-MB (U/L) TropI(μg/L) LDH (U/L)

Normal 149.13依15.86 299.38依23.97 0.03依0.01 695.30依5.95

ISP control 1307.50依80.04# 2137.83依82.85# 6.01依0.31# 1426.00依6.13#

HETA+ISP100 1072.50依64.18** 1831.50依172.63** 4.77依0.73** 1335.00依6.17

HETA+ISP200 1044.50依88.16** 1707.33依88.96** 4.52依0.53** 1305.00依5.76**

HETA+ISP400 908.14依73.54** 1590.00依94.25** 3.89依0.50** 1210.00依9.12**

Vit E+ISP 1032.57依65.49** 1628.43依66.65** 3.73依0.38** 1235.00依8.67**

SGOT: serum glutamate oxalloacitate transaminase; CK-MB: creatine phophokinase myocardial band; Trop-I: Troponin I; LDH: lactate dehydrogenase. Values are expressed as mean±SEM (n=8); # P≤0.001 vs. normal rats; **P≤0.01 vs. ISP control.

3.3. Effect of on histology of myocardium

Microscopic examination of heart sections from healthy control group presented normal architecture of the myocardium (Figure 2a). Sections from ISP control rat showed

Figure2. Representative photomicrographs of myocardial section stained with H&E (200暳) from different experimental groups. a: Healthy control rats showing normal architecture of myocardium; b: ISP control rats, showing myocardial necrosis with excessisive inflammatory cell infilteration; c: HETA 400 treated rats, showing normal myocardium with less inflammation; d: Vitamin E (alpha-tocopherol) treated rats, showing normal myocardium. Figures are representative of at least five separate experiments.

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Figure 1. HPTLC chromatogram.a: Arjunolic acid standard; b: hydroalcoholic extract of T. arjuna. showing the presence of arjunolic acid.

Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-7 5

marked focal myonecrosis, hyper-contracted myofibrils, vacuolar degeneration and lymphocytic infiltration (Figure 2b). Hydroalcoholic extract of bark of T. arjuna treated rats (400 mg/kg bodyweight) demonstrated structural improvement with regard to decrease degree of myonecrosis and contraction in myofibrils (Figure 2c). In addition, lesser vacuolization and inflammation in the myocytes was observed. Alpha-tocopherol treated rats also revealed reduction in myocardial abnormalities (Figure 2d). The histopathological changes during ischemic episode were graded and summarized (Table 3).

Table 3.Effect of Hydroalcoholic extract of bark of T. arjuna on the degree of histological changes in normal and isoproterenol induced cardio toxic rats.Groups Necrosis Inflammation EdemaNormal A A AISP control +++ +++ +++HETA+ISP100 ++ + ++HETA+ISP200 ++ ++ ++HETA+ISP400 + A AVit E+ISP + A +

Photomicrographs were used to evaluate the degree of damage in the heart tissues. A: No changes; +: Focal changes; ++: Mild changes; +++: Marked changes. ISP: Isoproterenol; HETA: hydroalcoholic extract of T. arjuna.

3.4. Effect of hydroalcoholic extract of bark of T. arjuna on myocardial apoptosis myocyte Bax/bcl2 protein expression and TUNEL positivity

3.4.1. Myocyte Bax protein expression

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Figure 3. Representative photomicrographs of ventricular tissue stained for Bax protein (400暳) from different experimental groups. The localization of Bax protein is indicated by purple positive immunoreactivity. a: Healthy control rats,showing slight Bax immunoreactivity; b: ISP control rats, showing increased expression for Bax protein; c: HETA 400 treated rats, attenuated Bax expression; d: Vitamin E treated rats, showing attenuation in Bax expression.

As shown in Figure 3a, slight Bax immunoreactivity (3.2%依

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Figure 4. Immunohistochemical findings of Bcl-2 protein in the rat myocardium (200暳) from different experimental groups. The localization of Bcl-2 protein is indicated by dark brown positive immunoreactivity. a: Healthy control rats, showing positive bcl-2 immunoreactivity in the myocytes; b: ISP control rats, shows lesser bcl-2 immunoreactivity; c: HETA 400 treated rats, showing up regulation in the expression of bcl-2; d: Vitamin E treated rats upregulated bcl-2 expression.

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Figure 5. Representative photomicrographs of ventricular tissue stained for nick-end labeling (TUNEL) for DNA breaks (200暳) from different experimental groups. TUNEL positive nuclei are stained as brown to black nuclei. a: Healthy control rats, showing no TUNEL positive cells; b: ISP control rats, showing TUNEL positive cells, indicated by brown to black staining; c: HETA 400 treated rats, reduced TUNEL positivity; d: Vitamin E treated rats.

0.7%) was observed in the myocytes of the healthy control group. ISP-induced myocardial injury significantly increased (P<0.001) the expression of Bax protein (9.3%依

Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-76

0.7%). However, Bax expression was found to be significantly attenuated (P<0.01) in the extract treated (5.05%依0.5%) and in alpha-tocopherol (5.03%依0.7%) treated groups (Figure 3b, 3c and 3d, respectively).

3.4.2. Myocyte bcl-2 protein expression Bcl-2 protein was expressed in the myocardium of healthy control group, as indicated by positive Bcl-2 immunoreactivity in the myocytes (1.75%依0.25%) (Figure 4a). ISP administration resulted in a reduction of Bcl-2 expression as decreased bcl-2 immunoreactivity was demonstrated in light microscopic evaluation of sections from ISP control group (Figure 4b). Sections from pretreatment rats with T. arjuna bark extract revealed significant up regulation (P<0.01) in the expression of Bcl-2 (8.15%依0.15%), (Figure 4c). Alpha-tocopherol group also showed improvement in Bcl-2 expression (Figure 4d).

3.4.3. TUNEL positivity No TUNEL positive cells were observed in the healthy control group (Figure 5a). In healthy control group, the cells were taken methyl green staining indicative of normal cells. However, TUNEL positive cells were seen indicated by significant red to brown staining (P<0.001) in the myocytes of ischemic control group (3.5%依0.5%) (Figure 5b). Pre-treatment with T. arjuna bark extract 400 mg/kg bodyweight significantly reduced (P<0.01) TUNEL positivity (0.7%依0.3%). Pre-treatment with alpha-tocopherol also reduced (P<0.01) the number of TUNEL positive cells vs. ischemic control group (0.65%依0.5%), (Figure 5c, 5d).

4. Discussion

ISP is well known cardiotoxic agent and mediates myocardial injury primarily via the β1-adrenergic receptor. Due to stimulation of β-adrenergic receptor, superoxide dismutase enzyme activity was down regulated and glutathione level was also reduced which further leads to the loss of membrane integrity and myocyte toxicity, a remarkable sign of myocardial necrosis[3,22]. In the present study, ISP administration showed significant elevation in lipid peroxidation and suppression in antioxidant defense cascade as evident by significant reduction in SOD and GSH levels. The prior administration of T. arjuna bark extract at 200 and 400 mg/kg doses decreased serum lipid peroxide levels, and improved the erythrocyte’s SOD activity and glutathione content. Since oxidative stress is the major deleterious factor, it can be suggested that antioxidant effect of hydroalcoholic extract of bark of T. arjuna significantly narrows down the development of MI produced by ISP. T. arjuna attributes antioxidant effect due to its flavonoid and other polyphenolic constituents, leading to radical scavenging of both free radicals and reactive oxygen species[23]. The quantitative analysis of the hydroalcoholic extract of bark of T. arjuna revealed the presence of arjunolic acid (0.394% w/w) in this study, which has been reported earlier for its beneficial cardioprotective effect in ISP-induced MI in rats[13]. ISP lead to the destruction of myocardial cells, as a result cytosolic enzymes are released into blood stream and thus serve as the diagnostic markers of myocardial tissue damage[24,25]. In the present study, ISP treated rats showed significant elevation in the serum

levels of cardiac function markers (SGOT, CK-MB, LDH and troponin I). The extract (100, 200 and 400 mg/kg) showed significant reduction in cardiac markers. Significant rise observed in the levels of diagnostic markers in the serum following ISP administration is an indication of the severity of the necrotic damage to the myocardial membrane[26]. The amount of these markers appears in serum in proportion to the number of necrotic cells[27]. Thus, reduction in cardiac markers reflects reduced extent of myocardial damage in rats pretreated with HETA. Two distinct types of cell death in myocardium i.e., necrosis and apoptosis, have been linked with cardiac damage. Although it has been reported that necrotic cell death leads to a destruction of a large group of cells, apoptosis may independently contribute to irreversible myocardial damage[28,29]. Induction of apoptosis is implicated in myocardial injury among various cardiovascular diseases[30]. The fact that apoptosis plays a role in the tissue damage after myocardial infarction has pathological and therapeutic implications. Cardiomyocyte apoptosis plays an important role in initiation and progression of cardiac diseases, drugs that effectively and specifically inhibit apoptosis might be useful therapeutic agents for attenuating myocardial injury[31]. Hence, the screening of hydroalcoholic extract of bark of T. arjuna for anti-apoptotic activity is of clinical importance. In this study, anti-apoptotic evaluation was carried out by means of TUNEL assay and the localization of Bax and Bcl-2 proteins to delineate the involvement of apoptosis in ISP-induced injury. Results of this study demonstrated that ISP administration triggers apoptotic cell death as evident by increase in infarct size, TUNEL positivity and Bax expression, and reduction in Bcl-2 expression in ISP control group compared to healthy control group. This observation receives support from earlier studies[32,33]. Pre-treatment with T. arjuna extract reduced apoptotic cells which are further confirmed by increased Bcl-2 and attenuated Bax expression and infarct size in extract-treated rats compared to ISP control rats. Various studies have demonstrated that not only ROS Per Se but also their oxidation products and other by-products generated by ROS can trigger the programmed cell death[29]. It has been reported that the antioxidants inhibited the programmed cell death[34]. Hence, it can be suggested that HETA exhibits anti-apoptotic potential due to its strong antioxidant property. Histopathologic evaluation further confirms the cardioprotective potential of hydroalcoholic extract of bark of T. arjuna. The rats received treatment with extract demonstrated improvement in structural myocardial morphology in contrast to ISP control group. Hence, hydroalcoholic bark extract of T. arjuna may have salvaged myocytes and prevented cell loss induced by apoptosis and necrosis. In conclusion, our study revealed that pre-treatment with hydroalcoholic extract of T. arjuna provide significant cardioprotection to ISP challenged myocardial infarcted rats. Our study demonstrated that hydroalcoholic extract of T. arjuna pre-treatment has better antioxidant and anti-apoptotic activity which further strengthens the cardioprotective potential of the extract. Hence it may be useful as adjuvant therapy along with conventional drugs.

Conflict of interest statement

Santosh Kumar Shukla et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-7 7

We declare that we have no conflict of interest.

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6

Journal of Advance Researches in Biological Sciences, 2009, 1 (2), 6-11

STUDIES ON ANTIOXIDANT ACTIVITY OF WATER AND HYDROALCOHOLIC EXTRACT OF TERMINALIA ARJUNA IN NORMAL MALE ALBINO RATS

Santosh Kumar Shukla1, Shridhar Dwivedi2, Usha Rani Singh3, Suman Bala Sharma1 Deptt. of Biochemistry1, Deptt. of Medicine and Preventive Cardiology Group2 & Deptt. of Pathology3 University College of Medical Sciences (University of Delhi), Delhi

Paper Submitted on:- 13/11/2009, Accepted on:- 29/11/2009

ABSTRACT

BACKGROUND AND OBJECTIVES:- The present study demonstrates the effect of water and hydroalcoholic extract of Terminalia arjuna bark on lipid peroxidation (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) to evaluate their antioxidant activity. METHOD: Wistar albino male rats (n=6) weighing 150-200g were randomly divided in to Normal Saline Control (Group I), Hot water extract of T. arjuna treated (Group II) ,Cold water extract of T. arjuna treated(Group III) and Hydroalcoholic extract of T. arjuna treated(Group IV). The extracts were orally administered for 21 days at a dose of100 mg/kg b.w. Fasting blood samples were taken prior to and after given the treatment to estimate MDA, GSH and SOD. After comparative analysis of water and hydroalcoholic extract, the potential extract was orally administered at dose of(100,200 and 400 mg/kg b.w) and MDA, GSH and SOD were evaluated. RESULTS: HWTA, CWTA and HETA produced significant depletion in MDA with a concomitant elevation in activity of SOD, however more prominent effect was seen in rats treated with HETA. The GSH level were significantly increased by HETA. Although HETA showed dose dependant response, no significant difference was observed with 200 and 400 mg/kg doses when compared with 100mg/kg b.w dose. CONCLUSIONS: This study shows that the hydroalcoholic extract of crude bark of T.arjuna augments endogenous antioxidant compounds in rats.

KEYWORDS: Antioxidant, Cardiovascular disease, Hydroalcoholic extract, Terminalia arjuna

INTRODUCTION

Cardiovascular diseases is one of the leading cause of morbidity and mortality in developed as well as in developing countries1, including India too2 and their prevention is a major public health challenge. Despite low incidence in developing countries, mortality from cardio vascular disease CVD) contributes a greater share of the global burden than industrialized nations3. The incidence of cardiovascular disease is on an increase in Asia4. By 2025, a large share of the incidence of cardiovascular disease worldwide will occur in Asia. The emphasis on prevention, early detection and advances in therapeutic modalities has

Corresponding Author:- Dr. S.B Sharma Department of Biochemistry University College of Medical Sciences & GTB Hospital, Delhi.

significantly reduced morbidity and mortality. Increasing public awareness and scientific interest has directed research towards the role of antioxidant in health promotion and disease treatment. Oxidative stress plays a potential role in CAD and it has been suggested to accelerate atherosclerosis. Terminalia arjuna is traditionally used for the treatment of heart disease.

The bark of Terminalia arjuna (TA), a deciduous tree of Combretaceae family, has been mentioned in ancient Indian medicinal literature to have beneficial effects in heart diseases5 . The crude bark of T. arjuna has strong antioxidant property and it augments endogenous antioxidant compound in rats6.T.arjuna bark extract have shown antioxidant effect on N-nitrosodiethylamine induced