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Journal of Ethnopharmacology 117 (2008) 123–129

Protective effects of Terminalia arjuna againstDoxorubicin-induced cardiotoxicity

Gurvinder Singh a, Anu T. Singh a,∗, Aji Abraham a, Beena Bhat a, Ashok Mukherjee a,Ritu Verma a, Shiv K. Agarwal a, Shivesh Jha b, Rama Mukherjee a, Anand C. Burman a

a Dabur Research Foundation, 22 Site IV, Sahibabad, Ghaziabad 201010, U.P., Indiab Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, India

Received 20 April 2007; received in revised form 7 January 2008; accepted 24 January 2008Available online 3 February 2008

bstract

Terminalia arjuna has been marked as a potential cardioprotective agent since vedic period. The present study was aimed to investigate theffects of butanolic fraction of Terminalia arjuna bark (TA-05) on Doxorubicin (Dox)-induced cardiotoxicity. Male wistar rats were used as in vivoodel for the study. TA-05 was administered orally to Wistar rats at different doses (0.42 mg/kg, 0.85 mg/kg, 1.7 mg/kg, 3.4 mg/kg and 6.8 mg/kg)

or 6 days/week for 4 weeks. Thereafter, all the animals except saline and TA-05-treated controls were administered 20 mg/kg Dox intraperitonially.here was a significant decrease in myocardial superoxide dismutase (38.94%) and reduced glutathione (23.84%) in animals treated with Dox.oncurrently marked increase in serum creatine kinase-MB (CKMB) activity (48.11%) as well as increase in extent of lipid peroxidation (2.55-fold)as reported. Co-treatment of TA-05 and Dox resulted in an increase in the cardiac antioxidant enzymes, decrease in serum CKMB levels and

eduction in lipid peroxidation as compared to Dox-treated animals. Electron microscopic studies in Dox-treated animals revealed mitochondrial

welling, Z-band disarray, focal dilatation of smooth endoplasmic reticulum (SER) and lipid inclusions, whereas the concurrent administration ofA-05 led to a lesser degree of Dox-induced histological alterations. These findings suggest that butanolic fraction of Terminalia arjuna bark hasrotective effects against Dox-induced cardiotoxicity and may have potential as a cardioprotective agent.

2008 Elsevier Ireland Ltd. All rights reserved.

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eywords: Terminalia arjuna; Doxorubicin; Cardiotoxicity; Wistar rats; Antio

. Introduction

The bark of Terminalia arjuna (Roxb.) Whight and Arnot, aeciduous tree of Combretaceae family, has been widely used

ince Vedic period for the treatment of various heart diseases. It isppropriately known as “Hridya”, as it possesses heart strength-ning and cardiotonic properties. It has long established use in

Abbreviations: CKMB, creatine kinase; Dox, doxorubicin; DTNB, [5,5-ithiobis(2-nitrobenzoic acid)]; DPPH, [1,1-diphenyl-2-picrylhydrazyl]; GSH,educed glutathione; MDA, malonyldialdehyde; mg, milligram; NADH, nicoti-amide adenine dinucleotide-reduced disodium salt; ROS, reactive oxygenpecies; SER, smooth endoplasmic reticulum; SDS, sodium dodecyl sulphate;OD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituriccid reactive substances; TCA, trichloroacetic acid; IR, ischemic reperfusion.∗ Corresponding author at: Dabur Research Foundation, Molecular Oncology

ab, 22, Site IV, Sahibabad, Ghaziabad 201010, U.P., India.el.: +91 120 4378505; fax: +91 120 2777303.

E-mail address: singhat@dabur.com (A.T. Singh).

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378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2008.01.022

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raditional medicine. About 1200 years ago, Vagabhatta was therst to use Terminalia arjuna bark powder mixed with milkor the relief of chest pain caused by heart (Vagabhatta, 1963).is observations were followed by eminent Hindu physicianhakrapani (Chakrapani, 1969). Sequels of studies were per-

ormed that strongly supported its usefulness in several cardiacomplications like cardiac failure, hypertension and other dig-talis unresponsive cardiac diseases (Chopra and Ghosh, 1926;hopra et al., 1969). Terminalia arjuna bark has been used inifferent forms such as Asava (alcoholic decoction), Ghrita (clar-fied butter), Kshirpka (boiled with milk) and dried bark powderor different medicinal purposes (Nandkarni and Nandkarni,951; Singh, 1969; Kurup et al., 1979).

The aqueous extract of Terminalia arjuna bark has been

eported to have positive ionotropic (Radhakrishnan et al., 1993)nd antianginal effects in heart patients (Tripathi, 1993). Termi-alia arjuna therapy was associated with significant decrease innginal episodes in patients (Bharani et al., 2002). The alcoholic

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24 G. Singh et al. / Journal of Ethn

xtract of Terminalia arjuna has also shown to be effective inecreasing blood pressure and heart rate in cats and dogs (Singht al., 1982). The beneficial effects of Terminalia arjuna barkowder and its extract were also established in ischemic heartiseases (Dwivedi, 1994; Gauthaman et al., 2001) and conges-ive heart failure (Colabawalla, 1951). The usefulness of Termi-alia arjuna is also recognized in other indications like obesity,ypertension and hyperglycemia (Dwivedi and Udupa, 1989).

The chronic oral administration of the crude bark of Termi-alia arjuna is reported to augment endogenous antioxidants ofat heart, resulting in the prevention of oxidative stress associ-ted with in vitro ischemic-reperfusion (IR) injury (Gauthamant al., 2001). In addition, Terminalia arjuna is also known to bendowed with potent free-radical scavenging activity (Maulikt al., 1997). As a consequence of these properties, Terminaliarjuna can have immense potential in preventing the oxidativeamage to the heart caused by anthracycline anticancer drugsuch as Doxorubicin (Dox). Dox is effective against malignan-ies such as leukemias, lymphomas and several solid tumors.owever, dose-dependent cardiotoxic effects limit its practical

herapeutic use. These cardiotoxic effects result from over-helming production of reactive oxygen species (ROS) and

oncomitant decrease in the levels of antioxidants like superox-de dismutase (SOD), catalase and reduced glutathione (GSH).n the present study butanolic fraction of Terminalia arjuna wasxtracted and its efficacy to prevent Dox-induced cardiotoxicityas studied.

. Materials and methods

.1. Plant material

Dried bark of Terminalia arjuna (Roxb.) Whight andrnot (Combretaceae), collected from foothills of Himalayasas procured from ayurvedic store and authenticated at theepartment of Botany at Dabur India Limited. A voucher

pecimen (no. 137642) has been deposited in the sameepartment.

.2. Extraction and fractionation

1000 g of pulverized bark powder was extracted with 95%lcohol using soxhlet. The extract was filtered and concen-rated under reduced pressure to afford a 267.5 g of ethanolicxtract (26.75%, w/w). The alcohol extract was resuspendedn water and successively partitioned with hexane, chloroform,thyl acetate and butanol, respectively. All the fractions wereried under reduced pressure to afford a 42.8 g (∼16%, w/w) ofutanolic extract. Fingerprinting of butanolic fraction (TA-05)as done by HPTLC-based method (data not shown). HPTLCngerprinting of the hydrolyzed fraction of TA-05 was carried

ut using Linomet V spotter and scanned on TLC scanner-IICAMAG with Cats 3.18 software) using silica gel 60 F254LC plates (Merck). The solvent system used was chloro-

orm:methanol:water (7:2:0.4) and the plate was scanned at54 nm.

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macology 117 (2008) 123–129

.3. Phytochemical analysis of TA-05

Phytochemical screening was performed on butanol fractionTA-05) to test the presence of phenolic compounds, tannins,lycosides, saponins, alkaloids and flavonoids in the bark oferminalia arjuna using appropriate tests (Kokate, 1994).

.4. Animals

Male Wistar albino rats weighing 110–140 g, 6–7-week oldere procured from National Center for Laboratory Animalsciences (NCLAS, NIN, Hyderabad, India). They were divided

nto 12 groups of 6 animals each and kept at 25 ± 5 ◦C in well-entilated animal house under 12 h light and dark cycle. Thenimals were allowed to adapt to the new housing environmentor 1 week before the experiment. They were provided with stan-ard food pellets and water ad libitum. All animal experimentsere performed according to the guidelines of the Animal Ethicsommittee of Dabur Research Foundation.

.5. Chemicals

Doxorubicin hydrochloride (2 mg/ml) was obtained fromabur, India. Creatine kinase (CKMB) kits were procured fromayer Diagnostics, India. All other chemicals were obtained

rom Sigma Chemicals Co. (St. Louis, MO, USA).

.6. Experimental procedure

To study the effect of chronic administration of TA-05 onox-induced cardiotoxicity, 12 groups of 6 animals each were

aken and treated as follows:

Group I. Saline (0.75 ml/animal), orally 6 days/week for 4weeks.Group II. Saline (0.75 ml/animal) + Dox 20 mg/kg, singleintraperitoneal injection after 4 weeks.Group III. TA-05 (0.42 mg/kg), orally 6 days/week for 4 weeks.Group IV. TA-05 (0.85 mg/kg), orally 6 days/week for 4 weeks.Group V. TA-05 (1.7 mg/kg), orally 6 days/week for 4 weeks.Group VI. TA-05 (3.4 mg/kg), orally 6 days/week for 4 weeks.Group VII. TA-05 (6.8 mg/kg), orally 6 days/week for 4 weeks.Group VIII. TA-05 (0.42 mg/kg) as in Group III + Dox(20 mg/kg) single intraperitoneal injection after 4 weeks.Group IX. TA-05 (0.85 mg/kg) as in Group IV + Dox(20 mg/kg) single intraperitoneal injection after 4 weeks.Group X. TA-05 (1.7 mg/kg) as in Group V + Dox (20 mg/kg)single intraperitoneal injection after 4 weeks.Group XI. TA-05 (3.4 mg/kg) as in Group VI + Dox (20 mg/kg)single intraperitoneal injection after 4 weeks.Group XII. TA-05 (6.8 mg/kg) as in Group VII + Dox(20 mg/kg) single intraperitoneal injection after 4 weeks.

8 h after Dox administration blood was collected for the estima-ion of serum CKMB levels. The animals were sacrificed after2 h of Dox administration and hearts were excised out. Cardiacuscle from lower third of the ventricle was visualized under

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G. Singh et al. / Journal of Ethn

lectron microscope and the remaining heart tissue was snaprozen in liquid nitrogen.

.7. Determination of radical scavenging activity by,1-diphenyl-2-picrylhydrazyl (DPPH) assay

Free-radical scavenging activity of different fractions ofxtract was analyzed by modified method of Lebeau et al.2000). Dilutions of Terminalia arjuna extracts were preparedn a concentration ranging from 0.9 �g/ml to 1 mg/ml. Fromhese dilutions 100 �l of sample was added to 100 �l of 0.2 mMPPH solution. The decrease in absorbance was recorded

fter 5 min at 492 nm. At the same time, a blank solution ofPPH with solvent used in dilutions was screened to esti-ate the DPPH decomposition during the time of measurement.he percent free-radical scavenging activity was calculated aselow:

Blank OD − Sample OD

Blank OD× 100

he activity of extracts was determined in terms of ECR50 value,hich is the ratio of antioxidant concentration to DPPH concen-

ration producing a 50% decrease in DPPH free radicals aftermin.

.8. Measurement of serum of serum creatine kinase-MBCKMB) level

CKMB levels were assayed using the CKMB test kit (Bayeriagnostics). Blood samples were collected and allowed to

oagulate at room temperature for 30 min followed by centrifu-ation for the extraction of serum. CKMB activity was measuredn freshly separated serum as per the manufacturer’s instruc-ions.

.9. Biochemical assays in myocardial tissue

.9.1. Myocardial superoxide dismutase activitySOD levels in the myocardial tissue of rats were determined

ccording to the modified method of Kakkar et al. (1984).riefly heart tissue was homogenized in 0.25 M, Tris sucroseuffer pH 7.4 and then centrifuged at 10,000 rpm for 15 mint 4 ◦C. 600 �l of supernatant was added to the solution con-aining 1.2 ml of sodium pyrophosphate buffer (0.052 M, pH.3), 0.1 ml of phenazine methosulphate solution (186 �M) and.3 ml of nitro blue tetrazolium solution (300 �M). Reactionas initiated by the addition of 0.2 ml of nicotinamide adenineinucleotide-reduced disodium salt (NADH) solution (780 �M).his reaction mixture was incubated for 90 s at room tempera-

ure and then stopped by the addition of 1 ml glacial acetic acid.eaction mixture was vortexed and absorbance was read spec-

rophotometrically at 560 nm. SOD activity was expressed as/mg protein.

.9.2. Myocardial reduced glutathioneMyocardial GSH was estimated according to the modified

ethod of Ellman (1959). The heart tissue was homogenized

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macology 117 (2008) 123–129 125

ith 10% trichloroacetic acid (TCA) buffer and centrifugedt 3000 rpm for 10 min at 4 ◦C. The reaction mixture con-ained 0.1 ml of supernatant, 2.0 ml of 0.3 M phosphate bufferpH 8.4), 0.4 ml of double-distilled water and 0.5 ml of DTNB5,5-dithiobis(2-nitrobenzoic acid)]. The reaction mixture wasncubated for 10 min and the absorbance was measured at12 nm within 15 min. The concentration of GSH was expresseds �g/g of heart tissue.

.9.3. Myocardial catalase activityLevels of catalase were estimated by the modified method

f Aebi (1984). Hearts were homogenized at 4 ◦C in 50 mMotassium phosphate buffer (pH 7.4) and centrifuged at000 rpm for 10 min. Ethanol equal to 0.01 ml/ml of super-atant was added and incubated for 30 min in ice. Triton00× was added to a final concentration of 1%. Super-atant (50 �l) was added to a cuvette containing 1.95 ml of0 mM phosphate buffer (pH 7.0). Then 1.0 ml of 30 mMydrogen peroxide was added and rate of decompositionf hydrogen peroxide was measured spectrophotometricallyt 240 nm. Catalase activity was expressed as U/mg pro-ein.

.9.4. Lipid peroxidationThiobarbituric acid reactive substances (TBARS) levels in

he hearts were determined by modified method of Okhawat al. (1979). Heart tissues were homogenized in 10% TCAuffer in ice. 0.2 ml of homogenate was pipetted into a testube, followed by the addition of 0.2 ml of 8.1% sodium dode-yl sulphate (SDS), 1.5 ml of 20% acetic acid (pH 3.5) and.5 ml of 0.8% thiobarbituric acid (TBA). Tubes were boiledt 95 ◦C for 60 min and then cooled. 1.0 ml of double-distilledater and 5.0 ml of n-butanol:pyridine (15:1, v/v) mixture were

dded to the tubes and centrifuged at 5000 rpm for 10 min.he absorbance of organic layer was measured at 540 nm.alonyldialdehyde (MDA), an end product of lipid perox-

dation forms pink color adducts with TBARS. The extentf lipid peroxidation was expressed as �M of MDA/g heartissue.

.10. Electron microscopy

The hearts were removed, washed immediately with cold glu-araldehyde buffer. Lower portion of ventricle was sliced offnd immersed in 3% glutaraldehyde in 0.1 M sodium cacody-ate buffer, pH 7.4 and sliced into small 1 mm3 pieces. Tissuesere post-fixed in osmium, processed and embedded in Aralditepon resin. Ultrathin sections from selected areas were stainedith uranium and lead salts. Stained sections were observednder PHILIPS Morgagni 268-D transmission electron micro-cope. Two samples from each treatment group were randomlyelected for analysis.

.11. Statistical analysis

All values were expressed as mean ± S.E. (n = 6 in eachroup). Statistical differences between each group were deter-

126 G. Singh et al. / Journal of Ethnophar

Table 1ECR50 values of different fractions of Terminalia arjuna bark extract

S. no. Extract code* ECR50 ± S.E.M.

1 Vitamin C 0.064 ± 0.0132 TA-01 0.104 ± 0.2813 TA-02 NA4 TA-03 1.64 ± 0.0835 TA-04 0.145 ± 0.0116 TA-05 0.071 ± 0.0057 TA-06 0.09 ± 0.011

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* TA-01: fraction obtained after soxhlet extraction of Terminalia arjuna withlcohol; TA-02: hexane fraction; TA-03: chloroform fraction; TA-04: ethylcetate fraction; TA-05: butanol fraction; TA-06: aqueous fraction.

ined with t-test or analysis of variance test. A value of p < 0.05as considered statistically significant.

. Results

.1. Phytochemical investigation

Phytochemical analysis revealed that TA-05 contains pheno-ics, tannins, triterpenoid saponins, anthraquinone glycosides,lkaloids and flavonoids. The HPTLC fingerprint combinedith phytochemical analysis strongly suggests that the peaksbserved correspond to phenolic compounds, tannins, glyco-ides, saponins, alkaloids and flavonoids.

.2. Free-radical scavenging ability of the extracts

DPPH is a stable free radical. In the presence of free-radicaluencher it undergoes one molecule reduction, which can beetected as its color changes from violet to yellow at 492 nm.adical scavenging activity of various fractions of Terminaliarjuna was quantitated by DPPH assay as discussed in Section. The results obtained are given in Table 1. TA-05 showed the

est radical scavenging activity among all the fractions in termsf lowest ECR50 value. Vitamin C was taken as a referencetandard for the study. TA-05 was taken into consideration forurther in vivo studies.

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able 2ffect of Dox and TA-05 on myocardial SOD, GSH, catalase, TBARS and serum CK

. no. Treatment group Markers evaluated (±S.E.M.)

CKMB (U/l) SOD (U/mg protein

1 Control 2230 ± 135.4 1.04 ± 0.1722 Dox (20 mg/kg) 3303 ± 84.3* 0.635 ± 0.046*

3 TA-05 (0.425 mg/kg) 2182 ± 147 0.96 ± 0.0694 TA-05 (0.85 mg/kg) 2201 ± 105.4 1.33 ± 0.0735 TA-05 (1.7 mg/kg) 2255 ± 176.8 1.16 ± 0.0646 TA-05 (3.4 mg/kg) 2277 ± 286.7 1.22 ± 0.0597 TA-05 (6.8 mg/kg) 2205 ± 216.3 1.26 ± 0.0898 TA-05 (0.42 mg/kg) + Dox 3155 ± 81.9 0.535 ± 0.1939 TA-05 (0.85 mg/kg) + Dox 3088 ± 458.1 0.769 ± 0.1930 TA-05 (1.7 mg/kg) + Dox 2515 ± 181.4** 1.48 ± 0.19***

1 TA-05 (3.4 mg/kg) + Dox 2813 ± 124.1** 1.43 ± 0.447***

2 TA-05 (6.8 mg/kg) + Dox 2147 ± 236.2*** 1.58 ± 0.24***

# p < 0.005; *p < 0.05 w.r.t. respective control; **p < 0.05; ***p < 0.005 w.r.t. Dox-tr

macology 117 (2008) 123–129

.3. Effect on serum CKMB levels

Dox treatment resulted in significant increase in serumKMB levels as compared to control group (p < 0.05) (Table 2).retreatment with TA-05 significantly reduced (p < 0.05,< 0.005) the release of CKMB at 1.7 mg/kg, 3.4 mg/kg and.8 mg/kg dose levels. Animals treated with lower doses ofA-05 (0.85 mg/kg and 0.42 mg/kg) showed no significantrotection in terms of CKMB activity. No noticeable differ-nce was seen in TA-05 alone-treated animals as comparedo saline-treated animals. These results indicate that chronicdministration of TA-05 reduces circulating CKMB levels inistar rats treated with Dox.

.4. Biochemical investigations

.4.1. Effect on myocardial SOD activityDox treatment in Wistar rats causes significant decrease

p < 0.05) in SOD activity in the myocardium as comparedo untreated animals. Pretreatment with TA-05 significantlyncreases the SOD activity (p < 0.005) at 1.7 mg/kg, 3.4 mg/kgnd 6.8 mg/kg as compared to Dox-treated animals. No signif-cant increase in SOD activity was noticed at 0.42 mg/kg and.85 mg/kg dose of TA-05. Animals treated with TA-05 alonehowed augmentation of myocardial SOD activity (Table 2).

.4.2. Effect on levels of myocardial reduced glutathioneMyocardial GSH levels were significantly reduced (p < 0.05)

n Dox-treated animals as compared to untreated animals. Pre-reatment with TA-05 showed significant increase (p < 0.005)n GSH levels in animals treated with 3.4 mg/kg dose group.reatment of animals with doses of 0.42 mg/kg, 0.85 mg/kg,.7 mg/kg and 6.8 mg/kg led to insignificant increase in the lev-ls of GSH. Animals treated with TA-05 alone showed GSHevels similar to saline-treated group (Table 2).

.4.3. Effect on myocardial catalase activityThere was an increase in catalase activity in animals treated

ith Dox as compared to the untreated animals. Treatment with

MB activity in different groups

) GSH (�g/g tissue) Catalase (U/mg protein) LP (�M/g tissue)

276 ± 14.6 43.7 ± 3.5 674.7 ± 71.32210.2 ± 16.73* 68.98 ± 3.46* 1725 ± 73.2#

249 ± 31.91 41.6 ± 3.7 679 ± 47.44268 ± 36.02 48.4 ± 5.5 703.8 ± 33.79279 ± 24.5 45.6 ± 2.7 514.6 ± 105.8280 ± 34.1 38.4 ± 3.6 516.4 ± 56.3284 ± 16.0 40.2 ± 6.5 617.1 ± 71.7231 ± 23.44 58.5 ± 7.2 1447 ± 251.9

247.8 ± 48.62 75.03 ± 8.5 1123 ± 68.49**

321.7 ± 68.05 86.03 ± 3.88** 731 ± 76.32***

515.6 ± 16.3*** 82.58 ± 3.83** 748.9 ± 102.7***

236.4 ± 16.95 83.93 ± 3.03** 692.5 ± 90.15***

eated group.

G. Singh et al. / Journal of Ethnopharmacology 117 (2008) 123–129 127

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A-05 before Dox administration further resulted in a significantncrease in catalase activity (p < 0.05) at 1.7 mg/kg, 3.4 mg/kgnd 6.8 mg/kg dose levels. Animals treated with TA-05 alonehowed catalase activity similar to the saline-treated animalsTable 2).

.4.4. Effect on myocardial lipid peroxidationMyocardial lipid peroxidation was significantly increased

p < 0.005) in Dox-treated animals as compared to untreated ani-als. Pretreatment with TA-05 showed significant protection of

ipid peroxidation (p < 0.05, p < 0.005) at a dose ranging from.85 mg/kg to 6.8 mg/kg. TBRS levels in TA-05 alone-treatednimals remained unchanged as compared with saline-treatednimals (Table 2).

.5. Terminalia arjuna attenuates Dox-inducedltrastructural changes

No change in water and food intake was seen in any ofhe treatment groups. At necropsy hearts were grossly normalnd no change was noticed in light microscopy. The myocar-ial tissue of the animals in control group (Group I) and inA-05-treated groups (Groups III–VII) demonstrated normalltrastructural appearance. The heart muscle samples from theroup that received Dox at a dose of 20 mg/kg (Group II) showedharacteristic features of cardiotoxicity manifested in the form

f mitochondrial swelling, Z-band disarray, focal dilatation ofmooth endoplasmic reticulum (SER) and lipid inclusions. Innimals treated with Dox all these ultrastructural alterations inyocardial tissues of animals could be seen in the samples exam-

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animals showing lipid inclusions, mitochondrial swelling and Z-band disarray

ned, indicative of widespread injury to the myocardial tissue,hereas the heart tissue from the groups treated with 20 mg/kgox and higher doses of TA-05 (Groups X–XII) showed lesseregree of cardiotoxicity with a single type of abnormal find-ng at one or two sites seen in each of the three groups. Hencehe group that received 1.7 mg/kg of TA-05 along with Doxhowed a focal mitochondrial swelling only while the grouphat received 3.4 mg/kg of TA-05 plus Dox showed a focus of Z-and disarray only, whereas the group that received 6.8 mg/kgf TA-05 plus Dox showed only focal lipid inclusions, whilehe group that received 0.42 mg/kg and 0.85 mg/kg TA-05 plusox showed greater toxic manifestations similar to Dox-treatedroup (Fig. 1).

. Discussion

The current study entails the cardioprotective potential of theutanolic fraction of alcoholic extract of Terminalia arjuna (TA-5) against anthracycline-induced cardiotoxicity for the firstime. Terminalia arjuna is a plant, well known for its cardiopro-ective properties in the traditional Indian system of medicine.n the present study cardioprotective effects of chronic oraldministration of Terminalia arjuna against Dox-induced acuteardiotoxicity were evaluated in male Wistar rats. Experimentalnd clinical evaluation of Terminalia arjuna suggests its bene-ts in the treatment of coronary artery diseases, heart failure,

nd hypercholestrolemia (Dwivedi, 1994; Ram et al., 1997).eported work on Terminalia arjuna suggests that its cardio-rotective activity is due to its free-radical scavenging activityKarthikeyan et al., 2003). It has been previously reported that

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28 G. Singh et al. / Journal of Ethn

he major chemical constituents present in the alcoholic extractf Terminalia arjuna (tannins, flavonoids and glycosides) areesponsible for the potent antioxidant activities (Karthikeyan etl., 2003).

The existing experimental evidence suggests that Dox-nduced oxidative stress is due to the generation of free radicalsn the heart tissue (Hardina et al., 2000; Naidu et al., 2002). Therinciple ROS generated are superoxide radicals and hydroxyladicals, which have the potential to cause damage to variousntracellular components. Cardiac muscle is particularly sus-eptible to free-radical injury, because it contains low levels ofree-radical detoxifying enzymes/molecules like superoxide dis-utase, GSH and catalase (Takacs et al., 1992). Furthermore,ox also has high affinity for the phospholipid component ofitochondrial membrane in cardiac myocyte, leading to accu-ulation of Dox in the heart tissue (Takacs et al., 1992).In this study we have investigated the cardioprotective effect

f butanolic fraction of Terminalia arjuna, TA-05 againstox-induced acute cardiotoxicity. We report here that the pre-

reatment of TA-05 was able to reduce the Dox-induced acuteardiotoxic manifestations in multiple ways. TA-05 showedotent free-radical scavenging activity in vitro, which was com-arable to that of vitamin C. The extract was found to containix constituents as revealed by HPTLC fingerprint (data nothown). The phytochemical investigations of TA-05 suggest thathese peaks may correspond to phenolics/tannins, glycosidesnd saponins, alkaloids and flavonoids, which are responsi-le for its potent antioxidant activity. Pretreatment with TA-05howed reduction in circulating CKMB levels with simultane-us increase in endogenous antioxidant components (SOD, GSHnd catalase). Concomitantly a decrease in extent of lipid per-xidation was also observed. Increase in catalase activity wasbserved in Dox-treated animals, which could be attributed tohe increased oxidative stress (Li and Singhal, 2000). These lev-ls were further increased significantly by TA-05 pretreatment.his cardioprotective activity was further supported by lower

ncidence of fine structural changes like Z-band disarray, vac-olization, lipid body inclusion, dilated SER and mitochondrialwelling in the animals treated with TA-05 plus Dox.

CKMB, an enzyme found primarily in the myocardium issed to evaluate the existence and extent of myocyte injury. It ishe WHO that recognized gold standard indicative of myocar-ial damage (Adams et al., 1994). TA-05 was found to inhibithe Dox-induced CKMB release in the serum of rats. It is widelyeported that Dox-induced free-radical generation triggers mem-rane peroxidation and disruption of cardiac myocytes, whichan lead to increased release of CKMB in the serum (Liu et al.,002). We have shown that TA-05 pretreatment led to inhibitionf CKMB release in a dose-dependent manner. There was a nearomplete inhibition of CKMB release in Dox-treated animals athe highest concentration of TA-05 evaluated by us.

This inhibition of serum CKMB levels with TA-05 isplendidly correlated with histopathological studies of rat

yocardium. In the present study electron microscopic eval-

ation of the rat myocardium revealed that Dox causeditochondrial swelling, Z-band disarray, focal dilatation of SER

nd lipid inclusions. Similar alterations in Dox-treated mice

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macology 117 (2008) 123–129

ave been reported earlier also (Liu et al., 2002). The Dox-nduced mitochondrial injury is critical to the heart becauset would presumably have extreme adverse effects on the con-ractile functioning of the cardiac myocytes by alterations inhe energy metabolism (Liu et al., 2002). The chronic pretreat-

ent of Wistar rats with TA-05 was able to protect the rat heartrom Dox-induced cardiotoxicity with significant reduction inhe intensity and extent of toxic changes.

Cardioprotective activity of TA-05 was further supported byncreased myocardial antioxidant enzyme activity and decreasedxtent of lipid peroxidation. The most abundant ROS generatedn living cells are superoxide anion and its derivatives, partic-larly highly reactive and damaging hydroxyl radical, whichnduces peroxidation of cell membrane lipids (Hemnani andarihar, 1998). Lipid peroxidation is known to cause cellularamage and is primarily responsible for ROS-induced organamage (Halliwell and Gutteridge, 1989). Our studies havehown that Dox-induced considerable increase in lipid peroxida-ion, which was significantly prevented by TA-05 pretreatment.

Redox cycling of Dox generates superoxide free radi-als (Hardina et al., 2000) due to conversion of quinone toemiquinone moiety, whereas SOD enzyme dismutates this freeadical to hydrogen peroxide. In this respect, any increase inOD activity of the organ appears to be beneficial in the eventf increased free-radical generation. Our studies showed that thectivity of SOD was significantly decreased in Dox-treated ani-als and the pretreatment with TA-05 reversed the SOD activity

n dose-dependent manner. However, it has been reported that aise in SOD activity, without a concomitant rise in the activity ofatalase/GSH might be detrimental (Herman, 1991). This is dueo the fact that SOD generates hydrogen peroxide as a metabolite,hich is cytotoxic and needs to be scavenged by catalase/GSH.hus a simultaneous increase in catalase/GSH activity is essen-

ial for an overall beneficial effect of increase in SOD activityMukherjee et al., 2003). Inhibition of Dox-induced oxidativetress and tissue injury might be due to an increase in GSH,yocardial SOD and catalase activities, following the chronic

dministration of TA-05. The observed increase in catalase activ-ty in Dox-treated animals supports the above hypothesis that thisncrease is possibly required to overcome excessive oxidativetress (Li and Singhal, 2000).

GSH levels were also lowered significantly in Dox-treatednimals, while pretreatment with TA-05 showed significantncrease in GSH levels in Dox-treated animals at doses of.7 mg/kg, 3.4 mg/kg and 6.8 mg/kg in male Wistar rats. Catalasectivity was increased after Dox treatment and pretreatment ofA-05 further increased its activity significantly at 1.7 mg/kg,.4 mg/kg and 6.8 mg/kg dose levels. The increase in cata-ase activity in Dox-treated animals could be indicative ofnhanced oxidative stress due to an adaptive myocardial mecha-ism. As already discussed, the increase in SOD activity wouldave to be followed by increase in levels of catalase/GSH.reviously it has been reported that myocardial adaptation

ccurs in response to various kinds of stimuli like ischemia,ertain endotoxins, ROS, etc. and protects the heart from sub-equent exposure to injuries of similar or more severe in natureKarthikeyan et al., 2003). These findings indicate the promising

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ole of TA-05 as a cardioprotective agent against Dox-inducedardiotoxicity.

. Conclusion

In conclusion, the present study shows that chronic admin-stration of butanolic fraction of alcoholic extract of Terminaliarjuna (TA-05) bark has cardioprotective potential againstox-induced cardiotoxicity. The identification of moleculesith cardioprotective potential from this fraction of Termina-

ia arjuna bark may provide new directions for identification ofardioprotectives, which could be given concomitantly duringox treatment. The cellular mechanisms mediating this car-ioprotective activity are currently under investigation in ouraboratory.

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