Aqueous bark extract of Terminalia arjuna protects against...

Journal of Pharmacy Research Vol.8 Issue 9. September 2014

Mousumi Dutta et al. / Journal of Pharmacy Research 2014,8(9),1285-1302

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Research ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author.Dr. Debasish BandyopadhyayOxidative Stress and Free Radical Biology Laboratory,Department of Physiology, University of CalcuttaUniversity College of Science and Technology92 APC Road, Kolkata 700 009, India

Aqueous bark extract of Terminalia arjuna protects against high fat diet aggravated arsenic-inducedoxidative stress in rat heart and liver: involvement of antioxidant mechanisms

Mousumi Dutta 1,2, Aindrila Chattopadhyay2, Gargi Bose 1 , Auroma Ghosh1, Adrita Banerjee1, Arnab Kumar Ghosh1, Sanatan Mishra1 , Sanjib K. Pattari3, Tridib Das4 and Debasish Bandyopadhyay1#*

1Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, University College of Scienceand Technology, 92, APC Road, Kolkata 700 009, India. #Principal Investigator, Centre with Potential for Excellence in Particular Area

(CPEPA), University of Calcutta, University College of Science and Technology, 92, APC Road, Kolkata 700009, India.2Department of Physiology, Vidyasagar College, Kolkata 700 006, India.

3RN Tagore International Institute of Cardiac Sciences, 124, Mukundapur, E.M. Bypass, Kolkata 700 099, India.4Acharya Prafulla Chandra Sikhsha Prangan, University of Calcutta, JD-2, Sector-III, Salt Lake City, Kolkata 700098, India.

Received on:02-08-2014; Revised on: 22-08-2014; Accepted on:20-09-2014

ABSTRACTBack ground: Terminalia arjuna (TA) Wight & Arn. (Combretaceae) is a tree having an extensive medicinal potential. The plant is usedtraditionally in the treatment of various ailments. Arsenic, a well known groundwater contaminant causes various disorders via oxidativestress. Nutrition can affect arsenic toxicity by several plausible mechanisms. The present study is aimed at investigating the effect of aqueousbark extract of TA against high fat diet aggravated arsenic-induced oxidative damages in rat liver and heart tissues. Methods: For the presentstudy, the average weight of rats remained within a range of 145g-170g. Finally, the animals were divided into ten groups. Each group ofanimals comprised of 6 rats. Control group of rats were fed with the standard laboratory diet that contained (for 100 g) 13.9 g protein (14.2%of total energy), 61.8 g carbohydrate (63.4%), 3.9 g fat (9%) and appropriate amounts of vitamins and minerals. The arsenic treated groups ofrats as well as the rats of the positive control groups were also fed with standard laboratory diet with the above mentioned composition. TheHFD contained (per 100 g) 11.1 g protein (11.4% of total energy), 32.8 g carbohydrate (33.6%) and fat 26.1 g (60%). The rats of the HFD groupswere pair fed in respect to the rats of the control group. The rats of group VIII, IX and X were also fed with HFD. After acclimatization tolaboratory environment, the rats of the treated groups (i.e., group V, VII, VIII, IX and X) were administered sodium arsenite at a dose of 0.75mg/kg body weight (5% of LD50), i.p. every day for a period of 8 days. The rats of the group VIII, IX and X were co-treated also with aqueous TAbark extract at the dose of 20, 40 and 60mg/kg of bw, respectively. Results: We have determined heart weight to body weight ratio as well asanalyzed various functional markers, oxidative stress bio-markers and also the activity of the antioxidant enzymes. Tissue morphologicalstudies confirmed the biochemical investigations. Conclusion: The results raise the possibility of the aqueous bark extract of TA beingconsidered for future use as a therapeutic antioxidant intervention.

KEY WORDS: Antioxidant, arsenic, heart, high fat diet, liver,.oxidative stress, rat, Terminalia arjuna

INTRODUCTIONArsenic pollution in the environment is becoming a major concern forenvironmental and occupational health, owing to its widespread toxicand multidimensional effects on humans and aquatic life and plantsthrough polluted ground water and food chains1. Groundwater con-tamination with arsenic in West Bengal, India, is reported to be thelargest arsenic calamity in the world1. Arsenic toxicity involves oxida-tive damage that plays a vital role for biochemical alteration2. Arsenicpossesses the ability to generate reactive radicals, resulting in cellu-

lar damage like depletion of enzyme activities, damage to lipid bilayerand DNA3. These reactive radical species include a wide variety ofoxygen-, carbon-, sulfur- and nitrogen- radicals, originating not onlyfrom superoxide radical, hydrogen peroxide, and lipid peroxides butalso in chelates of amino-acids, peptides, and proteins complexeswith the toxic metals. This metal generates reactive species, which inturn may cause neurotoxicity, hepatotoxicity and nephrotoxicity inhumans and animals3,4.

There is a wide variation in susceptibility to arsenic toxicity, which islikely to be related to factors such as variation in arsenic metabolism,nutrition, host-related defense mechanisms and genetic predisposi-tions. The main mechanisms of arsenic-nutrition interactions includearsenic-induced oxidative stress, which involves nutrient-dependentdefense systems and arsenic metabolism via 1-carbon metabolism,which requires methyl groups, folic acid, vitamin B-12, and betaine for



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the remethylation step. This step seems to be most critical from atoxicological point of view. A second mode of arsenic-nutrition inter-action involves epigenetic effects and fetal reprogramming via DNAmethylation5. Our previous study has established that high fat dietaggravates arsenic-induced oxidative damages in rat heart and liver6.But, till date there is no information in the scientific literature regard-ing protective effect of aqueous bark extract of TA against high fatdiet aggravated arsenic-induced oxidative damages in rat heart andliver. Aqueous extract of the bark of TA has earlier been demonstratedto have beneficial effect in various health situations. Herein, we pro-vide evidences that the aqueous bark extract of TA has the capabilityto protect against high fat diet aggravated arsenic-induced oxidativestress in vivo in rat heart and liver tissues and antioxidantmechanism(s) may be responsible for such protections.

MATERIALS AND METHODS

ChemicalsSodium arsenite was purchased from Sigma Aldrich, USA. All theother chemicals used including the solvents, were of analytical gradeand obtained from Sisco Research Laboratories (SRL), Mumbai, In-dia, Qualigens (India/Germany), SD fine chemicals (India), Merck Lim-ited, Delhi, India.

Preparation of aqueous TA bark extract Five gm of TA bark powder was dissolved in 25ml of double distilledwater. After proper mixing, it was kept overnight with cotton plugging(for approximately 16 hours). Then, it was centrifuged twice at 1300gfor 10 minutes. The supernatant, thus obtained, was collected, deepfrozen and lyophilized. The yield of the aqueous TA bark extract, inour case was 10%7.

AnimalsMale Wistar rats, weighing 140–200 g, were obtained from a CPCSEAregistered animal supplier. The animals were handled as per the guide-lines of the Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA), Ministry of Environment, Gov-ernment of India. The protocols using animals had the approval ofthe Institutional Anial Ethics Committee (IAEC), Department of Physi-ology, University of Calcutta.

Co-treatment of rats with high fat diet and arsenic and protection byaqueous TA bark extractThe rats were maintained in the animal room at a temperature of 25 ±1 °C, humidity 50 ± 10% and a 12-h light/dark cycle. The rats wereprovided with a standard diet containing 18% protein (casein) andwater ad libitum for 7 days (this is referred to as the quarantineperiod). The 18% protein diet was considered as an adequate dietaryprotein level, which was used on earlier occasions8.

For the present study, the average weight of rats remained within a

range of 145g-170g. Finally, the animals were divided into ten groups.Each group of animals comprised of 6 rats and the different groupsare listed below:

Group I: ControlGroup II: Rats treated only with aqueous TA bark extract at the doseof 20mg/kg of body weight (bw) (T20)Group III: Rats treated only with aqueous TA bark extract at the doseof 40mg/kg of bw (T40)Group IV: Rats treated only with aqueous TA bark extract at the doseof 60mg/kg of bw (T60)Group V: Rats treated with arsenic (As) onlyGroup VI: Rats fed on high fat diet (HFD) onlyGroup VII: Arsenic treated rats fed on high fat diet (As + HFD)Group VIII: Arsenic treated rats fed on high fat diet along with aqeousTA bark extract at the dose of 20mg/kg of bw (As+HFD+T20)Group IX: Arsenic treated rats fed on high fat diet along with aque-ous TA bark extract at the dose of 40mg/kg of body weight(As+HFD+T40)Group X: Arsenic treated rats fed on high fat diet along with aqueousTA bark extract at the dose of 60mg/kg of body weight (As+HFD+T60)Control group of rats were fed with the standard laboratory diet thatcontained (for 100 g) 13.9 g protein (14.2% of total energy), 61.8 gcarbohydrate (63.4%), 3.9 g fat (9%) and appropriate amounts of vita-mins and minerals. The arsenic treated groups of rats as well as therats of the positive control groups were also fed with standard labo-ratory diet with the above mentioned composition. The HFD con-tained (per 100 g) 11.1 g protein (11.4% of total energy), 32.8 g carbo-hydrate (33.6%) and fat 26.1 g (60%), a composition, as publishedelsewhere9. The rats of the HFD groups were pair fed in respect to therats of the control group. The rats of group VIII, IX and X were alsofed with HFD.

After acclimatization to laboratory environment, the rats of the treatedgroups (i.e., group V, VII, VIII, IX and X) were administered sodiumarsenite at a dose of 0.75mg/kg body weight (5% of LD50), i.p. everyday for a period of 8 days. The rats of the group VIII, IX and X wereco-treated also with aqueous TA bark extract at the dose of 20, 40 and60mg/kg of bw, respectively.

Collection of blood and tissue samplesAt the end of the treatment period, the rats were kept fasting over-night and sacrificed through cervical dislocation after subjecting themto mild ether anesthesia. The chest cavity was surgically openedthrough vertical incision and blood was carefully collected throughcardiac puncture for the preparation of serum and the heart surgicallyremoved. The liver was also removed surgically after carefully open-ing the abdominal cavity. The collected heart and liver tissues wererinsed well in cold saline, soaked properly with a piece of blottingpaper and stored in sterile plastic vials at -20°C for further biochemical



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trophotometer to zero with water. (Bio-Rad, Hercules, CA, USA).Theserum enzyme activities were expressed as IU/L10.

Measurement of serum total lactate dehydrogenase (LDH), lactatedehydrogenase 1 (LDH1) and lactate dehydrogenase 5 (LDH5) ac-tivitiesThe serum total LDH activity was obtained by measuring the oxida-tion of NADH (0.1 mM) to NAD+ at 340 nm using 1.0 mM sodiumpyruvate as substrate, after incubating the serum samples at 37°C for30 min according to the method of Strittmatter (1965) with somemodifications11, 12. The enzyme activity was expressed as IU/L.

The serum LDH1 activity was obtained by measuring the oxidation ofNADH (0.1 mM) to NAD+ at 340 nm using 1.0 mM sodium pyruvateas substrate, after incubating the serum samples at 65 °C which de-stroys all isoforms except LDH1 for 30 min according to the method ofStrittmatter (1965) with some modifications11, 12. The enzyme activitywas expressed as IU/L.

The serum LDH5 activity was obtained by measuring the oxidation ofNADH (0.1 mM) to NAD+ at 340 nm using 1.0 mM sodium pyruvateas the substrate, after incubating the serum samples at 55 °C whichdestroys all isoforms except LDH5 for 30 min according to the methodof Strittmatter (1965) with some modifications11, 12. The enzyme activ-ity was expressed as IU/L.

Measurement of cardiac and hepatic tissue lipid peroxidation (LPO)level, reduced glutathione (GSH) and protein carbonyl (PCO) con-tentsThe lipid peroxides in the cardiac and hepatic tissue homogenateswere determined separately as thiobarbituric acid reactive substances(TBARS) according to the method of Buege and Aust with somemodifications as adopted by Bandyopadhyay et al., (2004)13, 14. Briefly,the homogenates were mixed with thiobarbituric acid–trichloro aceticacid (TBA–TCA) reagent with thorough shaking and heated for 20min at 80°C. The samples were then cooled to room temperature. Theabsorbance of the pink chromogen present in the clear supernatantafter centrifugation at 12,000g for 10 min at room temperature wasmeasured at 532 nm using a UV–VIS spectrophotometer (Bio-Rad,Hercules, CA, USA). The values were expressed as nmoles of TBARS/mg of protein.

The GSH content (as acid soluble sulfhydryl) of both the cardiac andhepatic tissues were estimated separately by its reaction with DTNB(Ellman’s reagent) following the method of Sedlak et al., (1968) withsome modifications by Bandyopadhyay et al., (2004)15,14. Incubatedsample was mixed with Tris–HCl buffer, pH 9.0, followed by DTNB forcolor development. The absorbance was measured at 412 nm using aUV–VIS spectrophotometer to determine the GSH content. The val-ues were expressed as nmole GSH/ mg of protein.

analyses. For tissue morphological studies, a suitable amount of thecardiac and the hepatic tissues were placed immediately in appropri-ate fixatives. Each set of experiment was repeated at least two times.

Measurement of body weight and determination of heart weight tobody weight ratio

Isolation of mitochondria from heart and liver tissuesThe mitochondria from heart and liver tissues were isolated accord-ing to the procedure of Dutta et al., (2013)7. A portion of the heart andliver tissues were cleaned and cut into small pieces. Five hundred mgof 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 250Cfor 5min. The tissues were then homogenized separately in cold for 1minute at low speed by using a Potter Elvenjem glass homogenizer(Belco Glass Inc., Vineland, NJ, USA). The homogenates were centri-fuged at 1500rpm for 10 minutes at 4°C. The supernatant was pouredthrough several layers of cheesecloth and kept in ice. This fileterdsupernatant was centrifuged at 4000rpm for 5minutes at 4°C. Thesupernatant, thus,obtained, was further centrifuged at 14000rpm for20 minutes at 4°C. The final supernatant was discarded and the pelletwas re-suspended in sucrose buffer and stored at -200C until furtheranalysis. However, most of the enzymatic assays were carried outwith freshly prepared mitochondria.

Measurement of serum aspartate transaminase (AST) and serumalanine transaminase (ALT) activities Serum AST and serum ALT activities were measured by standardmethods. Non-hemolyzed serum was mixed with glutamate pyruvatetransaminase substrate and incubated for 30 min at 370 C. Then, 2, 4-dinitrophenyl hydrazine (DNPH) solution was added, mixed and keptfor 20 min at room temperature. Thereafter, 0.4(N) NaOH was added,mixed and kept at room temperature for 10 min. The intensity of thedeveloped colour was noted at 540 nm after setting the UV/VIS spec

Preparation of tissue homogenateA 10% tissue homogenate of each of heart and liver was prepared incold in two separate buffer solutions, viz., ice cold 0.1M phosphatebuffer (pH 7.4) and 1mM EDTA buffer, respectively, using a PotterElvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) for30 sec. The homogenates were kept in cold and processed for bio-chemical analyses within 30minutes of preparation.

The body weights of rats of all the groups before and after the treat-ment were noted. Heart weight of each of the rats was measuredfollowing sacrifice and heart weight to body weight ratio was deter-mined using the body weight of each of the rats on the day of sacri-fice6.



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initiated with 1 mM oxidized glutathione (GSSG). The decrease inNADPH absorption was monitored spectrophotometrically at 340 nm.The specific activity of the enzyme was calculated as units/ mg tissueprotein.

The GPx activity was measured according to the method of Paglia andValentine with some modifications as adopted by Dutta et al., (2014)22,23. A weighed amount of cardiac and hepatic tissues were separatelyhomogenized (10%) in ice cold 50 mM phosphate buffer containing 2mM EDTA, pH 7.0. The assay system contained, in a final volume of1 ml, 0.05 M phosphate buffer with 2 mM EDTA, pH 7.0, 0.025 mMsodium azide, 0.15 mM glutathione, and 0.25 mM NADPH. The reac-tion was started by the addition of 0.36 mM H2O2. The linear decreaseof absorbance at 340 nm was recorded using a UV/VIS spectropho-tometer. The specific activity was expressed as nmols of NADPHproduced/ mg tissue protein.

Measurement of the activities of pyruvate dehydrogenase (PDH) andsome of the Kreb’s cycle enzymesThe PDH activity of rat cardiac and hepatic tissues were measuredspectrophotometrically according to the method of Chretien et al.,(1995) with some modifications by following the reduction of NAD+

to NADH at 340 nm using 50 mM phosphate buffer, pH 7.4, 0.5 mMsodium pyruvate as the substrate and 0.5 mM NAD+ in addition tothe enzyme24. The enzyme activity was expressed as units/ mg tissueprotein.

Isocitrate dehydrogenase (ICDH) activity of rat cardiac and hepatictissues were measured according to the method of Duncan et al.,(1979) by measuring the reduction of NAD+ to NADH at 340 nm withthe help of a UV–VIS spectrophotometer25. One ml assay volumecontained 50 mM phosphate buffer, pH 7.4, 0.5 mM isocitrate, 0.1 mMMnSO4, 0.1 mM NAD+ and the suitable amount of mitochondria asthe source of enzyme. The enzyme activity was expressed as units/mg tissue protein.

Alpha-Ketoglutarate dehydrogenase (a-KGDH) activity of rat car-diac and hepatic tissues were measured spectrophotometrically ac-cording to the method of Duncan et al., (1979) by measuring thereduction of 0.35 mM NAD+ to NADH at 340 nm using 50 mM phos-phate buffer, pH 7.4 as the assay buffer, 0.1 mM a-ketoglutarate as thesubstrate and the suitable amount of mitochondria as the source ofenzyme25. The enzyme activity was expressed as units/ mg tissueprotein.

The PCO content was estimated by DNPH assay16. One fourth of amilliliter of the homogenates of cardiac and hepatic tissues were sepa-rately taken in respective tubes and 0.5 ml DNPH in 2.0 M HCl wasadded to the tubes. The tubes were vortexed every 10 min in the darkfor 1 h. Proteins were then precipitated with 30% TCA and centri-fuged at 4000g for 10 min at 4°C. The pellet was washed three timeswith 1.0 ml of ethanol: ethyl acetate (1:1, v/v). The final pellet wasdissolved in 1.0 ml of 6.0 M guanidine HCl in 20 mM potassiumdihydrogen phosphate (pH 2.3) solution. The absorbance was deter-mined spectrophotometrically at 370 nm. The PCO content was calcu-lated using a molar absorption coefficient of 2.2X 10-4 M-1 cm-1. Thevalues were expressed as nmoles /mg of protein.

Measurement of the activities of Cu-Zn superoxide dismutase (Cu-Zn SOD), catalase (CAT), glutathione reductase (GR) and glutathioneperoxidase (GPx) of rat cardiac and hepatic tissuesCopper–zinc superoxide dismutase (SOD1) activity was measured byhematoxylin autooxidation method of Martin et al., (1987) with somemodifications as adopted by Dutta et al., (2014)17,18. Briefly, the weighedamounts of cardiac and liver tissues were homogenized (10%) sepa-rately in ice-cold 50 mM phosphate buffer containing 0.1 mM EDTA,pH 7.4. The homogenates were centrifuged at 12,000g for 15 min at4°C and the supernatant was collected. Inhibition of hematoxylinauto-oxidation by the cell free supernatant was measured at 560 nmusing a UV–VIS spectrophotometer (Bio-Rad Smart spec Plus). Theenzyme activity was expressed as units/ mg of tissue protein.

The CAT activity was assayed by the method of Beers et al., (1952)with some modifications as adopted by Chattopadhyay et al.,(2003)19,20. A weighed amount of cardiac and liver tissue was homog-enized (5%) separately in ice-cold 50 mM phosphate buffer, pH 7.0.The homogenate was centrifuged in cold at 12,000g for 12 min at 4°C.The supernatants, thus obtained, were then collected and incubatedwith 0.01 ml of absolute ethanol at 4°C for 30 min, after which 10%Triton X-100 was added so as to have a final concentration of 1%.The samples, thus obtained, were used to determine CAT activity bymeasuring the breakdown of H2O2 spectrophotometrically at 240 nm.The enzyme activity was expressed as µmoles of H2O2 consumed/ mgtissue protein.

The GR activity was measured according to the method of Krohne-Ehrich et al., (1977)21. The assay mixture in a final volume of 3 mlcontained 50 mM phosphate buffer, 200 mM KCl, 1 mM EDTA andwater. The blank was set with this mixture. Then, 0.1 mM NADPH wasadded together with suitable amount of cardiac or liver tissue homo-genate into the cuvette as the source of enzyme. The reaction was



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cold at 12,000g for 20 min. The final supernatant was used as thesource of the enzyme, and the activity of the enzyme was measuredspectrophotometrically at 340 nm with 0.3 mM xanthine as the sub-strate (in 50 mM phosphate buffer, pH 7.5) and 0.7 mM NAD+ as anelectron donor. The enzyme activity was expressed as milli units/ mgtissue protein.

Measurement of nitric oxide (NO•) concentration in mitochondriaThe NO• concentration in mitochondria of both the cardiac and he-patic tissues were measured separately spectrophotometrically at548nm according to the method of Fiddler by using Griess reagent29,30.The reaction mixture in a spectrophotometer cuvette (1 cm path length)contained 100 µL of Griess Reagent, 700 µL of the sample and 700 µLof distilled water. The NO• concentration was expressed as µM/mg ofprotein.

Estimation of tissue proteinThe protein content of the different samples was determined by themethod of Lowry et al., (1951) 31.

Tissue morphological and histochemical studies

(a) Staining of tissue sections using hematoxylin-eosin, and peri-odic acid Schiff (PAS) stainsA portion of the extirpated rat heart and liver were fixed immediately in10% formalin and embedded in paraffin following routine procedureas used earlier by Dutta et al., (2014) 6. Sections of heart and livertissues (5 µm thick) were prepared. The cardiac tissue sections werestained with hematoxylin-eosin stain and the sections obtained fromthe liver tissue were stained with hematoxylin-eosin stain and peri-odic acid Schiff (PAS) stain. The stained tissue sections were exam-ined under Leica microscope and the images were captured with adigital camera attached to it.

(b) Determination of lipid content in cardiac and hepatic tissues byusing Sudan stainThe rat cardiac and hepatic tissue sections were stained with Sudanstain by following the method of Crason32. The tissue sections wereexamined under Leica microscope and the images were captured witha digital camera attached to it. The digitized images were then ana-lyzed using image analysis system (ImageJ, NIH Software, Bethesda,MI) and the total lipid content as represented in each image wasmeasured and expressed as the % lipid volume (% area).

(c) Quantification of fibrosis by confocal microscopyThe rat heart and liver tissue sections (5 µm thick) were stained with

Likewise, succinate dehydrogenase (SDH) activity of rat cardiac and

Indirect assessment of the generation of superoxide anion free radi-cal (O2

•-) by xanthine oxidase and xanthine dehydrogenaseXanthine oxidase (XO) activity of rat cardiac and hepatic tissueswere assayed by measuring the conversion of xanthine to uric acidfollowing the method of Greenlee et al., (1964) 28. Briefly, the weighedamounts of cardiac and hepatic tissues were separately homogenizedin cold (10%) in 50 mM phosphate buffer, pH 7.8. The homogenateswere centrifuged at 500g for 10 min at 4°C. The resulting supernatantswere further centrifuged at 12,000g for 20 min in cold. The superna-tant, thus obtained, was collected and used for spectrophotometricassay of the enzyme at 295 nm using 0.1 mM xanthine in 50 mMphosphate buffer, pH 7.8, as the substrate. The enzyme activity wasexpressed as milli Units/ mg tissue protein.

Xanthine dehydrogenase (XDH) activity was measured by followingthe reduction of NAD+ to NADH according to the method ofStrittmatter (1965) with some modifications11. In brief, the weighedamounts of rat cardiac and hepatic tissues were homogenized sepa-rately in cold (10%) in 50 mM phosphate buffer with 1 mM EDTA, pH7.2. The homogenates were centrifuged in cold at 500g for 10 min at4°C. The supernatants, thus obtained, were further centrifuged in

Measurement of the activities of some of the mitochondrial respira-tory chain enzymes NADH-Cytochrome c oxidoreductase activitywas measured spectrophotometrically by following the reduction ofoxidized cytochrome c at 565 nm according to the method of Goyal etal., (1995) 27. One ml of assay mixture contained in addition to theenzyme, 50 mM phosphate buffer, 0.1 mg BSA, 20 mM oxidized cyto-chrome c and 0.5 (M) NADH. The activity of the enzyme was ex-pressed as units/ mg tissue protein.

Cytochrome c oxidase activity was also determined spectrophoto-metrically by following the oxidation of reduced cytochrome c at 550nm according to the method of Goyal et al., (1995) 27. One ml of assaymixture contained 50 mM phosphate buffer, pH 7.4, 40 mM reducedcytochrome c and a suitable aliquot of the enzyme. The enzyme activ-ity was expressed as units /mg tissue protein.

hepatic tissues were measured spectrophotometrically by following

the reduction of potassium ferricyanide [K3Fe (CN) 6] at 420 nm ac-

cording to the method of Veeger et al., (1969) with some modifica-tions26. One ml assay mixture contained 50 mM phosphate buffer, pH7.4, 2% (w/v) BSA, 4 mM succinate, 2.5 mM K3Fe(CN)6 and a suitableamount of mitochondria as the source of enzyme. The enzyme activ-ity was expressed as units/ mg tissue protein.



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The heart weight/body weight ratio was also found to be increased inAs, HFD, As + HFD co-treated groups than control group by 50%,20% and 16% (P<0.001). Only 3.45%.10.34% and 13.79% decrease(P<0.001) were observed in TA protected groups compared toAs+HFD co-treated group (Fig. 1B). Furthermore, the heart weight tobody weight ratio of animals of HFD group was also found to beincreased significantly than the control group (P<0.001). AqueousTA bark extract, alone, at increasing concentrations did not show anyeffect on heart weight to body weight ratio.

Changes in activities of AST and ALTIn As treated group, the activities of AST and ALT were found to beincreased by 31.25% and 44% (P<0.001) respectively, compared tothe control group. Increase of 12.50% and 12% (P<0.001) in the activi-ties of AST and ALT from control group in high fat diet fed groupwere also observed. In case of animals co-treated with AS and HFD,the activities of AST and ALT were found to be increased by 66.88%and 72% (P<0.001) respectively, compared to the control group. Fol-lowed by treatment with increasing doses of TA bark extract, theactivities of AST and ALT were found to be protected from As+HFDco-treated group. In As+HFD+T20, the activities of AST and ALTwere found to be protected by 21.35% and 11.63% (P<0.001) fromAs+HFD co-treated group respectively. Similarly, the AST and ALTactivities were found to be protected by 40.07% and 37.21% (P<0.001)in As+HFD+T40 group and by 51.31% and 62.80% (P<0.001) inAs+HFD+T60 group, respectively, from the As+HFD co-treated group(Fig. 1C). Aqueous TA bark extract, alone, at increasing concentra-tions did not show any effect.

Changes in serum total LDH, LDH1 and LDH5 activitiesTotal LDH, LDH1 and LDH5 activities in serum were found to beincreased significantly by 57.14%, 142.86% and 159.26% (P<0.001)respectively, in As treated group compared to control group. In HFDfed group, the total LDH, LDH1 and LDH5 activities in serum werefound to be increased by 27.14%, 78.57% and 48.15% (P<0.001) re-spectively than control group. In As+HFD co-treated group, an in-crease of 157.14%, 321.43% and 225.93% (P<0.001) were found intotal LDH, LDH1, LDH5 activity compared to control animals. InAs+HFD+T20 group, the LDH, LDH1, LDH5 activities were found tobe protected by 16.67%, 23.73% and 67.80% (P<0.001) compared toAs+HFD co-treated group. The total LDH, LDH1 and LDH5 activitieswere also found to be protected in case of As+HFD+T40 andAs+HFD+T60 by 45.56%, 50.85%, 43.18% and 60%, 67.80%, 65.91%(P<0.001) respectively, compared to As+HFD co-treated group. Aque-ous TA bark extract, alone, at increasing concentrations did not showany effect on the activities of these enzymes. (Fig. 1D).

Sirius red (Direct Red 80; Sigma Chemical Co., St. Louis, MO, USA)according to the method of Dutta et al., (2014) and imaged with a laserscanning confocal system (Zeiss LSM 510 META, Germany) and thestacked images through multiple slices were captured. The digitizedimages were then analyzed using image analysis system (ImageJ,NIH Software, Bethesda, MI) and the total collagen area fraction ofeach image was measured and expressed as the % collagen volume6.

Scanning electron microscopy (SEM)Small pieces of heart and liver tissues were fixed overnight with 2.5%glutaraldehyde. After washing three times with PBS, the pieces weredehydrated for 10 min at each concentration of a graded ethanolseries (50, 70, 80, 90, 95 and 100%). The dehydrated pieces wereimmersed in pure tert-butyl alcohol and were then placed into a 4°Crefrigerator until the tert-butyl alcohol solidified. The frozen tissuepieces were dried by placing them into a vacuum bottle. The cardiacand liver tissue surface morphology was evaluated by scanning elec-tron microscopy (SEM; Zeiss Evo 18 model EDS 8100).

Statistical evaluationEach experiment was repeated at least two times with different rats.Data are presented as means ± S.E. Significance of mean values ofdifferent parameters between the treatments groups were analyzedusing one way post hoc tests (Tukey’s HSD test) of analysis ofvariances (ANOVA) after ascertaining the homogeneity of variancesbetween the treatments. Pairwise comparisons were done by calcu-lating the least significance. Statistical tests were performed usingMicrocal Origin version 7.0 for Windows.

RESULTS

Changes in body weight and heart weight to body weight ratio ofanimals of different groupsThe pre-experimental body weights of all the groups of rats remainedwithin the range of 145g-170g. In case of As-treated group, post-experiment body weight was found to be decreased by 4.29% com-pared to pre-experiment body weight. Similarly the post-experimentbody weight of animals of HFD group gradually was found to beincreased by 19.40% (P<0.001) compared to pre-experiment bodyweight. But in case of animals co-treated with arsenic and HFD, thepost-experiment body weight was also found to be decreased duringthe period of experiment by 1.41% compared to pre experiment. In As+ HFD + T20, As + HFD + T40 and As + HFD+ T60 groups, bodyweight was found to be increased by 1.41%, 1.39% and 0.69% (P<0.001)respectively than pre-experimental weight (Fig. 1A). Aqueous TAbark extract, alone, at increasing concentration did not show anyeffect on body weight.



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Figure 1: Protective effect of aqueous TA bark extract on changes of (A) body weight of rats, (B) heart weight to body weight ratio of rats,(C) serum aspertate transaminase (AST) activity and serum alanine transaminase (ALT) activity, (D) total lactate dehydrogenase (TotalLDH) activity, lactate dehydrogenase1 (LDH1) activity and lactate dehydrogenase5 (LDH5) activity of rats of various groups during theperiod of experiment. As: Arsenic treated group, HFD: High fat diet treated group, As+HFD: Arsenic and high fat diet co-treated group,T20= group treated with aqueous TA extract at the dose of 20mg/ml, T40= group treated with aqueous TA extract at the dose of 40mg/ml,T60= group treated with aqueous TA extract at the dose of 60mg/ml, As+HFD+T20= group co-treated with arsenic, high fat diet and aqueousTA extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fat diet and aqueous TA extract at the dose of 40mg/ml, As+HFD+T60= group co-treated with arsenic, high fat diet and aqueous TA extract at the dose of 60mg/ml. The values are expressed asMean ± S.E.; *P<0.001 compared to control values using ANOVA. # P<0.001 compared to arsenic only treated values using ANOVA. ̂P<0.001 compared to arsenic and high fat diet co-treated values using ANOVA.

LPO level in liver tissue was found to be protected by 31.82%, 25%and 47.73% (P<0.001) in As, HFD and As+HFD co-treated group re-spectively, in comparison with control group. The elevated level ofLPO was found to be protected by 3.08%, 20% and 26.15% (P<0.001)in liver tissue of As+HFD+T20, As+HFD+T40 and As+HFD+T60group compared to As+HFD co-treated group. Aqueous TA barkextract, alone, at increasing concentrations did not show any effecton LPO level (Fig. 2A).

Changes in LPO level, GSH and protein carbonyl content in ratheart and liver tissuesThe LPO level in heart tissue was found to be increased by 28.13%,18.75% and 81.25% (P<0.001) in As, HFD and As+HFD co-treatedgroup respectively, in comparison with the control group. The el-evated level of LPO was found to be protected by 3.45%, 17.24% and39.66% (P<0.001) in heart tissue of As+HFD+T20, As+HFD+T40 andAs+HFD+T60 groups compared to As+HFD co-treated group. The

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Figure 2: Protective effect of aqueous TA bark extract on changes of (A) lipid peroxidation (LPO) level, (B) reduced glutathione (GSH)content, (C) protein carbonylation (PCO) level of heart and liver tissues of rats of various groups during the period of experiment. As:Arsenic treated group, HFD: High fat diet treated group, As+HFD: Arsenic and high fat diet co-treated group, T20= group treated withaqueous TA bark extract at the dose of 20mg/ml, T40= group treated with aqueous TA bark extract at the dose of 40mg/ml, T60= grouptreated with aqueous TA bark extract at the dose of 60mg/ml, As+HFD+T20= group co-treated with arsenic, high fat diet and aqueous TAbark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of40mg/ml, As+HFD+T60= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 60mg/ml. The values areexpressed as Mean ± S.E.; *P < 0.001 compared to control values using ANOVA. # P < 0.001 compared to only arsenic treated values usingANOVA. ̂ P < 0.001 compared to arsenic and high fat diet co-treated values using ANOVA.

The PCO level in heart and liver tissues of As treated group weresignificantly found to be increased by 65.71% and 35.42% (P<0.001)respectively, compared to control animals. In HFD fed group, thePCO level was found to be increased by 37.14% and 16.67% (P<0.001)in heart and liver, respectively. A significant increase of PCO level by111.43% and 95.83% (P<0.001) were found in heart and liver tissues ofAs+HFD co-treated group compared to control group. The PCO lev-els of As+HFD+T60 group were found to be protected significantlyfrom As+HFD co-treated group in heart and liver tissues by 59.46%and 38.30% respectively, (P<0.001) (Fig. 2C). Aqueous TA bark ex-tract, alone, at increasing concentrations did not show any effect.

The level of GSH in heart and liver tissues in As treated group werefound to be decreased by 18.18% and 22.22% (P<0.001) respectively,compared to control group. In HFD and As+HFD co-treated group,the decrease in GSH content in heart and liver tissues were found by12.73%, 13.89% and 30.91%,31.94% (P<0.001) respectively, in com-parison with control group. The GSH content of As+HFD+T60 groupwas found to be protected significantly from As+HFD co-treatedgroup in heart and liver tissues by 42.11% and 51.02% respectively(P<0.001). TA aqueous bark extract, alone, at increasingconcentrations did not show any effect (Fig. 2B).

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Changes in antioxidant enzyme activities of rat heart and liver tis-suesIn As treated group, Cu-Zn SOD activity was found to be decreasedby 50% and 27.27% (P<0.001) in heart and liver tissues respectively,from the control groups. In high fat diet fed only group, Cu-Zn SODactivity was found to be decreased by 40% and 13.64% (P<0.001)respectively, in heart as well as in liver tissues compared to control. Inheart and liver tissues of As+HFD co-treated group, a decrease of60% and 63.64% (P<0.001) were found respectively, in Cu-Zn SODactivity compared to control group. The Cu-Zn SOD activities ofAs+HFD+T60 group were found to be protected significantly com-pared to As+HFD co-treated group in heart and liver tissues by 150%and 162.5%, respectively (P<0.001). Aqueous TA bark extract, alone,at increasing concentration did not show any effect on the enzymeactivity (Fig. 3A).

Glutathione reductase (GR) activity in As treated group was found tobe decreased from control group by 25.93% and 22.58% (P<0.001) inheart and liver, respectively. In HFD group GR activity was also foundto be decreased by 11.11% in heart and by 14.52% (P<0.001) in liverwhich was significantly different compared to control. GR activity inheart and liver tissues of As+HFD co-treated group were also foundto be decreased significantly by 44.44% and 38.71% (P<0.001) com-pared to what has been observed in control rats. The GR activities ofAs+HFD+T60 group were found to be protected significantly fromAs+HFD co-treated group in heart and liver tissues by 86.67% and52.63%, respectively (P<0.001) (Fig. 3A). Aqueous TA bark extract,alone, at increasing concentration did not show any effect on GRactivity of both the tissues.

Glutathione peroxidase (GPx) activity in heart and liver tissues of As,HFD, As+HFD groups were found to be decreased by 51.35%, 43.24%and 59.46% (heart) and also by26.32%, 15.79% and 44.74% (liver) (P<0.001), respectively, compared to control group of rats. In heart tis-sues of As+HFD+T20, As+HFD+T40 and As+HFD+T60 groups, GPxactivities were found to be protected by 26.67%, 80% and 123.33%(P<0.001) compared to As+HFD group. In liver tissues ofAs+HFD+T20, As+HFD+T40 and As+HFD+T60 groups, GPx activi-ties were found to be protected by 28.57%, 61.90% and 85.71%(P<0.001) compared to As+HFD group (Fig. 3A). Aqueous TA barkextract, alone, at increasing concentration did not show any effect onGPx activity in both the tissues studied.

Changes in pyruvate dehydrogenase (PDH) activity and some of theKreb’s cycle enzymes in rat heart and liver tissuesThe activity of PDH was found to be decreased significantly by13.33%, 8.33% and 18.33% (P<0.001) in heart tissues of As, HFD,

As+HFD groups of compared to control group. In liver tissue, PDHactivity of As, HFD, As+HFD groups was found be decreased by15.38%, 7.70% and 23.08% (P<0.001) than control. In heart tissues ofAs+HFD+T20, As+HFD+T40 and As+HFD+T60 group, the PDH ac-tivity was found to be protected by 2.04%, 12.24% and 22.45%(P<0.001) compared to As+HFD group. The PDH activity in liver tis-sue was found to be protected by 10%, 20% and 40% (P<0.001) inAs+HFD+T20, As+HFD+T40 and As+HFD+T60 groups, respectively,compared to As+HFD group. Aqueous TA bark extract, alone, at in-creasing concentration did not show any effect on PDH activity (Fig.3B).

Isocitrate Dehydrogenase (ICDH) activity was found to be decreasedsignificantly by 31.25%, 25% and 37.5% (P<0.001) in heart tissue ofAs, HFD, As+HFD treated groups of rats compared to control groupof animals. In liver tissue, ICDH activity of As, HFD, As+HFD treatedgroups were found to be decreased by 20%, 6% and 40% (P<0.001)compared to control group of rats. In heart tissues of As+HFD+T20,As+HFD+T40 and As+HFD+T60 groups of rats, the ICDH activitywas found to be protected by 6%, 16% and 80% (P<0.001) comparedto As+HFD group. The ICDH activity in liver tissues were found tobe protected by 50%, 60% and 70% (P<0.001) in As+HFD+T20,As+HFD+T40 and As+HFD+T60 groups of rats, respectively, com-pared to As+HFD group. Aqueous TA bark extract, alone, at increas-ing concentration did not show any effect on ICDH activity (Fig. 3B).Alpha-Ketoglutarate Dehydrogenase (a-KGDH) activity was foundto be decreased significantly by 12.73%, 7.27% and 30.91% (P<0.001)in heart tissues of As, HFD, As+HFD groups of rats compared tocontrol group. In liver tissue, a-KGDH activity of As, HFD, As+HFDgroups of rats were found to be decreased by 23.08%, 12.82% and48.72% (P<0.001) when compared to the activity of the rats of controlgroup. In heart tissues of As+HFD+T20, As+HFD+T40 andAs+HFD+T60 groups of rats, the a-KGDH activity was found to beprotected by 18.42%, 31.58% and 44.74% (P<0.001) compared toAs+HFD fed group of rats. The a-KGDH activity in liver tissue wasfound to be protected by 40% and 85% (P<0.001) in As+HFD+T40and As+HFD+T60 groups of rats, respectively when comparedAs+HFD fed group of rats (Fig. 3B). Aqueous TA bark extract, alone,at increasing concentration did not show any effect on the activity ofthis enzyme.

Succinate dehydrogenase (SDH) activity was found to be decreasedsignificantly by 31.25%, 25% and 37.5% (P<0.001) in heart tissues ofAs, HFD, As+HFD groups of rats compared to control group of rats.In liver tissue, SDH activity of As, HFD, As+HFD fed groups wasfound to be decreased by 20%, 36% and 40% (P< 0.001) compared tocontrol. In heart tissues of As+HFD+T20, As+HFD+T40 and



1285-1302

As+HFD+T60 groups of rats, the SDH activitiy was found to beprotected by 6%, 16% and 80% (P<0.001) compared to As+HFD groupof rats. SDH activity in liver tissues was found to be protected by

Figure 3: Protective effect of aqueous TA bark extract on changes of (A) Cu-Zn superoxide dismutase (Cu-Zn SOD) activity, glutathionereductase (GR) activity and glutathione peroxidase (GPx) activity, (B) pyruvate dehydrogenase (PDH) activity, isocitrate dehydrogenaseactivity (ICDH), a-ketoglutarate dehydrogenase (a-KGDH) activity and succinate dehydrogenase (SDH) activity of heart and liver tissuesof rats of various groups during the period of experiment. As: Arsenic treated group, HFD: High fat diet treated group, As+HFD: Arsenicand high fat diet co-treated group, T20= group treated with aqueous TA bark extract at the dose of 20mg/ml, T40= group treated withaqueous TA bark extract at the dose of 40mg/ml, T60= group treated with TA at the dose of 60mg/ml, As+HFD+T20= group co-treatedwith arsenic, high fat diet and aqueous TA bark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fatdiet and aqueous TA bark at the dose of 40mg/ml, As+HFD+T60= group co-treated with arsenic, high fat diet and aqueous TA bark extractat the dose of 60mg/ml. The values are expressed as Mean ± S.E.; *P < 0.001 compared to control values using ANOVA. # P < 0.001compared to only arsenic treated values using ANOVA. ̂ P < 0.001 compared to arsenic and high fat diet co-treated values using ANOVA.

50%, 60% and 70% (P<0.001) in As+HFD+T20, As+HFD+T40 andAs+HFD+T60 groups of rats, respectively compared to As+HFD groupof rats (Fig. 3B). Aqueous TA bark extract, alone, at increasing con-centration did not show any effect on SDH activity.

Changes in mitochondrial respiratory chain enzymes in heart andliver tissuesThe activity of cytochrome C oxidoreductase was found to be de-creased by 36.84%, 21.05% and 60.53% (P<0.001) in the heart tissuesand by 50%, 30% and 64% (P<0.001) in liver tissues of As, HFD,As+HFD treated group of rats, respectively,compared to control

group of rats. In As+HFD+T20, As+HFD+T40 and As+HFD+T60treated groups of rats the activity of cytochrome C oxidoreductasewas found to be protected by 73.33%, 100%, 140% (P<0.001) in hearttissues and by 94.44%, 166.67%, 216.67% (P<0.001) in liver tissues incomparison to As+HFD treated group of rats. Aqueous TA bark ex-

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tract, alone, at increasing concentration did not show any effect onthe activity of this enzyme (Fig. 4A).

The activity of cytochrome C oxidase was found to be decreased by45.45%, 27.07% and 68.18% (P<0.001) in the heart tissues and by37.5%, 18.75% and 53.13% (P<0.001) in liver tissues of As, HFD,As+HFD treated groups of rats, respectively, compared to the activ-ity observed in the control group of rats. In As+HFD+T20,As+HFD+T40 and As+HFD+T60 treated groups of rats, the cyto-chrome C oxidase activity was found to be protected by 14.29%,185.71%, 300% (P<0.001) in heart tissues and by 4%,60%,140%(P<0.001) in liver tissues in comparison to As+HFD treated group ofrats (Fig. 4A). Aqueous TA bark extract, alone, at increasing concen-tration did not show any effect on the activity of this enzyme..

Figure 4: Protective effect of aqueous TA bark extract on changes of (A) cytochrome C oxido-reductase (Cyt C oxidoreductase) activity andcytochrome C oxidase (Cyt C oxidase) activity, (B) NO concentration of heart and liver tissues of rats of various groups during the periodof experiment. As: Arsenic treated group, HFD: High fat diet treated group, As+HFD: Arsenic and high fat diet co-treated group, T20=group treated with aqueous TA bark extract at the dose of 20mg/ml, T40= group treated with aqueous TA bark extract at the dose of 40mg/ml, T60= group treated with aqueous TA bark extract at the dose of 60mg/ml, As+HFD+T20= group co-treated with arsenic, high fat diet andaqueous TA bark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fat diet and aqueous TA bark extractat the dose of 40mg/ml, As+HFD+T60= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 60mg/ml. Thevalues are expressed as Mean ± S.E.; *P < 0.001 compared to control values using ANOVA. # P < 0.001 compared to only arsenic treatedvalues using ANOVA. ̂ P < 0.001 compared to arsenic and high fat diet co-treated values using ANOVA.

Changes in NO• concentration in mitochondria of heart and livertissues of ratThe NO• concentration in As treated group of rats was found to beincreased by 50% and 45.45% (P<0.001) in heart and liver tissues,respectively,compared to control group of rats. In case of rats, co-treated with As and HFD, the NO concentration were found to besignificantly increased than that observed in control rats, by 78.13%and 100% (P<0.001) in heart and liver tissue, respectively. InAs+HFD+T20, As+HFD+T40 and As+HFD+T60 treated groups ofrats, the NO concentrations were found to be protected by 14.04%,22.81% and 43.86% in (P<0.001) heart tissue and by 20.45%, 36.36%and 43.18% (P<0.001) in liver tissue compared to As+HFD group ofrats. Aqueous TA bark extract, alone, at increasing concentration didnot show any effect on NO• concentration in both the tissues studied(Fig. 4B).

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Liver Cyt C oxidase

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Indirect assessment of superoxide radical generation (O2-•) in rat

heart and liver tissuesWe also examined whether arsenic and high fat diet administration torats induced the generation of ROS. The results presented in Fig.5A–E clearly indicate that there was an increase in the generation of(O2

-•) in vivo following treatment of rats with arsenic and high fat diet.The activities of xanthine oxidase (XO), xanthine dehydrogenase(XDH), total enzyme activity, i.e., XO plus XDH, XO - XDH ratio andXO/XO + XDH ratio all increased significantly following arsenic andhigh fat diet treatment of rats. All these parameters were found to

beincreased when the rats were co-treated with arsenic and high fatdiet. In the As+HFD +T60 group the XO, XDH, XO+XDH activitieswere found to be protected significantly by 33.33%, 41.67%, 34.48%(P<0.001) in heart tissues and by 26.09%, 33.90%, 31.03% (P<0.001)in liver tissues than that observed in the As+HFD treated group ofrats. On treatment with aqueous TA bark extract, XO/ (XO+XDH)activity was shown to be protected further in both tissues, in com-parison with As+HFD group of animals significantly. Aqueous TAbark extract, alone, at increasing concentration did not show anyeffect on the generation of this ROS.

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Figure 5: Protective effect of aqueous TA bark extract on changes in the activity of (A) xanthine oxidase (XO), (B) xanthine dehydrogenase(XDH), (C) XO+XDH, (D) XO-XDH ratio, (E) XO/XO+XDH of heart and liver tissues of rats of various groups. As: Arsenic treated group,HFD: High fat diet treated group, As+HFD: Arsenic and high fat diet co-treated group, T20= group treated with aqueous TA bark extract atthe dose of 20mg/ml, T40= group treated with aqueous TA bark extract at the dose of 40mg/ml, T60= group treated with aqueous TA barkextract at the dose of 60mg/ml, As+HFD+T20= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 40mg/ml, As+HFD+T60= groupco-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 60mg/ml. The values are expressed as Mean ± S.E.; *P <0.001 compared to control values using ANOVA. # P < 0.001 compared to only arsenic treated values using ANOVA. ̂ P < 0.001 comparedto arsenic and high fat diet co-treated values using ANOVA.

Tissue morphological and histochemical studies

Lipid content in rat heart and liver tissuesLipid contents were found to be decreased in heart as well as in-creased significantly in liver tissues, respectively, in case of arsenic

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treated group (P<0.001). But, in case of HFD treated group of rats, thelipid content was found to be increased compared to control group.In the rats co-treated with As+HFD, the lipid content was found to beprotected from being altered both the in cardiac and hepatic tissuessignificantly (P<0.001). In both tissues, aqueous TA bark extract de-creased the lipid content in a dose-dependent manner significantly

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A B



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Figure 6: Protective effect of aqueous TA bark extract on changes of lipid contents in rat cardiac (A) and liver (B) tissues and the graphicalrepresentation of the changes of lipid contents in heart (C) and liver (D) tissues in various groups and changes of rat cardiac tissuemorphology: (E)H and E stained (40X magnification), (H) acid Sirius stained (40X magnification) and changes of rat liver tissue morphology:(F) H and E stained (40X magnification) (G) PAS staining (40X magnification) (I) acid Sirius stained (40X magnification), The imagescaptured by confocal laser scanning microscope for quantification of fibrosis of heart (J) and liver (K) tissues, the graphical representationof the changes of collagen content of heart (L) and liver (M) tissues; As: Arsenic treated group, HFD: High fat diet treated group, As+HFD:Arsenic and high fat diet co-treated group, T20= group treated with aqueous TA bark extract at the dose of 20mg/ml, T40= group treatedwith aqueous TA bark extract at the dose of 40mg/ml, T60= group treated with aqueous TA bark extract at the dose of 60mg/ml, As+HFD+T20=group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated witharsenic, high fat diet and aqueous TA bark extract at the dose of 40mg/ml, As+HFD+T60= group co-treated with arsenic, high fat diet andaqueous TA bark extract at the dose of 60mg/ml. The values are expressed as Mean ± S.E.; *P < 0.001 compared to control values usingANOVA. # P < 0.001 compared to only arsenic treated values using ANOVA. ̂ P < 0.001 compared to arsenic and high fat diet co-treatedvalues using ANOVA.

Con T20 T40 T60 As HFD As+HFD

As+HFD+T

As+HFD+T

As+HFD+T

A

B

Con T40 T20 T60 HFD As As+HFD

As+HFD+T

As+HFD+T

As+HFD+T

E

G

H

I

J

K

F

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

Con T20 T40 T60 As

HFD

As+HFD

As+HFD

+T20

As+HFD

+T40

As+HFD

+T60

Perc

ent a

rea

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

Con T20 T40 T60 As

HFD

As+HFD

As+HFD

+T20

As+HFD

+T40

As+HFD

+T60

Perc

ent a

rea

0

0 .0 1

0 .0 2

0 .0 3

0 .0 40 .0 5

0 .0 6

0 .0 7

0 .0 8

0 .0 9

Con T20 T40 T60 As

HFD

As+HFD

As+HFD

+T20

As+HFD

+T40

As+HFD

+T60

Colla

gen

volu

me

(%)

0

0 .0 1

0 .0 2

0 .0 3

0 .0 4

0 .0 5

0 .0 6

0 .0 7

0 .0 8

Con T20 T40 T60 As

HFD

As+HFD

As+HFD

+T20

As+HFD

+T40

As+HFD

+T60

Colla

gen

volu

me

(%)

(P<0.001) (Fig. 6A-D). Aqueous TA bark extract, alone, at increasingconcentration did not show any effect on the lipid content of boththe tissues studied.

Studies using tissue sections stained with hematoxylin – eosin aswell as PAS stainsTissue morphological studies revealed that arsenic (As) mediate dam-

age to cyto-architecture of both cardiac and liver tissues. Degenera-tive changes in cardiac tissue and necrosis in liver tissue can beclearly demonstrated from the HE stained cardiac and liver tissue andPAS stained liver. Mild dilatation of central vein, mild inflammatorycell infiltration in portal tract and mild congestion of sinusoids andglycogen depletion were observed. In case of high fat diet treated

*

*

*

*#^*#^

*#^*#^

*

*

*

* * *

**

*

D

C

L

M



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Figure 7: Scanning electron micrograph (X6000) of heart and liver tissues of rats of various groups; As: Arsenic treated group, HFD:High fat diet treated group, As+HFD: Arsenic and high fat diet co-treated group, T20= group treated with aqueous TA bark extract at thedose of 20mg/ml, T40= group treated with aqueous TA bark extract at the dose of 40mg/ml, T60= group treated with aqueous TA barkextract at the dose of 60mg/ml, As+HFD+T20= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 20mg/ml, As+HFD+T40= group co-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 40mg/ml, As+HFD+T60= groupco-treated with arsenic, high fat diet and aqueous TA bark extract at the dose of 60mg/ml. The values are expressed as Mean ± S.E.; *P <0.001 compared to control values using ANOVA. # P < 0.001 compared to only arsenic treated values using ANOVA. ̂ P < 0.001 comparedto arsenic and high fat diet co-treated values using ANOVA.

creased and in case of hepatic tissues the collagen contents werefound to be decreased (P<0.001). Aqueous TA bark extract, alone, atincreasing concentration did not show any effect on tissue collagen(Fig.6H-M).

Studies on rat cardiac and hepatic tissue surface morphology byScanning Electron Microscopy (SEM)It was observed that in both heart and liver tissues of As treatedgroup, there was presence of furrow and a fuzzy appearance wasnoted. In HFD group of rats, in case of heart, there was mild furrowformation and in liver tissue, accumulation of large amount of fat hadbeen observed. In As+HFD co-treated group of rats, the fat deposi-tion was found to be increased and more prominent furrows wereobserved. However, in aqueous TA bark extract protected group ofrats, the fuzziness and furrows were found to be disappeared withrestoration of normalcy (Fig. 7). Aqueous TA bark extract, alone, atincreasing concentration did not show any effect on surface mor-phology of both the tissues studied.

group prominent dilatation of central vein, sinusoidal congestion,focal necrosis of hepatocytes and mild inflammatory cell infiltrationwere observed in HE stained liver tissues. High amount of collagendepletion can be observed by PAS stained liver tissue. These dam-ages became more prominent in case of As+HFD co-treated group ofrats. But, HE and PAS stain revealed that aqueous TA bark extractsuccessfully protected the tissue morphology and with increasingdose, the protection was found to be almost complete (Fig. 6E-G).Aqueous TA bark extract, alone, at increasing concentration did notshow any effect on tissue morphology.

Studies on collagen content by Sirius red stainUnder confocal microscopy, with the study of Sirius red stained tis-sues, it had been revealed that, collagen content in As, HFD andAs+HFD groups decreased in heart tissue and increased in liver tis-sue significantly (P<0.001). In heart tissues of the As+HFD+TA co-treated groups of rats the collagen contents were found to be in



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of oxidative stress. Arsenic may induce oxidative stress by enhanc-ing tissue LPO and by altering the antioxidant system in the organs.Increased oxidative stress due to accumulated fat leads to deregu-lated production of adipocytokines and selective increase in ROSproduction43.

A decrease in the activity of Cu–Zn SOD can be owed to an enhancedsuperoxide production during arsenic metabolism44. Exposure to ar-senic decreased the catalase activity45. Wang and Huang, (1994)showed that arsenic induced increase of micronuclei by overproduc-tion of H2O2

46. Catalase catalyzes the removal of H2O2 formed duringthe reaction catalyzed by SOD.

The depletion of reduced glutathione level in heart and liver of ratsafter exposure to arsenic may be accounted for by the decrease in GRactivity. Arsenic can inhibit the activity of GR47. The inhibition maybe due to the interaction of trivalent arsenic with critical thiol groupsin GR molecules. GPx reduces lipid hydroperoxide into lipid alcohols;this enzyme is coupled with GR. GSH serves as a substrate for GPx.Xanthine oxidoreductase, under normal conditions, exists in dehy-drogenase form and uses NAD+ and there is no or very little produc-tion of superoxide anion. Under ischemic conditions, there is deple-tion of ATP and subsequent loss of membrane Ca2+ gradient. In-creased Ca2+ levels activates Ca2+ dependent proteases which causeselective proteolysis of the dehydrogenase to convert it into xan-thine oxidase (XO) which acts both on hypoxanthine and xanthine atthe expense of molecular oxygen to produce superoxide anion freeradical48. Thus, XO in oxidative stress conditions may play an impor-tant role in contributing free radical mediated damage. The genera-tion of ROS in heart and liver tissues was aggravated after the co-treatment of rats with arsenic and high fat diet by the endogenousformation of superoxide anion free radical and .OH radical as evidentfrom our present studies.

Mitochondria are the major source of ROS production in cells49. Inour study, we found that the activities of pyruvate dehydrogenaseand the Kreb’s cycle enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinate dehydrogenase weredecreased after treatment of rats with arsenic.

The impairment of electron transfer through NADH:ubiquinone oxi-doreductase (complex I) and ubiquinol:cytochrome c oxidoreductase(complex III) may induce superoxide formation. Mitochondrial pro-duction of ROS is thought to play an adverse role in many pathologicdisorders. In our present study, copper-ascorbate induced oxidativestress inhibits NADH cytochrome c oxidoreductase and cytochromec oxidase enzymes of ETC. The activities of these enzymes were

DISCUSSIONOxidative stress is generally defined as an imbalance that favors theproduction of ROS over antioxidant defenses; however, the precisemechanisms by which ROS cause cellular injury remain elusive. Thecurrent study has shown that the aqueous bark extract of Terminaliaarjuna can protect high fat diet-induced aggravation of arsenic-in-duced cardiac and liver injuries in rats. The central hypothesis is thathigh fat diet could enhance arsenic-induced hepato-fibrogenesis aswell as obesity and hypertension which are also the two major riskfactors that lead to increased incidence of cardiac diseases includingcoronary artery disease, heart failure and cardiomyopathy33,34. Con-cerns over the safety of synthetic antioxidants have shifted the glo-bal interests towards exploration of antioxidant compounds from natu-ral sources35. A plethora of phenolics extracted from several plantspecies have been reported to possess strong antioxidant activi-ties36. Phenolics are ubiquitously present in plants, and when plantsare consumed as foods, these phytochemicals contribute to the in-take of natural antioxidants in the diets of human as well as animals.Out of all the phenolics, the flavonoids belong to a large family ofcompounds with different degrees of hydroxylation, oxidation andsubstitution. Our previous study showed that the aqueous extract ofTA contains tannins, cumarins and terpenoids and this extract re-vealed a higher amount of polyphenol which may be responsible forantioxidant activity of this extract7. Arsenic itself reduced body weightby interfering with various metabolic pathways. When high fat diet isadministered along with arsenic treatment, body weight also gradu-ally reduced further. Arsenic toxicity has been reported to cause car-diac hypertrophy and reduction of body weight37. Activities of bothAST and ALT were significantly higher in arsenic treated rats indicat-ing liver dysfunctions and increased activity of AST indicated car-diac disorders38,39. Increment of the activities of AST and ALT in se-rum may be mainly due to the leakage of these enzymes from the livercytosol into the blood stream, which gives an indication on the hepa-totoxic effect of arsenic40. High fat diet also elicited the activities ofAST and ALT41.

LDH oxidizes lactic acid to pyruvic acid, using NAD+ as the coen-zymes. It can also catalyze the reverse reaction. Of the five isozymes,LDH 1 predominates in the cardiac muscle and LDH 5 in the liver. Anysignificant increase in serum LDH 1activity specifically indicatesmyocardial damage and serum LDH 5 increases during liver diseasessuch as hepatic dysfunction due to oxidative stress42.

The arsenic-induced cardiac and liver tissue damages, in our experi-mental situation, is due to generation of oxidative stress as is evidentfrom elevated levels of tissue LPO and protein carbonyl content anda decreased tissue level of reduced GSH, the well known bio-markers



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Figure 8: Schematic diagram representing the antioxidant mechanism(s) of protection of aqueous TA bark extract against of arsenic-induced aggravation of high fat diet-induced oxidative damages in rat heart and liver tissues.

CONCLUSIONFrom the findings of our present study it can be said that co-treat-ment of rats with aqueous extract of bark of Terminalia arjuna pro-vides protective effects against arsenic-induced aggravation of highfat diet-induced oxidative stress mediated damages in cardiac andliver tissues in a dose-dependent manner and such protection may beexerted via various antioxidative mechanism(s).

ACKNOWLEDGEMENTMD is supported under Women Scientists Scheme-A (WOS-A), De-partment of Science and Technology, Govt. of India. AC is supportedby funds available to her from UGC Minor Research Project, Govt. ofIndia. AG is a UGC-JRF at University of Calcutta. AKG is an extendedSRF under DST-PURSE Program at University of Calcutta. GB andSM are supported from the funds available to Dr. DB from Teacher’sResearch Grant (BI 92) of University of Calcutta. Dr. Dr. SKP is sup-ported from the funds available to them from their respective insti

found to be protected when the mitochondria were co-treated withaqueous TA bark extract. This strongly indicates that the extract pos-sesses either some chelating property or is simply able to protectmitochondria from getting damaged due to ROS production, by itselfbeing a quencher of reactive oxygen species.

In our present study, it was established that the oxidative stress-induced damages due to arsenic and high fat diet co-treatment werefound to be protected when the rats were co-treated with aqueous TAbark extract. This is also clear from our tissue morphological as wellas histochemical studies of both the cardiac and hepatic tissues,using light, confocal and scanning electron microscopy. This, fur-ther, strongly indicates that the extract possesses either some chelat-ing property or is simply able to protect heart and liver tissues of ratfrom getting damaged due to ROS production, by itself being aquencher of reactive oxygen species or by various antioxidantmechanism(s) (Fig. 8).

Aqueous extract of bark ofTerminalia arjuna

Aggravation

Inhibition


Aggravation

Inhibition


Aggravation

Inhibition



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Source of support: UGC,India; Conflict of interest: None Declared

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